DNA encoding a cell membrane glycoprotein of a tick gut

TECHNICAL FIELD

This invention relates to an antigen isolated from the cattle tick

Boophilus microplus

and to the gene coding for that antigen and to the protein product of that gene. The antigen when used in part or in entirety as an immunogen administered to cattle as a vaccine results in the production by the cattle of an immune response which is capable of damaging ticks feeding on vaccinated cattle to such an extent that the survival of such ticks is decreased and/or the reproductive capacity of the ticks is decreased to such an extent that the antigen coded for by the gene can be used as an effective vaccine against said ticks.

BACKGROUND ART

On first infestation with ticks such-as the cattle tics,

Boophilus microplus

, animals such as cattle are very susceptible to the parasite. Typically about 50% of the tick larvae which attach, complete the full life cycle to eventually drop off as engorged adults. On prolonged exposure to the parasite, cattle acquire some degree of immunological resistance to it, but this resistance reaches a relatively stable level at which economically important losses to cattle production still occur. The losses to production are largely due to losses of blood and tissue fluid taken up by the parasite during feeding. Additional losses are due to the hypersensitive or allergic response which animals develop to tick salivary and cement antigens in conjunction with natural immunity, a condition known as tick worry.

A large number of approaches are used to control ticks. The most widely used is treatment of cattle with acaracides—chemicals which kill ticks. This approach has several short comings. For example resistance to the chemicals arises in the tick population and new classes of chemicals must be introduced frequently. The chemicals have little residual effect so cattle must be treated frequently in order to control the ticks effectively. The chemicals may have detrimental effects on the cattle, personnel and the environment. A second method for control of ticks is to breed for host resistance. Zebu breeds and Zebu cross breeds are more resistant to ticks than the highly susceptible British breeds. However Zebu crosses have behavioural problems, are less productive than pure British breeds and, even with the use of chemicals, the degree of resistance to ticks is far from ideal. Other methods of tick management such as pasture spelling and tick eradication present practical problems in most cattle producing areas throughout the world. An effective vaccine against ticks would provide a highly attractive alternative to the currently available methods of tick control.

Intermittent attempts have been made in the past to immunise animals against ticks. (1-5, see 13 for review). The majority of these studies have used tick-host systems in which strong immunity seems to develop naturally, and have usually used laboratory animals as hosts. Usually the effects observed have been some reduction in engorgement weights and egg masses of adult ticks and some decrease in the viability of those eggs (1-5) although in two reports some decrease in the viability of engorging adults has been reported (3,4). Many of these studies have used antigens derived from salivary glands in order to attempt to mimic natural immunity. However it is unlikely that a vaccine which mimics natural immunity would be of great commercial benefit due to the economic losses which still occur once natural immunity has been expressed and the deleterious effect of hypersensitivity responses to ticks.

The alternative approach is to vaccinate animals with “concealed” or “novel” antigens, “Concealed” or “novel” antigens are, in this context, components of the parasite which can be used to raise a protective immune response in animals when used (in a partially or fully purified form) to vaccinate those animals, but are antigens which are not involved in naturally acquired immunity.

The successful vaccination against ticks using concealed or novel antigens has been reported (2,5). Animals were immunised with extracts of whole ticks or tick midgut. Immunization led to reductions in tick engorgement weights, feeding period, egg masses and egg viability but no significant increase in tick mortality was observed. However, the antigen fractions used in these experiments were so complex that it was not possible to identify the individual tick antigens which were responsible for the effects noted and the reasons for the effects were not investigated in detail.

In a recent patent application (Australian Patent Application No. 59707/86), claims are made that antigens derived from the synganglia of ticks can act as effective vaccines against tick infestation. However, there is no evidence presented in that patent that synganglia antigens can be effective alone. In this work dissected guts and synganglia were isolated, the gut cells were lysed, centrifuged and both the supernatant and pellet were used to vaccinate the same animals together, in some cases, with a cell suspension of synganglia. All cattle in the experiments reported were vaccinated with tick gut components and some received synganglia in addition. Therefore, it is clearly implicit in the experimental design that gut damage as a result of an immune response against gut components of ticks such as the gut cell antigens described herein and in the CSIRO patent application (45936/85), is an essential prerequisite for any secondary protective effects which may possibly result from an immune response against synganglia-specific antigens.

In all of the examples cited above, the tick extracts which were used to vaccinate the animals were extremely complex. In the majority of the reports the fractions used were homogenates of tick organs and in some cases, the pellets derived therefrom by centrifugation. In this and the other studies, no data on the complexity of the fractions is presented but it is certain that they must contain many hundreds and probably thousands of components. In the one study where any purification and characterization of the protective fraction was carried out (Australian patent application No. 45936/85) the most highly purified fraction, GF ⅚ was still very complex as will be shown below and it was not possible from this work to identify the individual component(s) of this fraction which were responsible for the protective immune response. In the present invention one such antigen is purified and characterized.

Boophilus microplus

presents a particularly challenging problem. Since the naturally-acquired immunity is only partially effective, duplication of natural immunity by artificial immunization would be of comparatively little commercial value.

Boophilus microplus

is a parasite of cattle and does not feed readily on laboratory animals. The possibility of inducing “unnatural immunity” to

Boophilus microplus

has been examined and shown to be possible (6, 7, 8, Australian Patent Application No. 45936/85). The practical exploitation of this, however, would require as a first step the isolation of the antigen or antigens responsible, and as a second step, the development of means by which the effective antigens could be produced in quantities which would be sufficient for commercial uses.

The initial steps in the purification of the antigens in question and the demonstration of the efficacy of these antigens has been described previously (Australian Patent Application No. 45936/85). Briefly, ticks removed from cattle were disrupted, and sonicated, the cuticles and debris removed by low speed centrifugation, the supernatant was subjected to high speed centrifugation at 100 000×g for 1 hour, the membrane enriched pellet was extracted with a non-ionic detergent, the extract was subjected sequentially to chromatography on Sephacryl S-300 columns, broad range isoelectric focussing, narrow range isoelectric focussing and gel filtration chromatography on HPLC. At each step, fractions obtained were tested for efficacy as immunogens and the most highly protective fractions subjected to the next purification step. The most highly protective antigens were thus identified as being membranous, possessing an isoelectric point (pI) of between 5.05 and 5.65 and molecular weights in the range 205 to 79 kilodaltons. Other less highly protective fractions were also described and are of interest in both this and the preceding Australian Patent Application 459361/85.

Further development of the purification procedure as described herein has enabled the most highly protective antigens to be more clearly defined and characterised more precisely and has enabled animals to be vaccinated with more highly purified immunogen preparations. One such antigen has been purified to near homogeneity and it has been shown that when cattle are vaccinated with this tick component an immune response is generated in those cattle which results in the death of the majority of ticks used to challenge those vaccinated animals. The antigen isolated from ticks has been shown to be a glycoprotein with a molecular weight or approximately 89 kilodaltons and an isoelectric point in the range of 5.30 to 5.67. The method for the purification of this glycoprotein (referred to hereafter as the WGL

+

antigen or WGL

+

) has been improved and a method is disclosed herein which results in a much larger yield of the antigen than could be obtained by the method previously described (Aust. Patent Application No. 45936/85). During this and previous work, other fractions which give protection have been identified.

Having devised means by which the WGL

+

antigen can be obtained in larger amounts (not sufficient for commercial uses), experiments have been performed to analyse the structure of parts of the protein portion of the antigen. The purified preparation was reduced and carboxy-methylated and digested with endoproteinase lys-C. The peptide fragments so produced were purified and the partial amino acid sequence determined for some peptides. This amino acid sequence data has enabled the design of oligonucleotides which have been used to isolate bacterial cells containing cDNA coding for the WGL

+

antigen.

Analysis of the DNA from these bacterial cells leads to the unambiguous identification of the gene coding for one protective antigen and the production of recombinant proteins which can be used as effective vaccines against ticks. These developments are the subject of the present invention.

DEFINITIONS

Whilst the invention provides products and processes suitable for the protection of cattle against tick infestation, it is to be understood that the principles of the invention can be equally applied to the protection of other animals such as horses, deer, goats, sheep, dogs, cats and pigs against tick infestation.

It is recognised that the tick population worldwide is genetically diverse as is the case for all organisms which reproduce sexually. Each individual of a population differs subtly from the others in the population and these differences are a consequence of differences in the sequence of the DNA which each individual inherits from its parents.

Further, random mutational events which can occur in either sexually or asexually reproducing organisms are a further source of genetic variation.

Thus for each gene encoding a particular protein, there are likely to be differences in the sequence among the population of individuals.

Such related molecules are referred to herein as homologues of antigens according to the invention and to the extent that they fulfill the functions of immunogens as defined herein they are included within the scope of the invention.

Homologous antigens may be defined as antigens related by evolution but not necessarily by function. Similar but not necessarily identical DNA or protein sequences may be provided. It should be noted however that function in this sense relates to the natural in vivo function of the protein.

Illustration of this point is provided by considering:

1. WGL

+

from

Boophilus microplus

and other tick species

2. WGL

+

from variants or different individuals of the

Boophilus microplus

population

3. WGL

+

and related gut cell plasma membrane glycoproteins from ticks which are homologues of the WGL

+

antigen defined herein.

It is stressed that for the purposes of this invention, homologues include only those WGL+ related plasma membrane glycoproteins which function as immunogens as defined herein.

Such homologous WGL

+

related plasma membrane glycoproteins may exist in the tick population worldwide and will be capable, when incorporated into a vaccine, of eliciting in animals vaccinated with those antigens an immune response which is capable of killing ticks, by damaging tick gut cells and which additionally results in a reduction in tick engorgement weights or otherwise damaging the surviving ticks in such a way that for example egg production by those ticks is decreased to such an extent that the vaccine can be used commercially agains infestation by tick species such as Boophilus spp, Haemaphysalis spp, Otobius spp, Rhiphicephalus spp, Ambylomma spp, Dermacentor spp, Ixodes spp and Hyalomma spp, and especially from

B. annulatus, B. decoloratus, Otobius megnini, Rhiphicephalus appendiculatus, Dermacentor andersoni, D. variabilis, Haemaphysalis longicornis, Ambylomma variegatum

and

Ixodes holocyclus.

Further, it should be recognised that it is possible to generate chemicals which are not related to the WGL

+

antigen by evolution or necessarily by structure but which may serve as immunogens to generate an immune response against protective epitopes on the WGL

+

antigen and thereby act as effective vaccines. These molecules are referred to herein as analogues and to the extent that they fulfill the functions of immunogens as defined herein, they are included within the scope of the invention. Such analogues include chemically synthesized oligopeptide molecules with sequences corresponding to portions of the amino acid backbone of the WGL

+

molecule, oligopeptides which when used as immunogens elicit an immune response which recognises native WGL

+

antigen in ticks, carbohydrate structures from whatever source which when used as antigens elicit an immune response which recognises the WGL

+

antigen in ticks, and anti-idiotype antibodies raised against the variable region of antibodies which recognise the epitope(s) of the WGL

+

antigen.

DISCLOSURE OF THE INVENTION

In a first embodiment the invention provides an immunogen comprising an antigen derived from a tick species or ticks cell line which antigen is capable of inducing immunity to tick infestation of a mammalian host to which said immunogen has been administered characterised in that said immunity results in the mammalian host producing an immune response which is capable of damaging the plasma membrane of the gut cells of ticks feeding on said host to such an extent that the majority of said ticks fail to survive to adult stage or surviving ticks become red in colour and the reproductive capacity of said surviving ticks is substantially decreased wherein said immunogen includes immunogens displaying similar immunological activity to said antigen including parts, analogues, homologues, derivatives and combinations thereof of said antigen.

Preferably the antigen is derived from

Boophilus microplus.

In a preferred embodiment the immunity induced is immunity to infestation by a Boophilus species.

More preferably the immunity induced is immunity to

B. microplus

infestation.

However, immunity may also be induced to other species of ticks, including Haemaphysalis spp, Otobius spp, Rhiphicephalus spp, Ambylomma spp, Dermacentor spp, Ixodes spp and Hyalomma spp and especially to other species of Boophilus such as

B. annulatus

or

B. decoloratus.

Of the other species of ticks against which immunity can be induced preferred species include

Otobius megnini, Rhiphicephalus appendiculatus, Dermacentor andersoni, D. variabilis, Haemaphysalis longicornis, Ambylomma variegatum

and

Ixodes holocyclus.

By immunization with related antigens isolated from other species of ticks including Boophilus spp, Haemaphysalis spp, Otobius spp, Rhiphicephalus spp, Ambylomma sop, Dermacentor spp, Ixodes spp and Hyalomma spp., immunity to infestation by other ticks may also be induced. Preferred species from which the related antigens are isolated include

B. annulatus, B. decoloratus, Otobius megnini, Rhiphicephalus appendiculatus, Dermacentor andersoni, D. variabilis, Haemaphysalis longicornis, Ambylomma variegatum

and

Ixodes holocyclus

. By protecting against infestation with ticks, the antigen may also provide protection against diseases caused by agents such as

Babesia bovis, Babesia bigemina, Anaplasma marginale, Cowdria ruminantium, Theileria parva parva, T. parva lawrencii, T. annulata

and

T. hirci.

In a second embodiment the invention provides a polynucleotide sequence comprising a first polynucleotide sequence which acts as a coding sequence for amino acid sequences of an immunogen according to the invention, a polynucleotide sequence which hybridises to said first sequence or a polynucleotide sequence related to said first sequence or hybridising sequence by mutation including single or multiple base substitutions, deletions, insertions and inversions.

Preferably the polynucleotide sequence is a DNA sequence.

In a further preferred form of the invention the DNA sequence is a cDNA sequence.

The DNA sequence coding for part or all of the protective antigen isolated from

Boophilus microplus

can be used in DNA hybridization experiments to identify related DNA sequence from other species of ticks. These latter DNA sequences can be constructed by genetic engineering techniques to obtain the expression by bacterial or eukaryote cells such as yeast, plant, insect, tick or mammalian cell lines of all or parts of the antigen from other species of ticks and provide an effective vaccine against those tick species which are responsible for morbidity or economic losses to man or morbidity and productivity losses to animals.

The invention also provides a recombinant DNA molecule which comprises at least one DNA sequence according to the invention and vector DNA.

In a preferred form the vector DNA comprises plasmid, phage or viral DNA.

Preferred vectors include lambda gt11, pUR290, pUR291, pUR282, pUK270, pUC8, pUC9, baculovirus, pZipNeo, an SV40 based vector, lambda gt10, an EMBL vector, pBR327, pBR329, or pBR329 containing a par locus.

The invention further provides a transformant cell line, said transformant carrying at least one recombinant DNA molecule according to the invention.

In a further embodiment the invention provides a vaccine comprising at least one immunogen according to the invention together with a pharmaceutically acceptable carrier, adjuvant, immunopotentiator or diluent.

In accordance with the present invention an antigen derived from a tick species which antigen is capable of inducing a highly significant degree of immunity to tick challenge when used to vaccinate cattle has been purified and characterised. Further, bacterial cells which contain DNA sequences derived from a tick species have been produced and those bacterial cells which contain DNA sequences encoding portions of the tick protective antigen have been iaentified. The DNA sequence of the tick gene encoding that antigen has been determined, the resulting DNA sequence has been used to identify further bacterial cells containing related genes from other species of ticks. Expression of the antigen or portions of the antigen by bacteria or other mnicroorganisms or by eukaryotic cells such as yeast, insect, tick, plant and mammaliam cells grown in vitro provides a large amount of the antigen effective as an immunogen for the protection of cattle and other domestic animals against infestation by

Boophilis microplus

and other tick species.

The invention also includes within its scope the epitope or the epitopes of immunogens of the invention which are responsible for the protective immune response. These epitopes may be created artificially by the synthetic production of oligopeptides which contain sequences of portions of the protective antigen which can be predicted from the results of immunochemical tests on fragments of the protective antigen produced in bacteria or generated as a result of chemical or enzymatic cleavage of the native or recombinant peptides and includes relevant epitopes from those protective antigens, oligopeptides, idiotypes and anti-idiotypes which resemble or recognise those epitopes which may have protective effects when used to actively or passively immunise animals.

In a further embodiment the invention provides methods for the purification of immunogens according to the invention and particularly protective antigens derived from ticks.

The invention provides a process for the preparation of an immunogen according to the invention which process comprises a chromatographic step performed on wheat germ lectin or on a lectin having the same or similar terminal sugar specificity as wheat germ lectin.

Preferably the invention provides a process for the preparation of an immunogen according to the invention said process comprising extracting membrane enriched fractions obtained from homogenised ticks with detergent and subjecting the solubilised material to wheat germ lectin sepharose chromatography and elution with N-acetylglucosamine or chromatography using a lectin having the same or similar terminal sugar specificity to wheat germ lectin.

Preferably said detergent is selected from NP40, an NP40 derivative, Zwittergent 3-14 or SDS.

The process may further comprise Concanavalin-A sepharose chromatography and elution with methyl-α-D-mannopyranoside, a preparative isoelectrofocussing step or size exclusion chromatography.

In a preferred form said methods include preparation of an homogenate of ticks, centrifugation to produce membrane enriched fractions, treatment of those membranes with detergents such as Zwittergent 3-14, chromatography of the detergent soluble material on lectin affinity columns such as wheat germ lectin-Sepharose 68 columns, separation of the lectin binding antigens by isoelectric focusing in buffers containing detergent such as Zwittergent 3-14, chromatography of these antigens by size exclusion HPLC on columns such as Bio-Sil TSK 4,000 and PP 300 SW columns in series in buffers containing detergents and analysis of various fractions produced by SDS-polyacrylamide gel electrophoresis.

The invention also provides an immunogen produced by a process according to the invention. Included within the scope of an immunogen produced by a process according to the invention are those immunogens produced as a result of purification schemes performed on native materials and recombinant or synthetic immunogens produced as a result of recombinant DNA or chemical synthetic methods respectively.

In a further embodiment, the invention provides examples of methods for the treatment of the purified antigens with proteolytic enzymes such as endo lys-C, the purification of oligopeptide fragments produced as a result of proteinase digestion by HPLC chromatography on columns such as Aquapore RP-300 C-8 or Aquapore RP-318 columns and determination of the amino acid sequence or some of the oligopeotides so produced and purified.

The invention further provides the peptide sequence information for such peptide fragments including:

FRAGMENT NUMBER

F1 SEQ ID NO:1

(K) D P D P G K

F2 SEQ ID NO:2

(K) W Y E D (G) V L E A I (X) T S I G K

F3 SEQ ID NO:3

(K) (X) Q A C E (H) P I G E (W) C M M Y P K

(C)

F4 SEQ ID NO:4

(K) E A G F V Q K

(V) (V) (I)

F5 SEQ ID NO:5

(K) G (P) (D) G Q (C) I N (A) (C) K

(G)

F6 SEQ ID NO:6

(K) A (D) V S T N E N E Q L E Q A D K

(G)

F7 SEQ ID NO:7

(K) S (D) T Q (X) I D H I S K

(A) (A)

F8 SEQ ID NO:8

(K) D Q E (Y) (Y) Y

F9 SEQ ID NOS:9 and 10

[(K) C P C D N M Y F N A A E E I G C I E ]

A N Q C P P D T R R G E I G C I E

F10 SEQ ID NOS:11 and 12

[(K) A P R Q N M Y F N A A E E I G C I E ]

[ C N C D C P P D T R P G E I G C I E ]

F11 SEQ ID NO:13

(K) W Y E D R V L E A I R T S I G K

F12 SEQ ID NO:14

(K) E S S I C X D F G N E F C R N A E C E V V P

F13 SEQ ID NO:15

(K) T R E C S Y G R C V E S N P S K

F14 SEQ ID NO:16

(K) A Y E C T C P R A F T V A E D G I S/H C K

[(K) D E V D N A S/H L V C Q N A ]

F15 SEQ ID NOS:17-19

[(K) N V L Q S D G C G P Y ]

[(K) C L N P R P/L R L K H/S ]

F16 SEQ ID NO:20

(K) A X V L C E X P

C

G

F17 SEQ ID NO:21

(K) L Q A C E H P I

NOTE: Amino acids which were ascribed with low confidence are bracketed. X indicates no amino acid could be ascribed to this position; [ ] denotes mixed sequences.

In a preferred embodiment of the invention, these peptide sequences are:

F1 SEQ ID NO:1

K D P D G K

F2, F11 SEQ ID NO:13

K W Y E D R V L E A I R T S I G K

F3, F17 SEQ ID NO:22

K L Q A C E H P I G E W C M M Y P K

F4 SEQ ID NO:23

K E A G F V C K

F5 SEQ ID NO:24

K G P D G Q C I N A C K

F6 SEQ ID NO:25

K A G V S C N E N E Q S E C A D K

F8 SEQ ID NO:26

K D Q E A A Y K

F9, F10 SEQ ID NOS:27-29

K C P R D N M Y F N A A E K

K A N C Q C P P D T K P G E I G C I E

K A N C Q C P P D T R P G E I G C I E

F12 SEQ ID NO:30

A E S S L C S D F G N E F C R N A E C E V V P G

F13 SEQ ID NO:15

K T R E C S Y G R C V E S N P S K

F14 SEQ ID NOS:31 and 32

K A Y E C T C P S G S T V A E D G I T C K

K A Y E C T C P R A F T V A E D G I T C K

F15 SEQ ID NO:33

K N L L Q R D S R C C Q

F16 SEQ ID NO:34

K G T V L C E C P

The invention also provides examples of methods which can be used to design from the amino acid sequence data oligonucleotide sequences which are suitable for use as hybridization probes to identify nucleic acids sequences (DNA or RNA) coding for the polypeptide containing those amino acid sequences, methods for the construction of bacterial cells containing complementary DNA and genomic DNA fragments from ticks, the use of the oligonucleotides to identify bacterial cells containing complementary and genomic DNA fragments coding for that antigen, the DNA sequence of one such cDNA fragment, methods by which recombinant DNA technology can be used to produce bacterial or eukaryote cells which synthesize the protein or parts of that protein and methods for culturing those cells and for purification of the tick antigen or parts thereof to be incorporated into effective vaccines against ticks.

In a preferred model, the mechanism of action of the vaccine is one in which an immune response is generated in vaccinated animals which results in ticks feeding on those animals ingesting components of the host immune system such as antibodies which interact with the surface of tick gut cells and either alone, or together with other factors in the host blood such as components of complement result in damage occuring such as lysis of the tick gut cells which in turn results in the ticks becoming unable to effectively digest blood, the tick gut becoming permeable to host blood components, to such an extent that host blood components such as albumin, haemoglobin, immunoglobulin and blood cells can be identified in the haemoloymon of the ticks and the ticks appear red in colour. This gut damage in turn results in the death of the majority of the ticks feeding on vaccinated animals before they reach engorgement stage and those few which may survive are so badly damaged that their engorgement weight is decreased and/or reproductive capacity is impaired (6,7,8).

The invention also relates to antibodies generated against epitopes on the antigens according to the invention (so called idiotype antibodies) and to antibodies generated against the variable region of those first antibodies, (so called anti-idiotype antibodies) which mimic the protective epitopes on the antigen and may be used as effective vaccines in either passive protection of animals (idiotypes) or active immunization of animals (anti-idiotypes) and thereby result in effective protection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

is a schematic representation of the method for the isolation of the “WGL post IEF pool” and “LL Post IEF pool”.

FIG. 2

is a schematic representation of the fractionation procedure for the isolation of “LL

+

antigen”.

FIG. 3

is a schematic representation of the fractionation procedure for the isolation of “WGL

+

antigen”.

FIG. 4

is a simplified schematic representation of the purification procedure for the isolation of WGL

+

and LL

+

antigens.

FIG. 5

shows purity of the WGL

+

antigen.

FIGS.

6

-

6

(

2

) show the DNA sequence for the WGL+ gene (bases 1-2012 of SEQ ID NO:55 and SEQ ID NO:56).

FIG. 7

shows the translated amino acid sequence (residues 11-688 of SEQ ID NO: 57) for the WGL+ antigen deduced from the DNA sequence.

FIGS. 8A-8B

show a restriction enzyme map for part of the WGL+ gene showing an example of the expression strategy.

FIGS. 9A-9B

show a SDS polyacrylamide gel and immunoblot demonstrating expression of WGL+ by bacteria.

FIGS. 10A-10C

show hybridization of the

Boophilus microplus

DNA coding for WGL+ to DNA from other tick species.

FIGS.

11

(A)-

11

(C) show the DNA sequence SEQ ID NO:58 for the YBm017 gene and the translated amino acid sequence SEQ ID NO:59 deduced from the DNA sequence. YBm017 is an Australian isolate (Yeerongpilly, Queensland) of

Boophilus microplus.

FIGS.

12

(A)-

12

(C) show the DNA sequence SEQ ID NO: 60 for the YBm22M8 gene and the translated amino acid sequence SEQ ID NO:61 deduced from the DNA sequence. YBm22M8 is an Australian isolate (Yeerongpilly, Queensland) of

Boophilus microolus.

FIGS

13

(A)-

13

(C) (SEQ ID NO:62) show the DNA sequence (SEQ ID NO; 62) for the Bm023 gene and the translated amino acid sequence SEQ ID NO:63 deduced from the DNA sequence. Bm023 is another Australian isolate of

Boophilus microplus.

FIGS.

14

(A)-

14

(C) show the DNA sequence SEQ ID NO:64 for the Vbm021 gene and the translated amino acid sequence SEQ ID NO:65 deduced from the DNA sequence. VBm021 is a Venezuelan isolate of

Boophilus microplus.

FIGS.

15

(A)-

15

(C) show the DNA sequence SEQ ID NO:66 for the MexBm86 gene and the translated amino acid sequence SEQ ID NO:67 deduced from the DNA sequence. MexBm86 is a Mexican isolate of

Boophilus microplus.

FIG. 16

shows a partial DNA sequence SEQ ID NO:εfor the Ra442 gene and the translated amino acid sequence SEQ ID NO:69 deduced from the DNA sequence. Ra442 is a

Rhipicephalus appendiculatus

isolate.

BEST MODE OF CARRYING OUR THE INVENTION

The invention is further described in the following examples which are illustrative of the invention but in no way limiting on its scope.

SOURCE OF REAGENTS

Sephacryl

Pharmacia

Sepharose 6 MB

Pharmacia

Zwittergent 3-14

Calbiochem

Sephadex

Pharmacia

Brij 35

Sigma

Bio-gel

Bio Rad

Cyanogen Bromide

Sigma or Ajax

Sarkosyl

Sigma

Endoproteinase lys-c

Boehringer

Triflouroacetic acid

Pierce

HFBA

Pierce

Acetonitrile

Mallinckrodt

Columns for HPLC

Waters, BioRad, Beckman

Poly U Sepharose

Collaborative research

Oligo dT cellulose

Collaborative research

dATP, dCTP, dGTP and dTTP

Boehringer

32

P-labelled deoxynucleic acid triphosphates

Amersham

Spermidine

Calbiochem

PEI cellulose

Merck

Concanavalin A-Sepharose

Pharmacia

CNBr-Sepharose

Pharmacia

Other chemicals used were of reagent grade.

ABBREVIATIONS

HPLC

High performance liquid chromatography

SDS

Sodium dodecylsulfate

EDTA

Ethylenediaminetetraacetic acid

WGL

Wheat germ lectin

WGL 1

Wheat germ lectin bound antigen pool 1

WGL 2

Wheat germ lectin bound antigen pool 2

WGL

+

Wheat germ lectin bound antigen

WGL

−

Wheat germ lectin unbound

IEF

Iso electric focussing

LL

Lentil lectin

LL

+

Lentil lectin bound antigen

LL

−

Lentil lectin unbound

HEPES

N-2-Hydroxyethylpiperazine-N

1

-2-ethane-sulfonic acid

Endo lys C

Endoproteinase lys C

DTT

dithiothreitol

pI

isoelectric point

HFBA

heptafluorobutytic acid

BSA

bovine serum albumin

MMLV

murine maloney leukemia virus

dNTP

deoxy nucleotide triphosphate

dATP

deoxy adenosine triphosphate

dCTP

deoxy cytidine triphosphate

dGTP

deoxy guanidine triphosphate

dTTP

deoxy thymidine triphosphate

d(GCT)TP

a mixture of dGTP, dCTP, and dTTP

NAD

nicatinamide adenine dinucleotide

ATP

adenosine triphosphate

PEI

polyethyleneimine

BRL

Bethesda Research Laboratories

IBI

International Biotechnologies Inc.

A260, A280

Absorbance at 260 or 280 nm

cDNA

complementary DNA

ds

double stranded

g

gram

g

av

average gravity units

m, μ, n, p

milli, micro, nano, pico

(prefixes)

M

Molar

l

liter

U

Units of activity (restriction enzymes)

bp

base pairs

Kb

Kilobase pairs (thousand base pairs)

TLC

Thin layer chromatograph

ELISA

enzyme linked immunoabsorbent assay

BUFFERS

10 × 1st strand

0.5M Tris pH 1.5

0.75M KCl

0.03M MgCl

2

5 × 2nd strand (RNase H)

0.2M Tris pH 7.5

0.05M MgCl

2

0.1M (NH

4

)

2

SO

4

1M KCl

1.5 mm B-NAD

10 × Methylase Buffer

0.5M Tris pH 7.5

0.01M EDTA

10 × TA Buffer

0.33M Tris-Acetate pH 7.9

0.56M K-Acetate

0.1M Mg-Acetate

5 × Kinase Buffer

0.05M Tris pH 7.5

0.05M Mg Cl

2

0.05M DTT

0.5 mM Spermidine

10 × Ligation Buffer

0.3M Tris pH 8

17 mM EDTA

70 mM MgCl

2

10 mM ATP

0.1M DTT

20 ug/ml BSA

1 mM Spermidine

10 × High Salt Buffer

1M NaCl

0.5M Tris pH 7.5

0.1M MgCl

2

10 mM DTT

10 × S1 Buffer

0.3M NaAcetate pH 4.4

2.5M NaCl

10 mM ZnCl

2

TE

10 mM Tris pH 7.5

1 mM EDTA

PEI cellulose buffer

0.75M K

2

PO

4

pH 3.5

Buffer A

0.05M Tris

0.03M acetic acid

0.1M NaCl

TEAB buffer

TEAB is a solution of triethylamine equilibrated to pH7 by bubbling CO

2

through the solution. It is prepared as a 1M stock solution which is stored at 4° C. The pH is checked before use, the solution being re-equilibrated with a CO

2

pellet if required.

EXAMPLE 1

(a) Demonstration that at Least Some of the Protective Antigens are Glycoproteins.

Since the majority of plasma membrane proteins are glycoproteins, inital attempts at further characterization of tie protective antigen(s) focussed on lectin affinity.

It was found that wheat germ lectin and Concanavalin A bound to several components of tick antigen preparation B4/B5. Thus it appeared that a number of antigens in the tick preparation bear terminal N-acetylglucosamine residues and it is recognised that wheat germ lectin could be replaced in the purification scheme by other lectins with the same or similar terminal sugar specificity.

Approximately 2.1 mg of antigen B4/B5 purified by the narrow range isoelectric focussing procedure (described in Australian Patent Application No. 45936/85) was applied to a column of WGL-Sepharose 6 MB (14) in 0.05M Tris chloride buffer, 1% Zwittergent 3-14, pH8 and washed with the same buffer. Bound glycoproteins were then eluted with 100 mg/ml N-acetylglucosamine in the same buffer. Bound and unbound material was used to immunize sheep (Two vaccinations in Freunds incomplete adjuvant using five sheep per group). Induced immunity was estimated by applying freshly moulted adult ticks to the sheep and measuring the success of engorgement by the proportion of female ticks which finally engorged, relative to the number attached to the sheep skin three days after initial application of the young adults (Table 1).

TABLE 1

Immunization of Sheep with Glycoprotein Preparations

Percentage of Ticks

Group

Engorging

Controls

100, 100, 100, 100, 100

Material not binding to WGL

100, 6, 93, 100, 100

Material binding to WGL

0, 93, 0, 83, 28

It is clear that some animals in each vaccinated group were highly protected from tick challenge. Serum was obtained from each sheep in this experiment after vaccination but before tick challenge and the antibody titres of each serum sample against the antigens used in the vaccine were measured by radioimmunoassays. The animals in each group which showed tick damage had high antibody titres against the antigen preparation injected whereas those which had low titres allowed large numbers of ticks to engorge without any visible signs of damage (data not shown). It appears that protective antigens were present in both fractions used in this experiment but failure to observe tick damage with some animals was due to the failure of those animals to respond vigorously to vaccination for reasons which are currently unclear.

(b) In a subsequent experiment with sheep, fraction GF5 and 6, the more highly purified gel filtration fractions (Australian Patent Application No. 45936/85) were chromatographed on a WGL-sepharose affinity column and the specifically bound and the unbound material was used to vaccinate sheep in a similar way to that described above. Again, for some animals in each group, the immune response generated by vaccination with either fraction was capable of producing damage to ticks feeding on those animals as demonstrated by the lower numbers of viable ticks recovered from the sheep (% surviving), the percentage of those ticks which were red in colour (% damage) and the lower weight of those ticks which survived or engorged (Table 2).

TABLE 2

Number of Ticks

%

Animal

Surviving/Number

Surviving

%

Mean

Group

No.

Applied

Ticks

Damage

Weight

Controls

181

36/40

90%

0

254

182

45/50

90%

4

224

183

32/40

80%

3

214

WGL

121

27/40

67.5%

19

182

unbound

122

27/40

67.5%

41

179

123

30/40

75%

3

223

WGL

124

27/40

67.5%

52

156

bound

125

7/40

17.5%

100

11

180

9/40

22.5%

100

11

In particular the material which was specifically bound to the affinity column is to be characterized herein but the protective antigens in the inbound fraction are also clearly capable of giving protection.

(c) An experiment similar to that described above was performed in which cattle were vaccinated with material which had specifically bound and material which failed to bind to a WGL-Sepharose column (Table 3). Again, the immune response generated by both fractions following vaccination gave indications of damage to ticks feeding on vaccinated cattle. The material which failed to bind to the WGL-Sepharose column was particularly effective in this experiment.

TABLE 3

Group

Animal No.

Tick No.

% Damage

Weight (mg)

Controls

943

239

2

226

944

190

7

216

957

282

1

245

WGL bound

945

214

22

202

960

125

64

183

962

188

5

218

WGL unbound

938

19

84

148

950

10

97

164

959

25

92

140

NOTE: Tick no. in this and subsequent experiment refers to the average number of engorged female ticks dropping from each animal per day. Three weeks after vaccination, cattle are challenged with approximately 1000 larvae per day for a period of at least 16 days. When the ticks mature and engorged female ticks are observed, the engorged female ticks are collected each day and counted for a period of at least 16 days. This number is

# averaged over that period and presented in the Tick no. column. On each day during this period, the number of ticks which are visibly damaged are scored (red ticks) and that proportion listed in the % damage column. The average weight of the engorged females is also determined.

(d) Concurrently with this experiment, the material which had specifically bound to the WGL-sepharose column was fractionated on SDS polyacrylamide gels. Silver stains of these gels showed two major staining components which were excised (fractions S2 and S4) and these, as well as the intermediate portions of the gels (fractions S1, S3, S5 and S6) were used to vaccinate cattle. The most highly protective fraction was S2 (Table 4) which corresponds to one of the bands observed in stained gels which has an apparent molecular weight of approximately 80-90 kilodaltons in this gel system compared with Pharmacia and BRL molecular weight markers.

In this experiment, the number of ticks surviving on the cattle vaccinated with S2 was reduced compared with the other groups (Tick No column—the average number of engorged adult female ticks dropping from each animal per day over the 21 day period studied). In addition, the majority of the surviving ticks were red or appeared to be otherwise abnormal when examined visually (% damage) and the weight of those surviving ticks in the S2 group was reduced compared to the ticks from the animals in the other groups (Table 4).

TABLE 4

Group

Animal No.

Tick No.

% Damage

Weight

S1

947

196

10

215

951

194

1

230

963

243

30

198

S2

941

115

66

147

942

86

87

150

953

173

32

192

S3

961

166

3

212

967

240

4

243

968

193

4

233

S4

939

163

1

229

940

155

4

229

952

149

9

258

S5

937

276

3

248

955

232

2

225

956

160

5

221

S6

946

269

12

222

954

157

19

297

953

281

1

245

(e) In vitro experiments were conducted in which a range of lectins were tested to determine which were capable of reacting with the material not retained on the WGL-sepharose column. Lentil lectin was found to be reactive and therefore the material not bound to the WGL-Sepharose was fractional on a lentil lectin column(15). Cattle were vaccinated with these fractions, and the immune response generated against the material not bound to WGL-sepharose but bound to the LL-sepharose was found to result in some small indication of damage to ticks feeding on vaccinated cattle (Table 5). SDS gel analysis of this fraction shows a band which has a molecular weight which is in the same range as the S2 antigen identified in the previous experiment.

TABLE 5

Group

Animal No.

Tick No.

% Damage

Weight

Controls

990

195

0.7

252

980

220

0.7

243

979

248

0.6

252

WGL unbound

1006

183

6.2

196

LL unbound

1002

188

0.3

233

988

185

30.8

197

WGL unbound

1001

270

3.9

244

LL bound

996

267

1.1

251

994

249

16.1

206

Both fractions used in this experiment were capable of generating an immune response which was capable of giving some indication of protection in this experiment.

The lentil lectin chromatography step produced a far greater yield of material having a similar molecular weight to the S2 antigen than was produced by the wheat germ lectin chromatography step.

This similarity in molecular weight and difference in lectin affinity suggested that the molecules may have been related by a common peptide backbone but differed in glycosylation.

This was later disproved (Example 2g).

Due to the presumed similarity to S2 and greater abundance of the LL bound material it was proposed that this material be used as a starting material for further purification.

However, subsequent poor vaccination results with this material in the light of good vacation results with WGL bound material (Example 3) and demonstrated difference in amino acid composition have led to further purification schemes and cloning schemes being developed for the S2 or WGL

+

material.

EXAMPLE 2

Knowing from the above results the iso-electric point, molecular weight and lectin binding characteristics of the major protective antigen (referred to above as S2), a number of experiments were performed in order to improve the efficiency of the isolation procedure. The following method has been devised which yields at least 10 times more of the S2 antigen (later referred to as the wheat germ lectin bound antigen, WGL

+

antigen or WGL

+

) and lentil lectin bound antigen (later referred to as the LL

+

antigen or LL

+

) than the methods described in Australian Patent Application No. 45936/85.

The procedure is outlined in the flow charts (

FIGS. 1

,

2

,

3

, and

4

).

IMPROVEMENT OF THE PROCEDURES FOR ISOLATION OF THE MAJOR PROTECTIVE ANTIGEN

(a) Isolation and Extraction of Tick Membrane and Particulate Material

1290 grams of semi-engorged adult female

Boophilus microplus

were picked from cattle on the day prior to the completion of engorgement. They were homogenised in 0.5M Tris, 0.025M acetic acid, 0.1M sodium chloride, 1 mM EDTA, the homogenate strained through fine gauze and the retained material, which was mostly cuticle fragments, was rinsed with buffer. A total of 3 ml of buffer per gram of ticks was used in the extraction. The suspension of tick material was then mixed with 350 mg phenylmethanesulfonyl fluoride per liter and centrifuged at 600×g

av

for 15 min. The supernatant was then centrifuged at 20,000×g

av

for 30 min and the supernatant from that, centrifuged for 100,000×g

av

for 1 h. Precipitates were collected from each of these centrifugation steps and frozen at −20° C. until used.

The 600×g, 20,000×g and 100,000×g precipitates were thawed, suspended in buffer A (0.05M Tris, 0.03M acetic acid) and the protein concentration was measured. The suspension was diluted in buffer A containing Brij 35 to final protein and detergent concentrations of 5 and 10 mg/ml respectively. The tick material was extracted at 37° C. for 1 h then centrifuged at 3,300×g

av

for 30 min at 20° C. The precipitate was resuspended in buffer A and the protein concentration re-assayed. Extraction was repeated at the protein and detergent concentrations used before, substituting Zwittergent 3-14 for Brij 35, while the extraction time was lengthened to 90 min. The suspension was centrifuged as before and the supernatant was retained.

(b) Lectin Affinity Chromatography and Isoelectric Focussing (

FIG. 1

)

The supernatant from the Zwittergent 3-14 extraction, (3255 ml), was stirred with 90 ml of WGL Sepharose for 16 h at 20° C., filtered and the WGL-Sepharose conjugate was poured into an 18×2.5 cm column, washed with buffer A containing 1% Zwittergent 3-14 then eluted in buffer A containing 1% Zwittergent 3-14 and 100 mg/ml N-acetylglucosamine. Fractions were pooled on the basis of the A280 absorption of specifically eluted material to give wheat germ lectin bound pool 1 (WGL1).

The adsorption of the detergent supernatant with WGL-Sepharose and subsequent elution of bound material was repeated as described above to give WGL2. The two eluates were then pooled (WGL pool).

The WGL pool was dialysed against 2×2.5 liters of water, then against 0.05M Tris-chloride buffer pH7.5 containing 0.1M ammonium thiocyanate. Concanavalin A-Sepharose (Pharmacia) was poured as a 2.5×11 cm column and washed in buffer containing 0.05M Tris, 1% Zwittergent 3-14, 0.1 mM calcium chloride, 0.1 mM manganese chloride, 0.1M ammonium thiocyanate, adjusted to pH7.5 with hydrochloric acid. The WGL pool was loaded on this column, washed and the specifically bound material was eluted in the same buffer to which had been added 50 mg/ml methyl-α-D-mannopyranoside. Fractions were pooled, dialysed against water then subjected to preparative isoelectricfocussing.

Isoelectricfocussing was carried out in a flat bed of IEF Sephadex containing 1% (w/v) Zwittergent 3-14 and Pharmalyte 4-6.5 diluted 1 to 15 (v/v) for 10,000 Vhr. Individual fractions were analysed by SDS gel electrophoresis. The required protein appeared to be present in fractions with pI's of 5.3 to 5.7 though, for the sake of better purification, only those fractions with pI's of 5.4 to 5.6 were pooled to give “WGL post IEF pool”.

The Zwittergent 3-14 soluble material left after the second extraction with WGL-Sepharose was mixed with 70 ml of LL-Seoharose and stirred for 24h at 20° C., the suspension was filtered and the collected Sepharose conjugate was poured as a 2.5×14 cm column. This was then washed with Tris-acetate, 1% Zwittergent 3-14 buffer and eluted in the same buffer containing 50 mg/ml methyl-α-D-manno-pyranoside. Fractions were pooled on the basis of their A280 and dialysed against water. Further fractionation was carried out by preparative isoelectric focussing using the conditions already described for material which bound to WGL. Fractions were analysed by SDS polyacrylamide gel electrophoresis. The protein being isolated focussed over a pI range of 4.8 to 5.2 though the fractions which were pooled for further purification covered the range of 4.8 to 5.0.

The LL unbound material from the first affinity chromatography was readsorbed to LL-Sepharose and the material specifically eluted with methyl-α-D-mannopyranoside was separated by IEF. Material with the same pI range of 4.8 to 50 was pooled, then the products of the two experiments mixed to give “LL post IEF pool”.

The method for the isolation of “WGL post IEF pool” and “LL post IEF pool” is shown schematically in FIG.

1

.

(c) Hydrophobic Chromatography of LL Post IEF Pool (

FIG. 2

)

A 1.6×6.5 cm column of LL-Sepharose was equilibrated in 0.1M Tris-acetate buffer, 1% Zwittergent 3-14 pH8.0. The “LL post IEF pool” was adjusted to pH7.1 and applied to this column which was subsequently washed with buffer, then with 0.1M Tris-acetate buffer, 0.1% Brij pH7.5. Bound material was then eluted with 0.1M Tris-acetate-Brij buffer containing 50 mg/ml methyl-α-D-mannopyranoside.

Eluted material was dialysed against 0.1M Tris-acetate-Brij buffer then ammonium sulfate was added to a final concentration of 0.5M. The sample was applied to a 7.5×75 mm TSK phenyl-5-PW column which had been equilibrated in 0.1M Tris-acetate, 0.5M ammonium sulfate, 0.1% Brij, pH7.5 and, after washing, the column was resolved with a linear gradient from this starting ufrrer to a buffer containing 0.1M Tris-acetate, 0.1% Brie oH7.5. Fractions were analysed by SDS gel electrophoresis and those containing the required protein pooled to give “LL

+

antigen” or LL

+

.

This procedure is shown schematically in FIG.

2

.

(d) Size Exclusion Chromatography of WGL Post IEF Pool (

FIG. 3

)

The pH of the “WGL post IEF pool” was increased to 7.3 and the material then loaded on a column of WGL-Sepharose equilibrated in 0.05M Tris-chloride, 0.2% Zwittergent 3-14 pH7.5. The column was washed with 0.05M Tris-chloride, 0.1% SDS, then bound material eluted in Tris-chloride-SDS buffer containing 100 mg/ml N-acetylglucosamine. Fractions were analysed by SDS electrophoresis and those containing the required protein pooled, dialysed against 0.05M Tris-chloride buffer pH7.5 and concentrated on a Savant Speedvac.

Size exclusion chromatography was carried out using a Waters HPLC system and, in sequence, an Si200 Polyol guard column (Serva, Heidelberg), a 7.5×30 cm Bio-Sil TSK 4000 and a 7.5 mm×30cm PP 300 SW (Waters). Chromatography was carried out in a buffer containing 0.05M HEPES, 0.1M sodium thiocyanate, 0.1% SDS, the pH adjusted to 7.0 with sodium hydroxide, at a flow rate of 1 ml/min and a column temperature of 37° C. In this system, bovine serum albumin had an elution time of 13.8 min and ribonuclease A of 17.7 min. Fractions were analysed by SDS gel electrophoresis. The material of interest was found to elute from the HPLC column at between 14.0 and 15.0 min and these fractions were pooled.

The product of this step still contained some impurity of lower molecular weight. It was therefore loaded on a 0.6×10 cm column of WGL-Sepharose in 0.05M Tris-chloride, 0.1% SDS pH7.5, washed in this buffer, then the bound material was eluted in the same buffer containing firstly; 20 mg/ml then 100 mg/ml N-acetylglucosamine. Fractions were analysed by SDS gel electrophoresis and pooled on a basis of the amount and purity of the desired protein in each. They were concentrated and re-chromatographed on HPLC size exclusion chromatography as described above. The final pool of frections containing the desired antigen (“WGL

+

antigen”) was made after analysis by SDS gel electrophoresis as described above.

This procedure is shown schematically in FIG.

3

.

(e) Protein Determination

Four methods of protein determination were used during antigen isolation, the methods being chosen on a basis of sensitivity required and the nature of expected interfering substances. These methods, and the abbreviations used for them in

FIGS. 1

,

2

and

3

were:

1. Biuret method; abbreviated (B)

2. Spectrophotometric method, from A280 and A260 measurements; abreviated (S).

3. Fluorescence method, from the integrated fluorescence of high molecular weight material after derivatization with O-phthalaldehyde; abbreviated (F).

4. Absorbance method, based on the integrated A280 from HPLC chromatographic runs, assuming that a 1 mg/ml solution of the protein in a 1 cm light path had an absorbance at 280 nm of 1; abbreviated (A).

(f) Comments on the Isolation Procedure

The major residual problem with the procedure described above is that in some preparations of the WGL

+

antigen, a contaminant of lower molecular weight was observed as judged by SDS polyacrylamide gel electrophoresis. This contaminant could be partially, though not entirely, removed by repeating the affinity chromatography on WGL-Sepharose in SDS buffer and elution at two concentrations of N-acetylglucosamine.

The amounts of this impurity are variable from preparation to preparation. In a subsequent antigen isolation it was present in minor amounts and good antigen purity was obtained after pooling fractions with pI's in the range 5.30 to 5.67 on preparative isoelectricfocussing, followed by a single HPLC size exclusion chromatography. The yield of WGL

+

antigen was thus higher (approximately 300 μg from 1.3 μg of ticks).

FIG. 5

shows SDS-polyacrylamide gel profiles of fraction GF ⅚, the starting material in this work, (lane 2) and of the purified WGL

+

antigen (lanes 4 & 5) and LL

+

antigen (lanes 6 & 7) together with appropriate molecular weight markers (lanes 1, 3 & 8). It is clear from these gels that the GF ⅚ fraction is very impure and contains a large number of components in addition to the WGL

+

antigen which is in fact such a minor component that it can not be distinguished from the other components in the fraction. The WGL

+

and LL

+

antigens are highly purified. In lane 5 which is an overloaded sample of WGL

+

antigen, a small amount of the contaminating material at lower molecular weight can just be seen.

(g) Amino Acid Composition of WGL

+

and LL

+

Antigens

Samples of the WGL

+

and LL

+

antigens isolated by the new purification procedure were analysed by amino acid analysis. The HPLC plots and calculated amino acid compositions derived from the HPLC printout by integration of the areas under each peak (Table 6) indicate that the antigens have different amino acid compositions. In addition the antigens clearly have different terminal sugar residues accounting for the different lectin binding characteristics.

TABLE 6

Amino Acid Compositions of Tick Antigens

(mole %)

WGL

+

Antigen

LL

+

Antigen

Asp

7.4

11.0

Glu

6.8

10.3

Ser

9.7

7.4

Gly

7.4

10.5

His

2.9

2.9

Arg

5.0

5.2

Thr

9.0

5.6

Ala

9.1

6.8

Pro

5.9

5.2

Tyr

4.8

3.9

Val

7.9

6.5

Met

1.9

2.9

Cys

1.4

0.5

Ile

4.7

4.5

Leu

6.6

8.8

Phe

4.1

4.0

Lys

5.4

3.8

NOTES: Trp is destroyed in this assay. The results presented are obtained from samples taken after 24, 48 and 72 hours of acid hydrolysis.

EXAMPLE 3

Vaccinal Activities of WGL

+

and LL

+

Antigens

Samples of WGL

+

antigen (21 μg) and LL

+

antigen (400 μg) were homogenised in Freunds Complete adjuvant and used to vaccinate cattle ({fraction (1/10)} of each preparation per animal per vaccination) as described in Australian Patent Application No. 45936/85. Vaccinated animals, together with control cattle were challenged with ticks and the numbers of engorged female ticks drogging from the experimental animals was monitored over a 16 day period Table 7). It is clear that cattle vaccinated with very small amounts of WGL

+

antigen were strongly protected from infestation in that the number of ticks dropping from each animal per day was reduced, the weight of the surviving ticks was lower and a high proportion of the surviving ticks were visibly damaged as a result of gut damage allowing cattle blood components to pass into the haemolymph of the ticks (% Red column). In addition, the ticks which survived on the cattle vaccinated with the WGL

+

antigen had a greatly reduced capacity to produce eggs compared to the control animals.

TABLE 7

Animal

Wt. eggs/

No.

Wt. Ticks

Antigen

Tick No.

Tick Wt.

% Red

26

0.49

Controls

199

224

6

29

0.52

237

231

3

31

0.47

227

220

1

28

269

223

9

36

WGL

−

186

228

3

40

LL

−

170

199

4

27

272

224

2

35

LL

+

antigen

338

262

0

37

(130 μg)

238

233

1

30

0.16

25

152

86

32

0.25

WGL

+

antigen

135

175

79

34

0.22

(7 μg)

38

152

70

The LL

+

antigen at a higher dose failed to give significant protection to the cattle despite the fact that the cattle had mounted a strong immune response to the vaccine as determined by ELISA [data not shown].

Both WGL

+

and LL

+

antigens appeared to be largely pure by SDS gel electrophoresis (

FIG. 5

) and both have similar molecular weights of approximately 89 kd in the gel system used compared to the BRL molecular weight standards used.

The new purification procedure outlined above is an improvement over that used previously giving a yield of 33-300 μg WGL

+

antigen compared approximately 3 μg of “S2” antigen per 1.29 kg tick starting material. It is asserted that these two antigens (WGL

+

and S2) are the same glycoprotein based on similar molecular weight, isoelectric point, lectin binding properties, amino acid composition and vaccinal efficacy.

EXAMPLE 4

Digestion of WGL

+

antigen with endoproteinase lys-C, separation of oligopeptides, determination of the amino acid sequence of oligopeptides and design of oligonucleotide sequences suitable as hybridization probes to detect recombinant organisms containing DNA sequences coding for the WGL

+

peptide.

Approximately 40 μg of WGL

+

antigen purified as described in Example 2 was mixed with 100 μl of 0.1M Tris-chloride buffer pH 8.3 containing 20 mM dithiothreitol and 2% (w/v) SDS, then incubated at 56° C. for 30 min. The solution was then cooled to room temperature and sodium iodoacetate added to a final concentration of 0.14M. After 45 min. in the dark, cold methanol was added in a ratio 9:1 methanol:sample (v/v). The sample was stored at −20° C. overnight, centrifuged, the supernatant removed and the precipitate dried.

The precipitate was then dissolved in 76 μl of 0.1M Tris-chloride buffer containing 4M urea, pH 8.5, then 4 μl of endo lys C (6 units per ml) was added. After 2 hrs at 37° C., another 4 μl of enzyme was added and the digestion was continued for a further 17 hrs.

The digest was applied directly to an Aquapore RP-300 C-8 column in 0.1% trifluoroacetic acid and peptides were eluted in a linear gradient from 0-60% v/v acetonitrile/water in 0.1% trifluoroacetic acid. If necessary, peptides were rechromatographed in the same solvent system using an Aquapore 318 column. Peptides were collected, concentrated to 50-100 μl by rotary dessication in a rotary evaporator. The amino acid sequences of the oligopeptides were determined using an Applied Biosystems amino acid sequencer. The following peptide sequences were obtained. The one letter and 3 letter codes used for amino acids are shown in Table 8.

FRAGMENT NUMBER

F1 SEQ ID NO:1

(K)

1

D P D P G K (20-mer oligonucleotide)

F2 SEQ ID NO:2

(K)

1

W Y E D (G)

2

V L E A I (X)

3

T S I G K (50-mer oligonucleotide)

F3 SEQ ID NO:3

(K)

1

(X)

4

Q A C E (H)

2

P I G E (W)

2

C M M Y P K (53-mer oligonucleotide)

(C)

5

F4 SEQ ID NO:4

(K)

1

E A G F V Q K (23-mer oligonucleotide)

In addition, the following peptide sequences were deduced from mixed sequences which may assist in the characterization of the clones although there is a great deal of uncertainty in some of these sequences (especially F7).

(S) (V) (V) (I)

F5 SEQ ID NO:5

(K)

1

G (P) (D) G Q (C) I N (A) (C) K

(G)

F6 SEQ ID NO:6

(K)

1

A (D) V S T N E N E Q L E Q A D K

(G)

F7 SEQ ID NO:7

(K)

1

S (D) T Q (X)

5

I D H I S K

(N) (A) (A)

F8 SEQ ID NO:8

(K)

1

(D) Q E (Y) (Y) Y

F9 SEQ ID NOS:35 and 36

[(K)

1

C P C D N M Y F N A A E K ]

6

[(K)

1

A N R Q C P P D T R R G E I G C I E]

6

Oligonucleotides may be prepared using these amino acid sequences. For example the following could be used.

7

20-mer SEQ ID NO:37

5′

T T A C C T G G A T C T G G A T C C T T

3′

50-mer SEQ ID NO:38

TTA CCA ATG GAT GTA CAA ATA GCT TCA AGG ACA CCA TCT TCG TAC CAC TT

T

53-mer SEQ ID NO:39

TTTGGGTACATCATACACCATTCACCAATTGGGTGTTCACAAGCCTGAGGCTT

AC

NOTES: The following assumptions were made in interpreting the peptide sequences and in designing oligonucleotide probes:

Numbers 1-6 refer to superscripts in the peptide sequence listed above.

1. It was assumed that a lysine (K) preceded the first amino acid which was determined for each peotide based on the specificity of the endo lys-C.

2. These amino acids were assumed to be correct although they were detected at lower molar ratios than expected.

3. No amino acid could be confidently ascribed to the positions shown as X.

4. This position contained a number of amino acids. For the design of oligonucleotides, the correct amino acid was assumed to be either D, A or L but may be another.

5. More than one amino acid was detected in some sequences. The uncertainty is denoted by brackets.

6. These sequences were mixed (square brackets) and the relative molar abundance of the amino acids detected was approximately the same in each cycle.

7. A number of approaches known in the art can be used to design oligonucleotides suitable for use as hybridization probes. For example inosine base can be incorporated in positions where a number of deoxyribonucleotides are used in the third positions of redundant codons. The reverse complementary sequences to those presented can also be used equally well as hybridization probes. In the examples shown the codon usage was based on the sequence for the mRNA coding for the brine shrimp elongation factor (12).

TABLE 8

amino acid

three letter code

one letter code

alanine

ala

A

arginine

arg

R

asparagine

asn

N

aspartic acid

asp

D

cysteine

cys

C

glutamic acid

glu

E

glutamine

gln

Q

glycine

gly

G

histidine

his

H

isoleucine

ile

I

leucine

leu

L

lysine

lys

K

methionine

met

M

phenylalanine

phe

F

proline

pro

P

serine

ser

S

thrionine

thr

T

tryptophan

trp

W

tyrosine

tyr

Y

valine

val

V

EXAMPLE 5

Approximately 40 μg of WGL

+

antigen was digested with endo lys-C as described in Example 4. The digest products were applied to an Aquapore RP-300 C-8 column in 0.1% heptafluorobutyric acid (HFBA) and peptides were eluted in a linear gradient from 0-60% acetonitrile/water in 0.1% HFBA. Selected fractions were then re-chromatographed on Aquapore RP-300 C-8 or C-18 columns using trifluoroacetic acid in place of HFBA. The most symmetrical fractions were analysed for the presence of amino acids by hydrolysis of one tenth of the sample in hydrochloric acid vapour, derivatization with O-phthalaldehyde followed by reverse phase separation on HPLC and detection by fluorescence. The remaining portions of the samples were dessicated to 50-100 μl volumes in a rotary evaporator and the amino acid sequence was determined using an Applied Biosystems amino acid sequencer.

The following peptide sequences were obtained.

FRAGMENT NUMBER

F10 SEQ ID NOS:40 and 41

[(K) A P R Q N M Y F N A A E K ]

[(K) C N C D C P P D T R P G E I G C I E ]

F11 SEQ ID NO:13

(K) W Y E D R V L E A I R T S I G K

F12 SEQ ID NO:42

(K) E S S I C X

D F G N E F

C R N A E C E V V P (K) 72-mer

64 = 17 - mers

F13 SEQ ID NO:15

(K) T R E C S Y G R C V E S N P S K 51-mer

F14 SEQ ID NO:16

(K) A Y E C T C P

R A F T V A E D G I S/H C K 63-mer

64 = 17-mers

[(K) D E V D N A S/H L V C Q N - A ]

F15 SEQ NOS:17-19

[(K) N V L Q S D G C G P Y ]

[(K) C L N P R P/L R L K H/S ]

S

F16 SEQ ID NO:20

(K) A X V L C E X P

C

G

F17 SEQ ID NO:21

(K) L Q A C E H P I

NOTES: It was assumed that a lysine precedes each fragment (K). X indicates that no amino acid could be confidently ascribed to the position during the peptide sequencing. F10 and F15 were mixtures of two and three peptide fragments respectively (denoted by [ ]).

F10 is the sequence of the same mixture of two peptides as analysed for F9. It is surprising that these two oligopeptides co-purified on both occasions as the peptide fractionation procedure was different in the two examples.

F11 and F2 are likely to be the same fragment as the only differences are that the two uncertain amino adids in the F2 sequence are both R in the F11 sequence. A larger amount of material was present in F11 so this sequence is likely to be correct.

F17 and F3 appear to be sequences of the same peptide. F3 could be read further as more material was present but F17 contained less impurities so the first residue could be identified.

From these amino acid sequences, oligonucleotides can be prepared which would be suitable for screening cDNA and genomic DNA banks to identify the gene coding for the WGL

+

antigen. The following examples could be used (see note 7 in Example 4. In the following examples, the third position in the codons was chosen to minimise secondary structure, not on brine shrimp usage as used in Example 4).

72-mer SEQ ID NO:43

5′ TTT AGG TAC AAC CTC ACA TTC AGC ATT CCT ACA AAA TTC ATT ACC GAA ATC

AAA ACA AAT ACT ACT CTC CTT 3′

51-mer SEQ ID NO:44

5′ CTT CGA CGG ATT GGA TTC GAC GCA TCT GCC ATA GCT ACA TTC CCT CGT

CTT 3′

63-mer SEQ ID NO:45

5′ CTT GCA ATG GAT TCC ATC CTC GGC GAC AGT GAA AGC TCT AGG GCA AGT GCA

CTC ATA AGC CTT 3′

In addition, degenerate shorter oligonucleotides could be synthesized. For example 64 fold degenerate 17-mer oligonucleotides could be designed using the sequence DFGNEF from the F12 sequence and from the sequences KAYECT and YECTCP from the F14 sequence. 16 fold degenerate 17-mer oligonucleotide mixtures could also be designed using the sequence CMMYPK from the amino acid sequence of F3 shown in Example 4. These short degenerate sequences may be useful to confirm that clones isolated using long oligonucleotides contain the desired DNA sequences coding for the WGL

+

antigen.

EXAMPLE 6

Genetic Engineering of the WGL

+

(S2) Antigen

The major limitation to the development of commercial vaccines using the WGL

+

antigen is the limited availability of WGL

+

material which can be obtained from natural sources. Means by which this limitation can be overcome include the construction by genetic engineering techniques of bacteria, yeast or other readily cultivated cells including mammalian or insect cell lines which synthesize large amounts of all or part of the WGL

+

antigen. There are several means by which this goal can be achieved but they basically fall into a small number of steps which, by means of example only, are set out below.

In order to identify the recombinant organisms which contain the tick genetic information coding for the protein backbone of the WGL

+

antigen, appropriate reagents must first be generated. These may be antibodies from animals vaccinated with the purified protective antigen or with partially purified preparations containing that antigen preferably following denaturation of the antigen with a suitable detergent such as SDS. Bacterial. yeast or other cells which synthesize part or all of the WGL

+

antigen must then be constructed and screened with the antiserum.

Preferably the WGL

+

antigen is isolated in sufficient quantities in order to determine the amino acid sequence of the protein portion of the antigen or of fragments of the antigen produced as a result of endoproteolyic enzyme digestion using enzymes such as trypsin, endo lys C or pepsin or by chemical cleavage of the peptide using reagents such as cyanogen bromide. Fragments produced as a result of these treatments are separated and isolated using methods known in the art such as fractionation by HPLC reverse phase liquid chromatography or HPLC on columns containing hydrophobic resins, ion exchange resins or size fractionation resins. Preferably reverse phase resins such as C1, C3 or C18 are used singly or consecutively depending on the characteristics of the fragments produced as a result of the enzymatic or chemical treatment chosen.

Fragments of the WGL

+

peptide produced as a result of these treatments are then analysed on a gas phase amino acid sequenator using methods known in the art. From the amino acid sequence and the known DNA sequences which encode each amino acid, oligonucleotide sequences can be prepared which are complementary to the DNA sequence coding for the antigen and these can be used in hybridisation experiments to identify recombinant organisms containing the DNA coding for the antigen. The DNA sequence of these reacting clones can then be determined to confirm that the sequence is the one of interest. The DNA sequence can then be used to design the best means by which the microorganisms can be engineered to manufacture large amounts of the polypeptide.

(a) Construction of Gene Libraries

Readily cultivated microorganisms which contain the genetic information coding for the WGL

+

protective antigen can be constructed using synthesised DNA which is complementary to the RNA isolated from the appropriate developmental stage of ticks or using DNA fragments isolated from any stage of ticks, preferably eggs or larvae as they will not contain bovine blood.

If antibody probes are the only reagents available for detection of the clones containing the WGL antigen, cDNA or genomic DNA libraries must be constructed in a phage, viral or plasmid system which would result in the expression of the tick antigen or parts thereof. Such vectors include lambda gt11, bacterial plasmids such as pUR290, pUR291, pUR282, pUK270, pUC8, pUC9 or eukaryotic viral vectors such as the baculovirus, pZipNeo, or SV40-based vectors.

If oligonucleotide probes are available, clone libraries can be constructed using cDNA or genomic DNA fragments in a larger range of phage, plasmid and viral systems including, for example, lambda gt10, EMBL vectors, or plasmid vectors such as pBR327, pBR329 or pBR322.

Preferably cDNA libraries are generated since smaller numbers of clones have to be screened and any problems with expression through introns are avoided. Ideally the developmental stage of ticks which synthesise maximum levels of the WGL

+

0

antigen are first identified. If antibodies are the only means available to do this, in vitro translation of RNA isolated from ticks of various ages followed by immunoprecipitation of the translation products with antibodies and analysis by SDS gel electrophoresis and fluorography enables the identification of the most suitable stage of ticks for extraction of RNA for construction of cDNA banks. The apparently low abundance of the WGL

+

protein makes this approach very difficult. If oligonucleotides are available, hybridisation to the RNA isolated from ticks of various ages should enable the identification of the RNA source containing the highest abundance of mRNA coding for the WGL

+

antigen.

RNA can be isolated from ticks and cDNA synthesized and cloned a number of methods known in the art can be used. The following methods are outlined by means of example only.

(b) Ethanol precipitation

In the following methods, ethanol precipitation involves adding to the solution of nucleic acid, one tenth of the solution volume of 3M sodium acetate and three to four volumes of absolute ethanol. The mixture is then stored at −20° C. for at least 2 hrs or at −70° C. or in an ethanol/dry ice bath until the solution becomes viscous. The mixture is then centifuged usually at 12,000×g

av

for at least 10 minutes. The supernatant is carefully removed and the pellet containing the nucleic acid material (as well as other macro-molecules) is used in further manipulations.

(c) Ethanol precipitation from 2M ammonium acetate

In high salt solutions (e.g. 2M ammonium acetate), the majority of unincorporated deoxynucleotide triphosphates (and other small molecular weight material) will remain in the supernatant after an ethanol precipitation. The procedure is as described above except an equal volume of 4M ammonium acetate is added to the solution instead of the sodium acetate followed by 3-4 volumes of ethanol before cooling as described above.

(d) Phenol or phenol/chloroform extraction

Phenol or phenol/chloroform extraction involves the addition to the nucleic acid solution of an equal volume of redistilled phenol or a 1:1 (v/v) mixture of phenol and chloroform equilibrated with 0.1M Tris pH8. The contents of the tube are mixed and the phases separated by centrifugation. The upper (aqueous) phase is removed to a fresh tube and the phenol or phenol/chloroform is discarded. Usually the aqueous phase is re-extracted and then extracted with ether to remove remaining phenol. Optionally the phenol or phenol chloroform phase from the first extraction may be re-extracted by addition of TE, mixing and centrifugation. In this case, the two aqueous phases would be combined before ether extraction and further proccessing.

(e) PEI cellulose TLC

To monitor incorporation of radioactive dATP into nucleic acids during the various reactions in this procedure, thin layer chromatography on PEI cellulose was performed in 0.75M phosphate buffer pH 3.5. An aliquot of material to be monitored is applied toward one end of a strip of PEI cellulose and, after the chromatogram is resolved, the strip is exposed to an X-ray film. Following development of the autoradiograph, the areas of the PEI cellulose strip containing radioactivity are cut, placed in vials and the radioactivity in each determined by Cherenkov counting in a scintillation counter. The proportion of the radioactive material at the origin of the chromatograph can be used to determine the success of the reaction. This procedure is referred to as PEI cellulose chromatography.

(f) Extraction of DNA and RNA

High molecular weight DNA and RNA are isolated from ticks picked from the host at different developmental stages. Ticks are homogenised at room temperature in an Omnimixer for 2-3 minutes in a buffer containing guanidine isothiocyanate (4.7M), Sarkosyl (7.4%), is (5 mM) and β-mercaptoethanol (70 mM). The homogenate is centrifuged at 4° C. at

14000×g

av

for 10 minutes. Solid CsCl is added to the homogenate (1 g/2.5 ml) which is layered onto a CsCl cushion (2.5 g/ml) and centrifuged for 48 hours at 25,000 rpm in a SW28 rotor (Beckman). The upper layer is aspirated, the DNA band recovered and the RNA pellet recovered, precipitated with ethanol, Sashed several times with 70% ethanol and stored in TE at −70° C. until used.

Polyadenylated mRNA can be isolated by passage over oligo dT cellulose columns or poly U Sepharose columns using methods described by the manufacturers (Collaborative Research).

(g) cDNA Synthesis

Several methods can be used for the construction of cDNA banks in phage or plasmid vectors. The following method by means of example only is a modification of the “RNase H” method for construction of cCNA banks in lambda gt11. The method is outlined schematically in FIG.

6

.

(h) First Strand Synthesis

2 μg of poly A

+

RNA is dissolved in TE. Water is adder to give a final volume of 25 μl. The solution is heated at 70° C. for 3 minutes then rapidly cooled on ice. To the cooled solution is added 5 μi 10× is Strand Buffer, 5 μl 0.1M DTT, 5 μl Oligo-jT [Boehringer 100 mg/1 μl], 1.25 μl RNasin [Promega 40 U/μl], 2 μl BSA (5 mg/ml), 5 μl 10 mM d(GCT)TP, 0.5 μl 10 mM dATP and 3 μi M-MLV Reverse transcriptase [BRL 200 U/μl].

2.5 μl of the mixture is transferred to tube A (analytical reaction for monitoring synthesis) and 0.2 μl [

32

P]dATP is added (0.2 μCi).

To the remaining bulk reaction 0.5 μl 50 mM dATP is added. The tubes are incubated for 30 minutes at 42° C. 0.25 μl 10 mM dATP is then added to tube A and the incubations continued for a further 30 minutes. A 0.5 μl sample is taken from tube A and ethanol precipitated for gel analysis. A further 0.2 μl sample is taken from the tube to be monitored by TLC on PEI cellulose.

If all of the 2 μg of RNA added to the reaction was poly A-adenylated, it can be calculated that approximately 30% incorporation of [

32

P]dATP into nucleic acids is equivalent to 100% efficiency in first strand synthesis. Commonly RNA passaged over Oligo-dT cellulose once yields 6-10% incorporation.

To prepare a sample to monitor the second strand reaction, 2.5 μl of the bulk reaction is removed and precipitated with ethanol from 2M ammonium acetate. The sample is washed twice with 70% ethanol then resuspended in 2.5 μl 1×1st Strand Buffer in tube B.

(1) Second Strand (RNase H)

A solution of; 28 μl of water, 10 μl 10× RNase H Buffer; 1 μl 5 mg/μl BSA, 1.25 μl 10 mM d(GCT)TP, 0.5 μl 10 mM dATP, 1.6 μRNase H [BRL 20 U/μl], 5 μl DNA Polymerase 1 [holoenzyme (Biolabs) 100 U/μl ] and 2 μl

E. coli

DNA ligase, is prepared.

The solution is mixed, then 2.5 μl is dispensed into Tube B with 0.2 μl [

32

P]dATP, 1.8 μl is dispensed into Tube A and the remainder is dispensed into the bulk reaction tube. 0.75 μi of 10 mM dATP is added to the bulk reaction tube. The three tubes are incubated at 15° C. for 60 minutes, then at 22° C. for a further 60 minutes.

A 0.2 μl sample from tube B is chromatographed on PEI cellulose to monitor the reaction. A further sample from tube B is ethanol precipitated from 2M ammonium acetate for gel analysis. The tube A sample from the first strand synthesis and the tube B second strand synthesis sample are run on a 1.5%. agarose gel to determine the size of the cDNA which has been synthesized.

To prepare a sample to monitor the T

4

polymerase reaction, 0.5 μl is taken from the bulk reaction tube and placea in Tube C.

The remaining contents of Tubes A and B are pooled with the bulk reaction. The contents of both the bulk reaction, and Tube C are extracted with phenol/chloroform (1:1), precipitated with ethanol from 2M ammonium acetate and the precipitates are mashed twice with 70% ethanol.

(j) EcoR1 Methylation

A solution of 29.5 μl of water, 4 μl 0.1M DTT, 2 μl 10× EcoR1 Methylase buffer, 4 μl 1 mM S-adenosyl methonine [Biolabs] and 0.5 μl EcoR1 Methylase [Biolabs 2 U/μl] is prepared in a fresh tube. 2 μl of the mix is dispensed into Tube C and the remainder into the bulk reaction tube. The two tubes are incubated at 37° C. for 30 minutes then at 70° C. for a further 15 minutes then cooled in ice.

In a fresh tube, the following buffer is prepared: 4 μl 10× TA buffer, 2 μl 5 mg/ml BSA, 1.4 μl 0.1M DTT, 2 μl T

4

DNA polymerase [Biolabs 1 U/μl] and 29.5 μl of water. 2 μl is added to tube C which is then incubated at 37° C. for 10 minutes. 0.5 μl of a solution containing 10 mM d(GCTA)TP is added to the remainder of the solution and this is added to the bulk reaction tube which is then incubated at 37° C. for 50 minutes, 70° C. for 15 minutes then ice quenched.

To Tube C 0.2 μl of each of 50 μM d(GTC)TP, [

32

P]dATP [0.2 μCl] and 5 μM dATP are added and incubation is continued at 37° C. for a further 50 minutes, after which time 0.2 μl of the sample is spotted and chromatographed on PEI cellulose.

0.2 μl of 0.2 mM dATP represents approximately three times the amount of dATP required to add 2 adenosine residues to the 5′ ends of each molecule, assuming that there was a total of 2 μg of dscDNA of average size of 1 kb synthesized after 2nd strand synthesis.

(k) Kinase—There is some indication that the kinase step is not necessary and can probably be omitted. To the bulk reaction 20 μl 5× Kinase buffer, 0.2 μl 0.1M ATP, and 0.5 μl polynucleotide kinase [Biolabs 4 U/μl] is added. The mixture is incubated at 37° C. for 60 minutes. The reaction is extracted with an equal volume of a phenol/chloroform mixture (1:1), the aqueous phases are pooled, precipitated by ethanol from 2M ammonium acetate then washed twice with 70% ethanol.

(1) Linker Ligation

To monitor the linker ligation reaction, samples are prepared for agarose and polyacrylamide gel analysis.

Agarose gel: Samples from bulk reaction (

32

P cDNA)

Sample 1 cDNA before ligation to cold linkers

Sample 2 cDNA after ligation to cold linkers

Sample 3 cDNA after ligation to cold linkers and digestion with EcoR1

Polyacrylamide gel:

32

P linker+samples

Sample 4 Tube D

32

P linkers+cDNA before digestion with Eco R1

Sample 5 Tube D

32

P linkers+cDNA after digestion with Eco R1

Sample 6 Tube E

32

P linkers alone before digestion with Eco R1

Sample 7 Tube E

32

p linkers alone after digestion with Eco R1

The ligation mixture is prepared by adding to a fresh tube 9 μl EcoR1 linkers [Biolabs 200 ng/μl], and 1.7 μl of DNA ligase [IBI 3 U/μl]. 15 μl of ligation mixture is dispensed into the bulk reaction tube mixed quickly, then a 0.25 μl sample is removed and frozen on dry ice immediately for agarose gel analysis (Sample 1).

A 1 μl sample is taken from the bulk reaction tube and 0.2 μl

32

P labelled EcoR1 linkers is added (Tube D: cDNA linkers).

A 1 μl sample is taken from the remainder of the ligation mixture and 0.2 μl

32

P labelled EcoR1 linkers are added (Tube E: linkers alone).

The bulk reaction and tubes D and E are incubated at 25° C. for 4 hours. Samples of 0.25 μl from the bulk reaction tube and 0.6 μl from tubes D and E are removed for agarose or polyacrylamide gel analysis respectively (Samples 2, 4 and 6).

The remainder of the bulk reaction and tubes D and E are heated for 5 hours at 70° C. then cooled on ice.

(m) EcoR1 digestion

To a fresh tube 11 μl EcoR1 digestion buffer, 2 μl EcoR1 [IBI 18 U/μl ] and 82 μl of water are added. 4 μl of the mixture is dispensed into the bulk reaction tube. The three tubes are incubated at 37° C. for 60 minutes. A further 2 μl aliquot of EcoR1 [36U] is added to the bulk reaction tube and incubation is continued for a further 60 minutes. The remaining samples in tubes D and E are electrophoresed on agarose and acrylamide gels together with the samples taken from tubes D and E anove. Autoradiographs of those gels Demonstrate whether the reactions have worked.

A 1.4 μl sample is removed from the bulk reaction tube (Sample 3). The remainder of the bulk reaction is extracted with phenol/chloroform

A 1% agarose gel is run loaded with 0.25 μl each of samples 1, 2 and 3. Samples 4, 5, 6 and 7 are run on a 12% polyacrylamide gel. Both gels are autoradiographed to determine whether all reactions have succeeded.

(n) Separation of Linkers from cDNA

A 1.2 ×21 cm Sepharose 48 column is equilibrated with 0.1M TEAB. 150 μl samples of EcoR1 digested linkered cDNA are loaded on to the column and fractions collected in TEAB buffer (250-500 μl). Fractions containing cDNA fragments with sizes greater than 600 bp as determined by mobility on agarose or polyacrylamide gels are pooled, evaporated to dryness in a rotary evaporator suspended in TE and ligated to EcoR1 digested and phosphatased lambda gt11 or gt10. packaged in vitro and infected onto suitable host strains such as Y1090 or Y1089 in accordance with suppliers instructions (Promega or Integrated Sciences).

(o) Screening Clones with Oligonucleotides

From the amino acid sequence of the WGL

+

protein, peptide fragments derived from chemical cleavage of the WGL

+

protein or endoproteolytic digestion peptides derived from the WGL

+

protein, oligonucleotides coding for specific portions of the DNA coding for the protein can be designed and used in hybridisation experiments using procedures known in the art. The DNA sequence of hybridising fragments isolated from the library can then be determined and used to design strategies for engineering the gene for expression of the WGL

+

protein or portions thereof for incorporation into an effective vaccine.

A cDNA library was constructed in lambda gt 11 using RNA isolated from young adult

B microplus

which had been feeding on cattle for approximately 16 days. The phage were plated on

E.coli

strain RY1090 and grown at 37° C. for 16 hours. Nitrocellulose filters were placed on the plates and triplicate filters were taken from each plate. The DNA on the filters was denatured and fixed by baking at 80° C. under vacumn. The filters were incubated in prenybridization solution for 2-4 hours and then in hybridization solution for 16 hours essentially as described (10). The hybridization solution contained oligonucleotides which had been labelled with

32

P using polynucleotide kinase (10) and ψ

32

P-ATP (approximately 10

5

cpm/ml of each oligonucleotide used).

For each set of three filters, two were hybridized to the 63-mer oligonucleotide and the remaining replicate filter was hybridized to a mixture of 51-mer, 72-mer, 50-mer, and 53-mer oligonucleotldes. Following washing and autoradiography, plaques which gave rise to signals on all three filters were identified, picked and purified to single plaques.

EXAMPLE 7

Analysis of DNA Sequence of Gene Coding for WGL

+

Antigen

The DNA isolated from one clone will be described in detail. This lamoda gt11 clone contained three Eco R1 fragments of approximately 4 Kb, 1.5 Kb and 0.3 Kb. Southern hybridization (10) experiments showed that the 4 Kb fragment hybridized to the probes used. This fragment was therefore subcloned into a modified pUC 18 plasmid (giving pBTA 707) in host strain JM101 (recombinant host/plasmid referred to as BTA 1751 ATCC 67548). The 4 Kb fragment was then sonicated and subcloned into M13 mp18 for DNA sequence analysis.

M13 sub-clones were sequenced at random and the complete DNA sequence of the 4 kb inset compiled by assembly of the sequences of the sub-clones by use an alignment computer program.

FIGS.

6

-

6

(

2

) show the DNA sequence bases 1-2012 of SEQ ID NO:55 for the 4 kb DNA fragment and the amino acid sequence residues 11-688 of SEQ ID NO:56 which can be translated from one region of that DNA sequence into a protein sequence which is identified as the protein backbone of the WGL

+

antigen.

FIG. 8

shows that amino acid sequence using the one letter abbreviation code for amino acids (Table 8).

The peptide fragments identified during the peptide sequence analysis of endo lys-C digest products from the WGL

+

antigen isolated from ticks are identified in FIGS.

6

-

6

(

2

) and

8

by underlines and are tabulated in a summary in Table 9. References to aa numbers correspond to the numbering of amino acid residues shown in FIG.

7

and SEQ ID NO:56.

TABLE 9

F1 SEQ ID no: 1

(K) D P D P G K

aa 619-625

K D P D P G K

F2 SEQ ID no: 2

(K) W Y E D (G) V L E A I X T S I G K

aa 357-373

K W Y E D R V L E A I R T S I G K

F11 SEQ ID no: 13

(K) W Y E D R V L E A I R T S I G K

F3 SEQ ID no: 3

(K) X Q A C E (H) P I G E (W) C M M Y P K

aa 404-421

K L Q A C E H P I G E W C M M Y P K

F17 SEQ ID no: 21

(K) L Q A C E H P I

F4 SEQ ID no: 4

(K) E A G F V C/Q K

aa 212-219

K E A G F V C K

F5 SEQ ID no: 5

(K) G (P) (S/D) G Q (V/C) I N (V/A) (I/C) K

aa 199-210

K G P D G Q C I N A C K

F6 SEQ ID no: 6

(K) A (D/G) V S (T) N E N E Q (L) E (Q) A D K

aa 487-503

K A G V S C N E N E Q S E C A D K

N

F8 SEQ ID no: 8

(K) D Q E (

A

/Y)

A

/Y Y

aa 443-450

K D Q E A A Y K

F9 SEQ ID no: 27

(K) C P R D N M Y F N A A E K

aa 50-63

K C P R D N M Y F N A A E K

F10 SEQ ID no: 27

(K) C P R D N M Y F N A A E K

F9 b SEQ ID no: 46

(K) A N C Q C P P D T R R G E I G C I E

aa 513-531

K A N C Q C P P D T K P G E I G C I E

F10 b SEQ ID no: 29

(K) A N C Q C P P D T R P G E I G C I E

F12 SEQ ID no: 42

(K) E S S I C X D F G N E F C R N A E C E V V P (K)

aa 19-42

A E S S I C S D F G N E F C R N A E C E V V P G

F13 SEQ ID no: 15

(K) T R E C S Y G R C V E S N P S K

aa 72-88

K T R E C S Y G R C V E S N P S K

F14 SEQ ID no: 16

(K) A Y E C T C P R A F T V A E D G I S/H C K

aa 227-247

K A Y E C T C P S G S T V A E D G I T C K

F15 a SEQ ID no: 33

(K) N L L Q R D S — C C Q

aa 165-176

K N L L Q R D S R C C Q

F16 SEQ ID no: 47

K X X V L C E X P

aa 273-281

K G T V L C E C P

From the DNA sequence and the amino acid sequence deduced from that DNA sequence, it can be seen that the pre-pro-polypeptide of the WGL

+

antigen consists of 650 amino acids. SEQ ID NO:56.

The DNA sequence coding for peptide F12 SEQ ID NO:42 can be identified at the region 90-152 bp FIGS.

6

-

6

(

2

) of the DNA sequence and corresponds to amino acids 20-40 in the amino acid sequence (

FIG. 8

) residues 30-50 of SEQ ID NO:57 of the protein. The amino acid preceding the N-terminal glu residue identified in F12 is not a lysine (K) as would be expected if F12 was generated as a result of digestion by endo lys-C. Therefore it is assumed that the F12 peptide fragment was generated by the action of a proteinase other than endo lys-C. The 19 amino acid sequence preceding the F12 N-terminal glu residue begins with a methionine and has hydrophobicity properties which are very similar to leader sequences which precede other secreted and membrane-bound proteins in eukaryote cells (see 9 for review). In addition, the majority of peptide leader sequences are cleaved at positions following A residues (9). It appears therefore that the F12 sequence is the N-terminus of the mature WGL

+

polypeptide. This then indicates that the protein portion of the mature WGL

+

polypeptide is 631 amino acids long and which would have a molecular weight of 69 729 daltons.

Assuming that the consensus sequence for N-linked glycosylation is Asn X (Ser or Thr) in ticks as has been reported to be the case in other eukaryotic cells (10) 5 potential sites for N-linked glycosylation can be identified in the mature polypeptide sequence (FIGS.

6

-

6

(

2

)). Carbohydrate residues added to these residues or to other amino acids in the WGL

+

antigen produced by ticks would account for the differences in the observed molecular weight for the native antigen compared with that predicted from the DNA sequence.

By comparison of the amino acid sequence (Table 9) with the peptide sequences derived from the fractions from endo lys-C digestion, all of the peptides (F1-17) with the exception of F7 can be identified. In most cases, the amino acids which could not be confidently ascribed during the peptide sequence analysis can be shown to be correct following comparison with the sequence deduced from the DNA sequence.

The amino acid sequence for peptide fragments F1, F11, F13 and F17 SEQ ID NOS:1, 173, 15 and 21 respective all match precisely with the amino acid sequences deduced from one DNA sequence from the corresponding region of DNA (Table 9).

Peptide F2 SEQ ID NO:2 can be seen to be coded for by the DNA segment 1104-1152 bp. Table 9 shows that the G and the X tentatively ascribed to positions 5 and 11 in the F2 peptide sequence are both N. N is very difficult to detect during gas phase sequencing and there was very little material in the sample. Otherwise the match is precise. F2 is the same peptide as F11 and all amino acids were ascribed correctly during the sequence analyses of the F11 peptide fragment.

Peptides F3 SEQ ID NO:3 and F17 show sequences of the same peptide obtained from two different endo lys-C digests of WGL

+

(Examples 3 and 4). The amino acid sequence for F17 matches precisely with the translated sequence from amino acids 405 to 412 of the WGL

+

peptide. When sequencing F3, no amino acid could be ascribed to the first position (L from the DNA sequence) as there was a large amount of background but the rest of the amino acids match precisely with the amino acid sequence derived from the DNA sequence (amino acids 405-421

FIG. 8

) Residues 415-437 of SEQ ID NO:57.

F4 SEQ ID NO:4 is found at amino acids 213-219 of the WGL

+

protein (

FIG. 8

) Residues 223-229 of SEQ ID NO: 57. The sequence matches perfectly and the uncertain C/Q is shown from the DNA sequence to be C. Carboxy-methylated C migrates with a similar retention time to Q in the HPLC system used to separate the derivatized amino acids following the sequencing reactions.

Very small amounts of material were sequencable in fragment F5 SED ID NO:5 so there were several uncertainties. But it is clear that the sequence obtained corresponds to amino acids 200-210 in

FIG. 8

(Residues 210-220 of SEQ ID NO:57. One of the two amino acids tentatively ascribed to each peptide is present in the amino acid sequence derived from the DNA sequence and those ascribed with confidence appear in the expected order.

F6 SEQ ID NO:6 sequence corresponds to amino acids 488-503 in the WGL

+

protein sequence. The residues in the sequence derived for the F6 fragment differ from that derived from the DNA sequence. The F6 sequence presented was derived from a mixed sequence in which the amino acids shown to be correct from the DNA sequence were in fact present.

F7 SEQ ID NO:7 has not been identified with confidence in the amino acid sequence derived from the DNA sequence. As with F6, the F7 sequence was derived from a mixed sequence and very little confidence can be placed in it.

Small amounts of material were present in F8 SEQ ID NO:8 sample so there were several uncertainties in the sequence. However, the F8 amino acid sequence appears to correspond to amino acids 444-450in

FIG. 8

(Residues 454-469 of SEQ ID NO:57. Again all uncertain residues can be identified in the translated DNA sequence.

F9 SEQ ID NOS: 27 and 46 F10 SEQ ID NOS:27 and 29 were both mixtures of two amino acid sequences. It is apparent that one of those sequences corresponds to amino acids 51-63 in FIG.

8

(Residues 61-72 of SEQ ID NO:57) . In both cases, one of the two amino acids identified during the peptide sequence analysis can be ascribed to the amino acid sequence derived from the DNA sequence in the expected order.

The remaining peptide sequence from F9 and F10 corresponds to amino acids 514-531 in

FIG. 8

(Residues

524

-

541

of SEQ ID NO: 57. The R recorded for position 11 in the F9 sequence is P from the DNA sequence which is in agreement with the sequence obtained for F10. The DNA sequence shows K at what would be position 10 of this peptide. The DNA sequence shown is that coding for one molecule of the WGL

+

antigen and it is likely that different ticks have some variants of the sequence. This point will be expanded when discussing the F14 sequence.

The fragment sequenced as F12 (SEQ ID NO:42)clearly corresponds to amino acids 20-41 in

FIG. 8

(Residues 30-51 of SEQ ID NO:57) and, as discussed previously is assumed to be the N-terminal fragment of the mature WGL

+

peptide so the presumption that lysine preceded the first amino acids sequenced was incorrect in this case. The uncertain residue at position 6 of the peptide fragment is S from the DNA sequence. S is very difficult to detect during gas phase sequencing particularly as in this case, when the preceding amino acid is C and carboxy-methylated-C has a similar retention time to S in the HPLC system used to resolve the derivatized amino acids. Otherwise the F12 peptide sequence matches the sequence derived for WGL

+

exactly.

F14 SEQ ID NO:16 is very interesting (amino acids 228-247). The peptide sequence showed RAF for amino acids 8-10 in the peptide, whereas the DNA sequence, when translated, shows SGS in these positions. Both sequences appear to be correct retrospectively so there is a clear discrepancy between the two sequences. The most likely explanation is that both sequences are correct for the molecule (in the case of the cDNA) and the mixture of molecules (in the case of F12) which have been sequenced.

The tick population world wide is genetically diverse as is the case for all organisms which reproduce sexually. Each individual of a population differs subtly from the others in the population and these differences are a consequence of differences in the sequence of the DNA which each individual inherits from its parents. Thus for each gene coding for a particular protein, there are likely to be differences in the sequence among the population of individuals, referred to herein as homologues. In the particular example discussed here, the WGL

+

protein which was digested in Example 4 to give rise to F12 was extracted from a large number of ticks (60,000-70,000). The peptide sequence determined for F12 SEQ ID NO:42 (and the rest of the peptide fragments sequenced) is that of the majority of the population of WGL

+

molecules. Among that population of WGL

+

molecules. It is likely that minor variance (homologues) will exist at a level too low to be detected during the peptide sequence analysis. The cDNA sequence shown in

FIG. 7

bases 1-2012 of SEQ ID NO:55 and SEQ ID NO:56) and the amino acid sequence in

FIG. 8

are derived from one cDNA molecule from one individual in the population. This individual may have contained a DNA sequence coding for a minor variant of the WGL

+

molecule. It is of course understood that other cDNA molecules may be derived from other individuals of the tick population world wide which will similarly vary in some small way from the sequence shown in FIGS.

6

(A)-

6

(C) but still code for a protein which is essentially the same as that for the WGL

+

antigen molecule. These homologues are included within the scope of this invention.

If the differences such as the one above are found in regions of the WGL

+

molecule which are important epitopes for the protective immune response generated against the WGL

+

molecule following vaccination, it is possible that the ticks with a WGL

+

product which is a homologues of the sequence shown in FIGS.

6

-

6

(

2

) may survive feeding on vaccinated hosts. In this instance it is to be understood that cDNA can be synthesised or DNA isolated from these individuals as described above or by other methods known in the art. In hybridization experiments the 4 kb DNA fragment (or parts thereof) can be used as hybridization probes to identify clones containing DNA coding for the WGL

+

protein from those variants which can then be used to construct bacteria or other micro-organisms which synthesize the variant WGL

+

antigen to be incorporated into effective vaccine against the variant tick population. This principal extends to isolates of

Boophilus microplus

and to other species of ticks from anywhere in the world.

The other difference in the sequence of F14 SEQ ID NO:16 compared with the sequence for WGL

+

polypeptide derived from the DNA sequence is that the residue at position 18 (S or H in F12 which was ascribed with low confidence) is T from the DNA sequence.

F15 SEQ ID NO:17-19 was a mixture of at least three oligopeptides. Among those, one seems to be represented in the polypeptide at amino acids 166-176.

The F16 SEQ ID NO:20 sequence can be seen in the WGL

+

amino acid segment 274-281 (

FIG. 8

) Residues 284-291 of SEQ ID NO:57. The two uncertain residues in the peptide sequences are X=T in the second position and X=C in the seventh position of F16, both of which were due to the very small amounts of material which were present in this peptide sample.

In summary, it is clear that the DNA sequence shown in Table 9 codes for one homologue of the tick WGL

+

polypeptide as 16 of the 17 endo lys-C peptide fragments shown which were generated from the antigen isolated from ticks can be coded for by that DNA sequence. Evidence has been obtained that homologues of that gene may exist in the tick population and these are included within the scope of this invention whether the homologues originate from

Boophilus microplus

or other species of ticks found world wide.

The procedures outlined above refer to the WGL

30

antigen derived from

Boophilus microplus

. It is clear that this antigen or the equivalent antigen isolated from other tick species may well be effective against other species of Boophilus such as

B. annulatus

, other tick species such as Haemaphysalis spp, Otobius spp, Rhiphicephalus spp, Ambylomma spp. Dermacentor spp, Ixodes spp and Hyalomma spp, and particular species thereof including

Otobius megnini, Rhiphicephalus appendiculatus, Amblyomma variegatum, Haemaphysalis longicornis, Dermacentor andersoni, D. variabilis

and

Ixodes holocyclus

each of which causes significant economic loss throughout the world either as a result of infestation or as vectors of diseases such as

Babesia bovis, Babesia bigemina, Anaplasma marginale, Cowdria ruminatum, Theileria parva parva, T. parva lawrencii, T. annulata

and

T. hirci

. The WGL

+

gene product or the equivalent gene product from the related and other acarines would be expected to provide effective vaccines against the parasites as an extension of the work presented herein.

COMMENTS ON HYBRIDIZATION PROBES USED

The oligonucleotide probes which were chosen had several shortcomings which can be identified retrospectively. Some of these were due to incorrect choice of bases in the the third codon positions and, of course to the uncertainties in the peptide sequence analysis. The oligonucleotide which was relied upon most heavily was the 63-mer as it was based on the most reliable amino acid sequence obtained at that time. When isolating the clones it was surprising that the hybridization signal with this probe was weaker than expected from theoretical considerations and at one stage there was doubt that the clone isolated coded For the WGL

+

peptide. This uncertainty was alleviated to some extent by the use of the degenerate oligonucleotide sequences mentioned above as probes. These probes hybridized strongly to the DNA in the clone. The reason for the weaker than expected signal with the 63-mer can now be explained by the variation in the DNA sequence from that expected in this region. A large number of other clones were purified based on the hybridization signal obtained with one or two probes out these all turned out to be unrelated to the WGL

+

gene by DNA sequence analysis.

Therefore the strategy for isolating the clone by using triplicate filters and the use of the highly degenerate oligonucleotide sequences as hybridization probes to confirm the interest in the clone has been vindicated.

EXAMPLE 8

Construction of Recombinant Organisms Synthesizing WGL

+

Antigen

The major limitation to the development of a commercial vaccine based on the WGL

+

antigen or homologues thereof is the limited amount of the antigen which can be obtained from ticks. The means by which this shortage can be overcome include the use of recombinant DNA techniques to engineer bacteria or eukaryote cells to synthesize large amounts of the antigen. The following by means of example only outline some approaches which could be taken.

FIGS. 8A-8B

show a restriction enzyme map of the gene coding for the WGL

+

antigen isolated from

B. microplus

. In order to engineer bacteria which express the gene product at high levels, it would probably be desirable to remove the parts of the molecule which are hydrophobic. These include the hydrophobic leader sequence (amino acids 1-9) which is not found in the mature polypeptide, and the hydrophobic C-terminal sequence (amino acids 630-650) which is likely to be an anchor sequence involved in attaching the antigen to the outer surface of tick cells. In

FIGS. 8A-8B

, cleavage sites for the restriction enzymes XmnI (116 bases), PstI (1915 bases) and BamHI (1889 bases) are highlighted. DNA fragments produced by digestion of the WGL

+

gene with XmnI in addition to BamHI or Pstl will contain the coding region for the majority of the gene without the N-terminal hydrophobic sequence or the C-terminal hydrophobic sequences. These 1773 bp and 1799 bp fragments can be subcloned into a number of plasmids including plasmids pBTA603 and pBTA224 to yield recombinant plasmids which will direct the synthesis of fused proteins containing the majority of the WGL

+

peptide.

Plasmid pBTA603 has the PL promoter followed by a sequence from the N-terminus for the MS2 polymerase gene containing a multiple clone site vis SEQ ID NO:48

ATG TCG AAG ACA ACA AAG AAG TTC AAC TCT TTA TCG ATG/GAT CCC

Restriction endonuclease BamHl cuts the DNA where indicated (/) to give a 4 base 5′ single stranded overhang. When this is filled in with DNA polymerase 1, the sequence bases 34-43 of SEQ ID NO:48 MS2 - - TCG ATG GAT C is generated. When this is ligated to Xmnl cut WGL

+

DNA (Xmnl cuts a: the sequence SEQ ID NO:49 GAANNNNTTC i.e. following base 120) the sequence SEQ ID NO:50 MS2- - - - TCG ATG GAT CAG TTC TGT - - - WGL

+

is generated. The plasmid so constructed encodes a protein which contains 15N terminal amino acids from the MS2 polymerase and the cloning site sequences in place of the N-terminal 11 amino acids of the mature WGL

+

sequence followed by the WGL

+

amino acid sequence from amino acids 31 to 620 for the BamHl fragment or 31 to 628 for the Pstl fragment. When transformed into a suitable host such as N4830(10) which contains a mutation (cI

ts

) in the gene coding for the cI repressor, expression of the fused polypeptide is repressed at temperatures such as 30° C. but is active at temperatures such as 42° C. This temperature dependence of expression is advantageous in instances where the fused product is deleterious to the cells. Cells are grown at 30° C. to the desired cell density and the temperature is then increased to 42° C. to induce the synthesis of the fused protein. The expression vector pBTA224 was used to generate a strain capable of producing a β-galactosidase-WGL

+

fusion protein. pBTA224 was derived from pUR292 (EMBO J. 2, 1791-1794 (1983)) by eliminating the EcoR1 site that lies outside of the β-galactosidase-coding region. pBTA224 DNA was cut with the restriction endonucleases Sadcl and Pstl, and the resulting 4221 bp fragment was purified by agarose gel electrophoresis. Sacl cuts within the lac Z gene, 1181 bps from the 3′ end. Pstl cuts pBTA224 at the 3′ end of lacZ. A WGL

+

gene fragment suitable for expression in this vector was prepared by first inserting an Xmnl restriction fragment of about 2 Kb (position 116 to past 3′ end of WGL

+

gene) into the vector M13um31 (obtained from International Biotechnologies, Inc.). By cutting the new construct with Sacl and Pstl, a fragment encoding most of the WGL

+

and having Sacl and Pstl cohesive ends could be obtained. The sequences for the Sacl end are also shown in SEQ ID NO:51 and 52.

121 of WGL

+

sequence 1911

Sac1 5′ > > Pst1

end CGGTACCC AG TTC TGT AGT GCT GCA end

TCGAGCCATGGG TC AAG ACA TCA CG

3′ From M13um31 From WGL

+

gene

This fragment ligated to the large pBTA224 Sac1 Pst1 fragment described above

gives SEQ ID NOS:53 and 54

WGL

+

⅔

rds

lacZ gene AAC GAG CT CGGTACCCAG TCC ----

TTGC TC GA GCCATGGGTC AAG ----

The fusion protein expected to be produced after induction with IPTG consists of the first 651 amino acids of β galactosidase, 599 amino acids of WGL

+

and 19 amino acids that are encoded by other parts of the expression vector, such as the multiple cloning sites. The calculated molecular weight is 143,054 daltons.

The plasmid described above has been designated pBTA708. A suitable

E.coli

host containing the lacl

q

gene is JM101. BTA1752 is JM101 transformed with pBTA708.

Cell lysates prepared from IPTG induced and control cultures, were analysed by electrophoresis in SDS-polyacrylamide gels. One gel was stained with Coomassie brilliant blue and a band of about the expected size could be visualised (FIGS.

9

A-

9

B). The band was absent in the non-induced control. A duplicate SDS-polyacrylamide gel was also run and the proteins in the gel were transferred to nitrocellulose paper. The nitrocellulose paper was incubated in BLOTTO (a solution of 5% powdered milk in Tris-saline) for 2 hours, then in BLOTTO containing a {fraction (1/500)} dilution of serum from a rabbit vaccinated with the fractions GF5 and 6 (see above) for 13 hrs at 4° C., then washed three times with BLOTTO, then incubated in a solution containing goat-anti-rabbit immunoglobulin conjugated to alkaline phosphatase (Promega Biotec). Following incubation for lhr, the nitrocellulose was removed, washed twice in BLOTTO and incubated in buffer containing Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. A band appeared where the rabbit antibodies had bound to the β galactosidase-WGL

+

fused polypeptide synthesized by the bacteria (FIG.

10

). The position of the band corresponds to the position of the band seen in the coomassie stained gel.

EXAMPLE 9

Fermentation Purification and Formulation of Vaccines Based on the WGL

+

Antigen Produced by rDNA Techniques

Strains expressing the WGL

+

antigen or portions thereof are maintained as freeze-dried vials in the production culture collection. Cells from the storage vial are reconstituted and plated out on a selective medium, and the cells from this medium are used to prepare fermentor inocula. The inocula are used to seed fermentors containing a suitable growth medium and the fermentation proceeds under conditions appropriate for the production of the WGL

+

proteins. At the completion of the fermentation the cells are harvested and the product is released from the cells and undergoes purificaticn. The product is subjected to analyses and quality control, and is stored under conditions appropriate for good stability. The product is formulated for use by combination with other ingredients under conditions of strict hygiene.

The strains produce the WGL

+

fusion proteins in vivo as insoluble agglomerates termed inclusion bodies and can be produced and purified by the following procedure which is presented by means of example only.

Overnight cultures of BTA1752 is diluted 1:50 into 2×1 liter fresh LB (10 g tryp:one/5 g yeast extract/5 g NaCl per liter pH 7.5) in 2 liter baffled flasks and shaken at 30° C. until the culture density reached OD 0.3-0.4. IPTG is added to a final concentration of 10 mM and incubation continued for a further 10 hours. The cells are harvested and resuspended in 20 ml of water per liter of original culture and broken by use of a French Press. The suspension is made 0.1 mM in phenylmethylsulfonyl fluoride (PMSF) and 5% Triton X-100 then centrifuged at 12,000×gav for 10 minutes. The supernatant is discarded and the pellet resuspended bv ultrasound in 50 ml 1M NaCl/5% Triton X-100 and recentrifuged. This washing stage is repeated and the pellet finally resuspended using ultrasound in 2.5 ml 1M NaCl/5% Triton X-100 per liter original culture.

Purified inclusion bodies are dissolyed at 2 mg/ml in 8M urea/0.1M DTT/0.1M Tris HCl pH 8.0 under nitrogen at 37° C. for 2 hrs. The solution is centrifuged at 20,000×gav for 20 min and the supernatant passed through a 0.1 μm filter. The flow through is passed through a filter with a molecular weight cut-off of 30 kilodaltons and the retained material is applied to a DEAE resin which is poured into a column and washed with 0.1M tris buffer pH8. The column is then resolyed with a linear gradient of from 0-5M NaCl in 8M urea 0.1M Tris pH8.0 and the fractions analysed by SDS-Polyacrylamide gel electrophores. Those containing the desired protein are pooled, concentrated and desalted on a XM30 filter. The partially purified protein is emulsified in an adjuvant such as Marcol 52: Montanide 888 (9:1) or Freunds complete or incomplete adjuvant and administered to animals.

EXAMPLE 10

Identification of DNA Sequences Coding for the WGL

+

Antigen in Species of Tick Other than

Boophilus microplus

In various countries throughout the world, tick species other than

Boophilus microplus

are responsible for extensive productivity losses either due to the tics infestation or due to the other parasites which the ticks transmit or a combination of both. It would be highly desirable to develop vaccines against these tick species. This may be achieved by vaccinating animals with the WGL

+

antigen derived from

Boophilus microplus

or the other immunogenic protective fractions described in this and Australian patent application No. 45936/85. It may also be possible to vaccinate animals with the WGL

+

antigen produced by recombinant organisms described herein and elicit an immune response which protects against infestation of animals by other species of tick.

As discussed above, the other species of tick probably contain a molecule which is functionally related to the

Boophilus microplus

WGL

+

antigen but which differs in sequence from that shown in FIG.

8

. If those differences occur in areas eliciting protective immune responses, then the

Boophilus microplus

WGL

+

antigen may not be protective. However, the related gene product from the other species of tick is likely to be protective against those tick species, when incorporated into a vaccine.

One means by which this proposal can be tested is to conduct a series of vaccination/challenge experiments using fractions derived from homogenates of other ticks and purify the WGL

+

homologues from the other tick species. These can then be cleaved with proteinases, peptide fragments sequenced, oligonucleotides designed and used to identify recombinant organisms containing the genes in a similar way to that in which the

Boophilus microplus

WGL

+

gene has been identified in the present work.

A preferable approach is to construct cDNA or genomic DNA libraries from nucleic acids extracted from other tick species and to use the DNA fragment shown in FIGS.

6

-

6

(

2

) bases 1-2012 of SEQ ID NO:55 or portions thereof as hybridization probes to identify clones containing the homologous gene from the other tick species. Then, engineered recombinant microorganisms synthesizing the homologous gene product could be incorporated into an effective vaccine against the other species of ticks.

In order to demonstrate that this latter approach is feasible and to generate information concerning the conditions under which the hybridization to the clone libraries should be carried out, preliminary “Southern blot” hybridization experiments can be conducted. Briefly by way of example only DNA isolated from a number of species of tick is purified and digested with restriction endonucleases. The DNA fragments so produced are size fractionated by electrophoresis on agarose gels, denatured and transferred to nylon or nitrocellulose filters by capillary action. The filter is, incubated in a prehybridization solution and then in a hybridization solution containing radioactively labelled DNA fragments derived from the WGL

+

gene coding region. Following hybridization and washing of the filters, they are exposed to X-ray film and the resulting autoradiograph shows exposed areas which correspond to the DNA fragments from the various tick species which have hybridized to the WGL

+

DNA fragments. There are many variations of protocols for carrying out this procedure which will be known to individuals skilled in the art and the following is detailed by means of example only.

Eggs were obtained from female ticks of the species

Rhiphicephalus appendiculatus, Amblyomma variegatim, Boophilus decoloratus

and

Boophilus microplus

. They were incubated in a humidified incubator for 2-4 days then suspended in cold TE buffer and washed. They were then suspended in TE buffer containing 0.5% SDS in a loose fitting glass-homogeniser and gently homogenised to disrupt the eggs. Proteinase K was added to a final concentration of 50 μg/ml and the mixture was incubated at 37° C. for 1-2 h with gentle shaking. The viscous solution was gently extracted three times with phenol saturated with 0.1M Tris-HCl pH8.0 and then twice with ether (centrifugation at 5,000×gav for 10 minutes was used to resolve the phases during the phenol extractions). Sodium acetate was added to 0.3M and 2 volumes of ethanol was slowly added with stirring. The DNA which came out of solution as a fibrous precipitate was removed with a pasteur pipette, washed in ethanol, and gently redissolved in TE.

Aliquots (generally containing 10 μg) of these DNA samples were digested with restriction endonucleases according to the manufacturers instructions. Aliquots of the digest products were fractionated by electrophoresis on a 1.6% agarose gel in SEB buffer (10). The DNA was depurinated with 2 volumes of 0.25M HCl for 15 minutes. The DNA was transferred by capillary action to a nylon membrane (Zetaprobe, Biorad). The filters were incubated in prehybridization in a solution (10) containing herring sperm DNA for 2-4 hours at 55° C. Hybridization was carried out in the same solution containing heat denatured [

32

P] labelled DNA fragments from the WGL

+

gene (approximately 10

5

counts per minute/ml) for 20 hours at 68° C. The filters were washed at 55° C. for 30 minutes each in 2× SSC, 0.1% SDS then three times at 60° C. for 15 minutes. After exposure to X-ray film for at least 24 hours the size of the hybridizing fragments could be determined by comparison with marker DNA fragments of known size.

FIGS. 10A-10C

shows an autoradiogram of one such experiment. DNA was digested with restriction endonuclease Sau 3A. The WGL

+

DNA clearly hybridizes to the DNA from all four species of ticks. In this experiment, the DNA was not intact so a smear is observed in all cases but hybridization is specific as no hybridization to control DNA on same gel could be detected.

EXAMPLE 11

Isolation of Clones Coding for WGL

+

Homologous from other Tick Species

The DNA from each of the species tested possesses sequences which are similar to and homologous with the DNA coding for the WGL

+

antigen from

Boophilus microplus

. Clones containing those DNA sequences from other tick species can be isolated by constructing cDNA or genomic DNA libraries for the other tick species and hybridizing

Boophilus microplus

DNA fragments to those libraries, and purifying recombinant organisms containing the DNA sequences hybridizing to the homologous genes.

More specifically, the genomic DNA isolated from the tick species listed was subjected to partial digestion with the restriction enzyme Sau 3A to give fragments with an average size of 15-20 Kb as judged by gel analysis. These were ligated into the Bam HI site of lambda EMBL 3 arms essentially as described by the suppliers (Promega Biotech). The libraries were plated on a restrictive host K62 and incubated overnight at 37° C. The plaques were transferred to triplicate nitrocellulose filters, and the DNA denatured win 1.5M NaCl/0.5M NaOH, neutralised with 3M NaCl/0.5M Tris HCl pH 7.0. Then the filters were vacuum baked at 80° C. for 2 hours and hybridized to

Boophilus microplus

DNA probes labelled with

32

P. Following autoradiography, plaques which hybridized to the probes on both filters were identified, picked and purified to single plaques by repeated rounds of prehybridization.

DNA was isolated from one plaque from a

B. decoloratus

genomic library and digested with restriction endonucleases HaeIII and Apa 1. The fragments so produced were separated by electrophoresis on 1.6% agarose gels. One gel was stained with an ethidium bromide solution and the bands visualised under ultraviolet light (FIGS.

10

A-

10

C). A replicate gel was transferred to nylon membrane and hybridized to

Boophilus microplus

DNA coding for the WGL

−

antigen.

FIGS. 10A-10C

show that fragments from the

Boophilus decoloratus. Amblyomma variegatum

genomic clone hybridize to the WGL

−

gene.

The bands hybridising in the HaeIII digest are approximately 980, 630, and 340 bp and in the Apa 1 digest 27,300 bp when compared with fragments of DNA from bacteriophage lambda digested with Hind III.

The regions of the DNA in each plaque which codes for portions of the homologous gene for the WGL

−

antigen from each species of tick are sequenced and engineered for expression in recombinant organisms essentially as described above for the

Boophilus microplus

WGL

−

antigen. The same approach can also be taken to isolate cDNA clone from these and other tick species.

The homologous WGL

−

antigen proteins expressed by the microorganisms are then grown in fermenters, the expression of the recombinant antigen induced and the antigen is purified formulated with an adjuvant or carrier and used to vaccinate animals.

It is understood that this procedure can be equally applied to any species of tick to isolate clones coding for WGL

−

related antigens and the WGL

−

related proteins expressed by the so constructed genetically engineered microorganisms can be used as effective vaccines against a range of tick species which are responsible for productivity losses, morbidity and mortality to domestic animals and man.

EXAMPLE 12

RNA was extracted from ticks collected from different regions of the world and cDNA libraries were constructed using lambda vectors essentially as described in Example 6. Replicas of these cDNA libraries were hybridised with radioactively labelled restriction fragments derived from the DNA coding for the WGL

+

antigen using hybridisation conditions designed to detect nucleic acid sequences having a minimum of 70% homology to the hybridising sequence. The resulting plaques that reacted with the DNA hybridisation probes were then purified to single plaques. The DNA sequences of the genes were determined using standard sequencing techniques.

FIGS. 12-17

illustrate the DNA sequences and deduced amino acid sequences SEQ ID NOS:58-69.

The DNA sequence YBm017 (

FIG. 12

) SEQ ID NO:58, was derived from an Australian isolate of

Boophilus microplus

(Yeerongplly, Queensland). The WGL

+

antigen described in the preceding examples was also obtained from the same isolate. A comparison of the DNA sequences reveals:

(i) The DNA sequences are not identical. There is approximately 95% homology at the DNA level but it is clear that the two sequences code for the same antigen.

(ii) The translated amino acid sequence SEQ ID NO:59 has 8 differences between the two antigens. For example, the WGL

+

sequence encodes a phenylalanine at position 1551 while YBm017 encodes a cysteine at the corresponding position (nucleotide 1570 in

FIG. 12

) SEQ ID NO:58. In addition, the sequence serine glycine serine (encoded by nucleotides 735 through 743 in the WGL+ sequence) is arginine alanine phenylalanine in the corresponding position of YBm017 (nucleotides 754 to 762 in

FIG. 12

) SEQ ID NO:58.

A partial cDNA clone encoding a protein fragment with an amino acid sequence homologous to that of WGL+ is presented as YBm22M8 (

FIG. 13

) SEQ ID NO:60. This sequence extends from nucleotide position 276 to nucleotide position 1922 of the WGL+ sequence. There are 13 differences between the deduced amino acid sequences of YBm22M8 SEQ ID NO:61 and WGL+ SEQ ID NO:56.

These observations further illustrate that the tick population, even within one isolate of ticks is genetically diverse and that homologues of the antigen are found within that population.

A second form of the antigen consisting essentially of the sequence described for YBm22M8 but including the amino terminus of the original WGL+ clone has been expressed in recombinant bacteria and used to vaccinate cattle which were subsequently challenged with ticks. This recombinant antigen protects cattle as well as that encoded by the WGL+ antigen.

The DNA clone, Bm023 (

FIG. 14

) SEQ ID NO:62 was obtained from another Australian isolate of

Boophilus microplus

. The nucleotide sequence of this cDNA codes for a protein SEQ ID NO:63 that has 13 amino acids that are different from those encoded by the WGL+ cDNA. This demonstrates that the major form of the WGL+ antigen is similar for the two populations of ticks.

The VBm021 and MexBm86 cDNA molecules (

FIGS. 15

SEQ ID NO:64 is and 16 SEQ ID NO:66 respectively) were obtained from

Boophilus microplus

isolates from Venezuela and Mexico respectively. The VBm021 sequence is a partial cDNA clone in that the sequence does not extend to the start codon of the gene. The sequence begins at a position corresponding to amino acid 31 of the deduced WGL+ amino acid sequence (nucleotide position 123 in

FIG. 7

) bases 1-2012 of SEQ ID NO:55. The MexBm86 cDNA sequence extends through the start codon and into the 5′ untranslated region of the WGL+ sequence (

FIG. 7

) bases 1-2012 of SEQ ID NO:55. These sequences differ from the WGL+ deduced amino acid sequence by 28 (VBm021) SEQ ID NO:65 and 22 (MexBm86) SEQ ID NO:66 amino acids.

These results confirm that it is possible to isolate related genes from a diverse range of

Boophilus microplus

isolates using the WGL+ gene (or fragments derived from this gene) as hybridisation probes. The DNA sequences of the variants will enable the gene to be clearly identified as related to the WGL+ gene but the homology at the DNA sequence level may be no more than 50% over some regions. In addition the translated amino acid sequences of these genes clearly indicate that the genes code for proteins which are closely related to the WGL+ protein but may differ in amino acid sequence by as much as 30% over some stretches of the protein.

The Ra442 sequence (

FIG. 6

) SEQ ID NO:68 was obtained from

Rhipicephalus appendiculatus

. Comparison of this cDNA with WGL+ demonstrates that the Ra442 sequence codes for a protein fragment SEQ ID NO:69 which is homologous to the WGL+ sequence corresponding to nucleotides 1113 to 1553 (

FIG. 7

) bases 1-2012 of SEQ ID NO:55. It contains structural elements which are characteristic of these molecules. The homology over this region is approximately 85% at the DNA level and approximately 70% at the amino acid level, with particular regions having higher homology than others. This is clearly a molecule which is closely related in structure (and presumably in function) to the WGL+ antigen from

Boophilus microplus.

The nucleotide sequences presented in both YBm 017 SEQ ID NO:58 and VBm021 SEQ ID NO:64 contain two nucleotides each that could not be determined unambiguously when reading the sequencing gels. These are represented using the IUPAC ambiguity code and result in the translated amino acid, Xaa. These were not included when describing the number of amino acid differences between these clones and the WGL+ sequence.

DEPOSITION OF MICROORGANISMS

Strain BTA 1751 referred to herein has been deposited with the American Type Culture Collection of 12301 Parklawn Drive Rockville Md. 20852 USA in accordance with the provisions of the Budapest Treaty on October 12, 1987 under accession number ATCC 67548.

Strain BTA 1751 has also been deposited with the China Centre for Type Culture Collection under import licence IL-87044 and designated CTCC.

INDUSTRIAL APPLICATION

The current invention provides a means of vaccinating cattle against infestation with ticks such as

Boophilus microplus, Boophilus annulatus

; other species such as Haemaphysalis spp, Otobius spp, Rhiphicephalus spp, Ambylomma pp, Dermacentor spp, Ixodes spp and Hyalomma spp; and particular examples thereof including

Otobius megnini, Rhiphicephalus appendiculatus, Dermacentor andersoni, D. variabilis

and

Ixodes holocyclus

. Further it provides a means of protecting cattle against diseases such as those caused by

Babesia bovis, Cowdria ruminatum, Theleria parva parva, T. parva lawrencil, T. annulata

and

T. hirci

. Further it provides diagnostic tools for the identification and quantification of tick antigens.

REFERENCES

1. Brown. S. J., Shapiro, S. Z. and Askenase, P. W. J. Immunol. 133, 1984, 3319-3325.

2. Ackerman, S., Floyd, M. and Sonenshine, D. E. J. Med. Entomol. 17, 1980, 391-397.

3. McGowan, M. J., Barker, M. J, Homer, J. T., McNew, R. W. and Holscher, K. M. 1971, J. Med. Entomol. 18, 1981, 328.

4. Kikel, S. K. Am. J. Trop. Med. Hyg. 30, 1981, 284.

5. Allen, J. R. and Humphries, S. J. Nature, 280, 1979, 481-493.

6. Johnston, L. A. Y., Kemp. D. H. and Pearson, R. D. Int. J. Parasltol. 16, 27-34, 1986.

7. Kemp, D. H., Agbede, R. I. S., Johnston, L. A. Y. and Gough, J. M. Int. J.

Parasitol. 16, 121-130, 1986.

8. Agbede, R. I. S. and Kemp, D. H. Int. J. Parasitol. 16, 35-42, 1986.

9. Briggs M. S. and Gierasch, L. M. (1986), Molecular Mechanisms of Protein Secretion: The Role of the Signal Sequence, pages 110-180 in Advances in Protein Chemistry, vol. 38, Academic Press.

10. Maniatis, T., Fritsch, E. F. and Sambrook, J. (1982), Molecular cloning: A Laboratory Manual (Cold Spring Harbour Laboratory).

11. Kornfeld, R. and Kornfeld, S., 1985, Ann. Rev. Biochem 54, 631-664

12. Van Hemert, F. J., Amons, R., Pluijms, W. J. M., Van Crmondt, H. and Moeller, W. EMBO 3, 1109-1113 1984

13. Willadsan, P., Int. J. Parasitol, 17, 671-677 (1987)

14. Vretblad, P., Biochemica and Biophysic Acta, 434, 169-176 (1976).

15. Sage, H. J. and Green, R. W. In Methods in Enzymology, 28, Guinsburg, V., ed., 332-339(1972), London Academic Press.

7 amino acids

amino acid

linear

unknown

1
Lys Asp Pro Asp Pro Gly Lys
1 5

17 amino acids

amino acid

linear

unknown

2
Lys Trp Tyr Glu Asp Gly Val Leu Glu Ala Ile Xaa Thr Ser Ile Gly
1 5 10 15
Lys

18 amino acids

amino acid

linear

unknown

3
Lys Xaa Gln Ala Cys Glu His Pro Ile Gly Glu Trp Cys Met Met Tyr
1 5 10 15
Pro Lys

8 amino acids

amino acid

linear

unknown

Modified-site

/note= “Xaa at position 7
represents Cys or Gln”

4
Lys Glu Ala Gly Phe Val Xaa Lys
1 5

12 amino acids

amino acid

linear

unknown

Modified-site

/note= “Xaa at position 4
represents Ser or Asp”

Modified-site

/note= “Xaa at position 7
represents Val or Cys”

Modified-site

/note= “Xaa at position 10
represents Val or Ala”

Modified-site

/note= “Xaa at position 11
represents Ile or Cys”

5
Lys Gly Pro Xaa Gly Gln Xaa Ile Asn Xaa Xaa Lys
1 5 10

17 amino acids

amino acid

linear

unknown

Modified-site

/note= “Xaa at position 3
represents Gly or Asp”

6
Lys Ala Xaa Val Ser Thr Asn Glu Asn Glu Gln Leu Glu Gln Ala Asp
1 5 10 15
Lys

12 amino acids

amino acid

linear

unknown

Modified-site

/note= “Xaa at position 3
represents Gly or Asp”

7
Lys Ser Xaa Thr Gln Xaa Ile Asp His Ile Ser Lys
1 5 10

7 amino acids

amino acid

linear

unknown

Modified-site

/note= “Xaa at position 2
represents Asn or Asp”

Modified-site

/note= “Xaa at position 5
represents Ala or Tyr”

Modified-site

/note= “Xaa at position 6
represents Ala or Tyr”

8
Lys Xaa Gln Glu Xaa Xaa Tyr
1 5

19 amino acids

amino acid

linear

unknown

9
Lys Cys Pro Cys Asp Asn Met Tyr Phe Asn Ala Ala Glu Glu Ile Gly
1 5 10 15
Cys Ile Glu

17 amino acids

amino acid

linear

unknown

10
Ala Asn Gln Cys Pro Pro Asp Thr Arg Arg Gly Glu Ile Gly Cys Ile
1 5 10 15
Glu

19 amino acids

amino acid

linear

unknown

F10

11
Lys Ala Pro Arg Gln Asn Met Tyr Phe Asn Ala Ala Glu Glu Ile Gly
1 5 10 15
Cys Ile Glu

18 amino acids

amino acid

linear

unknown

F10

12
Cys Asn Cys Asp Cys Pro Pro Asp Thr Arg Pro Gly Glu Ile Gly Cys
1 5 10 15
Ile Glu

17 amino acids

amino acid

linear

unknown

F11

13
Lys Trp Tyr Glu Asp Arg Val Leu Glu Ala Ile Arg Thr Ser Ile Gly
1 5 10 15
Lys

23 amino acids

amino acid

linear

unknown

F12

14
Lys Glu Ser Ser Ile Cys Xaa Asp Phe Gly Asn Glu Phe Cys Arg Asn
1 5 10 15
Ala Glu Cys Glu Val Val Pro
20

17 amino acids

amino acid

linear

unknown

F13

15
Lys Thr Arg Glu Cys Ser Tyr Gly Arg Cys Val Glu Ser Asn Pro Ser
1 5 10 15
Lys

21 amino acids

amino acid

linear

unknown

F14

Modified-site

/note= “Xaa at position 19
represents Ser or His”

16
Lys Ala Tyr Glu Cys Thr Cys Pro Arg Ala Phe Thr Val Ala Glu Asp
1 5 10 15
Gly Ile Xaa Cys Lys
20

14 amino acids

amino acid

linear

unknown

F15

Modified-site

/note= “Xaa at position 8
represents Ser or His”

17
Lys Asp Glu Val Asp Asn Ala Xaa Leu Val Cys Gln Asn Ala
1 5 10

12 amino acids

amino acid

linear

unknown

F15

18
Lys Asn Val Leu Gln Ser Asp Gly Cys Gly Pro Tyr
1 5 10

11 amino acids

amino acid

linear

unknown

F15

Modified-site

/note= “Xaa at position 7
represents Pro or Leu”

Modified-site

/note= “Xaa at position 11
represents His or Ser”

19
Lys Cys Leu Asn Pro Arg Xaa Arg Leu Lys Xaa
1 5 10

9 amino acids

amino acid

linear

unknown

F16

Modified-site

/note= “Xaa at position 2
represents Ser, Ala, Cys or Gly”

20
Lys Xaa Xaa Val Leu Cys Glu Xaa Pro
1 5

9 amino acids

amino acid

linear

unknown

F17

21
Lys Leu Gln Ala Cys Glu His Pro Ile
1 5

18 amino acids

amino acid

linear

unknown

F3, F17

22
Lys Leu Gln Ala Cys Glu His Pro Ile Gly Glu Trp Cys Met Met Tyr
1 5 10 15
Pro Lys

8 amino acids

amino acid

linear

unknown

23
Lys Glu Ala Gly Phe Val Cys Lys
1 5

12 amino acids

amino acid

linear

unknown

24
Lys Gly Pro Asp Gly Gln Cys Ile Asn Ala Cys Lys
1 5 10

17 amino acids

amino acid

linear

unknown

25
Lys Ala Gly Val Ser Cys Asn Glu Asn Glu Gln Ser Glu Cys Ala Asp
1 5 10 15
Lys

8 amino acids

amino acid

linear

unknown

26
Lys Asp Gln Glu Ala Ala Tyr Lys
1 5

14 amino acids

amino acid

linear

unknown

27
Lys Cys Pro Arg Asp Asn Met Tyr Phe Asn Ala Ala Glu Lys
1 5 10

19 amino acids

amino acid

linear

unknown

F9, F10

28
Lys Ala Asn Cys Gln Cys Pro Pro Asp Thr Lys Pro Gly Glu Ile Gly
1 5 10 15
Cys Ile Glu

19 amino acids

amino acid

linear

unknown

F9, F10

29
Lys Ala Asn Cys Gln Cys Pro Pro Asp Thr Arg Pro Gly Glu Ile Gly
1 5 10 15
Cys Ile Glu

24 amino acids

amino acid

linear

unknown

F12

30
Ala Glu Ser Ser Ile Cys Ser Asp Phe Gly Asn Glu Phe Cys Arg Asn
1 5 10 15
Ala Glu Cys Glu Val Val Pro Gly
20

21 amino acids

amino acid

linear

unknown

F14

31
Lys Ala Tyr Glu Cys Thr Cys Pro Ser Gly Ser Thr Val Ala Glu Asp
1 5 10 15
Gly Ile Thr Cys Lys
20

21 amino acids

amino acid

linear

unknown

F14

32
Lys Ala Tyr Glu Cys Thr Cys Pro Arg Ala Phe Thr Val Ala Glu Asp
1 5 10 15
Gly Ile Thr Cys Lys
20

12 amino acids

amino acid

linear

unknown

F15

33
Lys Asn Leu Leu Gln Arg Asp Ser Arg Cys Cys Gln
1 5 10

9 amino acids

amino acid

linear

unknown

F16

34
Lys Gly Thr Val Leu Cys Glu Cys Pro
1 5

14 amino acids

amino acid

linear

unknown

35
Lys Cys Pro Cys Asp Asn Met Tyr Phe Asn Ala Ala Glu Lys
1 5 10

19 amino acids

amino acid

linear

unknown

36
Lys Ala Asn Arg Gln Cys Pro Pro Asp Thr Arg Arg Gly Glu Ile Gly
1 5 10 15
Cys Ile Glu

20 base pairs

nucleic acid

single

linear

unknown

37
TTACCTGGAT CTGGATCCTT 20

50 base pairs

nucleic acid

single

linear

unknown

38
TTACCAATGG ATGTACAAAT AGCTTCAAGG ACACCATCTT CGTACCACTT 50

53 base pairs

nucleic acid

single

linear

unknown

39
TTTGGGTACA TCATACACCA TTCACCAATT GGGTGTTCAC AAGCCTGADS CTT 53

14 amino acids

amino acid

linear

unknown

F10

40
Lys Ala Pro Arg Gln Asn Met Tyr Phe Asn Ala Ala Glu Lys
1 5 10

19 amino acids

amino acid

linear

unknown

F10

41
Lys Cys Asn Cys Asp Cys Pro Pro Asp Thr Arg Pro Gly Glu Ile Gly
1 5 10 15
Cys Ile Glu

24 amino acids

amino acid

linear

unknown

F12

42
Lys Glu Ser Ser Ile Cys Xaa Asp Phe Gly Asn Glu Phe Cys Arg Asn
1 5 10 15
Ala Glu Cys Glu Val Val Pro Lys
20

72 base pairs

nucleic acid

single

linear

unknown

43
TTTAGGTACA ACCTCACATT CAGCATTCCT ACAAAATTCA TTACCGAAAT CAAAACAAAT 60
ACTACTCTCC TT 72

51 base pairs

nucleic acid

single

linear

unknown

44
CTTCGACGGA TTGGATTCGA CGCATCTGCC ATAGCTACAT TCCCTCGTCT T 51

63 base pairs

nucleic acid

single

linear

unknown

45
CTTGCAATGG ATTCCATCCT CGGCGACAGT GAAAGCTCTA GGGCAAGTGC ACTCATAAGC 60
CTT 63

19 amino acids

amino acid

linear

unknown

F9 b

46
Lys Ala Asn Cys Gln Cys Pro Pro Asp Thr Arg Arg Gly Glu Ile Gly
1 5 10 15
Cys Ile Glu

9 amino acids

amino acid

linear

unknown

F16

47
Lys Xaa Xaa Val Leu Cys Glu Xaa Pro
1 5

45 base pairs

nucleic acid

single

linear

unknown

48
ATGTCGAAGA CAACAAAGAA GTTCAACTCT TTATCGATGG ATCCC 45

10 base pairs

nucleic acid

single

linear

unknown

49
GAANNNNTTC 10

18 base pairs

nucleic acid

single

linear

unknown

50
TCGATGGATC AGTTCTGT 18

16 base pairs

nucleic acid

single

linear

unknown

51
CGGTACCCAG TTCTGT 16

20 base pairs

nucleic acid

single

linear

YES

unknown

52
ACAGAACTGG GTACCGAGCT 20

21 base pairs

nucleic acid

single

linear

unknown

53
AACGAGCTCG GTACCCAGTC C 21

21 base pairs

nucleic acid

single

linear

YES

unknown

54
GAACTGGGTA CCGAGCTCGT T 21

2065 base pairs

nucleic acid

double

linear

unknown

Figure 7

CDS

33..1985

55
CCGCGACAGC TGCGGTGGTT CGACGCAGTG AG ATG CGT GGC ATC GCT TTG TTC 53
Met Arg Gly Ile Ala Leu Phe
1 5
GTC GCC GCT GTT TCA CTG ATT GTA GAG GGC ACA GCA GAA TCA TCC ATT 101
Val Ala Ala Val Ser Leu Ile Val Glu Gly Thr Ala Glu Ser Ser Ile
10 15 20
TGC TCT GAC TTC GGG AAC GAG TTC TGT CGC AAC GCT GAA TGT GAA GTG 149
Cys Ser Asp Phe Gly Asn Glu Phe Cys Arg Asn Ala Glu Cys Glu Val
25 30 35
GTG CCT GGT GCA GAG GAT GAT TTC GTG TGC AAA TGT CCG CGA GAT AAT 197
Val Pro Gly Ala Glu Asp Asp Phe Val Cys Lys Cys Pro Arg Asp Asn
40 45 50 55
ATG TAC TTC AAT GCT GCT GAA AAG CAA TGC GAA TAT AAA GAC ACG TGC 245
Met Tyr Phe Asn Ala Ala Glu Lys Gln Cys Glu Tyr Lys Asp Thr Cys
60 65 70
AAG ACA AGG GAG TGC AGC TAT GGA CGT TGC GTT GAA AGT AAC CCG AGC 293
Lys Thr Arg Glu Cys Ser Tyr Gly Arg Cys Val Glu Ser Asn Pro Ser
75 80 85
AAG GCT AGC TGC GTC TGC GAA GCA TCG GAC GAT CTA ACG CTA CAA TGC 341
Lys Ala Ser Cys Val Cys Glu Ala Ser Asp Asp Leu Thr Leu Gln Cys
90 95 100
AAA ATT AAA AAT GAC TAC GCA ACT GAC TGC CGA AAT CGA GGT GGC ACT 389
Lys Ile Lys Asn Asp Tyr Ala Thr Asp Cys Arg Asn Arg Gly Gly Thr
105 110 115
GCT AAG TTG CGC ACG GAT GGG TTT ATT GGC GCA ACG TGT GAC TGT GGT 437
Ala Lys Leu Arg Thr Asp Gly Phe Ile Gly Ala Thr Cys Asp Cys Gly
120 125 130 135
GAA TGG GGT GCG ATG AAC ATG ACC ACC CGG AAC TGT GTC CCT ACC ACG 485
Glu Trp Gly Ala Met Asn Met Thr Thr Arg Asn Cys Val Pro Thr Thr
140 145 150
TGT CTT CGT CCC GAC TTG ACC TGC AAA GAC CTC TGC GAG AAA AAC CTG 533
Cys Leu Arg Pro Asp Leu Thr Cys Lys Asp Leu Cys Glu Lys Asn Leu
155 160 165
CTT CAA AGG GAT TCT CGT TGT TGC CAG GGG TGG AAC ACA GCA AAC TGT 581
Leu Gln Arg Asp Ser Arg Cys Cys Gln Gly Trp Asn Thr Ala Asn Cys
170 175 180
TCA GCC GCT CCT CCA GCT GAC TCC TAT TGC TCT CCT GGG AGC CCC AAA 629
Ser Ala Ala Pro Pro Ala Asp Ser Tyr Cys Ser Pro Gly Ser Pro Lys
185 190 195
GGA CCG GAC GGA CAG TGT ATA AAT GCT TGC AAG ACG AAA GAA GCT GGG 677
Gly Pro Asp Gly Gln Cys Ile Asn Ala Cys Lys Thr Lys Glu Ala Gly
200 205 210 215
TTT GTC TGC AAG CAT GGA TGC AGG TCG ACC GGC AAG GCG TAC GAG TGC 725
Phe Val Cys Lys His Gly Cys Arg Ser Thr Gly Lys Ala Tyr Glu Cys
220 225 230
ACG TGC CCG AGT GGC TCT ACC GTC GCC GAA GAT GGC ATT ACC TGC AAA 773
Thr Cys Pro Ser Gly Ser Thr Val Ala Glu Asp Gly Ile Thr Cys Lys
235 240 245
AGT ATT TCG CAC ACA GTC AGC TGC ACT GCT GAG CAA AAA CAG ACC TGC 821
Ser Ile Ser His Thr Val Ser Cys Thr Ala Glu Gln Lys Gln Thr Cys
250 255 260
CGC CCA ACC GAA GAC TGT CGT GTG CAC AAA GGA ACT GTG TTG TGT GAG 869
Arg Pro Thr Glu Asp Cys Arg Val His Lys Gly Thr Val Leu Cys Glu
265 270 275
TGC CCG TGG AAT CAA CAT CTA GTG GGG GAC ACG TGC ATA AGT GAT TGC 917
Cys Pro Trp Asn Gln His Leu Val Gly Asp Thr Cys Ile Ser Asp Cys
280 285 290 295
GTC GAC AAG AAA TGC CAC GAA GAA TTT ATG GAC TGT GGC GTA TAT ATG 965
Val Asp Lys Lys Cys His Glu Glu Phe Met Asp Cys Gly Val Tyr Met
300 305 310
AAT CGA CAA AGC TGC TAT TGT CCA TGG AAA TCA AGG AAG CCG GGC CCA 1013
Asn Arg Gln Ser Cys Tyr Cys Pro Trp Lys Ser Arg Lys Pro Gly Pro
315 320 325
AAT GTC AAC ATC AAT GAA TGC CTA CTG AAT GAG TAT TAC TAC ACG GTG 1061
Asn Val Asn Ile Asn Glu Cys Leu Leu Asn Glu Tyr Tyr Tyr Thr Val
330 335 340
TCA TTC ACC CCA AAC ATA TCT TTT GAT TCT GAC CAT TGC AAA TGG TAT 1109
Ser Phe Thr Pro Asn Ile Ser Phe Asp Ser Asp His Cys Lys Trp Tyr
345 350 355
GAG GAT CGT GTT TTG GAA GCG ATA CGG ACC AGT ATC GGA AAA GAA GTT 1157
Glu Asp Arg Val Leu Glu Ala Ile Arg Thr Ser Ile Gly Lys Glu Val
360 365 370 375
TTT AAG GTT GAG ATA CTT AAC TGC ACG CAG GAC ATT AAG GCA AGA CTC 1205
Phe Lys Val Glu Ile Leu Asn Cys Thr Gln Asp Ile Lys Ala Arg Leu
380 385 390
ATA GCA GAG AAA CCA CTG TCA AAA CAC GTG CTC AGG AAA CTA CAA GCA 1253
Ile Ala Glu Lys Pro Leu Ser Lys His Val Leu Arg Lys Leu Gln Ala
395 400 405
TGC GAG CAT CCA ATC GGC GAA TGG TGC ATG ATG TAT CCG AAG TTG CTG 1301
Cys Glu His Pro Ile Gly Glu Trp Cys Met Met Tyr Pro Lys Leu Leu
410 415 420
ATC AAG AAA AAC TCT GCA ACA GAA ATC GAA GAA GAG AAC CTT TGC GAC 1349
Ile Lys Lys Asn Ser Ala Thr Glu Ile Glu Glu Glu Asn Leu Cys Asp
425 430 435
AGT CTG CTC AAG GAT CAG GAA GCT GCC TAC AAA GGT CAA AAC AAA TGC 1397
Ser Leu Leu Lys Asp Gln Glu Ala Ala Tyr Lys Gly Gln Asn Lys Cys
440 445 450 455
GTC AAG GTC GAC AAC CTC TTC TGG TTC CAG TGC GCT GAT GGT TAC ACA 1445
Val Lys Val Asp Asn Leu Phe Trp Phe Gln Cys Ala Asp Gly Tyr Thr
460 465 470
ACA ACT TAC GAG ATG ACA CGA GGT CGC CTA CGC CGC TCC GTG TGT AAA 1493
Thr Thr Tyr Glu Met Thr Arg Gly Arg Leu Arg Arg Ser Val Cys Lys
475 480 485
GCT GGA GTT TCT TGC AAC GAA AAC GAG CAG TCG GAG TGT GCT GAC AAA 1541
Ala Gly Val Ser Cys Asn Glu Asn Glu Gln Ser Glu Cys Ala Asp Lys
490 495 500
GGG CAA ATA TTT GTT TAC GAA AAC GGC AAA GCG AAT TGC CAA TGC CCA 1589
Gly Gln Ile Phe Val Tyr Glu Asn Gly Lys Ala Asn Cys Gln Cys Pro
505 510 515
CCA GAC ACT AAA CCT GGG GAG ATT GGC TGC ATT GAG CGT ACC ACA TGC 1637
Pro Asp Thr Lys Pro Gly Glu Ile Gly Cys Ile Glu Arg Thr Thr Cys
520 525 530 535
AAC CCT AAA GAA ATA CAA GAA TGC CAA GAC AAG AAG CTG GAG TGC GTT 1685
Asn Pro Lys Glu Ile Gln Glu Cys Gln Asp Lys Lys Leu Glu Cys Val
540 545 550
TAC AAA AAC CAT AAA GCA GAA TGC GAG TGT CCT GAT GAT CAC GAG TGT 1733
Tyr Lys Asn His Lys Ala Glu Cys Glu Cys Pro Asp Asp His Glu Cys
555 560 565
TAC AGG GAG CCT GCC AAA GAC TCT TGC AGT GAA GAG GAT AAT GGT AAA 1781
Tyr Arg Glu Pro Ala Lys Asp Ser Cys Ser Glu Glu Asp Asn Gly Lys
570 575 580
TGT CAA AGC AGT GGG CAG CGT TGT GTA ATA GAA AAC GGA AAG GCT GTT 1829
Cys Gln Ser Ser Gly Gln Arg Cys Val Ile Glu Asn Gly Lys Ala Val
585 590 595
TGC AAG GAA AAG TCT GAA GCA ACA ACA GCT GCG ACT ACA ACA ACG AAA 1877
Cys Lys Glu Lys Ser Glu Ala Thr Thr Ala Ala Thr Thr Thr Thr Lys
600 605 610 615
GCG AAA GAC AAG GAT CCA GAT CCT GGA AAG TCA AGT GCT GCA GCA GTA 1925
Ala Lys Asp Lys Asp Pro Asp Pro Gly Lys Ser Ser Ala Ala Ala Val
620 625 630
TCA GCT ACT GGG CTC TTG TTA CTG CTC GCA GCT ACT TCA GTC ACC GCA 1973
Ser Ala Thr Gly Leu Leu Leu Leu Leu Ala Ala Thr Ser Val Thr Ala
635 640 645
GCA TCG TTG TAAGGAAGAT GTCCAACTTG AATACGGAAC AGCTTGAATA 2022
Ala Ser Leu
650
TGTATATATA CATCACGCTT ACATCGAACA CCTAGCTTGG TTT 2065

650 amino acids

amino acid

linear

protein

unknown

56
Met Arg Gly Ile Ala Leu Phe Val Ala Ala Val Ser Leu Ile Val Glu
1 5 10 15
Gly Thr Ala Glu Ser Ser Ile Cys Ser Asp Phe Gly Asn Glu Phe Cys
20 25 30
Arg Asn Ala Glu Cys Glu Val Val Pro Gly Ala Glu Asp Asp Phe Val
35 40 45
Cys Lys Cys Pro Arg Asp Asn Met Tyr Phe Asn Ala Ala Glu Lys Gln
50 55 60
Cys Glu Tyr Lys Asp Thr Cys Lys Thr Arg Glu Cys Ser Tyr Gly Arg
65 70 75 80
Cys Val Glu Ser Asn Pro Ser Lys Ala Ser Cys Val Cys Glu Ala Ser
85 90 95
Asp Asp Leu Thr Leu Gln Cys Lys Ile Lys Asn Asp Tyr Ala Thr Asp
100 105 110
Cys Arg Asn Arg Gly Gly Thr Ala Lys Leu Arg Thr Asp Gly Phe Ile
115 120 125
Gly Ala Thr Cys Asp Cys Gly Glu Trp Gly Ala Met Asn Met Thr Thr
130 135 140
Arg Asn Cys Val Pro Thr Thr Cys Leu Arg Pro Asp Leu Thr Cys Lys
145 150 155 160
Asp Leu Cys Glu Lys Asn Leu Leu Gln Arg Asp Ser Arg Cys Cys Gln
165 170 175
Gly Trp Asn Thr Ala Asn Cys Ser Ala Ala Pro Pro Ala Asp Ser Tyr
180 185 190
Cys Ser Pro Gly Ser Pro Lys Gly Pro Asp Gly Gln Cys Ile Asn Ala
195 200 205
Cys Lys Thr Lys Glu Ala Gly Phe Val Cys Lys His Gly Cys Arg Ser
210 215 220
Thr Gly Lys Ala Tyr Glu Cys Thr Cys Pro Ser Gly Ser Thr Val Ala
225 230 235 240
Glu Asp Gly Ile Thr Cys Lys Ser Ile Ser His Thr Val Ser Cys Thr
245 250 255
Ala Glu Gln Lys Gln Thr Cys Arg Pro Thr Glu Asp Cys Arg Val His
260 265 270
Lys Gly Thr Val Leu Cys Glu Cys Pro Trp Asn Gln His Leu Val Gly
275 280 285
Asp Thr Cys Ile Ser Asp Cys Val Asp Lys Lys Cys His Glu Glu Phe
290 295 300
Met Asp Cys Gly Val Tyr Met Asn Arg Gln Ser Cys Tyr Cys Pro Trp
305 310 315 320
Lys Ser Arg Lys Pro Gly Pro Asn Val Asn Ile Asn Glu Cys Leu Leu
325 330 335
Asn Glu Tyr Tyr Tyr Thr Val Ser Phe Thr Pro Asn Ile Ser Phe Asp
340 345 350
Ser Asp His Cys Lys Trp Tyr Glu Asp Arg Val Leu Glu Ala Ile Arg
355 360 365
Thr Ser Ile Gly Lys Glu Val Phe Lys Val Glu Ile Leu Asn Cys Thr
370 375 380
Gln Asp Ile Lys Ala Arg Leu Ile Ala Glu Lys Pro Leu Ser Lys His
385 390 395 400
Val Leu Arg Lys Leu Gln Ala Cys Glu His Pro Ile Gly Glu Trp Cys
405 410 415
Met Met Tyr Pro Lys Leu Leu Ile Lys Lys Asn Ser Ala Thr Glu Ile
420 425 430
Glu Glu Glu Asn Leu Cys Asp Ser Leu Leu Lys Asp Gln Glu Ala Ala
435 440 445
Tyr Lys Gly Gln Asn Lys Cys Val Lys Val Asp Asn Leu Phe Trp Phe
450 455 460
Gln Cys Ala Asp Gly Tyr Thr Thr Thr Tyr Glu Met Thr Arg Gly Arg
465 470 475 480
Leu Arg Arg Ser Val Cys Lys Ala Gly Val Ser Cys Asn Glu Asn Glu
485 490 495
Gln Ser Glu Cys Ala Asp Lys Gly Gln Ile Phe Val Tyr Glu Asn Gly
500 505 510
Lys Ala Asn Cys Gln Cys Pro Pro Asp Thr Lys Pro Gly Glu Ile Gly
515 520 525
Cys Ile Glu Arg Thr Thr Cys Asn Pro Lys Glu Ile Gln Glu Cys Gln
530 535 540
Asp Lys Lys Leu Glu Cys Val Tyr Lys Asn His Lys Ala Glu Cys Glu
545 550 555 560
Cys Pro Asp Asp His Glu Cys Tyr Arg Glu Pro Ala Lys Asp Ser Cys
565 570 575
Ser Glu Glu Asp Asn Gly Lys Cys Gln Ser Ser Gly Gln Arg Cys Val
580 585 590
Ile Glu Asn Gly Lys Ala Val Cys Lys Glu Lys Ser Glu Ala Thr Thr
595 600 605
Ala Ala Thr Thr Thr Thr Lys Ala Lys Asp Lys Asp Pro Asp Pro Gly
610 615 620
Lys Ser Ser Ala Ala Ala Val Ser Ala Thr Gly Leu Leu Leu Leu Leu
625 630 635 640
Ala Ala Thr Ser Val Thr Ala Ala Ser Leu
645 650

688 amino acids

amino acid

linear

unknown

57
Ala Thr Ala Ala Val Val Arg Arg Ser Glu Met Arg Gly Ile Ala Leu
1 5 10 15
Phe Val Ala Ala Val Ser Leu Ile Val Glu Gly Thr Ala Glu Ser Ser
20 25 30
Ile Cys Ser Asp Phe Gly Asn Glu Phe Cys Arg Asn Ala Glu Cys Glu
35 40 45
Val Val Pro Gly Ala Glu Asp Asp Phe Val Cys Lys Cys Pro Arg Asp
50 55 60
Asn Met Tyr Phe Asn Ala Ala Glu Lys Gln Cys Glu Tyr Lys Asp Thr
65 70 75 80
Cys Lys Thr Arg Glu Cys Ser Tyr Gly Arg Cys Val Glu Ser Asn Pro
85 90 95
Ser Lys Ala Ser Cys Val Cys Glu Ala Ser Asp Asp Leu Thr Leu Gln
100 105 110
Cys Lys Ile Lys Asn Asp Tyr Ala Thr Asp Cys Arg Asn Arg Gly Gly
115 120 125
Thr Ala Lys Leu Arg Thr Asp Gly Phe Ile Gly Ala Thr Cys Asp Cys
130 135 140
Gly Glu Trp Gly Ala Met Asn Met Thr Thr Arg Asn Cys Val Pro Thr
145 150 155 160
Thr Cys Leu Arg Pro Asp Leu Thr Cys Lys Asp Leu Cys Glu Lys Asn
165 170 175
Leu Leu Gln Arg Asp Ser Arg Cys Cys Gln Gly Trp Asn Thr Ala Asn
180 185 190
Cys Ser Ala Ala Pro Pro Ala Asp Ser Tyr Cys Ser Pro Gly Ser Pro
195 200 205
Lys Gly Pro Asp Gly Gln Cys Ile Asn Ala Cys Lys Thr Lys Glu Ala
210 215 220
Gly Phe Val Cys Lys His Gly Cys Arg Ser Thr Gly Lys Ala Tyr Glu
225 230 235 240
Cys Thr Cys Pro Ser Gly Ser Thr Val Ala Glu Asp Gly Ile Thr Cys
245 250 255
Lys Ser Ile Ser His Thr Val Ser Cys Thr Ala Glu Gln Lys Gln Thr
260 265 270
Cys Arg Pro Thr Glu Asp Cys Arg Val His Lys Gly Thr Val Leu Cys
275 280 285
Glu Cys Pro Trp Asn Gln His Leu Val Gly Asp Thr Cys Ile Ser Asp
290 295 300
Cys Val Asp Lys Lys Cys His Glu Glu Phe Met Asp Cys Gly Val Tyr
305 310 315 320
Met Asn Arg Gln Ser Cys Tyr Cys Pro Trp Lys Ser Arg Lys Pro Gly
325 330 335
Pro Asn Val Asn Ile Asn Glu Cys Leu Leu Asn Glu Tyr Tyr Tyr Thr
340 345 350
Val Ser Phe Thr Pro Asn Ile Ser Phe Asp Ser Asp His Cys Lys Trp
355 360 365
Tyr Glu Asp Arg Val Leu Glu Ala Ile Arg Thr Ser Ile Gly Lys Glu
370 375 380
Val Phe Lys Val Glu Ile Leu Asn Cys Thr Gln Asp Ile Lys Ala Arg
385 390 395 400
Leu Ile Ala Glu Lys Pro Leu Ser Lys His Val Leu Arg Lys Leu Gln
405 410 415
Ala Cys Glu His Pro Ile Gly Glu Trp Cys Met Met Tyr Pro Lys Leu
420 425 430
Leu Ile Lys Lys Asn Ser Ala Thr Glu Ile Glu Glu Glu Asn Leu Cys
435 440 445
Asp Ser Leu Leu Lys Asp Gln Glu Ala Ala Tyr Lys Gly Gln Asn Lys
450 455 460
Cys Val Lys Val Asp Asn Leu Phe Trp Phe Gln Cys Ala Asp Gly Tyr
465 470 475 480
Thr Thr Thr Tyr Glu Met Thr Arg Gly Arg Leu Arg Arg Ser Val Cys
485 490 495
Lys Ala Gly Val Ser Cys Asn Glu Asn Glu Gln Ser Glu Cys Ala Asp
500 505 510
Lys Gly Gln Ile Phe Val Tyr Glu Asn Gly Lys Ala Asn Cys Gln Cys
515 520 525
Pro Pro Asp Thr Lys Pro Gly Glu Ile Gly Cys Ile Glu Arg Thr Thr
530 535 540
Cys Asn Pro Lys Glu Ile Gln Glu Cys Gln Asp Lys Lys Leu Glu Cys
545 550 555 560
Val Tyr Lys Asn His Lys Ala Glu Cys Glu Cys Pro Asp Asp His Glu
565 570 575
Cys Tyr Arg Glu Pro Ala Lys Asp Ser Cys Ser Glu Glu Asp Asn Gly
580 585 590
Lys Cys Gln Ser Ser Gly Gln Arg Cys Val Ile Glu Asn Gly Lys Ala
595 600 605
Val Cys Lys Glu Lys Ser Glu Ala Thr Thr Ala Ala Thr Thr Thr Thr
610 615 620
Lys Ala Lys Asp Lys Asp Pro Asp Pro Gly Lys Ser Ser Ala Ala Ala
625 630 635 640
Val Ser Ala Thr Gly Leu Leu Leu Leu Leu Ala Ala Thr Ser Val Thr
645 650 655
Ala Ala Ser Leu Xaa Gly Arg Cys Pro Thr Xaa Ile Arg Asn Ser Leu
660 665 670
Asn Met Tyr Ile Tyr Ile Thr Leu Thr Ser Asn Thr Xaa Leu Gly Phe
675 680 685

2259 base pairs

nucleic acid

double

linear

unknown

Figure 12

CDS

52..2004

58
GAATTCGCGG CCGCGAAAGT GCGACAGCTG CGGTGGTTCG ACGCAGTCGA G ATG CGT 57
Met Arg
1
GGC ATC GCT TTG TTC GTC GCC GCT GTT TCA CTG ATT GTA GAG GGC ACA 105
Gly Ile Ala Leu Phe Val Ala Ala Val Ser Leu Ile Val Glu Gly Thr
5 10 15
GCA GAA TCA TCC ATT TGC TCT GAC TTC GGG AAC GAG TTC TGT CGC AAC 153
Ala Glu Ser Ser Ile Cys Ser Asp Phe Gly Asn Glu Phe Cys Arg Asn
20 25 30
GCT GAA TGT GAA GTG GTG CCT GGT GCA GAG GAT GAT TTC GTG TGC AAA 201
Ala Glu Cys Glu Val Val Pro Gly Ala Glu Asp Asp Phe Val Cys Lys
35 40 45 50
TGT CCG CGA GAT AAT ATG TAC TTC AAT GCT GCT GAA AAG CAA TGC GAA 249
Cys Pro Arg Asp Asn Met Tyr Phe Asn Ala Ala Glu Lys Gln Cys Glu
55 60 65
TAT AAA GAC ACG TGC AAA ACA AGG GAG TGC AGC TAT GGA CGT TGC GTT 297
Tyr Lys Asp Thr Cys Lys Thr Arg Glu Cys Ser Tyr Gly Arg Cys Val
70 75 80
GAA AGT AAC CCG AGC AAG GCT AGC TGC GTC TGC GAA GCA TCG GAC GAT 345
Glu Ser Asn Pro Ser Lys Ala Ser Cys Val Cys Glu Ala Ser Asp Asp
85 90 95
CTA ACG CTA CAA TGC AAA ATT AAA AAT GAC TAC GCA ACT GAC TGC CGA 393
Leu Thr Leu Gln Cys Lys Ile Lys Asn Asp Tyr Ala Thr Asp Cys Arg
100 105 110
AAC CGA GGT GGC ACT GCT AAG TTG CGC ACG GAT GGG TTT ATT GGC GCA 441
Asn Arg Gly Gly Thr Ala Lys Leu Arg Thr Asp Gly Phe Ile Gly Ala
115 120 125 130
ACG TGT GAC TGT GGT GAA TGG GGT GCG ATG AAC ATG ACC ACC CGG AAC 489
Thr Cys Asp Cys Gly Glu Trp Gly Ala Met Asn Met Thr Thr Arg Asn
135 140 145
TGT GTC CCT ACC ACG TGT CTT CGT CCC GAC TTG AGC TGC AAA GAC CTC 537
Cys Val Pro Thr Thr Cys Leu Arg Pro Asp Leu Ser Cys Lys Asp Leu
150 155 160
TGC GAG AAA AAC CTG CTT CAA AGG GAT TCT CGT TGT TGC CAG GGG TGG 585
Cys Glu Lys Asn Leu Leu Gln Arg Asp Ser Arg Cys Cys Gln Gly Trp
165 170 175
AAC ACA GCA AAC TGT TCA GCC GCT CCT CCA GCT GAC TCC TAT TGC TCT 633
Asn Thr Ala Asn Cys Ser Ala Ala Pro Pro Ala Asp Ser Tyr Cys Ser
180 185 190
CCT GGG AGC CCC AAA GGA CCG GAC GGA CAG TGT ATA AAT GCT TGC AAG 681
Pro Gly Ser Pro Lys Gly Pro Asp Gly Gln Cys Ile Asn Ala Cys Lys
195 200 205 210
ATG AAA GAA GCT GGG TTT GTC TGC AAG CAT GGA TGC AGG TCG ACC GCC 729
Met Lys Glu Ala Gly Phe Val Cys Lys His Gly Cys Arg Ser Thr Ala
215 220 225
AAG GCG TAC GAG TGC ACG TGC CCA CGT GCC TTT ACC GTC GCG GAA GAT 777
Lys Ala Tyr Glu Cys Thr Cys Pro Arg Ala Phe Thr Val Ala Glu Asp
230 235 240
GGC ATT ACC TGC AAA AGT ATT TCG CAC ACA GTC AGC TGC ACT GCT GAG 825
Gly Ile Thr Cys Lys Ser Ile Ser His Thr Val Ser Cys Thr Ala Glu
245 250 255
CAA AAA CAG ACC TGC CGC CCA ACC GAA GAC TGT CGT GTG CAC AAA GGA 873
Gln Lys Gln Thr Cys Arg Pro Thr Glu Asp Cys Arg Val His Lys Gly
260 265 270
ACT GTG TTG TGT GAG TGC CCG TGG AAT CAA CAT CTA GTG GGG GAC ACG 921
Thr Val Leu Cys Glu Cys Pro Trp Asn Gln His Leu Val Gly Asp Thr
275 280 285 290
TGC ATA AGT GAT TGC GTC GAC AAG AAA TGC CAC GAA GAA TTT ATG GAC 969
Cys Ile Ser Asp Cys Val Asp Lys Lys Cys His Glu Glu Phe Met Asp
295 300 305
TGT GGC GTA TAT ATG AAT CGA CAA AGC TGC TAT TGT CCA TGG AAA TCA 1017
Cys Gly Val Tyr Met Asn Arg Gln Ser Cys Tyr Cys Pro Trp Lys Ser
310 315 320
AGG AAG CCG GGC CCA AAT GTC AAC ATC AAT GGA TGC CTA CTG AAT GAG 1065
Arg Lys Pro Gly Pro Asn Val Asn Ile Asn Gly Cys Leu Leu Asn Glu
325 330 335
TAT TAC TAC ACG GTG TCA TTC ACC CCA AAC ATA TCT TTT GAT TCT GAC 1113
Tyr Tyr Tyr Thr Val Ser Phe Thr Pro Asn Ile Ser Phe Asp Ser Asp
340 345 350
CAT TGC AAA TGG TAT GAG GAT CGT GTT TTG GAA GCG ATA CGG ACC AGT 1161
His Cys Lys Trp Tyr Glu Asp Arg Val Leu Glu Ala Ile Arg Thr Ser
355 360 365 370
ATC GGA AAA GAA GTT TTT AAG GTT GAG ATA CTT AAC TGC ACG CAG GAC 1209
Ile Gly Lys Glu Val Phe Lys Val Glu Ile Leu Asn Cys Thr Gln Asp
375 380 385
ATT AAG GCA AGA CTC ATA GCA GAG AAA TTA CTG TCA AAA CAC GTG CTC 1257
Ile Lys Ala Arg Leu Ile Ala Glu Lys Leu Leu Ser Lys His Val Leu
390 395 400
AGG AAA CTA CAA GCA TGC GAG CAT CCA ATC GGC GAA TGG TGC ATG ATG 1305
Arg Lys Leu Gln Ala Cys Glu His Pro Ile Gly Glu Trp Cys Met Met
405 410 415
TAT CCG AAG TTG CTG ATC AAG AAA AAC TCT GCA ACA GAA ATC GAA GAA 1353
Tyr Pro Lys Leu Leu Ile Lys Lys Asn Ser Ala Thr Glu Ile Glu Glu
420 425 430
GAG AAC CTT TGC GAC AGT CTG CTC AAG GAT CAG GAA GCT GCC TAC AAA 1401
Glu Asn Leu Cys Asp Ser Leu Leu Lys Asp Gln Glu Ala Ala Tyr Lys
435 440 445 450
GGT CAA AAC AAA TGC GTC AAG GTC GAC AAC CTC TTC TGG TTC CAG TGC 1449
Gly Gln Asn Lys Cys Val Lys Val Asp Asn Leu Phe Trp Phe Gln Cys
455 460 465
GCT GAT GGT TAC ACA ACA ACT TAC GAG ATG ACA CGA GGT CGC CTA CGC 1497
Ala Asp Gly Tyr Thr Thr Thr Tyr Glu Met Thr Arg Gly Arg Leu Arg
470 475 480
CGC TCC GTG TGT AAA GCT GGA GTT TCT TGC AAC GAA AAC GAG CAG TCG 1545
Arg Ser Val Cys Lys Ala Gly Val Ser Cys Asn Glu Asn Glu Gln Ser
485 490 495
GAG TGT GCT GAC AAA GGG CAA ATA TGT GTT TAC GAA AAC GGC AAA GCG 1593
Glu Cys Ala Asp Lys Gly Gln Ile Cys Val Tyr Glu Asn Gly Lys Ala
500 505 510
AAT TGC CAA TGC CCA CCA GAC ACT AAA CCT GGG GAG ATT GGC TGC ATT 1641
Asn Cys Gln Cys Pro Pro Asp Thr Lys Pro Gly Glu Ile Gly Cys Ile
515 520 525 530
GAG CGT ACC ACA TGC AAC CCT AAA GAG ATA CAA GAA TGC CAA GAC AAG 1689
Glu Arg Thr Thr Cys Asn Pro Lys Glu Ile Gln Glu Cys Gln Asp Lys
535 540 545
AAG CTG GAG TGC GTT TAC AAA AAC CAT AAA GCA GAA TSS AAG TGT CCT 1737
Lys Leu Glu Cys Val Tyr Lys Asn His Lys Ala Glu Xaa Lys Cys Pro
550 555 560
GAT GAT CAC GAG TGT TAC AGG GAG CCT GCC AAA GAC TCT TGC AGT GAA 1785
Asp Asp His Glu Cys Tyr Arg Glu Pro Ala Lys Asp Ser Cys Ser Glu
565 570 575
GAG GAT AAT GGT AAA TGT CAA AGC AGT GGG CAG CGT TGT GTA ATA GAA 1833
Glu Asp Asn Gly Lys Cys Gln Ser Ser Gly Gln Arg Cys Val Ile Glu
580 585 590
AAC GGA AAG GCT GTT TGC AAG GAA AAG TCT GAA GCA ACA ACA GCT GCG 1881
Asn Gly Lys Ala Val Cys Lys Glu Lys Ser Glu Ala Thr Thr Ala Ala
595 600 605 610
ACT ACA ACA ACG AAA GCG AAA GAC AAG GAT CCA GAT CCT GGA AAG TCA 1929
Thr Thr Thr Thr Lys Ala Lys Asp Lys Asp Pro Asp Pro Gly Lys Ser
615 620 625
AGT GCT GCA GCA GTA TCA GCT ACT GGG CTC TTG TTA CTG CTC GCA GCT 1977
Ser Ala Ala Ala Val Ser Ala Thr Gly Leu Leu Leu Leu Leu Ala Ala
630 635 640
ACT TCA GTC ACC GCA GCA TCG TTG TAAGGAAGAT GTCCAACTTG AATACGGAAC 2031
Thr Ser Val Thr Ala Ala Ser Leu
645 650
AGCTTGAATA TGTATATATA CATCACGCTT ACATCGAACA CCTAGCTTGG TTTTTGGAAT 2091
TTCAATATTG CGCATTGGTA CTCACGGCAA CATGAATGTA TTACTTTAGA ATGACAGGGA 2151
AGAGGGACGT GAAAGGAGTT TCCTTGTCTG AACATATCAA AGAAAATTTT CCCCTATCCG 2211
ACCGATGTCA AATAAAGATA GTTGGGTCTA AACAGCGGCC GCGAATTC 2259

650 amino acids

amino acid

linear

protein

unknown

59
Met Arg Gly Ile Ala Leu Phe Val Ala Ala Val Ser Leu Ile Val Glu
1 5 10 15
Gly Thr Ala Glu Ser Ser Ile Cys Ser Asp Phe Gly Asn Glu Phe Cys
20 25 30
Arg Asn Ala Glu Cys Glu Val Val Pro Gly Ala Glu Asp Asp Phe Val
35 40 45
Cys Lys Cys Pro Arg Asp Asn Met Tyr Phe Asn Ala Ala Glu Lys Gln
50 55 60
Cys Glu Tyr Lys Asp Thr Cys Lys Thr Arg Glu Cys Ser Tyr Gly Arg
65 70 75 80
Cys Val Glu Ser Asn Pro Ser Lys Ala Ser Cys Val Cys Glu Ala Ser
85 90 95
Asp Asp Leu Thr Leu Gln Cys Lys Ile Lys Asn Asp Tyr Ala Thr Asp
100 105 110
Cys Arg Asn Arg Gly Gly Thr Ala Lys Leu Arg Thr Asp Gly Phe Ile
115 120 125
Gly Ala Thr Cys Asp Cys Gly Glu Trp Gly Ala Met Asn Met Thr Thr
130 135 140
Arg Asn Cys Val Pro Thr Thr Cys Leu Arg Pro Asp Leu Ser Cys Lys
145 150 155 160
Asp Leu Cys Glu Lys Asn Leu Leu Gln Arg Asp Ser Arg Cys Cys Gln
165 170 175
Gly Trp Asn Thr Ala Asn Cys Ser Ala Ala Pro Pro Ala Asp Ser Tyr
180 185 190
Cys Ser Pro Gly Ser Pro Lys Gly Pro Asp Gly Gln Cys Ile Asn Ala
195 200 205
Cys Lys Met Lys Glu Ala Gly Phe Val Cys Lys His Gly Cys Arg Ser
210 215 220
Thr Ala Lys Ala Tyr Glu Cys Thr Cys Pro Arg Ala Phe Thr Val Ala
225 230 235 240
Glu Asp Gly Ile Thr Cys Lys Ser Ile Ser His Thr Val Ser Cys Thr
245 250 255
Ala Glu Gln Lys Gln Thr Cys Arg Pro Thr Glu Asp Cys Arg Val His
260 265 270
Lys Gly Thr Val Leu Cys Glu Cys Pro Trp Asn Gln His Leu Val Gly
275 280 285
Asp Thr Cys Ile Ser Asp Cys Val Asp Lys Lys Cys His Glu Glu Phe
290 295 300
Met Asp Cys Gly Val Tyr Met Asn Arg Gln Ser Cys Tyr Cys Pro Trp
305 310 315 320
Lys Ser Arg Lys Pro Gly Pro Asn Val Asn Ile Asn Gly Cys Leu Leu
325 330 335
Asn Glu Tyr Tyr Tyr Thr Val Ser Phe Thr Pro Asn Ile Ser Phe Asp
340 345 350
Ser Asp His Cys Lys Trp Tyr Glu Asp Arg Val Leu Glu Ala Ile Arg
355 360 365
Thr Ser Ile Gly Lys Glu Val Phe Lys Val Glu Ile Leu Asn Cys Thr
370 375 380
Gln Asp Ile Lys Ala Arg Leu Ile Ala Glu Lys Leu Leu Ser Lys His
385 390 395 400
Val Leu Arg Lys Leu Gln Ala Cys Glu His Pro Ile Gly Glu Trp Cys
405 410 415
Met Met Tyr Pro Lys Leu Leu Ile Lys Lys Asn Ser Ala Thr Glu Ile
420 425 430
Glu Glu Glu Asn Leu Cys Asp Ser Leu Leu Lys Asp Gln Glu Ala Ala
435 440 445
Tyr Lys Gly Gln Asn Lys Cys Val Lys Val Asp Asn Leu Phe Trp Phe
450 455 460
Gln Cys Ala Asp Gly Tyr Thr Thr Thr Tyr Glu Met Thr Arg Gly Arg
465 470 475 480
Leu Arg Arg Ser Val Cys Lys Ala Gly Val Ser Cys Asn Glu Asn Glu
485 490 495
Gln Ser Glu Cys Ala Asp Lys Gly Gln Ile Cys Val Tyr Glu Asn Gly
500 505 510
Lys Ala Asn Cys Gln Cys Pro Pro Asp Thr Lys Pro Gly Glu Ile Gly
515 520 525
Cys Ile Glu Arg Thr Thr Cys Asn Pro Lys Glu Ile Gln Glu Cys Gln
530 535 540
Asp Lys Lys Leu Glu Cys Val Tyr Lys Asn His Lys Ala Glu Xaa Lys
545 550 555 560
Cys Pro Asp Asp His Glu Cys Tyr Arg Glu Pro Ala Lys Asp Ser Cys
565 570 575
Ser Glu Glu Asp Asn Gly Lys Cys Gln Ser Ser Gly Gln Arg Cys Val
580 585 590
Ile Glu Asn Gly Lys Ala Val Cys Lys Glu Lys Ser Glu Ala Thr Thr
595 600 605
Ala Ala Thr Thr Thr Thr Lys Ala Lys Asp Lys Asp Pro Asp Pro Gly
610 615 620
Lys Ser Ser Ala Ala Ala Val Ser Ala Thr Gly Leu Leu Leu Leu Leu
625 630 635 640
Ala Ala Thr Ser Val Thr Ala Ala Ser Leu
645 650

1647 base pairs

nucleic acid

double

linear

unknown

Figure 13

CDS

1..1647

60
GTT GAA AGT AAC CCG AGC AAG GCT AGC TGC GTC TGC GAA CGA TCG GAC 48
Val Glu Ser Asn Pro Ser Lys Ala Ser Cys Val Cys Glu Arg Ser Asp
1 5 10 15
GAT CTA ACG CTA CAA TGC AAA ATT AAA AAT GAC TAC GCA ACT GAC TGC 96
Asp Leu Thr Leu Gln Cys Lys Ile Lys Asn Asp Tyr Ala Thr Asp Cys
20 25 30
CGA AAT CGA GGT GGC ACT GCT AAG TTG CGC ACG GAT GGG TTT ATT GGC 144
Arg Asn Arg Gly Gly Thr Ala Lys Leu Arg Thr Asp Gly Phe Ile Gly
35 40 45
GCA ACG TGT GAC TGT GGT GAA TGG GGT GCG ATG AAC ATG ACC ACC CGG 192
Ala Thr Cys Asp Cys Gly Glu Trp Gly Ala Met Asn Met Thr Thr Arg
50 55 60
AAC TGT GTC CCT ACC ACG TGT CTT CGT CCC GAC TTG ACC TGC AAA GAC 240
Asn Cys Val Pro Thr Thr Cys Leu Arg Pro Asp Leu Thr Cys Lys Asp
65 70 75 80
CTC TGC GAG AAA AAC CTG CTT CAA AGG GAT TCT CGT TGT TGC CAG GGG 288
Leu Cys Glu Lys Asn Leu Leu Gln Arg Asp Ser Arg Cys Cys Gln Gly
85 90 95
TGG AAC ACA GCA AAC TGT TCA GCC GCT CCT CCA GCT GAC TCC TAT TGC 336
Trp Asn Thr Ala Asn Cys Ser Ala Ala Pro Pro Ala Asp Ser Tyr Cys
100 105 110
TCT CCT GGG AGC CCC AAA GGA CCG GAC GGA CAG TGT ATA AAT GCT TGC 384
Ser Pro Gly Ser Pro Lys Gly Pro Asp Gly Gln Cys Ile Asn Ala Cys
115 120 125
AAG ATG AAA GAA GCT GGG TTT GTC TGC GAG CAT GGA TGC AGG TCG ACC 432
Lys Met Lys Glu Ala Gly Phe Val Cys Glu His Gly Cys Arg Ser Thr
130 135 140
GCC AAG GCG TAC GAG TGC ACG TGC CCA CGT GGC TTT ACC GTC GCG GAA 480
Ala Lys Ala Tyr Glu Cys Thr Cys Pro Arg Gly Phe Thr Val Ala Glu
145 150 155 160
GAT GGC ATT ACC TGC AAA AGT ATT TCG CAC ACA GTC AGC TGC ACT GCT 528
Asp Gly Ile Thr Cys Lys Ser Ile Ser His Thr Val Ser Cys Thr Ala
165 170 175
GAG CAA AAA CAG ACC TGC CGC CCA ACC GAA GAC TGT CGT GTG CAC AAA 576
Glu Gln Lys Gln Thr Cys Arg Pro Thr Glu Asp Cys Arg Val His Lys
180 185 190
GGA ACT GTG TTG TGT GAG TGC CCG TGG AAT CAA CAT CTA GTG GGG GAC 624
Gly Thr Val Leu Cys Glu Cys Pro Trp Asn Gln His Leu Val Gly Asp
195 200 205
ACG TGC ATA AGT GAT TGC GTC GAC AAG AAA TGC CAC GAA GAA TTT ATG 672
Thr Cys Ile Ser Asp Cys Val Asp Lys Lys Cys His Glu Glu Phe Met
210 215 220
GAC TGT GGC GTA TAT ATG AAT CGA CAA AGC TGC TAT TGT CCA TGG AAA 720
Asp Cys Gly Val Tyr Met Asn Arg Gln Ser Cys Tyr Cys Pro Trp Lys
225 230 235 240
TCA AGG AAG CCG GGC CCA AAT GTC AAC ATC AAT GGA TGC CTA CTG AAT 768
Ser Arg Lys Pro Gly Pro Asn Val Asn Ile Asn Gly Cys Leu Leu Asn
245 250 255
GAG TAT TAC TAC ACG GTG TCA TTC ACC CCA AAC ATA TCT TTT GAT TCT 816
Glu Tyr Tyr Tyr Thr Val Ser Phe Thr Pro Asn Ile Ser Phe Asp Ser
260 265 270
GAC CAT TGC AAA TGG TAT GAG GAT CGT GTT TTG GAA GCG ATA CGG ACC 864
Asp His Cys Lys Trp Tyr Glu Asp Arg Val Leu Glu Ala Ile Arg Thr
275 280 285
AGT ATC GGA AAA GAA GTT TTT AAG GTT GAG ATA CTT AAC TGC ACG CAG 912
Ser Ile Gly Lys Glu Val Phe Lys Val Glu Ile Leu Asn Cys Thr Gln
290 295 300
GAC ATT AAG GCA AGA CTC ATA GCA GAG AAA CCA CTG TCA AAC CAC GTG 960
Asp Ile Lys Ala Arg Leu Ile Ala Glu Lys Pro Leu Ser Asn His Val
305 310 315 320
CTC AGG AAA CTA CAA GCA TGC GAG CAT CCA ATC GGC GAA TGG TGC ATG 1008
Leu Arg Lys Leu Gln Ala Cys Glu His Pro Ile Gly Glu Trp Cys Met
325 330 335
ATG TAT CCG AAG TTG CTG ATC AAG AAA AAC TCT GCA ACA GAA ATC GAA 1056
Met Tyr Pro Lys Leu Leu Ile Lys Lys Asn Ser Ala Thr Glu Ile Glu
340 345 350
GAA GAG AAC CTT TGC GAC AGT CTG CTC AAG AAT CAG GAA GCT GCC TAC 1104
Glu Glu Asn Leu Cys Asp Ser Leu Leu Lys Asn Gln Glu Ala Ala Tyr
355 360 365
AAA GGT CAA AAC AAA TGC GTC AAG GTC GAC AAC CTC TTC TGG TTC CAG 1152
Lys Gly Gln Asn Lys Cys Val Lys Val Asp Asn Leu Phe Trp Phe Gln
370 375 380
TGC GCT GAT GGT TAC ACA ACA ACT TAC GAG ATG ACA CGA GGT CGC CTA 1200
Cys Ala Asp Gly Tyr Thr Thr Thr Tyr Glu Met Thr Arg Gly Arg Leu
385 390 395 400
CGC CGC TCC GTG TGT AAA GCT GGA GTT TCT TGC AAC GAA AAC GAG CAG 1248
Arg Arg Ser Val Cys Lys Ala Gly Val Ser Cys Asn Glu Asn Glu Gln
405 410 415
TTG GAG TGT GCT GAC AAA GGG CAA ATA TGT GTT TAC GAA AAC GGC AAA 1296
Leu Glu Cys Ala Asp Lys Gly Gln Ile Cys Val Tyr Glu Asn Gly Lys
420 425 430
GCG AAT TGC CAA TGC CCA CCA GAC ACT AAA CCT GGG GAG ATT GGC TGC 1344
Ala Asn Cys Gln Cys Pro Pro Asp Thr Lys Pro Gly Glu Ile Gly Cys
435 440 445
ATT GAG CGT ACC ACA TGC AAC CCT AAA GAG ATA CAA GAA TGC CAA GAC 1392
Ile Glu Arg Thr Thr Cys Asn Pro Lys Glu Ile Gln Glu Cys Gln Asp
450 455 460
AAG AAG CTG GAG TGC GTT TAC AAA AAC CAT AAA GCA GAA TGC AAG TGT 1440
Lys Lys Leu Glu Cys Val Tyr Lys Asn His Lys Ala Glu Cys Lys Cys
465 470 475 480
CCT GAT GAT CAC GAG TGT TCC AGG GAG CCT GCC AAA GAC TCT TGC AGT 1488
Pro Asp Asp His Glu Cys Ser Arg Glu Pro Ala Lys Asp Ser Cys Ser
485 490 495
GAA GAG GAT AAT GGT AAA TGT CAA AGC AGT GGG CAG CGT TGT GTA ATA 1536
Glu Glu Asp Asn Gly Lys Cys Gln Ser Ser Gly Gln Arg Cys Val Ile
500 505 510
GAA AAC GGA AAG GCT GTT TGC AAG GAA AAG TCT GAA GCA ACA ACA GCT 1584
Glu Asn Gly Lys Ala Val Cys Lys Glu Lys Ser Glu Ala Thr Thr Ala
515 520 525
GCG ACT ACA ACA ACG AAA GCG AAA GAC AAG GAT CCA GAT CCT GGA AAG 1632
Ala Thr Thr Thr Thr Lys Ala Lys Asp Lys Asp Pro Asp Pro Gly Lys
530 535 540
TCA AGT GCT GCA GCA 1647
Ser Ser Ala Ala Ala
545

549 amino acids

amino acid

linear

protein

unknown

61
Val Glu Ser Asn Pro Ser Lys Ala Ser Cys Val Cys Glu Arg Ser Asp
1 5 10 15
Asp Leu Thr Leu Gln Cys Lys Ile Lys Asn Asp Tyr Ala Thr Asp Cys
20 25 30
Arg Asn Arg Gly Gly Thr Ala Lys Leu Arg Thr Asp Gly Phe Ile Gly
35 40 45
Ala Thr Cys Asp Cys Gly Glu Trp Gly Ala Met Asn Met Thr Thr Arg
50 55 60
Asn Cys Val Pro Thr Thr Cys Leu Arg Pro Asp Leu Thr Cys Lys Asp
65 70 75 80
Leu Cys Glu Lys Asn Leu Leu Gln Arg Asp Ser Arg Cys Cys Gln Gly
85 90 95
Trp Asn Thr Ala Asn Cys Ser Ala Ala Pro Pro Ala Asp Ser Tyr Cys
100 105 110
Ser Pro Gly Ser Pro Lys Gly Pro Asp Gly Gln Cys Ile Asn Ala Cys
115 120 125
Lys Met Lys Glu Ala Gly Phe Val Cys Glu His Gly Cys Arg Ser Thr
130 135 140
Ala Lys Ala Tyr Glu Cys Thr Cys Pro Arg Gly Phe Thr Val Ala Glu
145 150 155 160
Asp Gly Ile Thr Cys Lys Ser Ile Ser His Thr Val Ser Cys Thr Ala
165 170 175
Glu Gln Lys Gln Thr Cys Arg Pro Thr Glu Asp Cys Arg Val His Lys
180 185 190
Gly Thr Val Leu Cys Glu Cys Pro Trp Asn Gln His Leu Val Gly Asp
195 200 205
Thr Cys Ile Ser Asp Cys Val Asp Lys Lys Cys His Glu Glu Phe Met
210 215 220
Asp Cys Gly Val Tyr Met Asn Arg Gln Ser Cys Tyr Cys Pro Trp Lys
225 230 235 240
Ser Arg Lys Pro Gly Pro Asn Val Asn Ile Asn Gly Cys Leu Leu Asn
245 250 255
Glu Tyr Tyr Tyr Thr Val Ser Phe Thr Pro Asn Ile Ser Phe Asp Ser
260 265 270
Asp His Cys Lys Trp Tyr Glu Asp Arg Val Leu Glu Ala Ile Arg Thr
275 280 285
Ser Ile Gly Lys Glu Val Phe Lys Val Glu Ile Leu Asn Cys Thr Gln
290 295 300
Asp Ile Lys Ala Arg Leu Ile Ala Glu Lys Pro Leu Ser Asn His Val
305 310 315 320
Leu Arg Lys Leu Gln Ala Cys Glu His Pro Ile Gly Glu Trp Cys Met
325 330 335
Met Tyr Pro Lys Leu Leu Ile Lys Lys Asn Ser Ala Thr Glu Ile Glu
340 345 350
Glu Glu Asn Leu Cys Asp Ser Leu Leu Lys Asn Gln Glu Ala Ala Tyr
355 360 365
Lys Gly Gln Asn Lys Cys Val Lys Val Asp Asn Leu Phe Trp Phe Gln
370 375 380
Cys Ala Asp Gly Tyr Thr Thr Thr Tyr Glu Met Thr Arg Gly Arg Leu
385 390 395 400
Arg Arg Ser Val Cys Lys Ala Gly Val Ser Cys Asn Glu Asn Glu Gln
405 410 415
Leu Glu Cys Ala Asp Lys Gly Gln Ile Cys Val Tyr Glu Asn Gly Lys
420 425 430
Ala Asn Cys Gln Cys Pro Pro Asp Thr Lys Pro Gly Glu Ile Gly Cys
435 440 445
Ile Glu Arg Thr Thr Cys Asn Pro Lys Glu Ile Gln Glu Cys Gln Asp
450 455 460
Lys Lys Leu Glu Cys Val Tyr Lys Asn His Lys Ala Glu Cys Lys Cys
465 470 475 480
Pro Asp Asp His Glu Cys Ser Arg Glu Pro Ala Lys Asp Ser Cys Ser
485 490 495
Glu Glu Asp Asn Gly Lys Cys Gln Ser Ser Gly Gln Arg Cys Val Ile
500 505 510
Glu Asn Gly Lys Ala Val Cys Lys Glu Lys Ser Glu Ala Thr Thr Ala
515 520 525
Ala Thr Thr Thr Thr Lys Ala Lys Asp Lys Asp Pro Asp Pro Gly Lys
530 535 540
Ser Ser Ala Ala Ala
545

2308 base pairs

nucleic acid

double

linear

unknown

Figure 14

CDS

58..2010

62
CCCCCTCGAG GTCGACGGTA TCGATAAGCT TGATATCGAA TTCCGCCGGC CGCCGAG 57
ATG CGT GGC ATC GCT TTG TTC GTC GCC GCT GTT TCA CTG ATT GTA GAG 105
Met Arg Gly Ile Ala Leu Phe Val Ala Ala Val Ser Leu Ile Val Glu
1 5 10 15
TGC ACA GCA GAA TCA TCC ATT TGC TCT GAC TTC GGG AAC GAG TTC TGT 153
Cys Thr Ala Glu Ser Ser Ile Cys Ser Asp Phe Gly Asn Glu Phe Cys
20 25 30
CGC AAC GCT GAA TGT GAA GTG GTG CCT GGT GCA GAG GAT GAT TTC GTG 201
Arg Asn Ala Glu Cys Glu Val Val Pro Gly Ala Glu Asp Asp Phe Val
35 40 45
TGC AAA TGT CCG CGA GAT AAT ATG TAC TTC AAT GCT GCT GAA AAG CAA 249
Cys Lys Cys Pro Arg Asp Asn Met Tyr Phe Asn Ala Ala Glu Lys Gln
50 55 60
TGC GAA TAT AAA GAC ACG TGC AAG ACA AGG GAG TGC AGC TAT GGA CGT 297
Cys Glu Tyr Lys Asp Thr Cys Lys Thr Arg Glu Cys Ser Tyr Gly Arg
65 70 75 80
TGC GTT GAA AGT AAC CCG AGC AAG GCT AGC TGC GTC TGC GAA GCA TCG 345
Cys Val Glu Ser Asn Pro Ser Lys Ala Ser Cys Val Cys Glu Ala Ser
85 90 95
GAC GAT CTA ACG CTA CAA TGC AAA ATT AAA AAT GAC TAC GCA ACT GAC 393
Asp Asp Leu Thr Leu Gln Cys Lys Ile Lys Asn Asp Tyr Ala Thr Asp
100 105 110
TGC CGA AAT CGA GGT GGC ACT GCT AAG TTG CGC ACG GAT GGG TTT ATT 441
Cys Arg Asn Arg Gly Gly Thr Ala Lys Leu Arg Thr Asp Gly Phe Ile
115 120 125
GGC GCA ACG TGT GAC TGT GGT GAA TGG GGT GCG ATG AAC ATG ACC ACC 489
Gly Ala Thr Cys Asp Cys Gly Glu Trp Gly Ala Met Asn Met Thr Thr
130 135 140
CGG AAC TGT GTC CCT ACC ACG TGT CTT CGT CCC GAC TTG ACC TGC AAA 537
Arg Asn Cys Val Pro Thr Thr Cys Leu Arg Pro Asp Leu Thr Cys Lys
145 150 155 160
GAC CTC TGC GAG AAA AAC CTG CTT CAA AGG GAT TCT CGT TGT TGC CAG 585
Asp Leu Cys Glu Lys Asn Leu Leu Gln Arg Asp Ser Arg Cys Cys Gln
165 170 175
GGG TGG AAC ACA GCA AAC TGT TCA GCC GCT CCT CCA GCT GAC TCC TAT 633
Gly Trp Asn Thr Ala Asn Cys Ser Ala Ala Pro Pro Ala Asp Ser Tyr
180 185 190
TGC TCT CCT GGG AGC CCC AAA GGA CCG GAC GGA CAG TGT ATA AAT GCT 681
Cys Ser Pro Gly Ser Pro Lys Gly Pro Asp Gly Gln Cys Ile Asn Ala
195 200 205
TGC AAG ATG AAA GAA GCT GGG TTT GTC TGC GAG CAT GGA TGC AGG TCG 729
Cys Lys Met Lys Glu Ala Gly Phe Val Cys Glu His Gly Cys Arg Ser
210 215 220
ACC GCC AAG GCG TAC GAG TGC ACG TGC CCA CGT GGC TTT ACC GTC GCG 777
Thr Ala Lys Ala Tyr Glu Cys Thr Cys Pro Arg Gly Phe Thr Val Ala
225 230 235 240
GAA GAT GGC ATT ACC TGC AAA AGT ATT TCG CAC ACA GTC AGC TGC ACT 825
Glu Asp Gly Ile Thr Cys Lys Ser Ile Ser His Thr Val Ser Cys Thr
245 250 255
GCT GAG CAA AAA CAG ACC TGC CGC CCA ACC GAA GAC TGT CGT GTG CAC 873
Ala Glu Gln Lys Gln Thr Cys Arg Pro Thr Glu Asp Cys Arg Val His
260 265 270
AAA GGA ACT GTG TTG TGT GAG TGC CCG TGG AAT CAA CAT CTA GTG GGG 921
Lys Gly Thr Val Leu Cys Glu Cys Pro Trp Asn Gln His Leu Val Gly
275 280 285
GAC ACG TGC ATA AGT GAT TGC GTC GAC AAG AAA TGC CAC GAA GAA TTT 969
Asp Thr Cys Ile Ser Asp Cys Val Asp Lys Lys Cys His Glu Glu Phe
290 295 300
ATG GAC TGT GGC GTA TAT ATG AAT CGA CAA AGC TGC TAT TGT CCA TGG 1017
Met Asp Cys Gly Val Tyr Met Asn Arg Gln Ser Cys Tyr Cys Pro Trp
305 310 315 320
AAA TCA AGG AAG CCG GGC CCA AAT GTC AAC ATC AAT GGA TGC CTA CTG 1065
Lys Ser Arg Lys Pro Gly Pro Asn Val Asn Ile Asn Gly Cys Leu Leu
325 330 335
AAT GAG TAT TAC TAC ACG GTG TCA TTC ACC CCA AAC ATA TCT TTT GAT 1113
Asn Glu Tyr Tyr Tyr Thr Val Ser Phe Thr Pro Asn Ile Ser Phe Asp
340 345 350
TCT GAC CAT TGC AAA TGG TAT GAG GAT CGT GTT TTG GAA GCG ATA CGG 1161
Ser Asp His Cys Lys Trp Tyr Glu Asp Arg Val Leu Glu Ala Ile Arg
355 360 365
ACC AGT ATC GGA AAA GAA GTT TTT AAG GTT GAG ATA CTT AAC TGC ACG 1209
Thr Ser Ile Gly Lys Glu Val Phe Lys Val Glu Ile Leu Asn Cys Thr
370 375 380
CAG GAC ATT AAG GCA AGA CTC ATA GCA GAG AAA CCA CTG TCA AAC CAC 1257
Gln Asp Ile Lys Ala Arg Leu Ile Ala Glu Lys Pro Leu Ser Asn His
385 390 395 400
GTG CTC AGG AAA CTA CAA GCA TGC GAG CAT CCA ATC GGC GAA TGG TGC 1305
Val Leu Arg Lys Leu Gln Ala Cys Glu His Pro Ile Gly Glu Trp Cys
405 410 415
ATG ATG TAT CCG AAG TTG CTG ATC AAG AAA AAC TCT GCA ACA GAA ATC 1353
Met Met Tyr Pro Lys Leu Leu Ile Lys Lys Asn Ser Ala Thr Glu Ile
420 425 430
GAA GAA GAG AAC CTT TGC GAC AGT CTG CTC AAG AAT CAG GAA GCT GCC 1401
Glu Glu Glu Asn Leu Cys Asp Ser Leu Leu Lys Asn Gln Glu Ala Ala
435 440 445
TAC AAA GGT CAA AAC AAA TGC GTC AAG GTC GAC AAC CTC TTC TGG TTC 1449
Tyr Lys Gly Gln Asn Lys Cys Val Lys Val Asp Asn Leu Phe Trp Phe
450 455 460
CAG TGC GCT GAT GGT TAC ACA ACA ACT TAC GAG ATG ACA CGA GGT CGC 1497
Gln Cys Ala Asp Gly Tyr Thr Thr Thr Tyr Glu Met Thr Arg Gly Arg
465 470 475 480
CTA CGC CGC TCC GTG TGT AAA GCT GGA GTT TCT TGC AAC GAA AAC GAG 1545
Leu Arg Arg Ser Val Cys Lys Ala Gly Val Ser Cys Asn Glu Asn Glu
485 490 495
CAG TTG GAG TGT GCT GAC AAA GGG CAA ATA TGT GTT TAC GAA AAC GGC 1593
Gln Leu Glu Cys Ala Asp Lys Gly Gln Ile Cys Val Tyr Glu Asn Gly
500 505 510
AAA GCG AAT TGC CAA TGC CCA CCA GAC ACT AAA CCT GGG GAG ATT GGC 1641
Lys Ala Asn Cys Gln Cys Pro Pro Asp Thr Lys Pro Gly Glu Ile Gly
515 520 525
TGC ATT GAG CGT ACC ACA TGC AAC CCT AAA GAG ATA CAA GAA TGC CAA 1689
Cys Ile Glu Arg Thr Thr Cys Asn Pro Lys Glu Ile Gln Glu Cys Gln
530 535 540
GAC AAG AAG CTG GAG TGC GTT TAC AAA AAC CAT AAA GCA GAA TGC AAG 1737
Asp Lys Lys Leu Glu Cys Val Tyr Lys Asn His Lys Ala Glu Cys Lys
545 550 555 560
TGT CCT GAT GAT CAC GAG TGT TCC AGG GAG CCT GCC AAA GAC TCT TGC 1785
Cys Pro Asp Asp His Glu Cys Ser Arg Glu Pro Ala Lys Asp Ser Cys
565 570 575
AGT GAA GAG GAT AAT GGT AAA TGT CAA AGC AGT GGG CAG CGT TGT GTA 1833
Ser Glu Glu Asp Asn Gly Lys Cys Gln Ser Ser Gly Gln Arg Cys Val
580 585 590
ATA GAA AAC GGA AAG GCT GTT TGC AAG GAA AAG TCT GAA GCA ACA ACA 1881
Ile Glu Asn Gly Lys Ala Val Cys Lys Glu Lys Ser Glu Ala Thr Thr
595 600 605
GCT GCG ACT ACA ACA ACG AAA GCG AAA GAC AAG GAT CCA GAT CCT GGA 1929
Ala Ala Thr Thr Thr Thr Lys Ala Lys Asp Lys Asp Pro Asp Pro Gly
610 615 620
AAG TCA AGT GCT GCA GCA GTA TCA GCT ACT GGG CTC TTG TTA CTG CTC 1977
Lys Ser Ser Ala Ala Ala Val Ser Ala Thr Gly Leu Leu Leu Leu Leu
625 630 635 640
GCA GCT ACT TCA GTC ACC GCA GCA TCG TTG TAAGGAAGMT GTCCAACTNC 2027
Ala Ala Thr Ser Val Thr Ala Ala Ser Leu
645 650
AATACGGAAC AGCTTGAATA TGTATATATA CATCACGCTT ACATCGAACA CCTAGCTTGG 2087
TTTTTGGAAT TTCAATATTG CGCATTGGTA CTCACNGCAA CATGAATGTA TTACTTTAGA 2147
ATGACAGGGA AGAGGGACGT GAAAGGAGTT TCCTTGTCTG AACATATCAA AGAAAATTTT 2207
CCCCTATCCG ACCGATGTCA GCGGCCGCGA ATTCCTGCAG CCCGGGGGAT CCACTAGTTC 2267
TAGAGCGGGC GGCCGCGTTA ACCACCGCGG TGGAGCTCCA G 2308

650 amino acids

amino acid

linear

protein

unknown

63
Met Arg Gly Ile Ala Leu Phe Val Ala Ala Val Ser Leu Ile Val Glu
1 5 10 15
Cys Thr Ala Glu Ser Ser Ile Cys Ser Asp Phe Gly Asn Glu Phe Cys
20 25 30
Arg Asn Ala Glu Cys Glu Val Val Pro Gly Ala Glu Asp Asp Phe Val
35 40 45
Cys Lys Cys Pro Arg Asp Asn Met Tyr Phe Asn Ala Ala Glu Lys Gln
50 55 60
Cys Glu Tyr Lys Asp Thr Cys Lys Thr Arg Glu Cys Ser Tyr Gly Arg
65 70 75 80
Cys Val Glu Ser Asn Pro Ser Lys Ala Ser Cys Val Cys Glu Ala Ser
85 90 95
Asp Asp Leu Thr Leu Gln Cys Lys Ile Lys Asn Asp Tyr Ala Thr Asp
100 105 110
Cys Arg Asn Arg Gly Gly Thr Ala Lys Leu Arg Thr Asp Gly Phe Ile
115 120 125
Gly Ala Thr Cys Asp Cys Gly Glu Trp Gly Ala Met Asn Met Thr Thr
130 135 140
Arg Asn Cys Val Pro Thr Thr Cys Leu Arg Pro Asp Leu Thr Cys Lys
145 150 155 160
Asp Leu Cys Glu Lys Asn Leu Leu Gln Arg Asp Ser Arg Cys Cys Gln
165 170 175
Gly Trp Asn Thr Ala Asn Cys Ser Ala Ala Pro Pro Ala Asp Ser Tyr
180 185 190
Cys Ser Pro Gly Ser Pro Lys Gly Pro Asp Gly Gln Cys Ile Asn Ala
195 200 205
Cys Lys Met Lys Glu Ala Gly Phe Val Cys Glu His Gly Cys Arg Ser
210 215 220
Thr Ala Lys Ala Tyr Glu Cys Thr Cys Pro Arg Gly Phe Thr Val Ala
225 230 235 240
Glu Asp Gly Ile Thr Cys Lys Ser Ile Ser His Thr Val Ser Cys Thr
245 250 255
Ala Glu Gln Lys Gln Thr Cys Arg Pro Thr Glu Asp Cys Arg Val His
260 265 270
Lys Gly Thr Val Leu Cys Glu Cys Pro Trp Asn Gln His Leu Val Gly
275 280 285
Asp Thr Cys Ile Ser Asp Cys Val Asp Lys Lys Cys His Glu Glu Phe
290 295 300
Met Asp Cys Gly Val Tyr Met Asn Arg Gln Ser Cys Tyr Cys Pro Trp
305 310 315 320
Lys Ser Arg Lys Pro Gly Pro Asn Val Asn Ile Asn Gly Cys Leu Leu
325 330 335
Asn Glu Tyr Tyr Tyr Thr Val Ser Phe Thr Pro Asn Ile Ser Phe Asp
340 345 350
Ser Asp His Cys Lys Trp Tyr Glu Asp Arg Val Leu Glu Ala Ile Arg
355 360 365
Thr Ser Ile Gly Lys Glu Val Phe Lys Val Glu Ile Leu Asn Cys Thr
370 375 380
Gln Asp Ile Lys Ala Arg Leu Ile Ala Glu Lys Pro Leu Ser Asn His
385 390 395 400
Val Leu Arg Lys Leu Gln Ala Cys Glu His Pro Ile Gly Glu Trp Cys
405 410 415
Met Met Tyr Pro Lys Leu Leu Ile Lys Lys Asn Ser Ala Thr Glu Ile
420 425 430
Glu Glu Glu Asn Leu Cys Asp Ser Leu Leu Lys Asn Gln Glu Ala Ala
435 440 445
Tyr Lys Gly Gln Asn Lys Cys Val Lys Val Asp Asn Leu Phe Trp Phe
450 455 460
Gln Cys Ala Asp Gly Tyr Thr Thr Thr Tyr Glu Met Thr Arg Gly Arg
465 470 475 480
Leu Arg Arg Ser Val Cys Lys Ala Gly Val Ser Cys Asn Glu Asn Glu
485 490 495
Gln Leu Glu Cys Ala Asp Lys Gly Gln Ile Cys Val Tyr Glu Asn Gly
500 505 510
Lys Ala Asn Cys Gln Cys Pro Pro Asp Thr Lys Pro Gly Glu Ile Gly
515 520 525
Cys Ile Glu Arg Thr Thr Cys Asn Pro Lys Glu Ile Gln Glu Cys Gln
530 535 540
Asp Lys Lys Leu Glu Cys Val Tyr Lys Asn His Lys Ala Glu Cys Lys
545 550 555 560
Cys Pro Asp Asp His Glu Cys Ser Arg Glu Pro Ala Lys Asp Ser Cys
565 570 575
Ser Glu Glu Asp Asn Gly Lys Cys Gln Ser Ser Gly Gln Arg Cys Val
580 585 590
Ile Glu Asn Gly Lys Ala Val Cys Lys Glu Lys Ser Glu Ala Thr Thr
595 600 605
Ala Ala Thr Thr Thr Thr Lys Ala Lys Asp Lys Asp Pro Asp Pro Gly
610 615 620
Lys Ser Ser Ala Ala Ala Val Ser Ala Thr Gly Leu Leu Leu Leu Leu
625 630 635 640
Ala Ala Thr Ser Val Thr Ala Ala Ser Leu
645 650

2131 base pairs

nucleic acid

double

linear

unknown

Figure 15

CDS

1..1863

64
TTC TGT CGC AAC GCT GAA TGC GAA GAG GTG CCT GGT GCC GAG GAT GAT 48
Phe Cys Arg Asn Ala Glu Cys Glu Glu Val Pro Gly Ala Glu Asp Asp
1 5 10 15
TTC GTG TGC AAA TGT CCG CGA TAT AAT ATG TAC TTC AAT GCT GCT GAA 96
Phe Val Cys Lys Cys Pro Arg Tyr Asn Met Tyr Phe Asn Ala Ala Glu
20 25 30
AAA CAA TGC GAA TAT AAA GAT ACG TGC AAG ACA AGA GAG TGC AGC TAT 144
Lys Gln Cys Glu Tyr Lys Asp Thr Cys Lys Thr Arg Glu Cys Ser Tyr
35 40 45
GGC CGT TGC GTT CAA AGT AAC CCG AGC AAG GCT AGC TGT GTC TGC GAA 192
Gly Arg Cys Val Gln Ser Asn Pro Ser Lys Ala Ser Cys Val Cys Glu
50 55 60
GCA TCT GAC ACT CTA ACG CTA CAA TGC AAC ATT AAC AAT GAC TAC GCA 240
Ala Ser Asp Thr Leu Thr Leu Gln Cys Asn Ile Asn Asn Asp Tyr Ala
65 70 75 80
ACT GAC TGC CGA AAC AGG GGT GGT ACC GCT AAG TTG CGC ACG GAT GGG 288
Thr Asp Cys Arg Asn Arg Gly Gly Thr Ala Lys Leu Arg Thr Asp Gly
85 90 95
TTT ATT GGC GCA ACG TGT GAC TGT GGT GAA TGG GGC GCA ATG AAC AAA 336
Phe Ile Gly Ala Thr Cys Asp Cys Gly Glu Trp Gly Ala Met Asn Lys
100 105 110
ACC ACC CGG AAC TGT GTC CCT ACC ACG TGT CTT CGT CCC GAC TTG ACC 384
Thr Thr Arg Asn Cys Val Pro Thr Thr Cys Leu Arg Pro Asp Leu Thr
115 120 125
TGC AAA GAC CTC TGC GAG AAA AAC CTG CTT CAA AGG GAT TCT CGT TGT 432
Cys Lys Asp Leu Cys Glu Lys Asn Leu Leu Gln Arg Asp Ser Arg Cys
130 135 140
TGC CAG GGG TGG AAC ACA GCA AAC TGT TTA GCC GCT CCT CCA GCT GAC 480
Cys Gln Gly Trp Asn Thr Ala Asn Cys Leu Ala Ala Pro Pro Ala Asp
145 150 155 160
TCC TAT TGC TCT CCT GGG AGC CCC AAA GGA CCG GAC GGA CAG TGT AAA 528
Ser Tyr Cys Ser Pro Gly Ser Pro Lys Gly Pro Asp Gly Gln Cys Lys
165 170 175
AAT GCT TGC AGG ACG AAA GAA GCT GGG TTT GTC TGC AAG CAT GGA TGC 576
Asn Ala Cys Arg Thr Lys Glu Ala Gly Phe Val Cys Lys His Gly Cys
180 185 190
AGG TCC ACC GAC AAG GCG TAC GAG TGC ACG TGC CCG AGT GGC TCT ACC 624
Arg Ser Thr Asp Lys Ala Tyr Glu Cys Thr Cys Pro Ser Gly Ser Thr
195 200 205
GTC GCC GAA GAT GGC ATT ACC TGC AAA AGT ATT TCG TAC ACA GTC AGC 672
Val Ala Glu Asp Gly Ile Thr Cys Lys Ser Ile Ser Tyr Thr Val Ser
210 215 220
TGC ACT GTT GAG CAA AAA CAG ACC TGC CGC CCA ACC GAA GAC TGT CGT 720
Cys Thr Val Glu Gln Lys Gln Thr Cys Arg Pro Thr Glu Asp Cys Arg
225 230 235 240
GTG CAG AAA GGA ACT GTG TTG TGT GAG TGC CCG TGG AAT CAA CAT CTA 768
Val Gln Lys Gly Thr Val Leu Cys Glu Cys Pro Trp Asn Gln His Leu
245 250 255
GTG GGG GAC AAG TGC ATA AGT GAT TGC GTC GAC AAG AAA TGT CAC GAA 816
Val Gly Asp Lys Cys Ile Ser Asp Cys Val Asp Lys Lys Cys His Glu
260 265 270
GAA TTT ATG GAC TGT GGC GTA TAT ATG AAT CGA CAA AGC TGC TAT TGT 864
Glu Phe Met Asp Cys Gly Val Tyr Met Asn Arg Gln Ser Cys Tyr Cys
275 280 285
CCA TGG AAA TCA AGG AAG CCG GGC CCA AAT GTC AAC ATC AAT GAA TGC 912
Pro Trp Lys Ser Arg Lys Pro Gly Pro Asn Val Asn Ile Asn Glu Cys
290 295 300
CTA CTG AAT GAG TAT TAC TAC ACG GTG TCA TTC ACC CCG AAC ATA TCT 960
Leu Leu Asn Glu Tyr Tyr Tyr Thr Val Ser Phe Thr Pro Asn Ile Ser
305 310 315 320
TTT GAT TCT GAC CAT TGC AAA CGG TAT GAG GAT CGT GTT TTG GAA GCG 1008
Phe Asp Ser Asp His Cys Lys Arg Tyr Glu Asp Arg Val Leu Glu Ala
325 330 335
ATA CGG ACC AGT ATC GGA AAA GAA GTT TTT AAG GTT GAG ATA CTT AAC 1056
Ile Arg Thr Ser Ile Gly Lys Glu Val Phe Lys Val Glu Ile Leu Asn
340 345 350
TGC ACG CAG GAC ATT AAG GCA AGA CTC ATA GCA GAG AAA CCA CTG TCA 1104
Cys Thr Gln Asp Ile Lys Ala Arg Leu Ile Ala Glu Lys Pro Leu Ser
355 360 365
AAA TAC GTG CTC AGG AAA CTA CAA GCA TGC GAG CAT CCA ATC GGC GAA 1152
Lys Tyr Val Leu Arg Lys Leu Gln Ala Cys Glu His Pro Ile Gly Glu
370 375 380
TGG TGC ATG ATG TAT CCG AAG TTG CTG ATC AAG AAA AAC TCT GCA ACA 1200
Trp Cys Met Met Tyr Pro Lys Leu Leu Ile Lys Lys Asn Ser Ala Thr
385 390 395 400
GAA ATT GAA GAA GAG AAC CTT TGC GAC AGT CTG CTC AAG AAT CAG GAA 1248
Glu Ile Glu Glu Glu Asn Leu Cys Asp Ser Leu Leu Lys Asn Gln Glu
405 410 415
GCT GCC TAC AAA GGT CAA AAC AAA TGC GTC AAG GTC GAC AAC CTC TTC 1296
Ala Ala Tyr Lys Gly Gln Asn Lys Cys Val Lys Val Asp Asn Leu Phe
420 425 430
TGG TTC CAG TGC GCT GAT GGT TAC ACA ACA ACT TAC GAG ATG ACA CGA 1344
Trp Phe Gln Cys Ala Asp Gly Tyr Thr Thr Thr Tyr Glu Met Thr Arg
435 440 445
GGT CGC CTA CGC CGC TCC GTG TGT AAA GCT GGA GTT TCT TGC AAC GAA 1392
Gly Arg Leu Arg Arg Ser Val Cys Lys Ala Gly Val Ser Cys Asn Glu
450 455 460
AAC GAG CAG TTG GAG TGT GCT AAC AAA GGT CAA ATA TGT GTC TAC GAA 1440
Asn Glu Gln Leu Glu Cys Ala Asn Lys Gly Gln Ile Cys Val Tyr Glu
465 470 475 480
AAC GGC AAA GCG AAT TGC CAA TGC CCA CCA GAC ACT AAA CCA GGG GAG 1488
Asn Gly Lys Ala Asn Cys Gln Cys Pro Pro Asp Thr Lys Pro Gly Glu
485 490 495
ATT GGC TGC ATT GAG CGT ACC ACA TGC AAC CCT AAA GAG ATA CAA GAA 1536
Ile Gly Cys Ile Glu Arg Thr Thr Cys Asn Pro Lys Glu Ile Gln Glu
500 505 510
TGC CAA GAC AAG AAG CTC GAG TGC GTT TAC AAA AAC CAT AAA GCA GAA 1584
Cys Gln Asp Lys Lys Leu Glu Cys Val Tyr Lys Asn His Lys Ala Glu
515 520 525
TSS AAG TGT CCT GAT GAT CAC GAG TGT TCT AGG GAG CCT GCC AAA GAC 1632
Xaa Lys Cys Pro Asp Asp His Glu Cys Ser Arg Glu Pro Ala Lys Asp
530 535 540
TCT TGC AGT GAA GAA GAT AAT GGT AAA TGT CAA AGC AGT GGG CAG CGT 1680
Ser Cys Ser Glu Glu Asp Asn Gly Lys Cys Gln Ser Ser Gly Gln Arg
545 550 555 560
TGT GTA ATG GAA AAC GGA AAT GCT GTT TGC AAA GAG AAG TCT GAT GCA 1728
Cys Val Met Glu Asn Gly Asn Ala Val Cys Lys Glu Lys Ser Asp Ala
565 570 575
ACA ACA GCT TCG ACT ACA ACA ACG AAA GCG AAA GAC AAG GAT CCA GAT 1776
Thr Thr Ala Ser Thr Thr Thr Thr Lys Ala Lys Asp Lys Asp Pro Asp
580 585 590
CCT GAA AAG TCA AGT GCT GCA GCA GTA TCA GCT ACT GGG CTC TTG TTA 1824
Pro Glu Lys Ser Ser Ala Ala Ala Val Ser Ala Thr Gly Leu Leu Leu
595 600 605
CTG CTC GCA GCT ACT TCA GTC ACC GCA GCA TCG TTG TAATGAAGAT 1870
Leu Leu Ala Ala Thr Ser Val Thr Ala Ala Ser Leu
610 615 620
GTCCAACTTG AATACGGAAC AGCTTGAAAA TGTATATATA CATCACGCTT ACATCGAACA 1930
TCTAGCTTGG TCTTTGGAAT TTAAATATTG CACATGGGTA CTCACGGCAA AATGGACGTA 1990
TTATTTTAGA ATGACAGGGA AGATGGACGT GAAAGGAGTT TCCTTGTCTG AAAATATCAA 2050
AGAAAAACTT TCCCTATCTG AATGATGTCA AATAAAGATA GTTGGGTCTA AACAAAAAAA 2110
AAAAAAAAAA AAAAGCGGCC G 2131

620 amino acids

amino acid

linear

protein

unknown

65
Phe Cys Arg Asn Ala Glu Cys Glu Glu Val Pro Gly Ala Glu Asp Asp
1 5 10 15
Phe Val Cys Lys Cys Pro Arg Tyr Asn Met Tyr Phe Asn Ala Ala Glu
20 25 30
Lys Gln Cys Glu Tyr Lys Asp Thr Cys Lys Thr Arg Glu Cys Ser Tyr
35 40 45
Gly Arg Cys Val Gln Ser Asn Pro Ser Lys Ala Ser Cys Val Cys Glu
50 55 60
Ala Ser Asp Thr Leu Thr Leu Gln Cys Asn Ile Asn Asn Asp Tyr Ala
65 70 75 80
Thr Asp Cys Arg Asn Arg Gly Gly Thr Ala Lys Leu Arg Thr Asp Gly
85 90 95
Phe Ile Gly Ala Thr Cys Asp Cys Gly Glu Trp Gly Ala Met Asn Lys
100 105 110
Thr Thr Arg Asn Cys Val Pro Thr Thr Cys Leu Arg Pro Asp Leu Thr
115 120 125
Cys Lys Asp Leu Cys Glu Lys Asn Leu Leu Gln Arg Asp Ser Arg Cys
130 135 140
Cys Gln Gly Trp Asn Thr Ala Asn Cys Leu Ala Ala Pro Pro Ala Asp
145 150 155 160
Ser Tyr Cys Ser Pro Gly Ser Pro Lys Gly Pro Asp Gly Gln Cys Lys
165 170 175
Asn Ala Cys Arg Thr Lys Glu Ala Gly Phe Val Cys Lys His Gly Cys
180 185 190
Arg Ser Thr Asp Lys Ala Tyr Glu Cys Thr Cys Pro Ser Gly Ser Thr
195 200 205
Val Ala Glu Asp Gly Ile Thr Cys Lys Ser Ile Ser Tyr Thr Val Ser
210 215 220
Cys Thr Val Glu Gln Lys Gln Thr Cys Arg Pro Thr Glu Asp Cys Arg
225 230 235 240
Val Gln Lys Gly Thr Val Leu Cys Glu Cys Pro Trp Asn Gln His Leu
245 250 255
Val Gly Asp Lys Cys Ile Ser Asp Cys Val Asp Lys Lys Cys His Glu
260 265 270
Glu Phe Met Asp Cys Gly Val Tyr Met Asn Arg Gln Ser Cys Tyr Cys
275 280 285
Pro Trp Lys Ser Arg Lys Pro Gly Pro Asn Val Asn Ile Asn Glu Cys
290 295 300
Leu Leu Asn Glu Tyr Tyr Tyr Thr Val Ser Phe Thr Pro Asn Ile Ser
305 310 315 320
Phe Asp Ser Asp His Cys Lys Arg Tyr Glu Asp Arg Val Leu Glu Ala
325 330 335
Ile Arg Thr Ser Ile Gly Lys Glu Val Phe Lys Val Glu Ile Leu Asn
340 345 350
Cys Thr Gln Asp Ile Lys Ala Arg Leu Ile Ala Glu Lys Pro Leu Ser
355 360 365
Lys Tyr Val Leu Arg Lys Leu Gln Ala Cys Glu His Pro Ile Gly Glu
370 375 380
Trp Cys Met Met Tyr Pro Lys Leu Leu Ile Lys Lys Asn Ser Ala Thr
385 390 395 400
Glu Ile Glu Glu Glu Asn Leu Cys Asp Ser Leu Leu Lys Asn Gln Glu
405 410 415
Ala Ala Tyr Lys Gly Gln Asn Lys Cys Val Lys Val Asp Asn Leu Phe
420 425 430
Trp Phe Gln Cys Ala Asp Gly Tyr Thr Thr Thr Tyr Glu Met Thr Arg
435 440 445
Gly Arg Leu Arg Arg Ser Val Cys Lys Ala Gly Val Ser Cys Asn Glu
450 455 460
Asn Glu Gln Leu Glu Cys Ala Asn Lys Gly Gln Ile Cys Val Tyr Glu
465 470 475 480
Asn Gly Lys Ala Asn Cys Gln Cys Pro Pro Asp Thr Lys Pro Gly Glu
485 490 495
Ile Gly Cys Ile Glu Arg Thr Thr Cys Asn Pro Lys Glu Ile Gln Glu
500 505 510
Cys Gln Asp Lys Lys Leu Glu Cys Val Tyr Lys Asn His Lys Ala Glu
515 520 525
Xaa Lys Cys Pro Asp Asp His Glu Cys Ser Arg Glu Pro Ala Lys Asp
530 535 540
Ser Cys Ser Glu Glu Asp Asn Gly Lys Cys Gln Ser Ser Gly Gln Arg
545 550 555 560
Cys Val Met Glu Asn Gly Asn Ala Val Cys Lys Glu Lys Ser Asp Ala
565 570 575
Thr Thr Ala Ser Thr Thr Thr Thr Lys Ala Lys Asp Lys Asp Pro Asp
580 585 590
Pro Glu Lys Ser Ser Ala Ala Ala Val Ser Ala Thr Gly Leu Leu Leu
595 600 605
Leu Leu Ala Ala Thr Ser Val Thr Ala Ala Ser Leu
610 615 620

2147 base pairs

nucleic acid

double

linear

unknown

Figure 16

CDS

49..2001

66
CAGGATCCGT GGAAAGTGCG ACAGCTGCGG TGGTTCGACG CAGTCGAG ATG CGT GGC 57
Met Arg Gly
1
ATC GCT TTG TTC GTC GCC GCT GTT TCA CTG ATT GTA GAG TGC ACA GCA 105
Ile Ala Leu Phe Val Ala Ala Val Ser Leu Ile Val Glu Cys Thr Ala
5 10 15
GAA TCA TCC ATT TGC TCT GAC TTC GGG AAC GAG TTC TGT CGC AAC GCT 153
Glu Ser Ser Ile Cys Ser Asp Phe Gly Asn Glu Phe Cys Arg Asn Ala
20 25 30 35
GAA TGT GAA GTG GTG CCT GGT GCA GAG GAT GAT TTC GTG TGC AAA TGT 201
Glu Cys Glu Val Val Pro Gly Ala Glu Asp Asp Phe Val Cys Lys Cys
40 45 50
CCG CGA GAT AAT ATG TAC TTC AAT GCT GCT GAA AAG CAA TGC GAA TAT 249
Pro Arg Asp Asn Met Tyr Phe Asn Ala Ala Glu Lys Gln Cys Glu Tyr
55 60 65
AAA GAT ACG TGC AAG ACA AGG GAG TGC AGC TAT GGA CGT TGC GTT GAA 297
Lys Asp Thr Cys Lys Thr Arg Glu Cys Ser Tyr Gly Arg Cys Val Glu
70 75 80
AGT AAC CCG AGC AAG GGT AGC TGC GTC TGC GAA GCA TCG GAC GAT CTA 345
Ser Asn Pro Ser Lys Gly Ser Cys Val Cys Glu Ala Ser Asp Asp Leu
85 90 95
ACG CTA CAA TGC AAA ATT AAA AAT GAC TTC GCA ACT GAC TGC CGA AAC 393
Thr Leu Gln Cys Lys Ile Lys Asn Asp Phe Ala Thr Asp Cys Arg Asn
100 105 110 115
CGA GGT GGC ACT GCT AAG TTG CGC ACG GAT GGG TTT ATT GGC CCA ACG 441
Arg Gly Gly Thr Ala Lys Leu Arg Thr Asp Gly Phe Ile Gly Pro Thr
120 125 130
TGT GAC TGT GGT GAA TGG GGT GCG ATG AAC AAG ACC ACA CGG AAC TGT 489
Cys Asp Cys Gly Glu Trp Gly Ala Met Asn Lys Thr Thr Arg Asn Cys
135 140 145
GTC CCT ACC ACG TGT CTT CGT CCC GAC TTG ACC TGC AAA GAC CTC TGC 537
Val Pro Thr Thr Cys Leu Arg Pro Asp Leu Thr Cys Lys Asp Leu Cys
150 155 160
GAG AAA AAC CTG CTT CAA AGG GAT TCT CGT TGT TGT CAG GGG TGG AAC 585
Glu Lys Asn Leu Leu Gln Arg Asp Ser Arg Cys Cys Gln Gly Trp Asn
165 170 175
ACA GCA AAC TGT TCA GCC GCT CCT CCA GCT GAC TCC TAT TGC TCT CCT 633
Thr Ala Asn Cys Ser Ala Ala Pro Pro Ala Asp Ser Tyr Cys Ser Pro
180 185 190 195
GGG AGC CCC AAA GGA CCG GAC GGA CAG TGT AAA AAT GCT TGC AGG ACG 681
Gly Ser Pro Lys Gly Pro Asp Gly Gln Cys Lys Asn Ala Cys Arg Thr
200 205 210
AAA GAA GCT GGG TTT GTC TGC AAG CAT GGA TGC AGG TCC ACC GAC AAG 729
Lys Glu Ala Gly Phe Val Cys Lys His Gly Cys Arg Ser Thr Asp Lys
215 220 225
GCG TAC GAG TGC ACG TGC CCG AGT GGC TCT ACC GTC GCC GAA GAT GGC 777
Ala Tyr Glu Cys Thr Cys Pro Ser Gly Ser Thr Val Ala Glu Asp Gly
230 235 240
ATT ACC TGC AAA AGT ATT TCG TAC ACA GTC AGC TGC ACT GTT GAG CAA 825
Ile Thr Cys Lys Ser Ile Ser Tyr Thr Val Ser Cys Thr Val Glu Gln
245 250 255
AAA CAG ACC TGC CGC CCA ACC GAA GAC TGT CGT GTG CAG AAA GGA ACT 873
Lys Gln Thr Cys Arg Pro Thr Glu Asp Cys Arg Val Gln Lys Gly Thr
260 265 270 275
GTG TTG TGT GAG TGC CCG TGG AAT CAA CAT CTA GTG GGG GAC ACG TGC 921
Val Leu Cys Glu Cys Pro Trp Asn Gln His Leu Val Gly Asp Thr Cys
280 285 290
ATA AGT GAT TGC GTC GAC AAG AAA TGT CAC GAA GAA TTT ATG GAC TGT 969
Ile Ser Asp Cys Val Asp Lys Lys Cys His Glu Glu Phe Met Asp Cys
295 300 305
GGC GTA TAT ATG AAT CGA CAA AGC TGC TAT TGT CCA TGG AAA TCA AGG 1017
Gly Val Tyr Met Asn Arg Gln Ser Cys Tyr Cys Pro Trp Lys Ser Arg
310 315 320
AAG CCG GGC CCA AAT GTC AAC ATC AAT GAA TGC CTA CTG AAT GAG TAT 1065
Lys Pro Gly Pro Asn Val Asn Ile Asn Glu Cys Leu Leu Asn Glu Tyr
325 330 335
TAC TAC ACG GTG TCA TTC ACC CCG AAC ATA TCT TTT GAT TCT GAC CAT 1113
Tyr Tyr Thr Val Ser Phe Thr Pro Asn Ile Ser Phe Asp Ser Asp His
340 345 350 355
TGC AAA CGG TAT GAG GAT CGT GTT TTG GAA GCG ATA CGG ACC AGT ATC 1161
Cys Lys Arg Tyr Glu Asp Arg Val Leu Glu Ala Ile Arg Thr Ser Ile
360 365 370
GGA AAA GAA GTT TTT AAG GTT GAG ATA CTT AAC TGC ACG CAG GAC ATT 1209
Gly Lys Glu Val Phe Lys Val Glu Ile Leu Asn Cys Thr Gln Asp Ile
375 380 385
AAG GCA AGA CTC ATA GCA GAG AAA CCA CTG TCA AAA TAC GTG CTC AGG 1257
Lys Ala Arg Leu Ile Ala Glu Lys Pro Leu Ser Lys Tyr Val Leu Arg
390 395 400
AAA CTA CAA GCA TGC GAG CAT CCA ATC GGC GAA TGG TGC ATG ATG TAT 1305
Lys Leu Gln Ala Cys Glu His Pro Ile Gly Glu Trp Cys Met Met Tyr
405 410 415
CCG AAG TTG CTG ATC AAG AAA AAC TCT GCA ACA GAA ATT GAA GAA GAG 1353
Pro Lys Leu Leu Ile Lys Lys Asn Ser Ala Thr Glu Ile Glu Glu Glu
420 425 430 435
AAC CTT TGC GAC AGT CTG CTC AAG AAT CAG GAA GCT GCC TAC AAA GGT 1401
Asn Leu Cys Asp Ser Leu Leu Lys Asn Gln Glu Ala Ala Tyr Lys Gly
440 445 450
CAA AAC AAA TGC GTC AAG GTC GAC AAC CTC TTC TGG TTC CAG TGC GCT 1449
Gln Asn Lys Cys Val Lys Val Asp Asn Leu Phe Trp Phe Gln Cys Ala
455 460 465
GAT GGT TAC ACA ACA ACT TAC GAG ATG ACA CGA GGT CGC CTA CGC CGC 1497
Asp Gly Tyr Thr Thr Thr Tyr Glu Met Thr Arg Gly Arg Leu Arg Arg
470 475 480
TCC GTG TGT AAA GCT GGA GTT TCT TGC AAC GAA AAC GAG CAG TTG GAG 1545
Ser Val Cys Lys Ala Gly Val Ser Cys Asn Glu Asn Glu Gln Leu Glu
485 490 495
TGT GCT AAC AAA GGT CAA ATA TGT GTC TAC GAA AAC GGC AAA GCG AAT 1593
Cys Ala Asn Lys Gly Gln Ile Cys Val Tyr Glu Asn Gly Lys Ala Asn
500 505 510 515
TGC CAA TGC CCA CCA GAC ACT AAA CCA GGG GAG ATT GGC TGC ATT GAG 1641
Cys Gln Cys Pro Pro Asp Thr Lys Pro Gly Glu Ile Gly Cys Ile Glu
520 525 530
CGT ACC ACA TGC AAC CCT AAA GAG ATA CAA GAA TGC CAA GAC AAG AAG 1689
Arg Thr Thr Cys Asn Pro Lys Glu Ile Gln Glu Cys Gln Asp Lys Lys
535 540 545
CTC GAG TGC GTT TAC AAA AAC CAT AAA GCA GAA TGC AAG TGT CCT GAT 1737
Leu Glu Cys Val Tyr Lys Asn His Lys Ala Glu Cys Lys Cys Pro Asp
550 555 560
GAT CAC GAG TGT TCT AGG CAG CCT GCC AAA GAC TCT TGC AGT GAA GAG 1785
Asp His Glu Cys Ser Arg Gln Pro Ala Lys Asp Ser Cys Ser Glu Glu
565 570 575
GAT AAT GGT AAA TGT CAA AGC AGT GGG CAG CGT TGT GTA ATG GAA AAC 1833
Asp Asn Gly Lys Cys Gln Ser Ser Gly Gln Arg Cys Val Met Glu Asn
580 585 590 595
GGA AAG GCT GTT TGC AAA GAG AAG TCT GAA GCA ACA ACA GCT GCG ACT 1881
Gly Lys Ala Val Cys Lys Glu Lys Ser Glu Ala Thr Thr Ala Ala Thr
600 605 610
ACA ACA ACG AAA GCG AAA GAC AAG GAT CCA GAT CCT GGA AAG TCA AGT 1929
Thr Thr Thr Lys Ala Lys Asp Lys Asp Pro Asp Pro Gly Lys Ser Ser
615 620 625
GCT GCA GCA GTA TCA GCT ACT GGG CTC TTG TTA CTG CTC GCA GCT ACT 1977
Ala Ala Ala Val Ser Ala Thr Gly Leu Leu Leu Leu Leu Ala Ala Thr
630 635 640
TCA GTC ACC GTA GCA TCG TTG TAATGAAGAT GTCCAACTTG AATACGGAAC 2028
Ser Val Thr Val Ala Ser Leu
645 650
AGCTTGAAAA TGTATATATA CATCGCGCTT ACATCGAACA CCTAGCTTGG TTTTTGGGAT 2088
TTCAATATTG CGCATGGGTA CTCACGTCAA CATGGGATGT ATTATTTGAG AATGACAAG 2147

650 amino acids

amino acid

linear

protein

unknown

67
Met Arg Gly Ile Ala Leu Phe Val Ala Ala Val Ser Leu Ile Val Glu
1 5 10 15
Cys Thr Ala Glu Ser Ser Ile Cys Ser Asp Phe Gly Asn Glu Phe Cys
20 25 30
Arg Asn Ala Glu Cys Glu Val Val Pro Gly Ala Glu Asp Asp Phe Val
35 40 45
Cys Lys Cys Pro Arg Asp Asn Met Tyr Phe Asn Ala Ala Glu Lys Gln
50 55 60
Cys Glu Tyr Lys Asp Thr Cys Lys Thr Arg Glu Cys Ser Tyr Gly Arg
65 70 75 80
Cys Val Glu Ser Asn Pro Ser Lys Gly Ser Cys Val Cys Glu Ala Ser
85 90 95
Asp Asp Leu Thr Leu Gln Cys Lys Ile Lys Asn Asp Phe Ala Thr Asp
100 105 110
Cys Arg Asn Arg Gly Gly Thr Ala Lys Leu Arg Thr Asp Gly Phe Ile
115 120 125
Gly Pro Thr Cys Asp Cys Gly Glu Trp Gly Ala Met Asn Lys Thr Thr
130 135 140
Arg Asn Cys Val Pro Thr Thr Cys Leu Arg Pro Asp Leu Thr Cys Lys
145 150 155 160
Asp Leu Cys Glu Lys Asn Leu Leu Gln Arg Asp Ser Arg Cys Cys Gln
165 170 175
Gly Trp Asn Thr Ala Asn Cys Ser Ala Ala Pro Pro Ala Asp Ser Tyr
180 185 190
Cys Ser Pro Gly Ser Pro Lys Gly Pro Asp Gly Gln Cys Lys Asn Ala
195 200 205
Cys Arg Thr Lys Glu Ala Gly Phe Val Cys Lys His Gly Cys Arg Ser
210 215 220
Thr Asp Lys Ala Tyr Glu Cys Thr Cys Pro Ser Gly Ser Thr Val Ala
225 230 235 240
Glu Asp Gly Ile Thr Cys Lys Ser Ile Ser Tyr Thr Val Ser Cys Thr
245 250 255
Val Glu Gln Lys Gln Thr Cys Arg Pro Thr Glu Asp Cys Arg Val Gln
260 265 270
Lys Gly Thr Val Leu Cys Glu Cys Pro Trp Asn Gln His Leu Val Gly
275 280 285
Asp Thr Cys Ile Ser Asp Cys Val Asp Lys Lys Cys His Glu Glu Phe
290 295 300
Met Asp Cys Gly Val Tyr Met Asn Arg Gln Ser Cys Tyr Cys Pro Trp
305 310 315 320
Lys Ser Arg Lys Pro Gly Pro Asn Val Asn Ile Asn Glu Cys Leu Leu
325 330 335
Asn Glu Tyr Tyr Tyr Thr Val Ser Phe Thr Pro Asn Ile Ser Phe Asp
340 345 350
Ser Asp His Cys Lys Arg Tyr Glu Asp Arg Val Leu Glu Ala Ile Arg
355 360 365
Thr Ser Ile Gly Lys Glu Val Phe Lys Val Glu Ile Leu Asn Cys Thr
370 375 380
Gln Asp Ile Lys Ala Arg Leu Ile Ala Glu Lys Pro Leu Ser Lys Tyr
385 390 395 400
Val Leu Arg Lys Leu Gln Ala Cys Glu His Pro Ile Gly Glu Trp Cys
405 410 415
Met Met Tyr Pro Lys Leu Leu Ile Lys Lys Asn Ser Ala Thr Glu Ile
420 425 430
Glu Glu Glu Asn Leu Cys Asp Ser Leu Leu Lys Asn Gln Glu Ala Ala
435 440 445
Tyr Lys Gly Gln Asn Lys Cys Val Lys Val Asp Asn Leu Phe Trp Phe
450 455 460
Gln Cys Ala Asp Gly Tyr Thr Thr Thr Tyr Glu Met Thr Arg Gly Arg
465 470 475 480
Leu Arg Arg Ser Val Cys Lys Ala Gly Val Ser Cys Asn Glu Asn Glu
485 490 495
Gln Leu Glu Cys Ala Asn Lys Gly Gln Ile Cys Val Tyr Glu Asn Gly
500 505 510
Lys Ala Asn Cys Gln Cys Pro Pro Asp Thr Lys Pro Gly Glu Ile Gly
515 520 525
Cys Ile Glu Arg Thr Thr Cys Asn Pro Lys Glu Ile Gln Glu Cys Gln
530 535 540
Asp Lys Lys Leu Glu Cys Val Tyr Lys Asn His Lys Ala Glu Cys Lys
545 550 555 560
Cys Pro Asp Asp His Glu Cys Ser Arg Gln Pro Ala Lys Asp Ser Cys
565 570 575
Ser Glu Glu Asp Asn Gly Lys Cys Gln Ser Ser Gly Gln Arg Cys Val
580 585 590
Met Glu Asn Gly Lys Ala Val Cys Lys Glu Lys Ser Glu Ala Thr Thr
595 600 605
Ala Ala Thr Thr Thr Thr Lys Ala Lys Asp Lys Asp Pro Asp Pro Gly
610 615 620
Lys Ser Ser Ala Ala Ala Val Ser Ala Thr Gly Leu Leu Leu Leu Leu
625 630 635 640
Ala Ala Thr Ser Val Thr Val Ala Ser Leu
645 650

441 base pairs

nucleic acid

double

linear

unknown

Figure 17

CDS

1..441

68
GCC CTT GTT TTG GAC GCG ATA AAG ACC AGT ATC GGA AGC GAA GTT TCT 48
Ala Leu Val Leu Asp Ala Ile Lys Thr Ser Ile Gly Ser Glu Val Ser
1 5 10 15
AAA CTT GAG ATA CTG AAC TGC ACG CAG GAT ATT AAG GCA AGG CTC ATA 96
Lys Leu Glu Ile Leu Asn Cys Thr Gln Asp Ile Lys Ala Arg Leu Ile
20 25 30
GTA CCG AAA CCG CTA TCA AAG CAC GTG CTC AAG AAG CTT CAA GCA TGC 144
Val Pro Lys Pro Leu Ser Lys His Val Leu Lys Lys Leu Gln Ala Cys
35 40 45
GAG CAT CCC GTC GGG GAC TTG TGT ATG CTG TAT CCG AAG TTG CCG ATC 192
Glu His Pro Val Gly Asp Leu Cys Met Leu Tyr Pro Lys Leu Pro Ile
50 55 60
AAG AAA AAC TCT GCG ACA GAA ATT GAA GAA GAG AAC CTT TGC GAC AGC 240
Lys Lys Asn Ser Ala Thr Glu Ile Glu Glu Glu Asn Leu Cys Asp Ser
65 70 75 80
CTC CTC AAG CGT CAG GAA GCT GCC TAC AAG GGT CAG AAC AAA TGC GTC 288
Leu Leu Lys Arg Gln Glu Ala Ala Tyr Lys Gly Gln Asn Lys Cys Val
85 90 95
AAG GTC GGT AAC ATT TTC TGG TTC CAG TGC GCT GAT GGT TAC AGA TCA 336
Lys Val Gly Asn Ile Phe Trp Phe Gln Cys Ala Asp Gly Tyr Arg Ser
100 105 110
GTT TAC GAC ATC ACA CAA GGT CGC CTA CGC CGC TCC GTG TGC GAA CGT 384
Val Tyr Asp Ile Thr Gln Gly Arg Leu Arg Arg Ser Val Cys Glu Arg
115 120 125
GGA ATT TCT TGC AGT GAT AAT GAA CAG TTG GAG TGT GCC AAG AAA GGA 432
Gly Ile Ser Cys Ser Asp Asn Glu Gln Leu Glu Cys Ala Lys Lys Gly
130 135 140
CAA ATA TGT 441
Gln Ile Cys
145

147 amino acids

amino acid

linear

protein

unknown

69
Ala Leu Val Leu Asp Ala Ile Lys Thr Ser Ile Gly Ser Glu Val Ser
1 5 10 15
Lys Leu Glu Ile Leu Asn Cys Thr Gln Asp Ile Lys Ala Arg Leu Ile
20 25 30
Val Pro Lys Pro Leu Ser Lys His Val Leu Lys Lys Leu Gln Ala Cys
35 40 45
Glu His Pro Val Gly Asp Leu Cys Met Leu Tyr Pro Lys Leu Pro Ile
50 55 60
Lys Lys Asn Ser Ala Thr Glu Ile Glu Glu Glu Asn Leu Cys Asp Ser
65 70 75 80
Leu Leu Lys Arg Gln Glu Ala Ala Tyr Lys Gly Gln Asn Lys Cys Val
85 90 95
Lys Val Gly Asn Ile Phe Trp Phe Gln Cys Ala Asp Gly Tyr Arg Ser
100 105 110
Val Tyr Asp Ile Thr Gln Gly Arg Leu Arg Arg Ser Val Cys Glu Arg
115 120 125
Gly Ile Ser Cys Ser Asp Asn Glu Gln Leu Glu Cys Ala Lys Lys Gly
130 135 140
Gln Ile Cys
145

14 amino acids

amino acid

linear

unknown

70
Lys Ala Asn Arg Gln Cys Pro Pro Asp Thr Arg Arg Gly Lys
1 5 10

14 amino acids

amino acid

linear

unknown

F10

71
Lys Cys Asn Cys Asp Cys Pro Pro Asp Thr Arg Pro Gly Lys
1 5 10

Number	Date	Country
PH9196	Nov 1986	AU
PH2570	Jun 1987	AU
PH4912	Oct 1987	AU

Number	Date	Country
1645983	Jan 1984	AU
59707	Jan 1986	AU
4593685	Feb 1986	AU
2 142 334	Jan 1985	GB

	Number	Date	Country
Parent	08/062109	May 1993	US
Child	08/325071		US

	Number	Date	Country
Parent	07/926368	Aug 1992	US
Child	08/062109		US
Parent	07/242196		US
Child	07/926368		US

DNA encoding a cell membrane glycoprotein of a tick gut

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

Priority Claims (3)

Parent Case Info

US Referenced Citations (1)

Foreign Referenced Citations (4)

Non-Patent Literature Citations (21)

Continuations (1)

Continuation in Parts (2)

Entry
Allen et al., “Immunization of Guinea Pigs and Cattle Against Ticks”, Nature, vol. 280, Aug. 1979, pp. 491-493.
Johnston et al., “Immunization of Cattle Against Boophilus microplus Using Extracts Derived from Adult Female Ticks: Effects of Induced Immunity on Tick Populations”, Inter. Journ. Parasitology, vol. 16, No. 1, 1984, pp. 27-34.
Brown et al., “Characterization of Tick Antigens Inducing Host Immune Resistance”, Journ. Immun., vol. 133, No. 6, Dec. 1984, pp. 3319-3325.
Ackerman et al., “Artificial Immunity to Dermacentor variabilis (Acari: Ixodidae): Vaccination Using Tick Antigens1”, J. Med. Entomol., vol. 17, No. 5, 1980, pp. 391-397.
McGowan et al., “Success of Tick Feeding on Calves Immunized with Amblyomma americanum (Acari: Ixodidae) Extract1”, J. Med. Entomol., vol. 18, No. 4, 1981, pp. 328-332.
Stephen K. Wikel, “The Induction of Host Resistance to Tick Infestation with a Salivary Gland Antigen”, Am. J. Trop. Med. Hyg., vol. 30, No. 1, 1981, pp. 284-288.
Briggs et al., “Molecular Mechanisms of Protein Secretion”, Advances in Protein Chemistry, Academic Press, vol. 38, pp. 110-180, 1986.
van Hemert et al., “The Primary Structure of Elongation Factor EF-1α from the Brine Shrimp Artemia”, EMBO Journ. vol. 3, No. 5, 1984, pp. 1109-1113.
P. Willadsen, “Immunological Approaches to the Control of Ticks”, Int. J. Parasit. vol. 17, pp. 671-677, 1987.
Vretblad, “Purification of Lectins by Biospecific Affinity Chromatography”, Bichimica et Biophysica Acta, vol. 434., 1976, pp. 169-176.
Sage et al., “Common Lentil (Lens culinaris) Phytohemagglutinin”, Methods in Enzymology 28 1972 Ginsburg. V. Ed., pp. 332-339.
Stephen K. Wikel, “Tick and Mite Toxicosis and Allergy”, Handbook of Natural Toxins, vol. 2, (Insect Poisons Allergens & Other Invertebrate Venoms A.T.TV, ed. New York: Marcel Dekker) 1984, pp. 371-396.
Stephen A. Wikel, “Immunomodulation of Host Responses to Ectoparasite Infestation—an Overview”, Vet., Parasit., vol. 14, 1984, pp. 321-339.
George et al., “Acquisition and Expression of Resistance by Bos indicus and Bos indicus Bos taurus Calves to Amblyomma americanum Infestation”, J. Parasit. vol. 71, No. 2, 1985, pp. 174-182.
S. K. Wikel., “Effects of Tick Infestation on the Plaque-Forming Cell Response to a Thymic Dependent Antigen”, Ann. Trop. Med. Parasit., vol. 79, No. 2, 1985, pp. 195-198.
S. K. Wikel, “Resistance to Ixodid Tick Infestation Induced by Administration of Tick-Tissue Culture Cells”, Ann. Trop. Med. Parasit., vol. 79, No. 5, 1985, pp. 513-518.
Wikel et al., “Ixodid-Host Immune Interaction. Identification and Characterization of Relevant Antigens and Tick-Induced Host Immunosuppression”, Vet. Parasit., vol. 20, 1986, pp. 149-174.
Whelen et al., “Dot-Elisa Assessment of Guinea Pig Antibody Responses to Repeated Dermacentor andersoni Infestations”, J. Parasit. vol. 72, No. 1, 1986, pp. 155-162.
Wikel et al., “Immunological Studies of Ixodid Tick-Host Interaction: Analysis of Immunogens”, J. Toxicol. Toxin Rev. vol. 5, No. 2, 1986, pp. 145-160.
Sharp et al., “Chromatography and Generation of Specific Antisera to Synthetic Peptides from a Protective Boophilus Microplus Antigen”, J. Chrom., vol. 512., 1990, pp. 189-202.
Old. Rev. & Primrose SB “Principles of Gene Manipulation”, 3rd. ed., Blackwell Scientific Publication, 1985, pp. 13, 111, 120, 288-89.