Papillomavirus polyprotein constructs

FIELD OF THE INVENTION

This invention relates to polyprotein constructs and in particular polyprotein constructs comprising a plurality of papillomavirus (PV) amino acid sequences which may be used in compositions for eliciting an immune response against PV, and particularly human papillomavirus (HPV), in, a host animal.

BACKGROUND OF THE INVENTION

Papillomaviruses induce benign hyperproliferative lesions in humans and in many animal species, some of which undergo malignant conversion. The biology of papillomavirus infection is summarised in a review by J. P. Sundberg, entitled “Papillomavirus Infections in Animals” In “Papillomaviruses and Human Disease” edited by K. Syrjanen, L. Gissmann and L. G. Koss, Springer Verlag (1987).

Papillomaviruses are a family of small DNA viruses encoding up to eight early (E1, E2, E3, E4, E5, E6, E7 and E8) and two late genes (L1 and L2). These viruses have been classified in several distinct groups such as HPV which are differentiated into types 1 to˜70 depending upon DNA sequence homology. A clinicopathological grouping of HPV and the malignant potential of the lesions with which they are most frequently associated are summarised in “Papillomaviruses and Human Cancer” by H. Pfister, CRC Press, Inc. (1990). For example, HPV type 1 (HPV-1) is present in plantar warts, HPV-6 or HPV-11 are associated with condylomata acuminata (anogenital warts), and HPV-16 or HPV-18 are common in pre-malignant and malignant lesions of the cervical squamous epithelium.

The immunological approach to the prevention of HPV disease requires a thorough analysis of the viral proteins against which humoral and cellular immune responses are mounted during and after infection. However, despite recent limited success (Kreider et al., 1986

, J. Virol

., 59, 369; Sterling et al., 1990

, J. Virol

., 64, 6305; Meyers et al., 1992

, Science

, 257, 971; Dollard et al., 1992

, Genes and Development

, 6, 1131), papillomaviruses are notoriously refractory to growth in cultured cells (Teichaman and LaPorta, 1987 In “The Papovaviridae”, Vol 2 edited by N. P. Salzman and P. M. Howley, p.109). As a consequence, the lack of viral reagents has delayed the analysis of the immune response to PV infection.

The recent advent of recombinant expression systems in vitro has allowed the production of viral proteins encoded by both early and late genes in relatively large amounts and in a purified form (Tindle et al., 1990

, J. Gen. Virol

., 71, 1347; Jarrett et al., 1991

, Virology

, 184, 33; Ghim etal., 1992

, Virology

, 190, 548; Stacey etal., 1991

, J. Gen. Virol

., 73, 2337). These systems have, for the first time, allowed the analysis of the host immune response to these viral proteins.

Interest in immune responses to the non-structural early open reading frame (ORF) proteins of HPV has centred on HPV-16 E7 because of an apparent association between serum antibodies to this protein and cervical cancer (for a review, see “Immune Response to Human Papillomaviruses and the Prospects of Human Papillomavirus-Specific Immunisation” by Tindle and Frazer In “Human Pathogenic Papillomaviruses” edited by H. zur Hausen, Current Topics in Microbioiogy Immunology, 186, Springer-Verlag, Berlin, 1994).

The immune responses to other HPV early ORF proteins have also been investigated including HPV-16 E6 (Stacey et al., 1992

, J. Cen. Virol

., 73, 2337; Bleul et al., 1991

, J. Clin. Microbiol

., 29, 1579; Dillner, 1990

, Int. J. Cancer

, 46, 703; and Mülleretal., 1992

, Virology

, 187,508), HPV-16 E2 (Dillner et al., 1989

Proc. Natl. Acad. Sci. USA

, 86, 3838; Dillner, 1990, supra; Lehtinen et al., 1992

, J. Med. Virol

., 37, 180; Mann et al., 1990

, Cancer Res

., 50, 7815; and Jenison et al., 1990

, J. Infect. Dis

., 162, 60) and HPV-16 E4 (Köchel et al., 1991

, Int. J. Cancer

, 48, 682; Jochmnus-Kudielka et al., 1989

, JNCI

, 81, 1698; and Barber et al., 1992

, Cancer Immunol. immunother

., 35, 33). However, comparison of these studies reveals a lack of correlation between the results of the various assays which have been used in assessing HPV early ORF protein reactivity in serum (Tindle and Frazer, 1994, supra).

In addition, antibodies to other HPV early ORF proteins have not yet been sought with sufficient rigour in large enough numbers of patients to determine their utility as disease markers or as indicators of HPV protein immunogenicity following HPV infection.

A problem associated with immunising animals with preparations of individual PV proteins is that most of these proteins are comparatively small and might therefore not comprise many reactive epitopes. In addition, immunodominance of particular B or T cell epitopes within a single PV protein would vary presumably between animals of different major histocompatibility (MHC) backgrounds. To this end, the efficacy of such immunogens, in respect of eliciting an immune response against PV, might be expected to differ between animals of diverse MHC background.

In addition, there is surprisingly little knowledge regarding which PV proteins are expressed by infected cells at various stages of differentiation, and hence it is not possible to predict which proteins will be responsible for defining appropriate immunological targets.

The present invention provides a polyprotein construct comprising a plurality of PV early ORF proteins in one fused or linked construct to improve the efficacy of immune stimulation against PV infection and to avoid the need to define specific immunological targets.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides as an isolated product, a polyprotein construct comprising at least two amino acid sequences fused directly or indirectly together, each of said sequences being the sequence of an early open reading frame (ORF) protein of papillomavirus (PV) or an immunogenic variant or fragment thereof, and at least one of said sequences being other than the E6 or E7 protein sequence or an immunogenic variant or fragment thereof.

In yet another aspect, the present invention provides a composition for eliciting a humoral and/or cellular immune response against PV in a host animal, said composition comprising an immunologically effective amount of a construct as described above, together with a pharmaceutically acceptable carrier and/or diluent.

In yet another aspect, this invention provides a method for eliciting a humoral and/or cellular response against PV in a host animal, which method comprises administering to the host animal an immunologically effective amount of a polyprotein construct as described above. In a related aspect, the invention also extends to use of such a polyprotein construct in eliciting an immune response against PV in a host animal. Preferably, the host animal is a human, however the host animal may also be a non-human mammal.

The present invention also extends to a nucleic acid molecule which encodes a polypeptide construct as broadly described above. Such a nucleic acid molecule may be delivered to a host animal in a nucleic acid vaccine composition with a pharmaceutically acceptable carrier and/or diluent, for expression of the encoded polyprotein construct in vivo in a host animal. Alternatively, the nucleic acid molecule may be included in a recombinant DNA molecule comprising an expression control sequence operatively linked to the nucleic acid molecule.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.”

DETAILED DESCRIPTION OF THE INVENTION

The term “polyprotein construct” as used herein is used to describe a protein construct made up of individual proteins that have been joined together in a sequence whereby they retain their original relevant biological activities.

The term “isolated” as used herein denotes that the polyprotein construct has undergone at least one purification or isolation step, and preferably is in a form suitable for administration to a host animal.

By use of the term “immunologically effective amount” herein in the context of treatment of PV infection, it is meant that the administration of that amount to an individual PV infected host, either in a single dose or as part of a series, that is effective for treatment of PV infection. By the use of the term “immunologically effective amount” herein in the context of prevention of PV infection, it is meant that the administration of that amount to an individual host, either in a single dose or as part of a series, that is effective to delay, inhibit, treat or prevent PV infection or disease. The effective amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the immunogen, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

Preferably, the amino acid sequences in the polyprotein construct substantially correspond to the sequences of wild-type early ORF proteins of PV, including allelic or other variants thereof. Suitable variants include variants having single or multiple amino acid substitutions or additions to the wild-type sequences, and may have at least 50-60%, more preferably at least 70-80%, and most preferably at least 90%, similarity to the wild-type amino acid sequences, provided the variant is capable of eliciting an immune response against PV in a host animal. The amino acid sequences may also be immunogenic fragments of the wild-type early ORF proteins, that is fragments of the proteins which are capable of eliciting an immune response in a host animal. Suitably, the immunogenic fragment will comprise at least five, and more preferably at least ten, contiguous amino acid residues of the particular protein. Such immunogenic fragments may also be recognised by PV-specific antibodies, particularly antibodies which have a protective or therapeutic effect in relation to PV infection. Preferably, the immunogenic fragment is a non-full length fragment of a wild-type amino acid sequence, which may for example comprise a deletion mutant of an early ORF protein corresponding to at least 50%, more preferably 60-70%, and even 80-90% of the full length wild-type amino acid sequence.

The amino acid sequences in the polyprotein construct of the present invention may be selected from the group consisting of the E1, E2, E3, E4, E5 (E5a, E5b), E6, E7 and E8 proteins of PV, and may be included in the construct in any desired order. By way of example, the construct may be selected from the group consisting of:

(a) E6/E4

(b) E6/E5a/E4

(c) E6/E 7/E4

(d) E6/E7/E5a/E4

(e) E6/E7/E1/E4

(f) E6/E7/E5a/E1/E4

(g) E6/E7/E5a/E1/E2/E4

(h) E6/E7/E5a/E5b/E1/E2/E4

(i) E2/E5b

(j) E2/E1/E5b

(k) E2/E5a/E5b

(I) E2/E1/E5a/E5b

(m) E2/E4/E5a/E5b/E6/E7/E1

(n) E2/E3/E4/E5/E8/E6/E7/E1.

As described above, at least one of the early ORF proteins is other than the E6 or E7 proteins. Preferably one of the early ORF proteins in the construct is the E4 protein.

The polyprotein constructs of this invention preferably comprise at least three, and more preferably three, four or five early ORF protein sequences. In addition, two or more different polyprotein constructs based on different combinations of early ORF proteins and/or different PV genotypes may be included in a single composition for prophylactic or therapeutic use.

In the polyprotein constructs of this invention, the amino acid sequences may be fused or linked directly together. Alternatively, they may be linked with a linker sequence of from 1 to 50, preferably 1 to 20, and more preferably 1 to 5, amino acid residues between the separate amino acid sequences. By way of example, such a linker sequence may be an amino acid sequence encoded by the nucleotide sequence comprising a restriction endonuclease site. Linker sequences as described above may also be provided before and/or after the amino acid sequences in the polyprotein constructs.

The polyprotein constructs of this invention may also comprise a tag protein or peptide moiety fused or otherwise coupled thereto to assist in purification of the polyprotein construct. Suitable tag moieties include, for example, (His)

6

, glutathione-S-transferase (GST) and FLAG (International Biotechnologies), with the (His)

6

tag moiety being preferred. The constructs may further comprise a component to enhance the immunogenicity of the polyprotein. The component may be an adjuvant such as diphtheria or cholera toxin or

E. coli

heat labile toxin (LT), or a non-toxic derivative thereof such as the holotoxoid or B subunit of cholera toxin or LT. In addition, the polyprotein construct of the invention may comprise a lipid binding region to facilitate incorporation into ISCOMs. Suitable lipid binding regions are disclosed by way of example in Australian Provisional Patent Application No. PN8867/96, dated Mar. 25, 1996. A preferred lipid binding region is an influenza haemagglutinin tail.

The present invention also provides a nucleic acid molecule comprising a sequence of nucleotides which encodes a polyprotein construct as broadly described above.

The nucleic acid molecule may be RNA or DNA, single stranded or double stranded, in linear or covalently closed circular form. It will be appreciated that the sequence of nucleotides of this aspect of the invention may be obtained from natural, synthetic or semi-synthetic sources; furthermore, this nucleotide sequence may be a naturally-occurring sequence, or it may be related by mutation, including single or multiple base substitutions, deletions, insertions and inversions, to such a naturally-occurring sequence, provided always that the nucleic acid molecule comprising such a sequence is capable of being expressed as a polyprotein construct as described herein.

The nucleotide sequence may have expression control sequences positioned adjacent to it, such control sequences being derived from either a homologous or a heterologous source.

Since nucleic acid molecules may be delivered directly as “naked DNA” to a host animal, (see, for example, Wolfe et al., 1990

, Science

247:1465 and Fynan er al., 1993

, Proc. Natl. Acad. Sci

. USA, 90:11478), the present invention also includes a nucleic acid vaccine composition comprising a nucleic acid molecule as described above, together with a pharmaceutically acceptable carrier and/or diluent.

Immunisation with an isolated nucleic acid molecule allows in vivo synthesis of the encoded polyprotein construct by the host animal in a manner similar to the manner in which PV proteins are expressed during infection by PV. In this aspect, the present invention also extends to a method for eliciting an immune response against PV in a host animal, which method comprises administering to the host animal an immunologically effective amount of a nucleic acid molecule as described above. The invention also extends to use of such a nucleic acid molecule in eliciting an immune response against PV in a host animal.

This invention also provides a recombinant DNA molecule comprising an expression control sequence having promoter and initiator sequences, the nucleotide sequence encoding the polyprotein construct being located 3′ to the promoter and initiator sequences and a terminator sequence located 3′ to this sequence of nucleotides. In yet another aspect, the invention provides a recombinant DNA cloning vehicle such as a plasmid capable of expressing the polyprotein construct, as well as a host cell containing a recombinant DNA cloning vehicle and/or a recombinant DNA molecule as described above.

Suitable expression control sequences and host cell/cloning vehicle combinations are well known in the art, and are described by way of example, in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press. Thus, the nucleotide sequence may be ligated into any suitable expression vector, which may be either a prokaryotic or eukaryotic expression vector. Preferably, the vector is a prokaryotic expression vector such as pTrcHisA or pGEX-STOP (a pGEX expression vector (Amrad/Pharmacia Biotech) which has been manipulated so as to result in truncation of the GST moiety, disclosed in Australian Provisional Patent Application No. PN8272/86, dated Feb. 26, 1996). Whilst the host cell is preferably a prokaryotic cell, more preferably a bacterium such as

E. coli

., it will be understood that the host cell may alternatively be a yeast or other eukaryotic cell, or insect cells infected with baculovirus or the like.

Once recombinant DNA cloning vehicles and/or host cells expressing a polyprotein construct of this invention have been identified, the expressed polypeptides synthesised by the host cells, for example, as a fusion protein, can be isolated substantially free of contaminating host cell components by techniques well known to those skilled in the art.

The polyprotein construct-encoding DNA sequence is formed by linking or “fusing” sequences encoding each of the individual protein moieties. The first sequence in the polyprotein DNA construction has a promoter element and a ribosome binding site. These elements assure that transcription of the polyprotein DNA into mRNA begins at a defined site and that the signal, the ribosome binding site, needed for translation of mRNA into protein is present. Synthesis of the polyprotein is made continuous from one protein component to the next by removing or altering any initiation or binding signals and stop codons from the subsequent proteincoding sequences. The stop codon, normally a signal for the ribosome to stop translation and to end the polypeptide, is not altered or removed from the last DNA sequence. The individual protein encoding sequences are jointed such that a proper phasing is made of the mRNA reading frames for translation of the sequence into the desired amino acids. Once a DNA sequence encoding a polyprotein construct or a “polyprotein gene” is made, it is necessary to demonstrate that the construction leads to production of a stable polyprotein construct. If the resulting protein is not stable, for example because the junctions between the proteins are vulnerable to proteolytic digestion, then the junction regions are modified. This can be done by inserting different amino acids at or near the junction or by building spacers of amino acids between the individual proteins. Linkers or spacers can also be introduced to modify the overall activity of the polyprotein. By adjusting the space between and orientation of the individual proteins it is possible to modify the total activity of the polyprotein construct. Further details of the preparation of polyprotein constructs of the present invention by recombinant DNA techniques are disclosed, by way of example, in U.S. Pat. No. 4,774,180, the disclosure of which is incorporated herein by reference.

Preferably, the polymerase chain reaction (PCR) is used to amplify the nucleotide sequences encoding each of the individual PV early ORF proteins. The nucleotide sequences which are amplified may be full length or non full-length fragments thereof. Restriction endonuclease sites may be incorporated in the oligonucleotide primers used for PCR to furnish directional ligation of the amplification products in the same translational frame and to enable directional cloning into a suitable expression vector. The primers may encode an artificial initiator codon or a termination codon.

The first nucleotide sequence has an initiator codon. This initiator codon may either be the normal wild-type initiator codon of the first sequence or may be inserted artificially at another chosen position of this sequence. Synthesis of the polyprotein construct is made continuous from one protein component to the next by removing or altering any initiation or binding signals and termination codons. The termination codon must be present in the last nucleotide sequence. This is effected normally by not altering or removing the termination codon of the last nucleotide sequence. However, this termination codon may be inserted artificially, by methods known to persons skilled in the art, by first removing the normal, wild-type termination codon of the last nucleotide sequence and inserting another, in the correct reading frame, at another position of this sequence.

The polyprotein construct-encoding DNA sequence may incorporate restriction sites at the flanking ends to facilitate insertion of the DNA sequence into a suitable expression vector.

The PV can be a human or an animal PV, and is preferably HPV. The HPV may be of any genotype, and may for example be selected from the group consisting of HPV-6, HPV-11, HPV-16, HPV-18, HPV-33, HPV-35, HPV-31 and HPV45. Preferably, the HPV is HPV-6 or HPV-11.

The present invention is particularly, but not exclusively, directed to polyprotein constructs comprising early ORF proteins of the HPV-6 and HPV-11 genotypes which are causative agents of condylomata acuminata, however it will be appreciated that the invention extends to variants of the corresponding proteins in other HPV genotypes, particularly the HPV-16 and HPV-18 genotypes, and other genotypes which have oncogenic potential of a type similarto HPV-16 and HPV-18.

The polyprotein constructs of the present invention may comprise early ORF proteins of a single HPV genotype, or alternatively they may comprise early ORF proteins from more than one HPV genotype. In addition, a combination of more than one polyprotein construct may be used in cases where not all early ORF proteins are represented in the one polyprotein construct, or where immune responses to more than one HPV genotype are desired.

The polyprotein constructs of the present invention are provided as isolated proteins, that is they are substantially free of other PV proteins, and find particular utility for the treatment of genital warts, cervical cancer or other conditions caused by HPV in man. The polyprotein constructs can be included in pharmaceutical compositions for the treatment or prevention of diseases involving HPV as well as the other conditions discussed above.

The polyprotein constructs of the invention may be used to raise antibodies and/or induce cellular immune responses, either in subjects for which protection against infection by PV is desired, i.e. as prophylactic vaccines, or to heighten the immune response to an PV infection already present, i.e. as therapeutic vaccines. They also can be injected into production species to obtain antisera. In lieu of the polyclonal antisera obtained in the production species, monoclonal antibodies may be produced using the standard methods or by more recent modifications thereof by immortalising spleen or other antibody-producing cells for injection into animals to obtain antibody-producing clones. The polyclonal or monoclonal antibodies obtained, corrected if necessary for species variations, can also be used as therapeutic agents.

Direct administration of the polyprotein constructs to a host animal such as a human can confer either protective immunity against PV or, if the subject is already infected, a boost to the subject's own immune response to more effectively combat the progress of the PV induced disease.

The magnitude of the prophylactic or therapeutic dose of a polyprotein constructs of this invention will, of course, vary with the group of patients (age, sex, etc.), the nature or the severity of the condition to be treated and with the particular polyprotein construct and its route of administration. In general, the weekly dose range for use lies within the range of from about 0.1 to about 5 μg per kg body weight of a mammal.

Any suitable route of administration may be employed for providing a mammal, especially a human, with an effective dosage of a polyprotein construct of this invention. For example, oral, rectal, vaginal, topical, parenteral, ocular, nasal, sublingual, buccal, intravenous and the like may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, suppositories, aerosols and the like. Said dosage forms also include injected or implanted slow releasing devices specifically designed for this purpose or other forms of implants modified to additionally act in this fashion.

If the polyprotein constructs are to be administered as vaccines, they are formulated according to conventional methods for such administration to the subject to be protected. The polyprotein constructs may be delivered in accordance with this invention in ISCOMS™ (immune stimulating complexes), liposomes or encapsulated in compounds such as acrylates or poly(DL-lactide-co-glycoside) to form microspheres. They may also be incorporated into oily emulsions and delivered orally.

Other adjuvants, as well as conventional pharmaceutically acceptable carriers, excipients, buffers or diluents, may also be included in vaccine compositions of this invention. Generally, a vaccine composition in accordance with the present invention will comprise an immunologically effective amount of the polyprotein construct, and optionally an adjuvant, in conjunction with one or more conventional pharmaceutically acceptable carriers and/or diluents. An extensive though not exhaustive list of adjuvants can be found in Coulter and Cox, “Advances in Adjuvant Technology and Application”, in

Animal Parasite Control Utilizing Biotechnology

, Chapter 4, Ed. Young, W. K., CRC Press, 1992. As used herein “pharmaceutically acceptable carriers and/or diluents” include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art and is described by way of example in

Remington's Pharmaceutical Sciences

, 18th Edition, Mack Publishing Company, Pennsylvania, U.S.A.

In practical use, a polyprotein construct of this invention can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. oral or parenteral (including intravenous and intraarterial). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water glycols, oils, alcohols, flavouring agents, preservatives, colouring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, capsules and tablets. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit torm, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.

In addition to the common dosage forms set out above, the polyprotein constructs of this invention may also be administered by controlled release means and/or delivery devices, including by way of example, the controlled release preparations disclosed in International Patent Specification No. PCT/AU93/00677 (Publication No. WO 94115636).

Pharmaceutical compositions of the present invention suitable for oral or parenteral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

Further features of the present invention are more fully described in the following Example(s). It is to be understood, however, that this detailed description is included solely for the purposes of exemplifying the present invention, and should not be understood in any way as a restriction on the broad description of the invention as set out above.

EXAMPLES

Example 1

Amplification and Cloning of Early Open Reading Frames (ORFS) of HPV6b

A clone containing the entire genome of HPV6b in pBR322 (de Villiers, 1981

, J. Virol

, 40:932) was used as the template for separate PCR amplifications of E6, E7, E5a, E5b, E1, E2 and E4 open reading frame (ORF) sequences.

Appropriate restriction enzyme recognition sequences were included in the oligonucleotides used for amplification (Table I; 1-7) to allow sequential assembly of these amplified early gene sequences into a “polyprotein” sequence as depicted in FIG.

1

A.

In this scheme, E6 was amplified with oligonucleotides containing a Smal site at the 5′ end and HindIII, Ncol and Xbal sites at the 3′ end. As well, E4 was amplified with oligonucleotides containing Xbal, Sacl, Kpnl and Spel sites 5′ and a Bg/II site 3′.

These amplified fragments were cloned as Smal/Xbal (E6) and Xbal/Bg/II (E4)

FIG. 1B

) in the vector pSP70 (Promega Corporation) which had been modified by the removal of an EcoRV/EcoRI fragment to contain a portion of the pGEM3Zf (Promega Corporation) polylinker—Hindul through EcoRI. As well, unwanted sites upstream of the Smal site were removed by cleaving with Smal/Xhol and insertion of a Smal/Sa/l/Xhol linker to create the vector pSP70 (MOD).

The E6/E4 cassette was able to be removed by cleavage with Smal/Bg/II and this was then cloned for expression into the pGEX-STOP vector which produces a non-fusion protein with a C-terminal six-histidine sequence for purification purposes.

Using the introduced restriction enzyme recognition sequences, other early ORF sequences were incorporated into the E6/E4 cassette cloned into pSP70 (MOD) and then the newly created cassette cloned as a Smal/BgII fragment into pGEX-STOP.

In this manner polyprotein constructs containing E6/E5a/E4, E6/E7/E4, E6/E7/E5a/E4, E6/E7/E1/E4 and E6/E7/E5a/E1/E4 were assembled. Complete DNA sequence data for the first three constructs is included and sequence data across the junctions of E1 is included for the latter two. DNA sequencing revealed the Spel site was inactivated by a single base change which occurred either during oligonucleotide synthesis, PCR or cloning.

As well the tetrafusion construct of E6/E7/E5a/E4 was cloned for expression into pET23b (Novagen) by firstly subcloning the tetramer as a Smal/BgfII fragment into the Smal/BamHI sites of the vector pRIT2T (AMRAD Pharmacia Biotech). The tetramer was then removed by restriction with Smal and Sa/I and cloned into the HincII/Xhol sites of the vector pET23b.

A further construct containing E2 and E5b, but which could also accommodate the addition of E1 and E5a, was created by amplifying E2 with oligonucleotides containing a Smal site at the 5′ end and Xbal, Ncol, Kpnl and Sadcl, sites at the 3′ end (Table 1; 8) and with E5b amplified using oligonucleotides with an Xbal site 5′ and Xhol, Bglll sites 3′ (Table 1; 9). These amplified fragments were then cloned into pSP70 (MOD) as depicted in FIG.

1

C.

TABLE 1

Oligonucleotides used for PCR

Early gene

Forward

Reverse

1 E6

5′

GCGCCCCGGGATGGAAAGTGC

5′

GCGCTCTAGACCATGGAAGCT

AAATGCCTC

3′

TGGGTAACATGTCTTCCATGC

3′

(SEQ ID No. 1)

(SEQ ID. No.2)

2 E4

5′

GCGCTCTAGAGAGCTCGGTACC

5′

GCGCAGATCTTAGGCGTAGCT

ACTAGTGGAGCACCAAACATTGG

GAACTGTTAC

3′

GAAG

3′

(SEQ ID No. 4)

(SEQ ID No. 3)

3 E5a

5′

GCGCCCATGGGAAGTGGTGCCT

5′

GCGCTCTAGATTGCTGTGTGG

GTACAAATAGC

3′

TAACAATATAG

3′

(SEQ ID No. 5)

(SEQ ID No. 6)

4 E7

5′

GCGCAAGCTTCATGGAAGACAT

5′

GCGCCCATGGGGTCTTCGGT

GTTACCCTAAAG

3′

GCGCAGATGG

3′

(SEQ ID No. 7)

(SEQ ID No. 8)

5 E1

5′

GCGCGAGCTCGCGGACGATTCA

5′

GCGCGGTACCTAAAGTTCTAA

GGTACAGAAAATG

3′

CAACTGTTCCTG

3′

(SEQ ID No. 9)

(SEQ ID No. 10)

6 E2

5′

GCGCGGTACCGAAGCAATAGCC

5′

GCGCACTAGTCAATAGGTGCA

AAGCGTTTAG

3′

GTGACATAAATC

3′

(SEQ ID No. 11)

(SEQ ID No. 12)

7 E5b

5′

GCGCTCTAGACTAACATGTCAAT

5′

GCGCGAGCTCATTCATATATA

TTAATGATG

3′

TATAATCACC

3′

(SEQ ID No. 13)

(SEQ ID No. 14)

8 E2

5′

GCGCCCCGGGATGGAAGCAATA

5′

GCGCTCTAGACCATGGGGTAC

GCCAAGCG

3′

CGAGCTCCAATAGGTGCAGTG

(SEQ ID No. 15)

ACATAAATC

3′

(SEQ ID No. 16)

9 E5 b

5′

GCGCTCTAGACTAACATGTCAAT

5′

GCGCAGATCTCTCGAGATTCA

TTAATGATG

3′

TATATATATAATCAC

3′

(SEQ ID No. 17)

(SEQ ID No. 18)

Example 2

Expression of Different Polyprotein Constructs

The following constructs in pGEX-STOP were expressed in

E. coli

strain BL21 and protein production was assayed by PAGE followed by Western blotting:

i) E6/E4

ii) E6/E5a/E4

iii) E6/E7/E4

iv) E6/E7/E5a/E4

Construct (iv) in pET23b, expressed in

E. coli

strains BL21(DE3)pLysS and D494(DE3)pLysS (Novagen), was also assayed for protein production by Western blotting and also by Coomassie Blue staining for the latter strain.

Cultures of 200 mL were grown in Terrific broth (Tartoff and Hobbs, Focus, 9:12, 1987) in the presence of 100 μg/mL ampicillin (BL21) and 34 μg/ml cloramphenicol [BL21(DE3)pLysS] and 15 μg/mL kanamycin [AD494(DE3)pLysS]. At OD

600

˜1 protein expression was induced by the addition of IPTG to 0.4 mM. Following induction samples were taken at 1, 2, 3, 4 and 5 hours and in some cases after overnight culture.

FIG. 2

show a Western blot result for the E6/E4 construct. This was probed with a polyclonal rabbit anti E4 antibody (MWE4—raised to the peptide LGNEHEESNSPLATPCVWPT conjugated to ovalbumin). An immunoreactive band of ˜30 kDa was present in the 4—hour-induced sample (lanes 2 & 4, arrow) which was not present in the uninduced sample (lane 3).

The same ˜30 kDa band can also be seen in the induced sample in

FIG. 3

, lane 3, arrow (lane 2-uninduced) while the E6/E5a/E4 trimer construct of ˜40kDa was poorly represented after a 4 hour induction period (lane 5, arrow; uninduced sample-lane 4) using the same anti-E4 antibody.

In contrast however, a trimer construct of E6/E7/E4 (˜41 kDa) could be easily detected after 5 hours induction using an anti-hexahistidine monoclonal antibody (Dianova) [

FIG. 4

, lane 4, arrow; uninduced sample—lane 3].

The same trimer construct was again easily visualised after 5 hours induction using the anti-E4 antibody MWE4 (

FIG. 5

, lane TRI, arrow; control sample—lane C) and the tetramer consisting of E6/E7/E5a/E4 (˜51 kDa) could also be detected (lane TET, arrow). Although this band is weak, it must be noted that a considerable amount of high molecular weight material is also immunoreactive, indicating the tetramer is reasonably well expressed but possibly prone to aggregation.

FIG. 6

indicates that an anti-E6 antibody (prepared as described below) was able to detect E6/E7/E4 after 5 hours induction (lane TRI, arrow) but not E6/E7/E5a/E4 (lane TET; lane C—uninduced). However, an anti-E7 antibody (prepared as described below) was able to detect after 5 hours induction both the trimer (

FIG. 7

, lane TRI, arrow; lane C—uninduced) and the tetramer (lane TET, arrow; lane C—uninduced), with the latter again showing indications of aggregation. A monoclonal antibody raised to an E4 peptide also recognised the trimer.

The phenomenon of aggregation was clearly apparent when the E6/E7/E5a/E4 tetramer was expressed in the pET23b plasmid in BL21 (DE3)pLysS (FIG.

8

—a Western blot probed with MWE4). Lanes 2-5 are 1 hour, 2 hour, 3 hour and overnight uninduced samples and lanes 69 represent 1 hour, 2 hour, 3 hour and overnight induced samples. After 1 hour induction a band of E6/E7/E5a/E4 can clearly be seen (arrow), but with increased times of induction this seems to decrease and aggregated forms are increased. In contrast, when strain AD494(DE3)pLysS was used to express the tetramer, a substantial signal was obtained at the˜50 kDa position on a Western blot of the insoluble fraction (

FIG. 9

, arrow) following 2 hours induction, which still persisted at 3 hours. This immunoreactive band was not present in control samples and no protein was detected in the samples from the soluble fractions.

FIG. 10

shows the Coomassie stained profile of an identical gel, indicating that the immunoreactive bands present after 2 and 3 hours induction (

FIG. 9

) can clearly be visualised as stained bands (arrow) which are not present in the control samples.

Example 3

DNA Sequencing of Polyprotein Constructs

Polyprotein constructs were sequenced in both directions by the dideoxy method using primers that generated overlapping sequence information. The

T7

Sequencing™ Kit (Pharmacia was used to generate

35

S-labelled chain-terminated fragments which were analysed on a Sequi-Gen™ (Biorad) electrophoretic gel apparatus. The DNA and corresponding amino acid sequences for E6/E5a/E4 (CSL690.SEQ), E6/E7/E4 (CSL760.SEQ) and E6/E7/E5a/E4 (CSL673.SEQ) are shown below. (SEQ ID Nos: 19 and 20, 21 and 22, and 23 and 24, respectively).

For constructs E6/E7/E1/E4 (CSL 791) and E6/E7/E5a/E1/E4 (CSL 762), which created from E6/E7/E4 and E6/E7/E5a/E4, respectively, DNA sequence analysis across the junctions of E1 with its neighbours is shown below (SEQ ID Nos. 25 and 26, 27 and 28, and 29 and 30, respectively).

File: CSL690.SEQ

Range: 1-11 Mode: Normal

Codon Table: Universal

E6/E5a/E4 - SEQ ID Nos, 19 (DNA) and 20 (amino acid)

9 18 27 36 45 54

5′

ATG GAA AGT GCA AAT GCC TCC ACG TCT GCA ACG ACC ATA GAC CAG TTG TGC AAG

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Met Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gln Leu Cys Lys

63 72 81 90 99 108

ACG TTT AAT CTA TCT ATG CAT ACG TTG CAA ATT AAT TGT GTG TTT TGC AAG AAT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn Cys Val Phe Cys Lys Asn

117 126 135 144 153 162

GCA CTG ACC ACA GCA GAG ATT TAT TCA TAT GCA TAT AAA CAC CTA AAG GTC CTG

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr Lys His Leu Lys Val Leu

171 180 189 198 207 216

TTT CGA GGC GGC TAT CCA TAT GCA GCC TGC GCG TGC TGC CTA GAA TTT CAT GGA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Phe Arg Gly Gly Tyr Pro Tyr ALa Ala Cys Ala Cys Cys Leu Glu Phe His Gly

225 234 243 252 261 270

AAA ATA AAC CAA TAT AGA CAC TTT GAT TAT GCT GGA TAT GCA ACA ACA GTT GAA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Lys Ile Asn Gln Tyr Arg His Phe Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu

279 288 297 306 315 324

GAA GAA ACT AAA CAA GAC ATC TTA GAC GTG CTA ATT CGG TGC TAC CTG TGT CAC

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Glu Glu Thr Lys Gln Asp Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu Cys His

333 342 351 360 369 378

AAA CCG CTG TGT GAA GTA GAA AAG GTA AAA CAT ATA CTA ACC AAG GCG CGG TTC

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Lys Pro Leu Cys Glu Val Glu Lys Val Lys His Ile Leu Thr Lys Ala Arg Phe

387 396 405 414 423 432

ATA AAG CTA AAT TGT ACG TGG AAG GGT CGC TGC CTA CAC TGC TGG ACA ACA TGC

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Ile Lys Leu Asn Cys Thr Trp Lys Gly Arg Cys Leu His Cys Trp Thr Thr Cys

441 450 459 468 477 486

ATG GAA GAC ATG TTA CCC AAG CTT CCA TGG GAA GTG GTG CCT GTA CAA ATA GCT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Met Glu Asp Met Leu Pro Lys Leu Pro Trp Glu Val Val Pro Val Gln Ile Ala

495 504 513 522 531 540

GCA GGA ACA ACC AGC ACA TTC ATA CTG CCT GTT ATA ATT GCA TTT GTT GTA TGT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Ala Gly Thr Thr Ser Thr Phe Ile Leu Pro Val Ile Ile Ala Phe Val Val Cys

549 558 567 576 585 594

TTT GTT AGC ATC ATA CTT ATT GTA TGG ATA TCT GAG TTT ATT GTG TAC ACA TCT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Phe Val Ser Ile Ile Leu Ile Val Trp Ile Ser Giu Phe Ile Val Tyr Thr Ser

603 612 621 630 639 648

GTG CTA GTA CTA ACA CTG CTT TTA TAT TTA CTA TTG TGG CTG CTA TTA ACA ACC

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Val Leu Val Leu Thr Leu Leu Leu Tyr Leu Leu Leu Trp Leu Leu Leu Thr Thr

657 666 675 684 693 702

CCC TTG CAA TTT TTC CTA CTA ACT CTA CTT GTG TGT TAC TGT CCC GCA TTG TAT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Pro Leu Gln Phe Phe Leu Leu Thr Leu Leu Val Cys Tyr Cys Pro Ala Leu Tyr

711 720 729 738 747 756

ATA CAC TAC TAT ATT GTT ACC ACA CAG CAA TCT AGA GAG CTC GGT ACC ACT AAT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Ile His Tyr Tyr Ile Val Thr Thr Gln Gln Ser Arg Glu Leu Gly Thr Thr Asn

765 774 783 792 801 810

GGA GCA CCA AAC ATT GGG AAG TAT GTT ATG GCA GCA CAG TTA TAT GTT CTC CTG

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Gly Ala Pro Asn Ile Gly Lys Tyr Val Met Ala Ala Gln Leu Tyr Val Leu Leu

819 828 837 846 855 864

CAT CTG TAT CTA GCA CTA CAC AAG AAG TAT CCA TTC CTG AAT CTA CTA CAT ACA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

His Leu Tyr Leu Ala Leu His Lys Lys Tyr Pro Phe Leu Asn Leu Leu His Thr

873 882 891 900 909 918

CCC CCG CAC AGA CCT CCA CCC TTG TGT CCT CAA GCA CCA AGG AAG ACG CAG TGC

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Pro Pro His Arg Pro Pro Pro Leu Cys Pro Gln Ala Pro Arq Lys Thr Gln Cys

927 936 945 954 963 972

AAA CGC CGC CTA GGA AAC GAG CAC GAG GAG TCC AAC AGT CCC CTT GCA ACG CCT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Lys Arg Arg Leu Gly Asn Glu His Glu Glu Ser Asn Ser Pro Leu Ala Thr Pro

981 990 999 1008 1017 1026

TGT GTG TGG CCC ACA TTG GAC CCG TGG ACA GTG GAA ACC ACA ACC TCA TCA CTA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Cys Val Trp Pro Thr Leu Asp Pro Trp Thr Val Glu Thr Thr Thr Ser Ser Leu

1035 1044 1053 1062 1071 1080

ACA ATC ACG ACC AGC ACC AAA GAC GGA ACA ACA GTA ACA GTT CAG CTA CGC CTA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Thr Ile Thr Thr Ser Thr Lys Asp Gly Thr Thr Val Thr Val Gln Leu Arg Leu

1089 1098 1107

AGA TCT CAT CAC CAT CAC CAT CAC TAA 3′

--- --- --- --- --- --- --- --- ---

Arg Ser His His His His His His ***

File: CSL760.SEQ

Range: 1-1128 Mode: Normal

Codon Table: Universal

E6/E7/E4 - SEQ ID Nos. 21 (DNA) and 22 (amino acid)

9 18 27 36 45 54

5′

ATG GAA AGT GCA AAT GCC TCC ACG TCT GCA ACG ACC ATA GAC CAG TTG TGC AAG

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Met Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gln Leu Cys Lys

63 72 81 90 99 108

ACG TTT AAT CTA TCT ATG CAT ACG TTG CAA ATT AAT TGT GTG TTT TGC AAG AAT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn Cys Val Phe Cys Lys Asn

117 126 135 144 153 162

GCA CTG ACC ACA GCA GAG ATT TAT TCA TAT GCA TAT AAA CAC CTA AAG GTC CTG

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr Lys His Leu Lys Val Leu

171 180 189 198 207 216

TTT CGA GGC GGC TAT CCA TAT GCA GCC TGC GCG TGC TGC CTA GAA TTT CAT GGA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Phe Arg Gly Gly Tyr Pro Tyr Ala Ala Cys Ala Cys Cys Leu Glu Phe His Gly

225 234 243 252 261 270

AAA ATA AAC CAA TAT AGA CAC TTT GAT TAT GCT GGA TAT GCA ACA ACA GTT GAA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Lys Ile Asn Gln Tyr Arg His Phe Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu

279 288 297 306 315 324

GAA GAA ACT AAA CAA GAC ATC TTA GAC GTG CTA ATT CGG TGC TAC CTG TGT CAC

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Glu Glu Thr Lys Gln Asp Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu Cys His

333 342 351 360 369 378

AAA CCG CTG TGT GAA GTA GAA AAG GTA AAA CAT ATA CTA ACC AAG GCG CGG TTC

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Lys Pro Leu Cys Glu Val Glu Lys Val Lys His Ile Leu Thr Lys Ala Arg Phe

387 396 405 414 423 432

ATA AAG CTA AAT TGT ACG TGG AAG GGT CGC TGC CTA CAC TGC TGG ACA ACA TGC

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Ile Lys Leu Asn Cys Thr Trp Lys Gly Arg Cys Leu His Cys Trp Thr Thr Cys

441 450 459 468 477 486

ATG GAA GAC ATG TTA CCC AAG CTT CAT GGA AGA CAT GTT ACC CTA AAG GAT ATT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Met Glu Asp Met Leu Pro Lys Leu His Gly Arg His Val Thr Leu Lys Asp Ile

495 504 513 522 531 540

GTA TTA GAC CTG CAA CCT CCA GAC CCT GTA GGG TTA CAT TGC TAT GAG CAA TTA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Val Leu Asp Leu Gln Pro Pro Asp Pro Val Gly Leu His Cys Tyr Glu Gln Leu

549 558 567 576 585 594

GTA GAC AGC TCA GAA GAT GAG GTG GAC GAA GTG GAC GGA CAA GAT TCA CAA CCT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Val Asp Ser Ser Glu Asp Glu Val Asp Glu Val Asp Gly Gln Asp Ser Gln Pro

603 612 621 630 639 648

TTA AAA CAA CAT TTC CAA ATA GTG ACC TGT TGC TGT GGA TGT GAC AGC AAC GTT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Leu Lys Gln His Phe Gln Ile Val Thr Cys Cys Cys Gly Cys Asp Ser Asn Val

657 666 675 684 693 702

CGA CTG GTT GTG CAG TGT ACA GAA ACA GAC ATC AGA GAA GTG CAA CAG CTT CTG

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Arg Leu Val Val Gln Cys Thr Glu Thr Asp Ile Arg Glu Val Gln Gln Leu Leu

711 720 729 738 747 756

TTG GGA ACA CTA AAC ATA GTG TGT CCC ATC TGC GCA CCG AAG ACC CCA TGG TCT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Leu Gly Thr Leu Asn Ile Val Cys Pro Ile Cys Ala Pro Lys Thr Pro Trp Ser

765 774 783 792 801 810

AGA GAG CTC GGT ACC ACT AAT GGA GCA CCA AAC ATT GGG AAG TAT GTT ATG GCA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Arg Glu Leu Gly Thr Thr Asn Gly Ala Pro Asn Ile Gly Lys Tyr Val Met Ala

819 828 837 846 855 864

GCA CAG TTA TAT GTT CTC CTG CAT CTG TAT CTA GCA CTA CAC AAG AAG TAT CCA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Ala Gln Leu Tyr Val Leu Leu His Leu Tyr Leu Ala Leu His Lys Lys Tyr Pro

873 882 891 900 909 918

TTC CTG AAT CTA CTA CAT ACA CCC CCG CAC AGA CCT CCA CCC TTG TGT CCT CAA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Phe Leu Asn Leu Leu His Thr Pro Pro His Arg Pro Pro Pro Leu Cys Pro Gln

927 936 945 954 963 972

GCA CCA AGG AAG ACG CAG TGC AAA CGC CGC CTA GGA AAC GAG CAC GAG GAG TCC

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Ala Pro Arg Lys Thr Gln Cys Lys Arg Arg Leu Gly Asn Glu His Glu Glu Ser

981 990 999 1008 1017 1026

AAC AGT CCC CTT GCA ACG CCT TGT GTG TGG CCC ACA TTG GAC CCG TGG ACA GTG

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Asn Ser Pro Leu Ala Thr Pro Cys Val Trp Pro Thr Leu Asp Pro Trp Thr Val

1035 1044 1053 1062 1071 1080

GAA ACC ACA ACC TCA TCA CTA ACA ATC ACG ACC AGC ACC AAA GAC GGA ACA ACA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Glu Thr Thr Thr Ser Ser Leu Thr Ile Thr Thr Ser Thr Lys Asp Gly Thr Thr

1089 1098 1107 1116 1125

GTA ACA GTT CAG CTA CGC CTA AGA TCT CAT CAC CAT CAC CAT CAC TAA 3′

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Val Thr Val Gln Leu Arg Leu Arg Ser His His His His His His ***

File: CSL673.DNA

Range: 1-1398 Mode: Normal

Codon Table: Universal

E6/E7/E5a/E4 - SEQ ID Nos. 23 (DNA) and 24 (amino acid)

9 18 27 36 45 54

5′

ATG GAA AGT GCA AAT GCC TCC ACG TCT GCA ACG ACC ATA GAC CAG TTG TGC AAG

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Met Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gln Leu Cys Lys

63 72 81 90 99 108

ACG TTT AAT CTA TCT ATG CAT ACG TTG CAA ATT AAT TGT GTG TTT TGC AAG AAT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn Cys Val Phe Cys Lys Asn

117 126 135 144 153 162

GCA CTG ACC ACA GCA GAG ATT TAT TCA TAT GCA TAT AAA CAC CTA AAG GTC CTG

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr Lys His Leu Lys Val Leu

171 180 189 198 207 216

TTT CGA GGC GGC TAT CCA TAT GCA GCC TGC GCG TGC TGC CTA GAA TTT CAT GGA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Phe Arg Gly Gly Tyr Pro Tyr Ala Ala Cys Ala Cys Cys Leu Glu Phe His Gly

225 234 243 252 261 270

AAA ATA AAC CAA TAT AGA CAC TTT GAT TAT GCT GGA TAT GCA ACA ACA GTT GAA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Lys Ile Asn Gln Tyr Arg His Phe Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu

279 288 297 306 315 324

GAA GAA ACT AAA CAA GAC ATC TTA GAC GTG CTA ATT CGG TGC TAC CTG TGT CAC

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Glu Glu Thr Lys Gln Asp Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu Cys His

333 342 351 360 369 378

AAA CCG CTG TGT GAA GTA GAA AAG GTA AAA CAT ATA CTA ACC AAG GCG CGG TTC

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Lys Pro Leu Cys Glu Val Glu Lys Val Lys His Ile Leu Thr Lys Ala Arg Phe

387 396 405 414 423 432

ATA AAG CTA AAT TGT ACG TGG AAG GGT CGC TGC CTA CAC TGC TGG ACA ACA TGC

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Ile Lys Leu Asn Cys Thr Trp Lys Gly Arg Cys Leu His Cys Trp Thr Thr Cys

441 450 459 468 477 486

ATG GAA GAC ATG TTA CCC AAG CTT CAT GGA AGA CAT GTT ACC CTA AAG GAT ATT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Met Glu Asp Met Leu Pro Lys Leu His Gly Arg His Val Thr Leu Lys Asp Ile

495 504 513 522 531 540

GTA TTA GAC CTG CAA CCT CCA GAC CCT GTA GGG TTA CAT TGC TAT GAG CAA TTA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Val Leu Asp Leu Gln Pro Pro Asp Pro Val Gly Leu His Cys Tyr Glu Gln Leu

549 558 567 576 585 594

GTA GAC AGC TCA GAA GAT GAG GTG GAC GAA GTG GAC GGA CAA GAT TCA CAA CCT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Val Asp Ser Ser Glu Asp Glu Val Asp Glu Val Asp Gly Gln Asp Ser Gln Pro

603 612 621 630 639 648

TTA AAA CAA CAT TTC CAA ATA GTG ACC TGT TGC TGT GGA TGT GAC AGC AAC GTT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Leu Lys Gln His Phe Gln Ile Val Thr Lys Cys Cys Gly Cys Asp Ser Asn Val

657 666 675 684 693 702

CGA CTG GTT GTG CAG TGT ACA GAA ACA GAC ATC AGA GAA GTG CAA CAG CTT CTG

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Arg Leu Val Val Gln Cys Thr Glu Thr Asp Ile Arg Glu Val Gln Gln Leu Leu

711 720 729 738 747 756

TTG GGA ACA CTA AAC ATA GTG TGT CCC ATC TGC GCA CCG AAG ACC CCA TGG GAA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Leu Gly Thr Leu Asn Ile Val Cy5 Pro Ile Cys Ala Pro Lys Thr Pro Trp Glu

765 774 783 792 801 810

GTG GTG CCT GTA CAA ATA GCT GCA GGA ACA ACC AGC ACA TTC ATA CTG CCT GTT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Val Val Pro Val Gln Ile Ala Ala Gly Thr Thr Ser Thr Phe Ile Leu Pro Val

819 828 837 846 855 864

ATA ATT GCA TTT GTT GTA TGT TTT GTT AGC ATC ATA CTT ATT GTA TGG ATA TCT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Ile Ile Ala Phe Val Val Cys Phe Val Ser Ile Ile Leu Ile Val Trp Ile Ser

873 882 891 900 909 918

GAG TTT ATT GTG TAC ACA TCT GTG CTA GTA CTA ACA CTG CTT TTA TAT TTA CTA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Glu Phe Ile Val Tyr Thr Ser Val Leu Val Leu Thr Leu Leu Leu Tyr Leu Leu

927 936 945 954 963 972

TTG TGG CTG CTA TTA ACA ACC CCC TTG CAA TTT TTC CTA CTA ACT CTA CTT GTG

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Leu Trp Leu Leu Leu Thr Thr Pro Leu Gln Phe Phe Leu Leu Thr Leu Leu Val

981 990 999 1008 1017 1026

TGT TAC TGT CCC GCA TTG TAT ATA CAC TAC TAT ATT GTT ACC ACA CAG CAA TCT

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Cys Tyr Cys Pro Ala Leu Tyr Ile His Tyr Tyr Ile Val Thr Thr Gln Gln Ser

1035 1044 1053 1062 1071 1080

AGA GAG CTC GGT ACC ACT AAT GGA GCA CCA AAC ATT GGG AAG TAT GTT ATG GCA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Arg Glu Leu Gly Thr Thr Asn Gly Ala Pro Asn Ile Gly Lys Tyr Val Met Ala

1089 1098 1107 1116 1125 1134

GCA CAG TTA TAT GTT CTC CTG CAT CTG TAT CTA GCA CTA CAC AAG AAG TAT CCA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Ala Gln Leu Tyr Val Leu Leu His Leu Tyr Leu Ala Leu His Lys Lys Tyr Pro

1143 1152 1161 1170 1179 1188

TTC CTG AAT CTA CTA CAT ACA CCC CCG CAC AGA CCT CCA CCC TTG TGT CCT CAA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Phe Leu Asn Leu Leu His Thr Pro Pro His Arg Pro Pro Pro Leu Cys Pro Gln

1197 1206 1215 1224 1233 1242

GCA CCA AGG AAG ACG CAG TGC AAA CGC CGC CTA GGA AAC GAG CAC GAG GAG TCC

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Ala Pro Arg Lys Thr Gln Cys Lys Arg Arg Leu Gly Asn Glu His Glu Glu Ser

1251 1260 1269 1278 1287 1296

AAC AGT CCC CTT GCA ACG CCT TGT GTG TGG CCC ACA TTG GAC CCG TGG ACA GTG

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Asn Ser Pro Leu Ala Thr Pro Cys Val Trp Pro Thr Leu Asp Pro Trp Thr Val

1305 1314 1323 1332 1341 1350

GAA ACC ACA ACC TCA TCA CTA ACA ATC ACG ACC AGC ACC AAA GAC GGA ACA ACA

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Glu Thr Thr Thr Ser Ser Leu Thr Ile Thr Thr Ser Thr Lys Asp Gly Thr Thr

1359 1368 1377 1386 1395

GTA ACA GTT CAG CTA CGC CTA AGA TCT CAT CAC CAT CAC CAT CAC TAA 3′

--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

Val Thr Val Gln Leu Arg Leu Arg Ser His His His His His His ***

Junction of E1 and E4 ORFs for CSL791 and CSL762

SEQ ID Nos. 25 (DNA) and 26 (amino acid)

Modified

kpn1 Spe1

5′ GAG GAA GAT GGA AGC AAT AGC CAA GCG TTT AGA TGC GTG CCA GGA ACA GTT GTT AGA ACT TTA GGT ACC ACT AAT GGA GCA CCA AAC ATT GGG AAG TAT GTT ATG GCA 3′

Glu Glu Asp Gly Ser Asn Ser Gln Ala Phe Arg Cys Val Pro Gly Thr Val Val Arg Thr Leu Gly Thr Thr Asn Gly Ala Pro Asn Ile Gly Lys Tyr Val Met Ala

E1 E4

Junction of E5a and E1 for CSL762

SEQ ID Nos. 27 (DNA) and 28(amino acid)

Xba1 Sac1

5′ TGT CCC GCA TTG TAT ATA CAC TAC TAT ATT GTT ACC ACA CAG CAA TCT AGA GAG CTC GCG GAC GAT TCA GGT ACA GAA AAT GAG GGG TCT GGG TGT ACA GGA 3′

Cys Pro Ala Leu Tyr Ile His Tyr Tyr Ile Val Thr Thr Gln Gln Ser Arg Glu Leu Ala Asp Asp Ser Gly Thr Glu Asn Glu Gly Ser Gly Cys Thr Gly

E5a E1

Junction of E7 and E1 for CSL791

SEQ ID Nos. 29 (DNA) and 30 (amino acid)

Nco1 Xba1 Sac1

5′ TTG GGA ACA CTA AAC ATA GTG TGT CCC ATC TGC GCA CCG AAG ACC CCA TGG TCT AGA GAG CTC GCG GAC GAT TCA GGT ACA GAA AAT GAG GGG TCT GGG TGT ACA GGA 3′

Leu Gly Thr Leu Asn Ile Val Cys Pro Ile Cys Ala Pro Lys Thr Pro Trp Ser Arg Glu Leu Ala Asp Asp Ser Gly Thr Glu Asn Glu Gly Ser Gly Cys Thr Gly

E7 E1

Example 4

Preparation of Antibodies to HPV6b Early ORF Protein Products

The following peptides corresponding to portions of the sequence of the relevant E proteins, were synthesised and conjugated to diphtheria toxoid:

E6 dip.tox-CYRHFDYAQYATTVEEETKQDILD

E7 MHGRHVTLKDIVLDLQPPD-C-dip.tox

For the E6 peptide two rabbits (following prebleeding) were each inoculated with approximately 54 μg peptide/104 μg diphtheria toxoid in Freund's complete adjuvant followed at 3-weekly intervals by a similar dose of peptide conjugate in Freund's incomplete adjuvant. Bleeds were taken one week after the second dose and one week following the third dose. The same regime was used for the E7 peptide using 45 μg peptide/103 μg diphtheria toxoid.

Serum derived from the bleeds were tested for specific antibody in a solid phase EIA against biotin-conjugated peptide which had been bound to plates coated with strepavidin.

Example 5

Purification of Polyprotein E6/E7/E4

The trimer polyprotein E6/E7/E4 was expressed in

E. coli

BL21 cells by induction of cells at OD

600

˜1 using 0.4 mM IPTG. The cells were harvested by centrifugation (4,000 g, 20 minutes), resuspended in 30 mM Tris pH 8.0, disrupted by sonication (MSE, amplitude 18 μm, 4×30 seconds) and inclusion bodies pelleted by centrifugation (12,000 g, 30 minutes). The pellet containing the trimer was solubilized in 8 M Urea, 30 mM Tris pH 8.0 for 16 hours in the presence of protease inhibitors (Boehringer Cat. No. 1697498) and then centrifuged at 12,000 g for 30 minutes and the supernatant collected. To this, Tris2-carboxyethyl)phosphine TCEP) [Pierce] was added to 1,2 mM final concentration. The supernatant was applied to Q-sepharose HP (Pharmacia) and the column washed with one column volume of 8 M Urea, 1.2 mM TCEP, 30 mM Tris pH 8.0. Fractions were then eluted using a gradient containing 0 to 1 M NaCl in 10 column volumes of the washing buffer. The fractions obtained were examined by Western blot from 4 to 20% SDS-PAGE probed with the anti-E4 antibody MWE4.

FIG. 11

shows a Western blot of material obtained from Q-sepharose. An immunoreactive band of ˜41 kDa is evident in supernatant material from the urea solubilisation lane 3, and in fractions corresponding to 120 to 150 mM NaCl (lanes 8 and 9, arrow).

Supernatant from the urea solublisation was also applied to a column containing Chelating Sepharose Fast Flow (Pharmacia) to take advantage of the C-terminal six histidine sequence. Relatively poor binding of the trimer to the Nickel column was observed under the conditions described. The trimer was eluted from the column using a 0 to 500 mM imidazole gradient.

Example 6

In a further example of the present invention, a DNA sequence coding for a single polyprotein (

FIG. 12

) is formed by fusion of DNA fragments encoding HPV-6 early ORF proteins wherein the order of the ORFs is E2, E4, E5a, E5b, E6, E7 and E1.

The DNA sequences encoding the early ORF proteins are amplified individually by PCR using HPV-6 genomic DNA using the primers set out in Table 2.

TABLE 2

Gene

Oligonucleotides

E2

(a)

5′-GTG TGT GAG CTC ATG GAA GCA ATA GCC AAG-3′

(SEQ ID No. 31) and

(b)

5′-GTG TGT GTC GAC CAA TAG GTG CAG TGA CAT-3′

(SEQ ID No. 32)

E4

(c)

5′-GTG TGT GTC GAC ATG GGA GCA CCA AAC ATT-3′

(SEQ ID No. 33) and

5′-GTG TGT AGA TCT TAG GCG TAG CTG AAC TGT-3′

(SEQ ID No. 34)

E5a

(e)

5′-GTG TGT AGA TCT ATG GAA GTG GTG CCT GTA-3′

(SEQ ID No. 35) and

(f)

5′-GTG TGT CTT AAG TTG CTG TGT GGT AAC AAT-3′

(SEQ ID No. 36)

E5b

(g)

5′-GTG TGT CTT AAG ATG ATG CTA ACA TGT CAA-3′

(SEQ ID No. 37) and

(h)

5′-GTG TGT CCG CGG ATT CAT ATA TAT ATA ATC-3′

(SEQ ID No. 38)

E6

(i)

5′-GTG TGT CCG CGG ATG GAA AGT GCA AAT GCC-3′

(SEQ ID No. 39) and

(j)

5′-GTG TGT GCT AGC GGG TAA CAT GTC TTC CTA-3′

(SEQ ID No. 40)

E7

(k)

5′-GTG TGT GCT AGC ATG CAT GGA AGA CAT GTT-3′

(SEQ ID NO. 41) and

(l)

5′-GTG TGT CGA TCG GGT CTT CGG TGC GCA GAT-3′

(SEQ ID No. 42)

E1

(m

5′-GTG TGT CGA TCG ATG GCG GAC GAT TCA GGT-3′

)

(SEQ ID No. 43) and

(n)

5′-GTG TGT GGT ACC TCA TAA AGT TCT AAC AAC-3′

(SEQ ID No. 44)

The primers are synthesised to incorporate artificial restriction enzyme sites at the 5′ and 3′ termini of the amplification products. These restriction enzyme sites are used to facilitate the fusion of PCR products encoding the appropriate early ORF proteins in the desired order and in the correct translational frame. The restriction enzyme sites are also used to aid the cloning of the PCR products into the expression vector pTrcHisA. When cloned into this vector, the polyprotein construct is expressed as an N-terminal hexaHis fusion. The nucleotide sequence and deduced amino acid sequence of this fusion are shown below (SEQ ID Nos. 45 and 46, respectively).

30 base pairs

nucleic acid

single

linear

DNA (genomic)

1
GCGCCCCGGG ATGGAAAGTG CAAATGCCTC 30

42 base pairs

nucleic acid

single

linear

DNA (genomic)

2
GCGCTCTAGA CCATGGAAGC TTGGGTAACA TGTCTTCCAT GC 42

49 base pairs

nucleic acid

single

linear

DNA (genomic)

3
GCGCTCTAGA GAGCTCGGTA CCACTAGTGG AGCACCAAAC ATTGGGAAG 49

31 base pairs

nucleic acid

single

linear

DNA (genomic)

4
GCGCAGATCT TAGGCGTAGC TGAACTGTTA C 31

33 base pairs

nucleic acid

single

linear

DNA (genomic)

5
GCGCCCATGG GAAGTGGTGC CTGTACAAAT AGC 33

32 base pairs

nucleic acid

single

linear

DNA (genomic)

6
GCGCTCTAGA TTGCTGTGTG GTAACAATAT AG 32

34 base pairs

nucleic acid

single

linear

DNA (genomic)

7
GCGCAAGCTT CATGGAAGAC ATGTTACCCT AAAG 34

30 base pairs

nucleic acid

single

linear

DNA (genomic)

8
GCGCCCATGG GGTCTTCGGT GCGCAGATGG 30

35 base pairs

nucleic acid

single

linear

DNA (genomic)

9
GCGCGAGCTC GCGGACGATT CAGGTACAGA AAATG 35

33 base pairs

nucleic acid

single

linear

DNA (genomic)

10
GCGCGGTACC TAAAGTTCTA ACAACTGTTC CTG 33

32 base pairs

nucleic acid

single

linear

DNA (genomic)

11
GCGCGGTACC GAAGCAATAG CCAAGCGTTT AG 32

33 base pairs

nucleic acid

single

linear

DNA (genomic)

12
GCGCACTAGT CAATAGGTGC AGTGACATAA ATC 33

32 base pairs

nucleic acid

single

linear

DNA (genomic)

13
GCGCTCTAGA CTAACATGTC AATTTAATGA TG 32

31 base pairs

nucleic acid

single

linear

DNA (genomic)

14
GCGCGAGCTC ATTCATATAT ATATAATCAC C 31

30 base pairs

nucleic acid

single

linear

DNA (genomic)

15
GCGCCCCGGG ATGGAAGCAA TAGCCAAGCG 30

51 base pairs

nucleic acid

single

linear

DNA (genomic)

16
GCGCTCTAGA CCATGGGGTA CCGAGCTCCA ATAGGTGCAG TGACATAAAT C 51

32 base pairs

nucleic acid

single

linear

DNA (genomic)

17
GCGCTCTAGA CTAACATGTC AATTTAATGA TG 32

36 base pairs

nucleic acid

single

linear

DNA (genomic)

18
GCGCAGATCT CTCGAGATTC ATATATATAT AATCAC 36

1107 base pairs

nucleic acid

single

linear

DNA (genomic)

CDS

1..1104

19
ATG GAA AGT GCA AAT GCC TCC ACG TCT GCA ACG ACC ATA GAC CAG TTG 48
Met Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gln Leu
1 5 10 15
TGC AAG ACG TTT AAT CTA TCT ATG CAT ACG TTG CAA ATT AAT TGT GTG 96
Cys Lys Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn Cys Val
20 25 30
TTT TGC AAG AAT GCA CTG ACC ACA GCA GAG ATT TAT TCA TAT GCA TAT 144
Phe Cys Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr
35 40 45
AAA CAC CTA AAG GTC CTG TTT CGA GGC GGC TAT CCA TAT GCA GCC TGC 192
Lys His Leu Lys Val Leu Phe Arg Gly Gly Tyr Pro Tyr Ala Ala Cys
50 55 60
GCG TGC TGC CTA GAA TTT CAT GGA AAA ATA AAC CAA TAT AGA CAC TTT 240
Ala Cys Cys Leu Glu Phe His Gly Lys Ile Asn Gln Tyr Arg His Phe
65 70 75 80
GAT TAT GCT GGA TAT GCA ACA ACA GTT GAA GAA GAA ACT AAA CAA GAC 288
Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu Glu Glu Thr Lys Gln Asp
85 90 95
ATC TTA GAC GTG CTA ATT CGG TGC TAC CTG TGT CAC AAA CCG CTG TGT 336
Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu Cys His Lys Pro Leu Cys
100 105 110
GAA GTA GAA AAG GTA AAA CAT ATA CTA ACC AAG GCG CGG TTC ATA AAG 384
Glu Val Glu Lys Val Lys His Ile Leu Thr Lys Ala Arg Phe Ile Lys
115 120 125
CTA AAT TGT ACG TGG AAG GGT CGC TGC CTA CAC TGC TGG ACA ACA TGC 432
Leu Asn Cys Thr Trp Lys Gly Arg Cys Leu His Cys Trp Thr Thr Cys
130 135 140
ATG GAA GAC ATG TTA CCC AAG CTT CCA TGG GAA GTG GTG CCT GTA CAA 480
Met Glu Asp Met Leu Pro Lys Leu Pro Trp Glu Val Val Pro Val Gln
145 150 155 160
ATA GCT GCA GGA ACA ACC AGC ACA TTC ATA CTG CCT GTT ATA ATT GCA 528
Ile Ala Ala Gly Thr Thr Ser Thr Phe Ile Leu Pro Val Ile Ile Ala
165 170 175
TTT GTT GTA TGT TTT GTT AGC ATC ATA CTT ATT GTA TGG ATA TCT GAG 576
Phe Val Val Cys Phe Val Ser Ile Ile Leu Ile Val Trp Ile Ser Glu
180 185 190
TTT ATT GTG TAC ACA TCT GTG CTA GTA CTA ACA CTG CTT TTA TAT TTA 624
Phe Ile Val Tyr Thr Ser Val Leu Val Leu Thr Leu Leu Leu Tyr Leu
195 200 205
CTA TTG TGG CTG CTA TTA ACA ACC CCC TTG CAA TTT TTC CTA CTA ACT 672
Leu Leu Trp Leu Leu Leu Thr Thr Pro Leu Gln Phe Phe Leu Leu Thr
210 215 220
CTA CTT GTG TGT TAC TGT CCC GCA TTG TAT ATA CAC TAC TAT ATT GTT 720
Leu Leu Val Cys Tyr Cys Pro Ala Leu Tyr Ile His Tyr Tyr Ile Val
225 230 235 240
ACC ACA CAG CAA TCT AGA GAG CTC GGT ACC ACT AAT GGA GCA CCA AAC 768
Thr Thr Gln Gln Ser Arg Glu Leu Gly Thr Thr Asn Gly Ala Pro Asn
245 250 255
ATT GGG AAG TAT GTT ATG GCA GCA CAG TTA TAT GTT CTC CTG CAT CTG 816
Ile Gly Lys Tyr Val Met Ala Ala Gln Leu Tyr Val Leu Leu His Leu
260 265 270
TAT CTA GCA CTA CAC AAG AAG TAT CCA TTC CTG AAT CTA CTA CAT ACA 864
Tyr Leu Ala Leu His Lys Lys Tyr Pro Phe Leu Asn Leu Leu His Thr
275 280 285
CCC CCG CAC AGA CCT CCA CCC TTG TGT CCT CAA GCA CCA AGG AAG ACG 912
Pro Pro His Arg Pro Pro Pro Leu Cys Pro Gln Ala Pro Arg Lys Thr
290 295 300
CAG TGC AAA CGC CGC CTA GGA AAC GAG CAC GAG GAG TCC AAC AGT CCC 960
Gln Cys Lys Arg Arg Leu Gly Asn Glu His Glu Glu Ser Asn Ser Pro
305 310 315 320
CTT GCA ACG CCT TGT GTG TGG CCC ACA TTG GAC CCG TGG ACA GTG GAA 1008
Leu Ala Thr Pro Cys Val Trp Pro Thr Leu Asp Pro Trp Thr Val Glu
325 330 335
ACC ACA ACC TCA TCA CTA ACA ATC ACG ACC AGC ACC AAA GAC GGA ACA 1056
Thr Thr Thr Ser Ser Leu Thr Ile Thr Thr Ser Thr Lys Asp Gly Thr
340 345 350
ACA GTA ACA GTT CAG CTA CGC CTA AGA TCT CAT CAC CAT CAC CAT CAC 1104
Thr Val Thr Val Gln Leu Arg Leu Arg Ser His His His His His His
355 360 365
TAA 1107

368 amino acids

amino acid

linear

protein

20
Met Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gln Leu
1 5 10 15
Cys Lys Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn Cys Val
20 25 30
Phe Cys Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr
35 40 45
Lys His Leu Lys Val Leu Phe Arg Gly Gly Tyr Pro Tyr Ala Ala Cys
50 55 60
Ala Cys Cys Leu Glu Phe His Gly Lys Ile Asn Gln Tyr Arg His Phe
65 70 75 80
Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu Glu Glu Thr Lys Gln Asp
85 90 95
Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu Cys His Lys Pro Leu Cys
100 105 110
Glu Val Glu Lys Val Lys His Ile Leu Thr Lys Ala Arg Phe Ile Lys
115 120 125
Leu Asn Cys Thr Trp Lys Gly Arg Cys Leu His Cys Trp Thr Thr Cys
130 135 140
Met Glu Asp Met Leu Pro Lys Leu Pro Trp Glu Val Val Pro Val Gln
145 150 155 160
Ile Ala Ala Gly Thr Thr Ser Thr Phe Ile Leu Pro Val Ile Ile Ala
165 170 175
Phe Val Val Cys Phe Val Ser Ile Ile Leu Ile Val Trp Ile Ser Glu
180 185 190
Phe Ile Val Tyr Thr Ser Val Leu Val Leu Thr Leu Leu Leu Tyr Leu
195 200 205
Leu Leu Trp Leu Leu Leu Thr Thr Pro Leu Gln Phe Phe Leu Leu Thr
210 215 220
Leu Leu Val Cys Tyr Cys Pro Ala Leu Tyr Ile His Tyr Tyr Ile Val
225 230 235 240
Thr Thr Gln Gln Ser Arg Glu Leu Gly Thr Thr Asn Gly Ala Pro Asn
245 250 255
Ile Gly Lys Tyr Val Met Ala Ala Gln Leu Tyr Val Leu Leu His Leu
260 265 270
Tyr Leu Ala Leu His Lys Lys Tyr Pro Phe Leu Asn Leu Leu His Thr
275 280 285
Pro Pro His Arg Pro Pro Pro Leu Cys Pro Gln Ala Pro Arg Lys Thr
290 295 300
Gln Cys Lys Arg Arg Leu Gly Asn Glu His Glu Glu Ser Asn Ser Pro
305 310 315 320
Leu Ala Thr Pro Cys Val Trp Pro Thr Leu Asp Pro Trp Thr Val Glu
325 330 335
Thr Thr Thr Ser Ser Leu Thr Ile Thr Thr Ser Thr Lys Asp Gly Thr
340 345 350
Thr Val Thr Val Gln Leu Arg Leu Arg Ser His His His His His His
355 360 365

1128 base pairs

nucleic acid

single

linear

DNA (genomic)

CDS

1..1125

21
ATG GAA AGT GCA AAT GCC TCC ACG TCT GCA ACG ACC ATA GAC CAG TTG 48
Met Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gln Leu
1 5 10 15
TGC AAG ACG TTT AAT CTA TCT ATG CAT ACG TTG CAA ATT AAT TGT GTG 96
Cys Lys Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn Cys Val
20 25 30
TTT TGC AAG AAT GCA CTG ACC ACA GCA GAG ATT TAT TCA TAT GCA TAT 144
Phe Cys Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr
35 40 45
AAA CAC CTA AAG GTC CTG TTT CGA GGC GGC TAT CCA TAT GCA GCC TGC 192
Lys His Leu Lys Val Leu Phe Arg Gly Gly Tyr Pro Tyr Ala Ala Cys
50 55 60
GCG TGC TGC CTA GAA TTT CAT GGA AAA ATA AAC CAA TAT AGA CAC TTT 240
Ala Cys Cys Leu Glu Phe His Gly Lys Ile Asn Gln Tyr Arg His Phe
65 70 75 80
GAT TAT GCT GGA TAT GCA ACA ACA GTT GAA GAA GAA ACT AAA CAA GAC 288
Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu Glu Glu Thr Lys Gln Asp
85 90 95
ATC TTA GAC GTG CTA ATT CGG TGC TAC CTG TGT CAC AAA CCG CTG TGT 336
Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu Cys His Lys Pro Leu Cys
100 105 110
GAA GTA GAA AAG GTA AAA CAT ATA CTA ACC AAG GCG CGG TTC ATA AAG 384
Glu Val Glu Lys Val Lys His Ile Leu Thr Lys Ala Arg Phe Ile Lys
115 120 125
CTA AAT TGT ACG TGG AAG GGT CGC TGC CTA CAC TGC TGG ACA ACA TGC 432
Leu Asn Cys Thr Trp Lys Gly Arg Cys Leu His Cys Trp Thr Thr Cys
130 135 140
ATG GAA GAC ATG TTA CCC AAG CTT CAT GGA AGA CAT GTT ACC CTA AAG 480
Met Glu Asp Met Leu Pro Lys Leu His Gly Arg His Val Thr Leu Lys
145 150 155 160
GAT ATT GTA TTA GAC CTG CAA CCT CCA GAC CCT GTA GGG TTA CAT TGC 528
Asp Ile Val Leu Asp Leu Gln Pro Pro Asp Pro Val Gly Leu His Cys
165 170 175
TAT GAG CAA TTA GTA GAC AGC TCA GAA GAT GAG GTG GAC GAA GTG GAC 576
Tyr Glu Gln Leu Val Asp Ser Ser Glu Asp Glu Val Asp Glu Val Asp
180 185 190
GGA CAA GAT TCA CAA CCT TTA AAA CAA CAT TTC CAA ATA GTG ACC TGT 624
Gly Gln Asp Ser Gln Pro Leu Lys Gln His Phe Gln Ile Val Thr Cys
195 200 205
TGC TGT GGA TGT GAC AGC AAC GTT CGA CTG GTT GTG CAG TGT ACA GAA 672
Cys Cys Gly Cys Asp Ser Asn Val Arg Leu Val Val Gln Cys Thr Glu
210 215 220
ACA GAC ATC AGA GAA GTG CAA CAG CTT CTG TTG GGA ACA CTA AAC ATA 720
Thr Asp Ile Arg Glu Val Gln Gln Leu Leu Leu Gly Thr Leu Asn Ile
225 230 235 240
GTG TGT CCC ATC TGC GCA CCG AAG ACC CCA TGG TCT AGA GAG CTC GGT 768
Val Cys Pro Ile Cys Ala Pro Lys Thr Pro Trp Ser Arg Glu Leu Gly
245 250 255
ACC ACT AAT GGA GCA CCA AAC ATT GGG AAG TAT GTT ATG GCA GCA CAG 816
Thr Thr Asn Gly Ala Pro Asn Ile Gly Lys Tyr Val Met Ala Ala Gln
260 265 270
TTA TAT GTT CTC CTG CAT CTG TAT CTA GCA CTA CAC AAG AAG TAT CCA 864
Leu Tyr Val Leu Leu His Leu Tyr Leu Ala Leu His Lys Lys Tyr Pro
275 280 285
TTC CTG AAT CTA CTA CAT ACA CCC CCG CAC AGA CCT CCA CCC TTG TGT 912
Phe Leu Asn Leu Leu His Thr Pro Pro His Arg Pro Pro Pro Leu Cys
290 295 300
CCT CAA GCA CCA AGG AAG ACG CAG TGC AAA CGC CGC CTA GGA AAC GAG 960
Pro Gln Ala Pro Arg Lys Thr Gln Cys Lys Arg Arg Leu Gly Asn Glu
305 310 315 320
CAC GAG GAG TCC AAC AGT CCC CTT GCA ACG CCT TGT GTG TGG CCC ACA 1008
His Glu Glu Ser Asn Ser Pro Leu Ala Thr Pro Cys Val Trp Pro Thr
325 330 335
TTG GAC CCG TGG ACA GTG GAA ACC ACA ACC TCA TCA CTA ACA ATC ACG 1056
Leu Asp Pro Trp Thr Val Glu Thr Thr Thr Ser Ser Leu Thr Ile Thr
340 345 350
ACC AGC ACC AAA GAC GGA ACA ACA GTA ACA GTT CAG CTA CGC CTA AGA 1104
Thr Ser Thr Lys Asp Gly Thr Thr Val Thr Val Gln Leu Arg Leu Arg
355 360 365
TCT CAT CAC CAT CAC CAT CAC TAA 1128
Ser His His His His His His
370 375

375 amino acids

amino acid

linear

protein

22
Met Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gln Leu
1 5 10 15
Cys Lys Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn Cys Val
20 25 30
Phe Cys Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr
35 40 45
Lys His Leu Lys Val Leu Phe Arg Gly Gly Tyr Pro Tyr Ala Ala Cys
50 55 60
Ala Cys Cys Leu Glu Phe His Gly Lys Ile Asn Gln Tyr Arg His Phe
65 70 75 80
Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu Glu Glu Thr Lys Gln Asp
85 90 95
Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu Cys His Lys Pro Leu Cys
100 105 110
Glu Val Glu Lys Val Lys His Ile Leu Thr Lys Ala Arg Phe Ile Lys
115 120 125
Leu Asn Cys Thr Trp Lys Gly Arg Cys Leu His Cys Trp Thr Thr Cys
130 135 140
Met Glu Asp Met Leu Pro Lys Leu His Gly Arg His Val Thr Leu Lys
145 150 155 160
Asp Ile Val Leu Asp Leu Gln Pro Pro Asp Pro Val Gly Leu His Cys
165 170 175
Tyr Glu Gln Leu Val Asp Ser Ser Glu Asp Glu Val Asp Glu Val Asp
180 185 190
Gly Gln Asp Ser Gln Pro Leu Lys Gln His Phe Gln Ile Val Thr Cys
195 200 205
Cys Cys Gly Cys Asp Ser Asn Val Arg Leu Val Val Gln Cys Thr Glu
210 215 220
Thr Asp Ile Arg Glu Val Gln Gln Leu Leu Leu Gly Thr Leu Asn Ile
225 230 235 240
Val Cys Pro Ile Cys Ala Pro Lys Thr Pro Trp Ser Arg Glu Leu Gly
245 250 255
Thr Thr Asn Gly Ala Pro Asn Ile Gly Lys Tyr Val Met Ala Ala Gln
260 265 270
Leu Tyr Val Leu Leu His Leu Tyr Leu Ala Leu His Lys Lys Tyr Pro
275 280 285
Phe Leu Asn Leu Leu His Thr Pro Pro His Arg Pro Pro Pro Leu Cys
290 295 300
Pro Gln Ala Pro Arg Lys Thr Gln Cys Lys Arg Arg Leu Gly Asn Glu
305 310 315 320
His Glu Glu Ser Asn Ser Pro Leu Ala Thr Pro Cys Val Trp Pro Thr
325 330 335
Leu Asp Pro Trp Thr Val Glu Thr Thr Thr Ser Ser Leu Thr Ile Thr
340 345 350
Thr Ser Thr Lys Asp Gly Thr Thr Val Thr Val Gln Leu Arg Leu Arg
355 360 365
Ser His His His His His His
370 375

1398 base pairs

nucleic acid

single

linear

DNA (genomic)

CDS

1..1395

23
ATG GAA AGT GCA AAT GCC TCC ACG TCT GCA ACG ACC ATA GAC CAG TTG 48
Met Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gln Leu
1 5 10 15
TGC AAG ACG TTT AAT CTA TCT ATG CAT ACG TTG CAA ATT AAT TGT GTG 96
Cys Lys Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn Cys Val
20 25 30
TTT TGC AAG AAT GCA CTG ACC ACA GCA GAG ATT TAT TCA TAT GCA TAT 144
Phe Cys Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr
35 40 45
AAA CAC CTA AAG GTC CTG TTT CGA GGC GGC TAT CCA TAT GCA GCC TGC 192
Lys His Leu Lys Val Leu Phe Arg Gly Gly Tyr Pro Tyr Ala Ala Cys
50 55 60
GCG TGC TGC CTA GAA TTT CAT GGA AAA ATA AAC CAA TAT AGA CAC TTT 240
Ala Cys Cys Leu Glu Phe His Gly Lys Ile Asn Gln Tyr Arg His Phe
65 70 75 80
GAT TAT GCT GGA TAT GCA ACA ACA GTT GAA GAA GAA ACT AAA CAA GAC 288
Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu Glu Glu Thr Lys Gln Asp
85 90 95
ATC TTA GAC GTG CTA ATT CGG TGC TAC CTG TGT CAC AAA CCG CTG TGT 336
Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu Cys His Lys Pro Leu Cys
100 105 110
GAA GTA GAA AAG GTA AAA CAT ATA CTA ACC AAG GCG CGG TTC ATA AAG 384
Glu Val Glu Lys Val Lys His Ile Leu Thr Lys Ala Arg Phe Ile Lys
115 120 125
CTA AAT TGT ACG TGG AAG GGT CGC TGC CTA CAC TGC TGG ACA ACA TGC 432
Leu Asn Cys Thr Trp Lys Gly Arg Cys Leu His Cys Trp Thr Thr Cys
130 135 140
ATG GAA GAC ATG TTA CCC AAG CTT CAT GGA AGA CAT GTT ACC CTA AAG 480
Met Glu Asp Met Leu Pro Lys Leu His Gly Arg His Val Thr Leu Lys
145 150 155 160
GAT ATT GTA TTA GAC CTG CAA CCT CCA GAC CCT GTA GGG TTA CAT TGC 528
Asp Ile Val Leu Asp Leu Gln Pro Pro Asp Pro Val Gly Leu His Cys
165 170 175
TAT GAG CAA TTA GTA GAC AGC TCA GAA GAT GAG GTG GAC GAA GTG GAC 576
Tyr Glu Gln Leu Val Asp Ser Ser Glu Asp Glu Val Asp Glu Val Asp
180 185 190
GGA CAA GAT TCA CAA CCT TTA AAA CAA CAT TTC CAA ATA GTG ACC TGT 624
Gly Gln Asp Ser Gln Pro Leu Lys Gln His Phe Gln Ile Val Thr Cys
195 200 205
TGC TGT GGA TGT GAC AGC AAC GTT CGA CTG GTT GTG CAG TGT ACA GAA 672
Cys Cys Gly Cys Asp Ser Asn Val Arg Leu Val Val Gln Cys Thr Glu
210 215 220
ACA GAC ATC AGA GAA GTG CAA CAG CTT CTG TTG GGA ACA CTA AAC ATA 720
Thr Asp Ile Arg Glu Val Gln Gln Leu Leu Leu Gly Thr Leu Asn Ile
225 230 235 240
GTG TGT CCC ATC TGC GCA CCG AAG ACC CCA TGG GAA GTG GTG CCT GTA 768
Val Cys Pro Ile Cys Ala Pro Lys Thr Pro Trp Glu Val Val Pro Val
245 250 255
CAA ATA GCT GCA GGA ACA ACC AGC ACA TTC ATA CTG CCT GTT ATA ATT 816
Gln Ile Ala Ala Gly Thr Thr Ser Thr Phe Ile Leu Pro Val Ile Ile
260 265 270
GCA TTT GTT GTA TGT TTT GTT AGC ATC ATA CTT ATT GTA TGG ATA TCT 864
Ala Phe Val Val Cys Phe Val Ser Ile Ile Leu Ile Val Trp Ile Ser
275 280 285
GAG TTT ATT GTG TAC ACA TCT GTG CTA GTA CTA ACA CTG CTT TTA TAT 912
Glu Phe Ile Val Tyr Thr Ser Val Leu Val Leu Thr Leu Leu Leu Tyr
290 295 300
TTA CTA TTG TGG CTG CTA TTA ACA ACC CCC TTG CAA TTT TTC CTA CTA 960
Leu Leu Leu Trp Leu Leu Leu Thr Thr Pro Leu Gln Phe Phe Leu Leu
305 310 315 320
ACT CTA CTT GTG TGT TAC TGT CCC GCA TTG TAT ATA CAC TAC TAT ATT 1008
Thr Leu Leu Val Cys Tyr Cys Pro Ala Leu Tyr Ile His Tyr Tyr Ile
325 330 335
GTT ACC ACA CAG CAA TCT AGA GAG CTC GGT ACC ACT AAT GGA GCA CCA 1056
Val Thr Thr Gln Gln Ser Arg Glu Leu Gly Thr Thr Asn Gly Ala Pro
340 345 350
AAC ATT GGG AAG TAT GTT ATG GCA GCA CAG TTA TAT GTT CTC CTG CAT 1104
Asn Ile Gly Lys Tyr Val Met Ala Ala Gln Leu Tyr Val Leu Leu His
355 360 365
CTG TAT CTA GCA CTA CAC AAG AAG TAT CCA TTC CTG AAT CTA CTA CAT 1152
Leu Tyr Leu Ala Leu His Lys Lys Tyr Pro Phe Leu Asn Leu Leu His
370 375 380
ACA CCC CCG CAC AGA CCT CCA CCC TTG TGT CCT CAA GCA CCA AGG AAG 1200
Thr Pro Pro His Arg Pro Pro Pro Leu Cys Pro Gln Ala Pro Arg Lys
385 390 395 400
ACG CAG TGC AAA CGC CGC CTA GGA AAC GAG CAC GAG GAG TCC AAC AGT 1248
Thr Gln Cys Lys Arg Arg Leu Gly Asn Glu His Glu Glu Ser Asn Ser
405 410 415
CCC CTT GCA ACG CCT TGT GTG TGG CCC ACA TTG GAC CCG TGG ACA GTG 1296
Pro Leu Ala Thr Pro Cys Val Trp Pro Thr Leu Asp Pro Trp Thr Val
420 425 430
GAA ACC ACA ACC TCA TCA CTA ACA ATC ACG ACC AGC ACC AAA GAC GGA 1344
Glu Thr Thr Thr Ser Ser Leu Thr Ile Thr Thr Ser Thr Lys Asp Gly
435 440 445
ACA ACA GTA ACA GTT CAG CTA CGC CTA AGA TCT CAT CAC CAT CAC CAT 1392
Thr Thr Val Thr Val Gln Leu Arg Leu Arg Ser His His His His His
450 455 460
CAC TAA 1398
His
465

465 amino acids

amino acid

linear

protein

24
Met Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gln Leu
1 5 10 15
Cys Lys Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn Cys Val
20 25 30
Phe Cys Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr
35 40 45
Lys His Leu Lys Val Leu Phe Arg Gly Gly Tyr Pro Tyr Ala Ala Cys
50 55 60
Ala Cys Cys Leu Glu Phe His Gly Lys Ile Asn Gln Tyr Arg His Phe
65 70 75 80
Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu Glu Glu Thr Lys Gln Asp
85 90 95
Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu Cys His Lys Pro Leu Cys
100 105 110
Glu Val Glu Lys Val Lys His Ile Leu Thr Lys Ala Arg Phe Ile Lys
115 120 125
Leu Asn Cys Thr Trp Lys Gly Arg Cys Leu His Cys Trp Thr Thr Cys
130 135 140
Met Glu Asp Met Leu Pro Lys Leu His Gly Arg His Val Thr Leu Lys
145 150 155 160
Asp Ile Val Leu Asp Leu Gln Pro Pro Asp Pro Val Gly Leu His Cys
165 170 175
Tyr Glu Gln Leu Val Asp Ser Ser Glu Asp Glu Val Asp Glu Val Asp
180 185 190
Gly Gln Asp Ser Gln Pro Leu Lys Gln His Phe Gln Ile Val Thr Cys
195 200 205
Cys Cys Gly Cys Asp Ser Asn Val Arg Leu Val Val Gln Cys Thr Glu
210 215 220
Thr Asp Ile Arg Glu Val Gln Gln Leu Leu Leu Gly Thr Leu Asn Ile
225 230 235 240
Val Cys Pro Ile Cys Ala Pro Lys Thr Pro Trp Glu Val Val Pro Val
245 250 255
Gln Ile Ala Ala Gly Thr Thr Ser Thr Phe Ile Leu Pro Val Ile Ile
260 265 270
Ala Phe Val Val Cys Phe Val Ser Ile Ile Leu Ile Val Trp Ile Ser
275 280 285
Glu Phe Ile Val Tyr Thr Ser Val Leu Val Leu Thr Leu Leu Leu Tyr
290 295 300
Leu Leu Leu Trp Leu Leu Leu Thr Thr Pro Leu Gln Phe Phe Leu Leu
305 310 315 320
Thr Leu Leu Val Cys Tyr Cys Pro Ala Leu Tyr Ile His Tyr Tyr Ile
325 330 335
Val Thr Thr Gln Gln Ser Arg Glu Leu Gly Thr Thr Asn Gly Ala Pro
340 345 350
Asn Ile Gly Lys Tyr Val Met Ala Ala Gln Leu Tyr Val Leu Leu His
355 360 365
Leu Tyr Leu Ala Leu His Lys Lys Tyr Pro Phe Leu Asn Leu Leu His
370 375 380
Thr Pro Pro His Arg Pro Pro Pro Leu Cys Pro Gln Ala Pro Arg Lys
385 390 395 400
Thr Gln Cys Lys Arg Arg Leu Gly Asn Glu His Glu Glu Ser Asn Ser
405 410 415
Pro Leu Ala Thr Pro Cys Val Trp Pro Thr Leu Asp Pro Trp Thr Val
420 425 430
Glu Thr Thr Thr Ser Ser Leu Thr Ile Thr Thr Ser Thr Lys Asp Gly
435 440 445
Thr Thr Val Thr Val Gln Leu Arg Leu Arg Ser His His His His His
450 455 460
His
465

108 base pairs

nucleic acid

single

linear

DNA (genomic)

CDS

1..108

25
GAG GAA GAT GGA AGC AAT AGC CAA GCG TTT AGA TGC GTG CCA GGA ACA 48
Glu Glu Asp Gly Ser Asn Ser Gln Ala Phe Arg Cys Val Pro Gly Thr
1 5 10 15
GTT GTT AGA ACT TTA GGT ACC ACT AAT GGA GCA CCA AAC ATT GGG AAG 96
Val Val Arg Thr Leu Gly Thr Thr Asn Gly Ala Pro Asn Ile Gly Lys
20 25 30
TAT GTT ATG GCA 108
Tyr Val Met Ala
35

36 amino acids

amino acid

linear

protein

26
Glu Glu Asp Gly Ser Asn Ser Gln Ala Phe Arg Cys Val Pro Gly Thr
1 5 10 15
Val Val Arg Thr Leu Gly Thr Thr Asn Gly Ala Pro Asn Ile Gly Lys
20 25 30
Tyr Val Met Ala
35

102 base pairs

nucleic acid

single

linear

DNA (genomic)

CDS

1..102

27
TGT CCC GCA TTG TAT ATA CAC TAC TAT ATT GTT ACC ACA CAG CAA TCT 48
Cys Pro Ala Leu Tyr Ile His Tyr Tyr Ile Val Thr Thr Gln Gln Ser
1 5 10 15
AGA GAG CTC GCG GAC GAT TCA GGT ACA GAA AAT GAG GGG TCT GGG TGT 96
Arg Glu Leu Ala Asp Asp Ser Gly Thr Glu Asn Glu Gly Ser Gly Cys
20 25 30
ACA GGA 102
Thr Gly

34 amino acids

amino acid

linear

protein

28
Cys Pro Ala Leu Tyr Ile His Tyr Tyr Ile Val Thr Thr Gln Gln Ser
1 5 10 15
Arg Glu Leu Ala Asp Asp Ser Gly Thr Glu Asn Glu Gly Ser Gly Cys
20 25 30
Thr Gly

108 base pairs

nucleic acid

single

linear

DNA (genomic)

CDS

1..108

29
TTG GGA ACA CTA AAC ATA GTG TGT CCC ATC TGC GCA CCG AAG ACC CCA 48
Leu Gly Thr Leu Asn Ile Val Cys Pro Ile Cys Ala Pro Lys Thr Pro
1 5 10 15
TGG TCT AGA GAG CTC GCG GAC GAT TCA GGT ACA GAA AAT GAG GGG TCT 96
Trp Ser Arg Glu Leu Ala Asp Asp Ser Gly Thr Glu Asn Glu Gly Ser
20 25 30
GGG TGT ACA GGA 108
Gly Cys Thr Gly
35

36 amino acids

amino acid

linear

protein

30
Leu Gly Thr Leu Asn Ile Val Cys Pro Ile Cys Ala Pro Lys Thr Pro
1 5 10 15
Trp Ser Arg Glu Leu Ala Asp Asp Ser Gly Thr Glu Asn Glu Gly Ser
20 25 30
Gly Cys Thr Gly
35

30 base pairs

nucleic acid

single

linear

DNA (genomic)

31
GTGTGTGAGC TCATGGAAGC AATAGCCAAG 30

30 base pairs

nucleic acid

single

linear

DNA (genomic)

32
GTGTGTGTCG ACCAATAGGT GCAGTGACAT 30

30 base pairs

nucleic acid

single

linear

DNA (genomic)

33
GTGTGTGTCG ACATGGGAGC ACCAAACATT 30

30 base pairs

nucleic acid

single

linear

DNA (genomic)

34
GTGTGTAGAT CTTAGGCGTA GCTGAACTGT 30

30 base pairs

nucleic acid

single

linear

DNA (genomic)

35
GTGTGTAGAT CTATGGAAGT GGTGCCTGTA 30

30 base pairs

nucleic acid

single

linear

DNA (genomic)

36
GTGTGTCTTA AGTTGCTGTG TGGTAACAAT 30

30 base pairs

nucleic acid

single

linear

DNA (genomic)

37
GTGTGTCTTA AGATGATGCT AACATGTCAA 30

30 base pairs

nucleic acid

single

linear

DNA (genomic)

38
GTGTGTCCGC GGATTCATAT ATATATAATC 30

30 base pairs

nucleic acid

single

linear

DNA (genomic)

39
GTGTGTCCGC GGATGGAAAG TGCAAATGCC 30

30 base pairs

nucleic acid

single

linear

DNA (genomic)

40
GTGTGTGCTA GCGGGTAACA TGTCTTCCTA 30

30 base pairs

nucleic acid

single

linear

DNA (genomic)

41
GTGTGTGCTA GCATGCATGG AAGACATGTT 30

30 base pairs

nucleic acid

single

linear

DNA (genomic)

42
GTGTGTCGAT CGGGTCTTCG GTGCGCAGAT 30

30 base pairs

nucleic acid

single

linear

DNA (genomic)

43
GTGTGTCGAT CGATGGCGGA CGATTCAGGT 30

30 base pairs

nucleic acid

single

linear

DNA (genomic)

44
GTGTGTGGTA CCTCATAAAG TTCTAACAAC 30

4770 base pairs

nucleic acid

single

linear

DNA (genomic)

CDS

1..4761

45
ATG GGG GGT TCT CAT CAT CAT CAT CAT CAT GGT ATG GCT AGC ATG ACT 48
Met Gly Gly Ser His His His His His His Gly Met Ala Ser Met Thr
1 5 10 15
GGT GGA CAG CAA ATG GGT CGG GAT CTG TAC GAC GAT GAC GAT AAG GAT 96
Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp
20 25 30
CGA TGG GGA TCC GAG CTC ATG GAA GCA ATA GCC AAG CGT TTA GAT GCG 144
Arg Trp Gly Ser Glu Leu Met Glu Ala Ile Ala Lys Arg Leu Asp Ala
35 40 45
TGC CAG GAA CAG TTG TTA GAA CTT TAT GAA GAA AAC AGT ACT GAC CTA 192
Cys Gln Glu Gln Leu Leu Glu Leu Tyr Glu Glu Asn Ser Thr Asp Leu
50 55 60
CAC AAA CAT GTA TTG CAT TGG AAA TGC ATG AGA CAT GAA AGT GTA TTA 240
His Lys His Val Leu His Trp Lys Cys Met Arg His Glu Ser Val Leu
65 70 75 80
TTA TAT AAA GCA AAA CAA ATG GGC CTA AGC CAC ATA GGA ATG CAA GTA 288
Leu Tyr Lys Ala Lys Gln Met Gly Leu Ser His Ile Gly Met Gln Val
85 90 95
GTG CCA CCA TTA AAG GTG TCC GAA GCA AAA GGA CAT AAT GCC ATT GAA 336
Val Pro Pro Leu Lys Val Ser Glu Ala Lys Gly His Asn Ala Ile Glu
100 105 110
ATG CAA ATG CAT TTA GAA TCA TTA TTA AGG ACT GAG TAT AGT ATG GAA 384
Met Gln Met His Leu Glu Ser Leu Leu Arg Thr Glu Tyr Ser Met Glu
115 120 125
CCG TGG ACA TTA CAA GAA ACA AGT TAT GAA ATG TGG CAA ACA CCA CCT 432
Pro Trp Thr Leu Gln Glu Thr Ser Tyr Glu Met Trp Gln Thr Pro Pro
130 135 140
AAA CGC TGT TTT AAA AAA CGG GGC AAA ACT GTA GAA GTT AAA TTT GAT 480
Lys Arg Cys Phe Lys Lys Arg Gly Lys Thr Val Glu Val Lys Phe Asp
145 150 155 160
GGC TGT GCA AAC AAT ACA ATG GAT TAT GTG GTA TGG ACA GAT GTG TAT 528
Gly Cys Ala Asn Asn Thr Met Asp Tyr Val Val Trp Thr Asp Val Tyr
165 170 175
GTG CAG GAC AAT GAC ACC TGG GTA AAG GTG CAT AGT ATG GTA GAT GCT 576
Val Gln Asp Asn Asp Thr Trp Val Lys Val His Ser Met Val Asp Ala
180 185 190
AAG GGT ATA TAT TAC ACA TGT GGA CAA TTT AAA ACA TAT TAT GTA AAC 624
Lys Gly Ile Tyr Tyr Thr Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn
195 200 205
TTT GTA AAA GAG GCA GAA AAG TAT GGG AGC ACC AAA CAT TGG GAA GTA 672
Phe Val Lys Glu Ala Glu Lys Tyr Gly Ser Thr Lys His Trp Glu Val
210 215 220
TGT TAT GGC AGC ACA GTT ATA TGT TCT CCT GCA TCT GTA TCT AGC ACT 720
Cys Tyr Gly Ser Thr Val Ile Cys Ser Pro Ala Ser Val Ser Ser Thr
225 230 235 240
ACA CAA GAA GTA TCC ATT CCT GAA TCT ACT ACA TAC ACC CCC GCA CAG 768
Thr Gln Glu Val Ser Ile Pro Glu Ser Thr Thr Tyr Thr Pro Ala Gln
245 250 255
ACC TCC ACC CTT GTG TCC TCA AGC ACC AAG GAA GAC GCA GTG CAA ACG 816
Thr Ser Thr Leu Val Ser Ser Ser Thr Lys Glu Asp Ala Val Gln Thr
260 265 270
CCG CCT AGG AAA CGA GCA CGA GGA GTC CAA CAG TCC CCT TGC AAC GCC 864
Pro Pro Arg Lys Arg Ala Arg Gly Val Gln Gln Ser Pro Cys Asn Ala
275 280 285
TTG TGT GTG GCC CAC ATT GGA CCC GTG GAC AGT GGA AAC CAC AAC CTC 912
Leu Cys Val Ala His Ile Gly Pro Val Asp Ser Gly Asn His Asn Leu
290 295 300
ATC ACT AAC AAT CAC GAC CAG CAC CAA AGA CGG AAC AAC AGT AAC AGT 960
Ile Thr Asn Asn His Asp Gln His Gln Arg Arg Asn Asn Ser Asn Ser
305 310 315 320
TCA GCT ACG CCT ATA GTG CAA TTT CAA GGT GAA TCC AAT TGT TTA AAG 1008
Ser Ala Thr Pro Ile Val Gln Phe Gln Gly Glu Ser Asn Cys Leu Lys
325 330 335
TGT TTT AGA TAT AGG CTA AAT GAC AGA CAC AGA CAT TTA TTT GAT TTA 1056
Cys Phe Arg Tyr Arg Leu Asn Asp Arg His Arg His Leu Phe Asp Leu
340 345 350
ATA TCA TCA ACG TGG CAC TGG GCC TCC TCA AAG GCA CCA CAT AAA CAT 1104
Ile Ser Ser Thr Trp His Trp Ala Ser Ser Lys Ala Pro His Lys His
355 360 365
GCC ATT GTA ACT GTA ACA TAT GAT AGT GAG GAA CAA AGG CAA CAG TTT 1152
Ala Ile Val Thr Val Thr Tyr Asp Ser Glu Glu Gln Arg Gln Gln Phe
370 375 380
TTA GAT GTT GTA AAA ATA CCC CCT ACC ATT AGC CAC AAA CTG GGA TTT 1200
Leu Asp Val Val Lys Ile Pro Pro Thr Ile Ser His Lys Leu Gly Phe
385 390 395 400
ATG TCA CTG CAC CTA TTG GTC GAC ATG GGA GCA CCA AAC ATT GGG AAG 1248
Met Ser Leu His Leu Leu Val Asp Met Gly Ala Pro Asn Ile Gly Lys
405 410 415
TAT GTT ATG GCA GCA CAG TTA TAT GTT CTC CTG CAT CTG TAT CTA GCA 1296
Tyr Val Met Ala Ala Gln Leu Tyr Val Leu Leu His Leu Tyr Leu Ala
420 425 430
CTA CAC AAG AAG TAT CCA TTC CTG AAT CTA CTA CAT ACA CCC CCG CAC 1344
Leu His Lys Lys Tyr Pro Phe Leu Asn Leu Leu His Thr Pro Pro His
435 440 445
AGA CCT CCA CCC TTG TGT CCT CAA GCA CCA AGG AAG ACG CAG TGC AAA 1392
Arg Pro Pro Pro Leu Cys Pro Gln Ala Pro Arg Lys Thr Gln Cys Lys
450 455 460
CGC CGC CTA GGA AAC GAG CAC GAG GAG TCC AAC AGT CCC CTT GCA ACG 1440
Arg Arg Leu Gly Asn Glu His Glu Glu Ser Asn Ser Pro Leu Ala Thr
465 470 475 480
CCT TGT GTG TGG CCC ACA TTG GAC CCG TGG ACA GTG GAA ACC ACA ACC 1488
Pro Cys Val Trp Pro Thr Leu Asp Pro Trp Thr Val Glu Thr Thr Thr
485 490 495
TCA TCA CTA ACA ATC ACG ACC AGC ACC AAA GAC GGA ACA ACA GTA ACA 1536
Ser Ser Leu Thr Ile Thr Thr Ser Thr Lys Asp Gly Thr Thr Val Thr
500 505 510
GTT CAG CTA CGC CTA AGA TCT ATG GAA GTG GTG CCT GTA CAA ATA GCT 1584
Val Gln Leu Arg Leu Arg Ser Met Glu Val Val Pro Val Gln Ile Ala
515 520 525
GCA GGA ACA ACC AGC ACA TTC ATA CTG CCT GTT ATA ATT GCA TTT GTT 1632
Ala Gly Thr Thr Ser Thr Phe Ile Leu Pro Val Ile Ile Ala Phe Val
530 535 540
GTA TGT TTT GTT AGC ATC ATA CTT ATT GTA TGG ATA TCT GAG TTT ATT 1680
Val Cys Phe Val Ser Ile Ile Leu Ile Val Trp Ile Ser Glu Phe Ile
545 550 555 560
GTG TAC ACA TCT GTG CTA GTA CTA ACA CTG CTT TTA TAT TTA CTA TTG 1728
Val Tyr Thr Ser Val Leu Val Leu Thr Leu Leu Leu Tyr Leu Leu Leu
565 570 575
TGG CTG CTA TTA ACA ACC CCC TTG CAA TTT TTC CTA CTA ACT CTA CTT 1776
Trp Leu Leu Leu Thr Thr Pro Leu Gln Phe Phe Leu Leu Thr Leu Leu
580 585 590
GTG TGT TAC TGT CCC GCA TTG TAT ATA CAC TAC TAT ATT GTT ACC ACA 1824
Val Cys Tyr Cys Pro Ala Leu Tyr Ile His Tyr Tyr Ile Val Thr Thr
595 600 605
CAG CAA CTT AAG ATG ATG CTA ACA TGT CAA TTT AAT GAT GGA GAT ACC 1872
Gln Gln Leu Lys Met Met Leu Thr Cys Gln Phe Asn Asp Gly Asp Thr
610 615 620
TGG CTG GGT TTG TGG TTG TTA TGT GCC TTT ATT GTA GGG ATG TTG GGG 1920
Trp Leu Gly Leu Trp Leu Leu Cys Ala Phe Ile Val Gly Met Leu Gly
625 630 635 640
TTA TTA TTG ATG CAC TAT AGA GCT GTA CAA GGG GAT AAA CAC ACC AAA 1968
Leu Leu Leu Met His Tyr Arg Ala Val Gln Gly Asp Lys His Thr Lys
645 650 655
TGT AAG AAG TGT AAC AAA CAC AAC TGT AAT GAT GAT TAT GTA ACT ATG 2016
Cys Lys Lys Cys Asn Lys His Asn Cys Asn Asp Asp Tyr Val Thr Met
660 665 670
CAT TAT ACT ACT GAT GGT GAT TAT ATA TAT ATG AAT CCG CGG ATG GAA 2064
His Tyr Thr Thr Asp Gly Asp Tyr Ile Tyr Met Asn Pro Arg Met Glu
675 680 685
AGT GCA AAT GCC TCC ACG TCT GCA ACG ACC ATA GAC CAG TTG TGC AAG 2112
Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gln Leu Cys Lys
690 695 700
ACG TTT AAT CTA TCT ATG CAT ACG TTG CAA ATT AAT TGT GTG TTT TGC 2160
Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn Cys Val Phe Cys
705 710 715 720
AAG AAT GCA CTG ACC ACA GCA GAG ATT TAT TCA TAT GCA TAT AAA CAC 2208
Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr Lys His
725 730 735
CTA AAG GTC CTG TTT CGA GGC GGC TAT CCA TAT GCA GCC TGC GCG TGC 2256
Leu Lys Val Leu Phe Arg Gly Gly Tyr Pro Tyr Ala Ala Cys Ala Cys
740 745 750
TGC CTA GAA TTT CAT GGA AAA ATA AAC CAA TAT AGA CAC TTT GAT TAT 2304
Cys Leu Glu Phe His Gly Lys Ile Asn Gln Tyr Arg His Phe Asp Tyr
755 760 765
GCT GGA TAT GCA ACA ACA GTT GAA GAA GAA ACT AAA CAA GAC ATC TTA 2352
Ala Gly Tyr Ala Thr Thr Val Glu Glu Glu Thr Lys Gln Asp Ile Leu
770 775 780
GAC GTG CTA ATT CGG TGC TAC CTG TGT CAC AAA CCG CTG TGT GAA GTA 2400
Asp Val Leu Ile Arg Cys Tyr Leu Cys His Lys Pro Leu Cys Glu Val
785 790 795 800
GAA AAG GTA AAA CAT ATA CTA ACC AAG GCG CGG TTC ATA AAG CTA AAT 2448
Glu Lys Val Lys His Ile Leu Thr Lys Ala Arg Phe Ile Lys Leu Asn
805 810 815
TGT ACG TGG AAG GGT CGC TGC CTA CAC TGC TGG ACA ACA TGC ATG GAA 2496
Cys Thr Trp Lys Gly Arg Cys Leu His Cys Trp Thr Thr Cys Met Glu
820 825 830
GAC ATG TTA CCC GCT AGC ATG CAT GGA AGA CAT GTT ACC CTA AAG GAT 2544
Asp Met Leu Pro Ala Ser Met His Gly Arg His Val Thr Leu Lys Asp
835 840 845
ATT GTA TTA GAC CTG CAA CCT CCA GAC CCT GTA GGG TTA CAT TGC TAT 2592
Ile Val Leu Asp Leu Gln Pro Pro Asp Pro Val Gly Leu His Cys Tyr
850 855 860
GAG CAA TTA GTA GAC AGC TCA GAA GAT GAG GTG GAC GAA GTG GAC GGA 2640
Glu Gln Leu Val Asp Ser Ser Glu Asp Glu Val Asp Glu Val Asp Gly
865 870 875 880
CAA GAT TCA CAA CCT TTA AAA CAA CAT TTC CAA ATA GTG ACC TGT TGC 2688
Gln Asp Ser Gln Pro Leu Lys Gln His Phe Gln Ile Val Thr Cys Cys
885 890 895
TGT GGA TGT GAC AGC AAC GTT CGA CTG GTT GTG CAG TGT ACA GAA ACA 2736
Cys Gly Cys Asp Ser Asn Val Arg Leu Val Val Gln Cys Thr Glu Thr
900 905 910
GAC ATC AGA GAA GTG CAA CAG CTT CTG TTG GGA ACA CTA AAC ATA GTG 2784
Asp Ile Arg Glu Val Gln Gln Leu Leu Leu Gly Thr Leu Asn Ile Val
915 920 925
TGT CCC ATC TGC GCA CCG AAG ACC CGA TCG ATG GCG GAC GAT TCA GGT 2832
Cys Pro Ile Cys Ala Pro Lys Thr Arg Ser Met Ala Asp Asp Ser Gly
930 935 940
ACA GAA AAT GAG GGG TCT GGG TGT ACA GGA TGG TTT ATG GTA GAA GCT 2880
Thr Glu Asn Glu Gly Ser Gly Cys Thr Gly Trp Phe Met Val Glu Ala
945 950 955 960
ATA GTG CAA CAC CCA ACA GGT ACA CAA ATA TCA GAC GAT GAG GAT GAG 2928
Ile Val Gln His Pro Thr Gly Thr Gln Ile Ser Asp Asp Glu Asp Glu
965 970 975
GAG GTG GAG GAC AGT GGG TAT GAC ATG GTG GAC TTT ATT GAT GAC AGC 2976
Glu Val Glu Asp Ser Gly Tyr Asp Met Val Asp Phe Ile Asp Asp Ser
980 985 990
AAT ATT ACA CAC AAT TCA CTG GAA GCA CAG GCA TTG TTT AAC AGG CAG 3024
Asn Ile Thr His Asn Ser Leu Glu Ala Gln Ala Leu Phe Asn Arg Gln
995 1000 1005
GAG GCG GAC ACC CAT TAT GCG ACT GTG CAG GAC CTA AAA CGA AAG TAT 3072
Glu Ala Asp Thr His Tyr Ala Thr Val Gln Asp Leu Lys Arg Lys Tyr
1010 1015 1020
TTA GGT AGT CCA TAT GTT AGT CCT ATA AAC ACT ATA GCC GAG GCA GTG 3120
Leu Gly Ser Pro Tyr Val Ser Pro Ile Asn Thr Ile Ala Glu Ala Val
1025 1030 1035 1040
GAA AGT GAA ATA AGT CCA CGA TTG GAC GCC ATT AAA CTT ACA AGA CAG 3168
Glu Ser Glu Ile Ser Pro Arg Leu Asp Ala Ile Lys Leu Thr Arg Gln
1045 1050 1055
CCA AAA AAG GTA AAG CGA CGG CTG TTT CAA ACC AGG GAA CTA ACG GAC 3216
Pro Lys Lys Val Lys Arg Arg Leu Phe Gln Thr Arg Glu Leu Thr Asp
1060 1065 1070
AGT GGA TAT GGC TAT TCT GAA GTG GAA GCT GGA ACG GGA ACG CAG GTA 3264
Ser Gly Tyr Gly Tyr Ser Glu Val Glu Ala Gly Thr Gly Thr Gln Val
1075 1080 1085
GAG AAA CAT GGC GTA CCG GAA AAT GGG GGA GAT GGT CAG GAA AAG GAC 3312
Glu Lys His Gly Val Pro Glu Asn Gly Gly Asp Gly Gln Glu Lys Asp
1090 1095 1100
ACA GGA AGG GAC ATA GAG GGG GAG GAA CAT ACA GAG GCG GAA GCG CCC 3360
Thr Gly Arg Asp Ile Glu Gly Glu Glu His Thr Glu Ala Glu Ala Pro
1105 1110 1115 1120
ACA AAC AGT GTA CGG GAG CAT GCA GGC ACA GCA GGA ATA TTG GAA TTG 3408
Thr Asn Ser Val Arg Glu His Ala Gly Thr Ala Gly Ile Leu Glu Leu
1125 1130 1135
TTA AAA TGT AAA GAT TTA CGG GCA GCA TTA CTT GGT AAG TTT AAA GAA 3456
Leu Lys Cys Lys Asp Leu Arg Ala Ala Leu Leu Gly Lys Phe Lys Glu
1140 1145 1150
TGC TTT GGG CTG TCT TTT ATA GAT TTA ATT AGG CCA TTT AAA AGT GAT 3504
Cys Phe Gly Leu Ser Phe Ile Asp Leu Ile Arg Pro Phe Lys Ser Asp
1155 1160 1165
AAA ACA ACA TGT TTA GAT TGG GTG GTA GCA GGG TTT GGT ATA CAT CAT 3552
Lys Thr Thr Cys Leu Asp Trp Val Val Ala Gly Phe Gly Ile His His
1170 1175 1180
AGC ATA TCA GAG GCA TTT CAA AAA TTA ATT GAG CCA TTA AGT TTA TAT 3600
Ser Ile Ser Glu Ala Phe Gln Lys Leu Ile Glu Pro Leu Ser Leu Tyr
1185 1190 1195 1200
GCA CAT ATA CAA TGG CTA ACA AAT GCA TGG GGA ATG GTA TTG TTA GTA 3648
Ala His Ile Gln Trp Leu Thr Asn Ala Trp Gly Met Val Leu Leu Val
1205 1210 1215
TTA TTA AGA TTT AAA GTA AAT AAA AGT AGA AGT ACC GTT GCA CGT ACA 3696
Leu Leu Arg Phe Lys Val Asn Lys Ser Arg Ser Thr Val Ala Arg Thr
1220 1225 1230
CTT GCA ACG CTA TTA AAT ATA CCT GAA AAC CAA ATG TTA ATA GAG CCA 3744
Leu Ala Thr Leu Leu Asn Ile Pro Glu Asn Gln Met Leu Ile Glu Pro
1235 1240 1245
CCA AAA ATA CAA AGT GGT GTT GCA GCC CTG TAT TGG TTT CGT ACA GGT 3792
Pro Lys Ile Gln Ser Gly Val Ala Ala Leu Tyr Trp Phe Arg Thr Gly
1250 1255 1260
ATA TCA AAT GCC AGT ACA GTT ATA GGG GAA GCA CCA GAA TGG ATA ACA 3840
Ile Ser Asn Ala Ser Thr Val Ile Gly Glu Ala Pro Glu Trp Ile Thr
1265 1270 1275 1280
CGC CAA ACA GTT ATT GAA CAC GGG TTG GCA GAC AGT CAG TTT AAA TTA 3888
Arg Gln Thr Val Ile Glu His Gly Leu Ala Asp Ser Gln Phe Lys Leu
1285 1290 1295
ACA GAA ATG GTG CAG TGG GCG TAT GAT AAT GAC ATA TGC GAG GAG AGT 3936
Thr Glu Met Val Gln Trp Ala Tyr Asp Asn Asp Ile Cys Glu Glu Ser
1300 1305 1310
GAA ATT GCA TTT GAA TAT GCA CAA AGG GGA GAT TTT GAT TCT AAT GCA 3984
Glu Ile Ala Phe Glu Tyr Ala Gln Arg Gly Asp Phe Asp Ser Asn Ala
1315 1320 1325
CGA GCA TTT TTA AAT AGC AAT ATG CAG GCA AAA TAT GTG AAA GAT TGT 4032
Arg Ala Phe Leu Asn Ser Asn Met Gln Ala Lys Tyr Val Lys Asp Cys
1330 1335 1340
GCA ACT ATG TGT AGA CAT TAT AAA CAT GCA GAA ATG AGG AAG ATG TCT 4080
Ala Thr Met Cys Arg His Tyr Lys His Ala Glu Met Arg Lys Met Ser
1345 1350 1355 1360
ATA AAA CAA TGG ATA AAA CAT AGG GGT TCT AAA ATA GAA GGC ACA GGA 4128
Ile Lys Gln Trp Ile Lys His Arg Gly Ser Lys Ile Glu Gly Thr Gly
1365 1370 1375
AAT TGG AAA CCA ATT GTA CAA TTC CTA CGA CAT CAA AAT ATA GAA TTC 4176
Asn Trp Lys Pro Ile Val Gln Phe Leu Arg His Gln Asn Ile Glu Phe
1380 1385 1390
ATT CCT TTT TTA ACT AAA TTT AAA TTA TGG CTG CAC GGT ACG CCA AAA 4224
Ile Pro Phe Leu Thr Lys Phe Lys Leu Trp Leu His Gly Thr Pro Lys
1395 1400 1405
AAA AAC TGC ATA GCC ATA GTA GGC CCT CCA GAT ACT GGG AAA TCG TAC 4272
Lys Asn Cys Ile Ala Ile Val Gly Pro Pro Asp Thr Gly Lys Ser Tyr
1410 1415 1420
TTT TGT ATG AGT TTA ATA AGC TTT CTA GGA GGT ACA GTT ATT AGT CAT 4320
Phe Cys Met Ser Leu Ile Ser Phe Leu Gly Gly Thr Val Ile Ser His
1425 1430 1435 1440
GTA AAT TCC AGC AGC CAT TTT TGG TTG CAA CCG TTA GTA GAT GCT AAG 4368
Val Asn Ser Ser Ser His Phe Trp Leu Gln Pro Leu Val Asp Ala Lys
1445 1450 1455
GTA GCA TTG TTA GAT GAT GCA ACA CAG CCA TGT TGG ATA TAT ATG GAT 4416
Val Ala Leu Leu Asp Asp Ala Thr Gln Pro Cys Trp Ile Tyr Met Asp
1460 1465 1470
ACA TAT ATG AGA AAT TTG TTA GAT GGT AAT CCT ATG AGT ATT GAC AGA 4464
Thr Tyr Met Arg Asn Leu Leu Asp Gly Asn Pro Met Ser Ile Asp Arg
1475 1480 1485
AAG CAT AAA GCA TTG ACA TTA ATT AAA TGT CCA CCT CTG CTA GTA ACG 4512
Lys His Lys Ala Leu Thr Leu Ile Lys Cys Pro Pro Leu Leu Val Thr
1490 1495 1500
TCC AAC ATA GAT ATT ACT AAA GAA GAT AAA TAT AAG TAT TTA CAT ACT 4560
Ser Asn Ile Asp Ile Thr Lys Glu Asp Lys Tyr Lys Tyr Leu His Thr
1505 1510 1515 1520
AGA GTA ACA ACA TTT ACA TTT CCA AAT CCA TTC CCT TTT GAC AGA AAT 4608
Arg Val Thr Thr Phe Thr Phe Pro Asn Pro Phe Pro Phe Asp Arg Asn
1525 1530 1535
GGG AAT GCA GTG TAT GAA CTG TCA AAT ACA AAC TGG AAA TGT TTT TTT 4656
Gly Asn Ala Val Tyr Glu Leu Ser Asn Thr Asn Trp Lys Cys Phe Phe
1540 1545 1550
GAA AGA CTG TCG TCA AGC CTA GAC ATT CAG GAT TCT GAG GAC GAG GAA 4704
Glu Arg Leu Ser Ser Ser Leu Asp Ile Gln Asp Ser Glu Asp Glu Glu
1555 1560 1565
GAT GGA AGC AAT AGC CAA GCG TTT AGA TGC GTG CCA GGA ACA GTT GTT 4752
Asp Gly Ser Asn Ser Gln Ala Phe Arg Cys Val Pro Gly Thr Val Val
1570 1575 1580
AGA ACT TTA TGAGGTACC 4770
Arg Thr Leu
1585

1587 amino acids

amino acid

linear

protein

46
Met Gly Gly Ser His His His His His His Gly Met Ala Ser Met Thr
1 5 10 15
Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp
20 25 30
Arg Trp Gly Ser Glu Leu Met Glu Ala Ile Ala Lys Arg Leu Asp Ala
35 40 45
Cys Gln Glu Gln Leu Leu Glu Leu Tyr Glu Glu Asn Ser Thr Asp Leu
50 55 60
His Lys His Val Leu His Trp Lys Cys Met Arg His Glu Ser Val Leu
65 70 75 80
Leu Tyr Lys Ala Lys Gln Met Gly Leu Ser His Ile Gly Met Gln Val
85 90 95
Val Pro Pro Leu Lys Val Ser Glu Ala Lys Gly His Asn Ala Ile Glu
100 105 110
Met Gln Met His Leu Glu Ser Leu Leu Arg Thr Glu Tyr Ser Met Glu
115 120 125
Pro Trp Thr Leu Gln Glu Thr Ser Tyr Glu Met Trp Gln Thr Pro Pro
130 135 140
Lys Arg Cys Phe Lys Lys Arg Gly Lys Thr Val Glu Val Lys Phe Asp
145 150 155 160
Gly Cys Ala Asn Asn Thr Met Asp Tyr Val Val Trp Thr Asp Val Tyr
165 170 175
Val Gln Asp Asn Asp Thr Trp Val Lys Val His Ser Met Val Asp Ala
180 185 190
Lys Gly Ile Tyr Tyr Thr Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn
195 200 205
Phe Val Lys Glu Ala Glu Lys Tyr Gly Ser Thr Lys His Trp Glu Val
210 215 220
Cys Tyr Gly Ser Thr Val Ile Cys Ser Pro Ala Ser Val Ser Ser Thr
225 230 235 240
Thr Gln Glu Val Ser Ile Pro Glu Ser Thr Thr Tyr Thr Pro Ala Gln
245 250 255
Thr Ser Thr Leu Val Ser Ser Ser Thr Lys Glu Asp Ala Val Gln Thr
260 265 270
Pro Pro Arg Lys Arg Ala Arg Gly Val Gln Gln Ser Pro Cys Asn Ala
275 280 285
Leu Cys Val Ala His Ile Gly Pro Val Asp Ser Gly Asn His Asn Leu
290 295 300
Ile Thr Asn Asn His Asp Gln His Gln Arg Arg Asn Asn Ser Asn Ser
305 310 315 320
Ser Ala Thr Pro Ile Val Gln Phe Gln Gly Glu Ser Asn Cys Leu Lys
325 330 335
Cys Phe Arg Tyr Arg Leu Asn Asp Arg His Arg His Leu Phe Asp Leu
340 345 350
Ile Ser Ser Thr Trp His Trp Ala Ser Ser Lys Ala Pro His Lys His
355 360 365
Ala Ile Val Thr Val Thr Tyr Asp Ser Glu Glu Gln Arg Gln Gln Phe
370 375 380
Leu Asp Val Val Lys Ile Pro Pro Thr Ile Ser His Lys Leu Gly Phe
385 390 395 400
Met Ser Leu His Leu Leu Val Asp Met Gly Ala Pro Asn Ile Gly Lys
405 410 415
Tyr Val Met Ala Ala Gln Leu Tyr Val Leu Leu His Leu Tyr Leu Ala
420 425 430
Leu His Lys Lys Tyr Pro Phe Leu Asn Leu Leu His Thr Pro Pro His
435 440 445
Arg Pro Pro Pro Leu Cys Pro Gln Ala Pro Arg Lys Thr Gln Cys Lys
450 455 460
Arg Arg Leu Gly Asn Glu His Glu Glu Ser Asn Ser Pro Leu Ala Thr
465 470 475 480
Pro Cys Val Trp Pro Thr Leu Asp Pro Trp Thr Val Glu Thr Thr Thr
485 490 495
Ser Ser Leu Thr Ile Thr Thr Ser Thr Lys Asp Gly Thr Thr Val Thr
500 505 510
Val Gln Leu Arg Leu Arg Ser Met Glu Val Val Pro Val Gln Ile Ala
515 520 525
Ala Gly Thr Thr Ser Thr Phe Ile Leu Pro Val Ile Ile Ala Phe Val
530 535 540
Val Cys Phe Val Ser Ile Ile Leu Ile Val Trp Ile Ser Glu Phe Ile
545 550 555 560
Val Tyr Thr Ser Val Leu Val Leu Thr Leu Leu Leu Tyr Leu Leu Leu
565 570 575
Trp Leu Leu Leu Thr Thr Pro Leu Gln Phe Phe Leu Leu Thr Leu Leu
580 585 590
Val Cys Tyr Cys Pro Ala Leu Tyr Ile His Tyr Tyr Ile Val Thr Thr
595 600 605
Gln Gln Leu Lys Met Met Leu Thr Cys Gln Phe Asn Asp Gly Asp Thr
610 615 620
Trp Leu Gly Leu Trp Leu Leu Cys Ala Phe Ile Val Gly Met Leu Gly
625 630 635 640
Leu Leu Leu Met His Tyr Arg Ala Val Gln Gly Asp Lys His Thr Lys
645 650 655
Cys Lys Lys Cys Asn Lys His Asn Cys Asn Asp Asp Tyr Val Thr Met
660 665 670
His Tyr Thr Thr Asp Gly Asp Tyr Ile Tyr Met Asn Pro Arg Met Glu
675 680 685
Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gln Leu Cys Lys
690 695 700
Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn Cys Val Phe Cys
705 710 715 720
Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr Lys His
725 730 735
Leu Lys Val Leu Phe Arg Gly Gly Tyr Pro Tyr Ala Ala Cys Ala Cys
740 745 750
Cys Leu Glu Phe His Gly Lys Ile Asn Gln Tyr Arg His Phe Asp Tyr
755 760 765
Ala Gly Tyr Ala Thr Thr Val Glu Glu Glu Thr Lys Gln Asp Ile Leu
770 775 780
Asp Val Leu Ile Arg Cys Tyr Leu Cys His Lys Pro Leu Cys Glu Val
785 790 795 800
Glu Lys Val Lys His Ile Leu Thr Lys Ala Arg Phe Ile Lys Leu Asn
805 810 815
Cys Thr Trp Lys Gly Arg Cys Leu His Cys Trp Thr Thr Cys Met Glu
820 825 830
Asp Met Leu Pro Ala Ser Met His Gly Arg His Val Thr Leu Lys Asp
835 840 845
Ile Val Leu Asp Leu Gln Pro Pro Asp Pro Val Gly Leu His Cys Tyr
850 855 860
Glu Gln Leu Val Asp Ser Ser Glu Asp Glu Val Asp Glu Val Asp Gly
865 870 875 880
Gln Asp Ser Gln Pro Leu Lys Gln His Phe Gln Ile Val Thr Cys Cys
885 890 895
Cys Gly Cys Asp Ser Asn Val Arg Leu Val Val Gln Cys Thr Glu Thr
900 905 910
Asp Ile Arg Glu Val Gln Gln Leu Leu Leu Gly Thr Leu Asn Ile Val
915 920 925
Cys Pro Ile Cys Ala Pro Lys Thr Arg Ser Met Ala Asp Asp Ser Gly
930 935 940
Thr Glu Asn Glu Gly Ser Gly Cys Thr Gly Trp Phe Met Val Glu Ala
945 950 955 960
Ile Val Gln His Pro Thr Gly Thr Gln Ile Ser Asp Asp Glu Asp Glu
965 970 975
Glu Val Glu Asp Ser Gly Tyr Asp Met Val Asp Phe Ile Asp Asp Ser
980 985 990
Asn Ile Thr His Asn Ser Leu Glu Ala Gln Ala Leu Phe Asn Arg Gln
995 1000 1005
Glu Ala Asp Thr His Tyr Ala Thr Val Gln Asp Leu Lys Arg Lys Tyr
1010 1015 1020
Leu Gly Ser Pro Tyr Val Ser Pro Ile Asn Thr Ile Ala Glu Ala Val
1025 1030 1035 1040
Glu Ser Glu Ile Ser Pro Arg Leu Asp Ala Ile Lys Leu Thr Arg Gln
1045 1050 1055
Pro Lys Lys Val Lys Arg Arg Leu Phe Gln Thr Arg Glu Leu Thr Asp
1060 1065 1070
Ser Gly Tyr Gly Tyr Ser Glu Val Glu Ala Gly Thr Gly Thr Gln Val
1075 1080 1085
Glu Lys His Gly Val Pro Glu Asn Gly Gly Asp Gly Gln Glu Lys Asp
1090 1095 1100
Thr Gly Arg Asp Ile Glu Gly Glu Glu His Thr Glu Ala Glu Ala Pro
1105 1110 1115 1120
Thr Asn Ser Val Arg Glu His Ala Gly Thr Ala Gly Ile Leu Glu Leu
1125 1130 1135
Leu Lys Cys Lys Asp Leu Arg Ala Ala Leu Leu Gly Lys Phe Lys Glu
1140 1145 1150
Cys Phe Gly Leu Ser Phe Ile Asp Leu Ile Arg Pro Phe Lys Ser Asp
1155 1160 1165
Lys Thr Thr Cys Leu Asp Trp Val Val Ala Gly Phe Gly Ile His His
1170 1175 1180
Ser Ile Ser Glu Ala Phe Gln Lys Leu Ile Glu Pro Leu Ser Leu Tyr
1185 1190 1195 1200
Ala His Ile Gln Trp Leu Thr Asn Ala Trp Gly Met Val Leu Leu Val
1205 1210 1215
Leu Leu Arg Phe Lys Val Asn Lys Ser Arg Ser Thr Val Ala Arg Thr
1220 1225 1230
Leu Ala Thr Leu Leu Asn Ile Pro Glu Asn Gln Met Leu Ile Glu Pro
1235 1240 1245
Pro Lys Ile Gln Ser Gly Val Ala Ala Leu Tyr Trp Phe Arg Thr Gly
1250 1255 1260
Ile Ser Asn Ala Ser Thr Val Ile Gly Glu Ala Pro Glu Trp Ile Thr
1265 1270 1275 1280
Arg Gln Thr Val Ile Glu His Gly Leu Ala Asp Ser Gln Phe Lys Leu
1285 1290 1295
Thr Glu Met Val Gln Trp Ala Tyr Asp Asn Asp Ile Cys Glu Glu Ser
1300 1305 1310
Glu Ile Ala Phe Glu Tyr Ala Gln Arg Gly Asp Phe Asp Ser Asn Ala
1315 1320 1325
Arg Ala Phe Leu Asn Ser Asn Met Gln Ala Lys Tyr Val Lys Asp Cys
1330 1335 1340
Ala Thr Met Cys Arg His Tyr Lys His Ala Glu Met Arg Lys Met Ser
1345 1350 1355 1360
Ile Lys Gln Trp Ile Lys His Arg Gly Ser Lys Ile Glu Gly Thr Gly
1365 1370 1375
Asn Trp Lys Pro Ile Val Gln Phe Leu Arg His Gln Asn Ile Glu Phe
1380 1385 1390
Ile Pro Phe Leu Thr Lys Phe Lys Leu Trp Leu His Gly Thr Pro Lys
1395 1400 1405
Lys Asn Cys Ile Ala Ile Val Gly Pro Pro Asp Thr Gly Lys Ser Tyr
1410 1415 1420
Phe Cys Met Ser Leu Ile Ser Phe Leu Gly Gly Thr Val Ile Ser His
1425 1430 1435 1440
Val Asn Ser Ser Ser His Phe Trp Leu Gln Pro Leu Val Asp Ala Lys
1445 1450 1455
Val Ala Leu Leu Asp Asp Ala Thr Gln Pro Cys Trp Ile Tyr Met Asp
1460 1465 1470
Thr Tyr Met Arg Asn Leu Leu Asp Gly Asn Pro Met Ser Ile Asp Arg
1475 1480 1485
Lys His Lys Ala Leu Thr Leu Ile Lys Cys Pro Pro Leu Leu Val Thr
1490 1495 1500
Ser Asn Ile Asp Ile Thr Lys Glu Asp Lys Tyr Lys Tyr Leu His Thr
1505 1510 1515 1520
Arg Val Thr Thr Phe Thr Phe Pro Asn Pro Phe Pro Phe Asp Arg Asn
1525 1530 1535
Gly Asn Ala Val Tyr Glu Leu Ser Asn Thr Asn Trp Lys Cys Phe Phe
1540 1545 1550
Glu Arg Leu Ser Ser Ser Leu Asp Ile Gln Asp Ser Glu Asp Glu Glu
1555 1560 1565
Asp Gly Ser Asn Ser Gln Ala Phe Arg Cys Val Pro Gly Thr Val Val
1570 1575 1580
Arg Thr Leu
1585

6 amino acids

amino acid

linear

peptide

47
His His His His His His
1 5

20 amino acids

amino acid

linear

protein

48
Leu Gly Asn Glu His Glu Glu Ser Asn Ser Pro Leu Ala Thr Pro Cys
1 5 10 15
Val Trp Pro Thr
20

24 amino acids

amino acid

linear

protein

49
Gln Tyr Arg His Phe Asp Tyr Ala Gln Tyr Ala Thr Thr Val Glu Glu
1 5 10 15
Glu Thr Lys Gln Asp Ile Leu Asp
20

19 amino acids

amino acid

linear

protein

50
Met His Gly Arg His Val Thr Leu Lys Asp Ile Val Leu Asp Leu Gln
1 5 10 15
Pro Pro Asp

Number	Name	Date	Kind
5955087	Whittle et al.	Sep 1999	A
6004557	Edwards et al.	Dec 1999	A
6123948	Whittle et al.	Sep 2000	A

Number	Date	Country
44 35 907	Apr 1996	DE
WO 9205248	Apr 1992	WO
WO 9211290	Jul 1992	WO
WO 9216636	Oct 1992	WO
WO 9412629	Jun 1994	WO

Papillomavirus polyprotein constructs

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Term Extension

Abstract

Description

Claims

Priority Claims (1)

CROSS REFERENCE TO RELATED APPLICATIONS

US Referenced Citations (3)

Foreign Referenced Citations (5)

Non-Patent Literature Citations (9)

Entry
Taniguchi et al., “A Major Transcript of Human Papillomavirus Type 16 in Transformed NIH 3T3 Cells contains Polycistronic MRNA Encoding E7, E5, and E1{circumflex over ( )}E4 Fusion Gene, ” Virus Genes (1990), vol. 3, No. 3, pp. 221-233.
Rohlfs et al., “Viral Trans. Human Keratinocyte Cell Lines Immortalized by Human Papillomavirus Type-16,” Virology (1991), vol. 183, pp. 331-342.
Chiang et al., “An E1M{circumflex over ( )}E2C Fusion Protein Encoded by Human Papillomavirus Type 11 is a Sequence-Specific Transcription Repressor,” Journal of Virology ; (1991), vol. 65, pp. 3317-3329.
Lamberti et al. “Transcriptional Activation by the Papillomabirus E6 Zinc Finger Oncoprotein,” The EMBO Journal (1990), vol. 9, pp. 1907-1913.
Abstract of WO 96/11272, “Papilloma Virus-Like Particles, Fusion Proteins and Process for Producing the Same, ” (Apr. 1996).
Stauffer et al., “Experimentia: 26th Annual Meeting of Swiss Societies for Experimental Biology,” XP002101770, Abstract Only, Feb. 1994.
Selvakumar et al, “Immunization with Nonstructural Proteins E1 and E2 of Cottontail Rabbit papillomavirus Stimulates Regression of Virus-Induced Papillomas,” J. of Virology (Jan. 1995), vol. 69, pp. 602-605.
Meneuzzi et al., “Immunization against Human Papillomavirus Type 16 Tumor Cells with Recombinant Vaccinia Viruses Expressing E6 and E7,” Virology (1991), Vo. 69, pp. 62-69.
Tomita et al., “Translational Properties of the Human Papillomavirus Type-6L1-Coding mRNA,” Virology (1991), vol. 183, pp. 331-342.