Polynucleotides and polypeptides in pathogenic mycobacteria and their use as diagnostics, vaccines and targets for chemotherapy

This invention relates to the novel polynucleotide sequence we have designated "GS" which we have identified in pathogenic mycobacteria. GS is a pathogenicity island within 8 kb of DNA comprising a core region of 5.75 kb and an adjacent transmissable element within 2.25 kb. GS is contained within Mycobacterium paratuberculosis, Mycobacterium avium subsp. silvaticum and some pathogenic isolates of M.avium. Functional portions of the core region of GS are also represented by regions with a high degree of homology that we have identified in cosmids containing genomic DNA from Mycobacterium tuberculosis.
BACKGROUND TO THE INVENTION
Mycobacterium tuberculosis (Mtb) is a major cause of global diseases of humans as well as animals. Although conventional methods of diagnosis including microscopy, culture and skin testing exist for the recognition of these diseases, improved methods particularly new immunodiagnostics and DNA-based detection systems are needed. Drugs used to treat tuberculosis are increasingly encountering the problem of resistant organisms. New drugs targeted at specific pathogenicity determinants as well as new vaccines for the prevention and treatment of tuberculosis are required. The importance of Mtb as a global pathogen is reflected in the commitment being made to sequencing the entire genome of this organism. This has generated a large amount of DNA sequence data of genomic DNA within cosmid and other libraries. Although the DNA sequence is known in the art, the functions of the vast majority of these sequences, the proteins they encode, the biological significance of these proteins, and the overall relevance and use of these genes and their products as diagnostics, vaccines and targets for chemotherapy for tuberculous disease, remains entirely unknown.
Mycobacterium avium subsp. silvaticum (Mavs) is a pathogenic mycobacterium causing diseases of animals and birds, but it can also affect humans. Mycobacterium paratuberculosis (Mptb) causes chronic inflammation of the intestine in many species of animals including primates and can also cause Crohn's disease in humans. Mptb is associated with other chronic inflammatory diseases of humans such as sarcoidosis. Subclinical Mptb infection is widespread in domestic livestock and is present in milk from infected animals. The organism is more resistant to pasteurisation than Mtb and can be conveyed to humans in retail milk supplies. Mptb is also present in water supplies, particularly those contaminated with run-off from heavily grazed pastures. Mptb and Mavs contain the insertion elements IS900 and IS902 respectively, and these are linked to pathogenicity in these organisms. IS900 and IS902 provide convenient highly specific multi-copy DNA targets for the sensitive detection of these organisms using DNA-based methods and for the diagnosis of infections in animals and humans. Much improvement is however required in the immunodiagnosis of Mptb and Mavs infections in animals and humans. Mptb and Mavs are in general, resistant in vivo to standard anti-tuberculous drugs. Although substantial clinical improvements in infections caused by Mptb, such as Crohn's disease, may result from treatment of patients with combinations of existing drugs such as Rifabutin, Clarithromycin or Azithromycin, additional effective drug treatments are required. Furthermore, there is an urgent need for effective vaccines for the prevention and treatment of Mptb and Mavs infections in animals and humans based upon the recognition of specific pathogenicity determinants.
Pathogenicity islands are, in general, 7-9 kb regions of DNA comprising a core domain with multiple ORFs and an adjacent transmissable element. The transmissable element also encodes proteins which may be linked to pathogenicity, such as by providing receptors for cellular recognition. Pathogenicity islands are envisaged as mobile packages of DNA which, when they enter an organism, assist in bringing about its convertion from a non-disease-causing to a disease-causing strain.

DESCRIPTION OF THE DRAWINGS
FIGS. 1(a) and (b) shows a linear map of the pathogenicity island GS in Mavs (FIG. 1a) and in Mptb (FIG. 1b). The main open reading frames are illustrated as ORFs A to H. ORFs A to F are found within the core region of GS. ORFs G and H are encoded by the adjacent transmissable element portion of GS.

DISCLOSURE OF THE INVENTION
Using a DNA-based differential analysis technology we have discovered and characterised a novel polynucleotide in Mptb (isolates 0022 from a Guernsey cow and 0021 from a red deer). This polynucleotide comprises the gene region we have designated GS. GS is found in Mptb using the identifier DNA sequences Seq. ID. No 1 and 2 where the Seq. ID No2 is the complementary sequence of Seq. ID No 1. GS is also identified in Mavs. The complete DNA sequence incorporating the positive strand of GS from an isolate of Mavs comprising 7995 nucleotides, including the core region of GS and adjacent transsmissable element, is given in Seq. ID No.3. DNA sequence comprising 4435 bp of the positive strand of GS obtained from an isolate of Mptb including the core region of GS (nucleotides 1614 to 6047 of GS in Mavs) is given in Seq. ID No 4. The DNA sequence of GS from Mptb is highly (99.4%) homologous to GS in Mavs. The remaining portion of the DNA sequence of GS in Mptb, is readily obtainable by a person skilled in the art using standard laboratory procedures. The entire functional DNA sequence including core region and transmisable element of GS in Mptb and Mavs as described above, comprise the polynucleotide sequences of the invention.
There are 8 open reading frames (ORFs) in GS. Six of these designated GSA, GSB, GSC, GSD, GSE and GSF are encoded by the core DNA region of GS which, characteristically for a pathogenicity island, has a different GC content than the rest of the microbial genome. Two ORFs designated GSG and GSH are encoded by the transmissable element of GS whose GC content resembles that of the rest of the mycobacterial genome. The ORF GSH comprises two sub-ORFs H.sub.1 H.sub.2 on the complementary DNA strand linked by a programmed frameshifting site so that a single polypeptide is translated from the ORF GSH. The nucleotide sequences of the 8 ORFs in GS and their translations are shown in Seq. ID No 5 to Seq. ID No 29 as follows:
ORF A: Seq. ID No 5 Nucleotides 50 to 427 of GS from Mavs Seq. ID No 6 Amino acid sequence encoded by Seq. ID No 5.
ORF B: Seq. ID No 7 Nucleotides 772 to 1605 of GS from Mavs Seq. ID No 8 Amino acid sequence encoded by Seq. ID No 7.
ORF C: Seq. ID No 9 Nucleotides 1814 to 2845 of GS from Mavs Seq. ID No 10 Amino acid sequence encoded by Seq. ID No 9. Seq. ID No 11 Nucleotides 201 to 1232 of GS from Mptb Seq. ID No 12 Amino acid sequence encoded by Seq. ID No 11
ORF D: Seq. ID No 13 Nucleotides 2785 to 3804 of GS from Mavs Seq. ID No 14 Amino acid sequence encoded by Seq. ID No 13. Seq. ID No 15 Nucleotides 1172 to 2191 of GS from Mptb Seq. ID No 16 Amino acid sequence encoded by Seq. ID No 15.
ORF E: Seq. ID No 17 Nucleotides 4080 to 4802 of GS from Mavs Seq. ID No 18 Amino acid sequence encoded by Seq. ID No 17. Seq. ID No 19 Nucleotides 2467 to 3189 of GS from Mptb Seq. ID No 20 Amino acid sequence encoded by Seq. ID No 19.
ORF F: Seq. ID No 21 Nucleotides 4947 to 5747 of GS from Mavs Seq. ID No 22 Amino acid sequence encoded by Seq. ID No 21. Seq. ID No 23 Nucleotides 3335 to 4135 of GS from Mptb Seq. ID No 24 Amino acid sequence encoded by Seq. ID No 23.
ORF G: Seq. ID No 25 Nucleotides 6176 to 7042 of GS from Mavs Seq. ID No 26 Amino acid sequence encoded by Seq. ID No 25.
ORF H: Seq. ID No 27 Nucleotides 7953 to 6215 from Mavs.
ORF H.sub.1 : Seq. ID No 28 Amino acid sequence encoded by nucleotides 7953 to 7006 of Seq. ID No 27
ORF H.sub.2 : Seq. ID No 29 Amino acid sequence encoded by nucleotides 7009 to 6215 of Seq. ID No 27
The polynucleotides in Mtb with homology to the ORFs B, C, E and F of GS in Mptb and Mavs, and the polypeptides they are now known to encode as a result of our invention, are as follows:
ORF B: Seq. ID No 30 Cosmid MTCY277 nucleotides 35493 to 34705 Seq. ID No 31 Amino acid sequence encoded by Seq. ID No 30.
ORF C: Seq. ID No 32 Cosmid MTCY277 nucleotides 31972 to 32994 Seq. ID No 33 Amino acid sequence encoded by Seq. ID No 32.
ORF E: Seq. ID No 34 Cosmid MTCY277 nucleotides 34687 to 33956 Seq. ID No 35 Amino acid sequence encoded by Seq. ID No 34.
ORF E: Seq. ID No 36 Cosmid MTO24 nucleotides 15934 to 15203 Seq. ID No 37 Amino acid sequence encoded by Seq. ID No 36.
ORF F: Seq. ID No 38 Cosmid MTO24 nucleotides 15133 to 14306 Seq. ID No 39 Amino acid sequence encoded by Seq. ID No 38.
The proteins and peptides encoded by the ORFs A to H in Mptb and Mavs and the amino acid sequences from homologous genes we have discovered in Mtb given in Seq. ID Nos 31, 33, 35, 37 and 39, as described above and fragments thereof, comprise the polypeptides of the invention. The polypeptides of the invention are believed to be associated with specific immunoreactivity and with the pathogenicity of the host micro-organisms from which they were obtained.
The present invention thus provides a polynucleotide in substantially isolated form which is capable of selectively hybridising to sequence ID Nos 3 or 4 or a fragment thereof. The polynucleotide fragment may alternatively comprise a sequence selected from the group of Seq. ID.No: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27. The invention further provides a polynucleotide in substantially isolated form whose sequence consists essentially of a sequence selected from the group Seq ID Nos. 30, 32, 34, 36 and 38, or a corresponding sequence selectively hybridizable thereto, or a fragment of said sequence or corresponding sequence.
The invention further provides diagnostic probes such as a probe which comprises a fragment of at least 15 nucleotides of a polynucleotide of the invention, or a peptide nucleic acid or similar synthetic sequence specific ligand, optionally carrying a revealing label. The invention also provides a vector carrying a polynucleotide as defined above, particularly an expression vector.
The invention further provides a polypeptide in substantially isolated form which comprises any one of the sequences selected from the group consisting Seq. ID.No: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 29, 31, 33, 35, 37 and 39, or a polypeptide substantially homologous thereto. The invention additionally provides a polypeptide fragment which comprises a fragment of a polypeptide defined above, said fragment comprising at least 10 amino acids and an epitope. The invention also provides polynucleotides in substantially isolated form which encode polypeptides of the invention, and vectors which comprise such polynucleotides, as well as antibodies capable of binding such polypeptides. In an additional aspect, the invention provides kits comprising polynucleotides, polypeptides, antibodies or synthetic ligands of the invention and methods of using such kits in diagnosing the presence or absence of mycobacteria in a sample. The invention also provides pharmaceutical compositions comprising polynucleotides of the invention, polypeptides of the invention or antisense probes and the use of such compositions in the treatment or prevention of diseases caused by mycobacteria. The invention also provides polynucleotihe prevention and treatment of infections due to GS-containing pathogenic mycobacteria in animals and humans and as a means of enhacing in vivo susceptibility of said mycobacteria to antimicrobial drugs. The invention also provides bacteria or viruses transformed with polynucleotides of the invention for use as vaccines. The invention further provides Mptb or Mavs in which all or part or the polynucleotides of the invention have been deleted or disabled to provide mutated organisms of lower pathogenicity for use as vaccines in animals and humans. The invention further provides Mtb in which all or part of the polynucleotides encoding polypeptides of the invention have been deleted or disabled to provided mutated organisms or lower pathogenicity for use as vaccines in animals and humans.
A further aspect of the invention is our discovery of homologies between the ORFs B, C and E in GS on the one hand, and Mtb cosmid MTCY277 on the other (data from Genbank database using the computer programmes BLAST and BLIXEM). The homologous ORFs in MTCY277 are adjacent to one another consistent with the form of another pathogenicity island in Mtb. A further aspect of the invention is our discovery of homologies between ORFs E and F in GS, and Mtb cosmid MTO24 (also Genbank, as above) with the homologous ORFs close to one another. The use of polynucleotides and polypeptides from Mtb (Seq. ID Nos 30, 31, 32, 33, 34, 35, 36, 37, 38 and 39) in substantially isolated form as diagnostics, vaccines and targets for chemotherapy, for the management and prevention of Mtb infections in humans and animals, and the processes involved in the preparation and use of these diagnostics, vaccines and new chemotherapeutic agents, comprise further aspects of the invention.
DETAILED DESCRIPTION OF THE INVENTION
A. Polynucleotides
Polynucleotides of the invention as defined herein may comprise DNA or RNA. They may also be polynucleotides which include within them synthetic or modified nucleotides or peptide nucleic acids. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to couple the said polynucleotide to a solid phase or to enhance the recognition, the in vivo activity, or the lifespan of polynucleotides of the invention.
A number of different types of polynucleotides of the invention are envisaged. In the broadest aspect, polynucleotides and fragments thereof capable of hybridizing to SEQ ID NO:3 or 4 form a first aspect of the invention. This includes the polynucleotide of SEQ ID NO: 3 or 4. Within this class of polynucleotides various sub-classes of polynucleotides are of particular interest.
One sub-class of polynucleotides which is of interest is the class of polynucleotides encoding the open reading frames A, B, C, D, E, F, G and H, including SEQ ID NOs:5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27. As discussed below, polynucleotides encoding ORF H include the polynucleotide sequences 7953 to 7006 and 7009 to 6215 within SEQ ID NO: 27, as well as modified sequences in which the frame-shift has been modified so that the two sub-reading frames are placed in a single reading frame. This may be desirable where the polypeptide is to be produced in recombinant expression systems.
The invention thus provides a polynucleotide in substantially isolated form which encodes any one of these ORFs or combinations thereof. Combinations thereof includes combinations of 2, 3, 4, 5 or all of the ORFs. Polynucleotides may be provided which comprise an individual ORF carried in a recombinant vector including the vectors described herein. Thus in one preferred aspect the invention provides a polynucleotide in substantially isolated form capable of selectively hybridizing to the nucleic acid comprising ORFs A to F of the core region of the Mptb and Mavs pathogenicity islands of the invention. Fragments thereof corresponding to ORFs A to E, B to F, A to D, B to E, A to C, B to D or any two adjacent ORFs are also included in the invention.
Polynucleotides of the invention will be capable of selectively hybridizing to the corresponding portion of the GS region, or to the corresponding ORFs of Mtb described herein. The term "selectively hybridizing" indicates that the polynucleotides will hybridize, under conditions of medium to high stringency (for example 0.03 M sodium chloride and 0.03 M sodium citrate at from about 50.degree. C. to about 60.degree. C.) to the corresponding portion of SEQ ID NO:3 or 4 or the complementary strands thereof but not to genomic DNA from mycobacteria which are usually non-pathogenic including non-pathogenic species of M.avium. Such polynucleotides will generally be generally at least 68%, e.g. at least 70%, preferably at least 80 or 90% and more preferably at least 95% homologous to the corresponding DNA of GS. The corresponding portion will be of over a region of at least 20, preferably at least 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.
By "corresponding portion" it is meant a sequence from the GS region of the same or substantially similar size which has been determined, for example by computer alignment, to have the greatest degree of homology to the polynucleotide.
Any combination of the above mentioned degrees of homology and minimum sizes may be used to define polynucleotides of the invention, with the more stringent combinations (i.e. higher homology over longer lengths) being preferred. Thus for example a polynucleotide which is at least 80% homologous over 25, preferably 30 nucleotides forms one aspect of the invention, as does a polynucleotide which is at least 90% homologous over 40 nucleotides.
A further class of polynucleotides of the invention is the class of polynucleotides encoding polypeptides of the invention, the polypeptides of the invention being defined in section B below. Due to the redundancy of the genetic code as such, polynucleotides may be of a lower degree of homology than required for selective hybridization to the GS region. However, when such polynucleotides encode polypeptides of the invention these polynucleotides form a further aspect. It may for example be desirable where polypeptides of the invention are produced recombinantly to increase the GC content of such polynucleotides. This increase in GC content may result in higher levels of expression via codon usage more appropriate to the host cell in which recombinant expression is taking place.
An additional class of polynucleotides of the invention are those obtainable from cosmids MTCY277 and MTO24 (containing Mtb genomic sequences), which polynucleotides consist essentially of the fragment of the cosmid containing an open reading frame encoding any one of the homologous ORFs B, C, E or F respectively. Such polynucleotides are referred to below as Mtb polynucleotides. However, where reference is made to polynucleotides in general such reference includes Mtb polynucleotides unless the context is explicitly to the contrary. In addition, the invention provides polynucleotides which encode the same polypeptide as the abovementioned ORFs of Mtb but which, due to the redundancy of the genetic code, have different nucleotide sequences. These form further Mtb polynucleotides of the invention. Fragments of Mtb polynucleotides suitable for use as probes or primers also form a further aspect of the invention.
The invention further provides polynucleotides in substantially isolated form capable of selectively hybridizing (where selectively hybridizing is as defined above) to the Mtb polynucleotides of the invention.
The invention further provides the Mtb polynucleotides of the invention linked, at either the 5' and/or 3' end to polynucleotide sequences to which they are not naturally contiguous. Such sequences will typically be sequences found in cloning or expression vectors, such as promoters, 5' untranslated sequence, 3' untranslated sequence or termination sequences. The sequences may also include further coding sequences such as signal sequences used in recombinant production of proteins.
Further polynucleotides of the invention are illustrated in the accompanying examples.
Polynucleotides of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels or a probe linked covalently to a solid phase, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 or more nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.
Primers of the invention which are preferred include primers directed to any part of the ORFs defined herein. The ORFs from other isolates of pathogenic mycobacteria which contain a GS region may be determined and conserved regions within each individual ORF may be identified. Primers directed to such conserved regions form a further preferred aspect of the invention. In addition, the primers and other polynucleotides of the invention may be used to identify, obtain and isolate ORFs capable of selectively hybridizing to the polynucleotides of the invention which are present in pathogenic mycobacteria but which are not part of a pathogenicity island in that particular species of bacteria. Thus in addition to the ORFs B, C, E and F which have been identified in Mtb, similar ORFs may be identified in other pathogens and ORFs corresponding to the GS ORFs C, D, E, F and H, may also be identified.
Polynucleotides such as DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
In general, primers will be produced by synthetic means, involving a step-wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art. Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair or primers (e.g. of about 15-30 nucleotides) to a region of GS, which it is desired to clone, bringing the primers into contact with genomic DNA from a mycobacterium or a vector carrying the GS sequence, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.
Such techniques may be used to obtain all or part of the GS or ORF sequences described herein, as well as further genomic clones containing full open reading frames. Although in general such techniques are well known in the art, reference may be made in particular to Sambrook J., Fritsch E F., Maniatis T (1989). Molecular cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory.
Polynucleotides which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways.
Other isolates or strains of pathogenic mycobacteria will be expected to contain allelic variants of the GS sequences described herein, and these may be obtained for example by probing genomic DNA libraries made from such isolates or strains of bacteria using GS or ORF sequences as probes under conditions of medium to high stringency (for example 0.03 M sodium chloride and 0.03 M sodium citrate at from about 50.degree. C. to about 60.degree. C.).
A particularly preferred group of pathogenic mycobacteria are isolates of M.paratuberculosis. Polynucleotides based on GS regions from such bacteria are particularly preferred. Preferred fragments of such regions include fragments encoding individual open reading frames including the preferred groups and combinations of open reading frames discussed above.
Alternatively, such polynucleotides may be obtained by site directed mutagenesis of the GS or ORF sequences or allelic variants thereof. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides of the invention. Such altered property or function will include the addition of amino acid sequences of consensus signal peptides known in the art to effect transport and secretion of the modified polypeptide of the invention. Another altered property will include metagenesis of a catalytic residue or generation of fusion proteins with another polypeptide. Such fusion proteins may be with an enzyme, with an antibody or with a cytokine or other ligand for a receptor, to target a polypeptide of the invention to a specific cell type in vitro or in vivo.
The invention further provides double stranded polynucleotides comprising a polynucleotide of the invention and its complement.
Polynucleotides or primers of the invention may carry a revealing label. Suitable labels include radioisotopes such as .sup.32 P or .sup.35 S, enzyme labels, other protein labels or smaller labels such as biotin or fluorophores. Such labels may be added to polynucleotides or primers of the invention and may be detected using by techniques known per se.
Polynucleotides or primers of the invention or fragments thereof labelled or unlabelled may be used by a person skilled in the art in nucleic acid-based tests for the presence or absence of Mptb, Mavs, other GS-containing pathogenic mycobacteria, or Mtb applied to samples of body fluids, tissues, or excreta from animals and humans, as well as to food and environmental samples such as river or ground water and domestic water supplies.
Human and animal body fluids include sputum, blood, serum, plasma, saliva, milk, urine, csf, semen, faeces and infected discharges. Tissues include intestine, mouth ulcers, skin, lymph nodes, spleen, lung and liver obtained surgically or by a biopsy technique. Animals particularly include commercial livestock such as cattle, sheep, goats, deer, rabbits but wild animals and animals in zoos may also be tested.
Such tests comprise bringing a human or animal body fluid or tissue extract, or an extract of an environmental or food sample, into contact with a probe comprising a polynucleotide or primer of the invention under hybridising conditions and detecting any duplex formed between the probe and nucleic acid in the sample. Such detection may be achieved using techniques such as PCR or by immobilising the probe on a solid support, removing nucleic acid in the sample which is not hybridized to the probe, and then detecting nucleic acid which has hybridized to the probe. Alternatively, the sample nucleic acid may be immobilized on a solid support, and the amount of probe bound to such a support can be detected. Suitable assay methods of this any other formats can be found in for example WO89/03891 and WO90/13667.
Polynucleotides of the invention or fragments thereof labelled or unlabelled may also be used to identify and characterise different strains of Mptb, Mavs, other GS-containing pathogenic mycobacteria, or Mtb, and properties such as drug resistance or susceptibility.
The probes of the invention may conveniently be packaged in the form of a test kit in a suitable container. In such kits the probe may be bound to a solid support where the assay format for which the kit is designed requires such binding. The kit may also contain suitable reagents for treating the sample to be probed, hybridising the probe to nucleic acid in the sample, control reagents, instructions, and the like.
The use of polynucleotides of the invention in the diagnosis of inflammatory diseases such as Crohn's disease or sarcoidosis in humans or Johne's disease in animals form a preferred aspect of the invention. The polynucleotides may also be used in the prognosis of these diseases. For example, the response of a human or animal subject in response to antibiotic, vaccination or other therapies may be monitored by utilizing the diagnostic methods of the invention over the course of a period of treatment and following such treatment.
The use of Mtb polynucleotides (particularly in the form of probes and primers) of the invention in the above-described methods form a further aspect of the invention, particularly for the detection, diagnosis or prognosis of Mtb infections.
B. Polypeptides
Polypeptides of the invention include polypeptides in substantially isolated form encoded by GS. This includes the full length polypeptides encoded by the positive and complementary negative strands of GS. Each of the full length polypeptides will contain one of the amino acid sequences set out in Seq ID NOs:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 29. Polypeptides of the invention further include variants of such sequences, including naturally occurring allelic variants and synthetic variants which are substantially homologous to said polypeptides. In this context, substantial homology is regarded as a sequence which has at least 70%, e.g. 80%, 90%, 95% or 98% amino acid homology (identity) over 30 or more, e.g 40, 50 or 100 amino acids. For example, one group of substantially homolgous polypeptides are those which have at least 95% amino acid identity to a polypeptide of any one of Seq ID NOs:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 29 over their entire length. Even more preferably, this homology is 98%.
Polypeptides of the invention further include the polypeptide sequences of the homologous ORFs of Mtb, namely Seq ID Nos. 31, 33, 35, 37 and 39. Unless explicitly specified to the contrary, reference to polypeptides of the invention and their fragments include these Mtb polypeptides and fragments, and variants thereof (substanially homologous to said sequences) as defined herein.
Polypeptides of the invention may be obtained by the standard techniques mentioned above. Polypeptides of the invention also include fragments of the above mentioned full length polypeptides and variants thereof, including fragments of the sequences set out in SEQ ID NOs:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 29, 31, 33, 35, 37 and 39. Such fragments for example of 8, 10, 12, 15 or up to 30 or 40 amino acids may also be obtained synthetically using standard techniques known in the art.
Preferred fragments include those which include an epitope, especially an epitope which is specific to the pathogenicity of the mycobacterial cell from which the polypeptide is derived. Suitable fragments will be at least about 5, e.g. 8, 10, 12, 15 or 20 amino acids in size, or larger. Epitopes may be determined either by techniques such as peptide scanning techniques as described by Geysen et al, Mol. Immunol., 23; 709-715 (1986), as well as other techniques known in the art.
The term "an epitope which is specific to the pathogenicity of the mycobacterial cell" means that the epitope is encoded by a portion of the GS region, or by the corresponding ORF sequences of Mtb which can be used to distinguish mycobacteria which are pathogenic by from related non-pathogenic mycobacteria including non-pathogenic species of M.avium. This may be determined using routine methodology. A candidate epitope from an ORF may be prepared and used to immunise an animal such as a rat or rabbit in order to generate antibodies. The antibodies may then be used, to detect the presence of the epitope in pathogenic mycobacteria and to confirm that non-pathogenic mycobacteria do not contain any proteins which react with the epitope. Epitopes may be linear or conformational.
Polypeptides of the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated. A polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the polypeptide in the preparation is a polypeptide of the invention.
Polypeptides of the invention may be modified to confer a desired property or function for example by the addition of Histidine residues to assist their purification or by the addition of a signal sequence to promote their secretion from a cell.
Thus, polypeptides of the invention include fusion proteins which comprise a polypeptide encoding all or part of one or more of an ORF of the invention fused at the N- or C-terminus to a second sequence to provide the desired property or function. Sequences which promote secretion from a cell include, for example the yeast .alpha.-factor signal sequence.
A polypeptide of the invention may be labelled with a revealing label. The revealing label may be any suitable label which allows the polypeptide to be detected. Suitable labels include radioisotopes, e.g. .sup.125 I, .sup.35 S enzymes, antibodies, polynucleotides and ligands such as biotin. Labelled polypeptides of the invention may be used in diagnostic procedures such as immunoassays in order to determine the amount of a polypeptide of the invention in a sample. Polypeptides or labelled polypeptides of the invention may also be used in serological or cell mediated immune assays for the detection of immune reactivity to said polypeptides in animals and humans using standard protocols.
A polypeptide or labelled polypeptide of the invention or fragment thereof may also be fixed to a solid phase, for example the surface of an immunoassay well, microparticle, dipstick or biosensor. Such labelled and/or immobilized polypeptides may be packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.
Such polypeptides and kits may be used in methods of detection of antibodies or cell mediated immunoreactivity, to the mycobacterial proteins and peptides encoded by the ORFs of the invention and their allelic variants and fragments, using immunoassay. Such host antibodies or cell mediated immune reactivity will occur in humans or animals with an immune system which detects and reacts against polypeptides of the invention. The antibodies may be present in a biological sample from such humans or animals, where the biological sample may be a sample as defined above particularly blood, milk or saliva.
Immunoassay methods are well known in the art and will generally comprise:
(a) providing a polypeptide of the invention comprising an epitope bindable by an antibody against said mycobacterial polypeptide;
(b) incubating a biological sample with said polypeptide under conditions which allow for the formation of an antibody-antigen complex; and
(c) determining whether antibody-antigen complex comprising said polypeptide is formed.
Immunoassay methods for cell mediated immune reactivity in animals and humans are also well known in the art (e.g. as described by Weir et al 1994, J. Immunol Methods 176; 93-101) and will generally comprise
(a) providing a polypeptide of the invention comprising an epitope bindable by a lymphocyte or macrophage or other cell receptor;
(b) incubating a cell sample with said polypeptide under conditions which allow for a cellular immune response such as release of cytokines or other mediator to occur; and
(c) detecting the presence of said cytokine or mediator in the incubate.
Polypeptides of the invention may be made by standard synthetic means well known in the art or recombinantly, as described below.
Polypeptides of the invention or fragments thereof labelled or unlabelled may also be used to identify and characterise different strains of Mptb, Mavs, other GS-containing pathogenic mycobacteria, or Mtb, and properties such as drug resistance or susceptibility.
The polypeptides of the invention may conveniently be packaged in the form of a test kit in a suitable container. In such kits the polypeptide may be bound to a solid support where the assay format for which the kit is designed requires such binding. The kit may also contain suitable reagents for treating the sample to be examined, control reagents, instructions, and the like.
The use of polypeptides of the invention in the diagnosis of inflammatory diseases such as Crohn's disease or sarcoidosis in humans or Johne's disease in animals form a preferred aspect of the invention. The polypeptides may also be used in the prognosis of these diseases. For example, the response of a human or animal subject in response to antibiotic or other therapies may be monitored by utilizing the diagnostic methods of the invention over the course of a period of treatment and following such treatment.
The use of Mtb polypeptides of the invention in the above-described methods form a further aspect of the invention, particularly for the detection, diagnosis or prognosis of Mtb infections.
Polypeptides of the invention may also be used in assay methods for identifying candidate chemical compounds which will be useful in inhibiting, binding to or disrupting the function of said polypeptides required for pathogenicity. In general, such assays involve bringing the polypeptide into contact with a candidate inhibitor compound and observing the ability of the compound to disrupt, bind to or interfer with the polypeptide.
There are a number of ways in which the assay may be formatted. For example, those polypeptides which have an enzymatic function may be assayed using labelled substrates for the enzyme, and the amount of, or rate of, conversion of the substrate into a product measured, e.g by chromatograpy such as HPLC or by a colourimetric assay. Suitable labels include .sup.35 S, .sup.125 I, biotin or enzymes such as horse radish peroxidase.
For example, the gene product of ORF C is believed to have GDP-mannose dehydratase activty. Thus an assay for inhbitors of the gene product may utilise for example labelled GDP-mannose, GDP or mannose and the activity of the gene product followed. ORF D encodes a gene related to the synthesis and regulation of capuslar polysaccharides, which are often associated with invasiveness and pathogenicity. Labelled polysaccharide substrates may be used in assays of the ORF D gene product. The gene product of ORF F encodes a protein with putative glucosyl transferase activity and thus labelled amino sugars such as .beta.-1-3-N-acetylglucosamine may be used as substrates in assays.
Candidate chemical compounds which may be used may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants which contain several characterised or uncharacterised components may also be used.
Alternatively, the a polypeptide of the invention may be screened against a panel of peptides, nucleic acids or other chemical functionalities which are generated by combinatorial chemistry. This will allow the definition of chemical entities which bind to polypeptides of the invention. Typically, the polypeptide of the invention will be brought into contact with a panel of compounds from a combinantorial library, with either the panel or the polypeptide being immobilized on a solid phase, under conditions suitable for the polypeptide to bind to the panel. The solid phase will then be washed under conditions in which only specific interactions between the polypeptide and individual members of the panel are retained, and those specific members may be utilized in further assays or used to design further panels of candidate compounds.
For example, a number of assay methods to define peptide interaction with peptides are known. For example, WO86/00991 describes a method for determining mimotopes which comprises making panels of catamer preparations, for example octamers of amino acids, at which one or more of the positions is defined and the remaining positions are randomly made up of other amino acids, determining which catamer binds to a protein of interest and re-screening the protein of interest against a further panel based on the most reactive catamer in which one or more additional designated positions are systematically varied. This may be repeated throughout a number of cycles and used to build up a sequence of a binding candidate compound of interest.
WO89/03430 describes screening methods which permit the preparation of specific mimotopes which mimic the immunological activity of a desired analyte. These mimotopes are identified by reacting a panel of individual peptides wherein said peptides are of systematically varying hydrophobicity, amphipathic characteristics and charge patterns, using an antibody against an antigen of interest. Thus in the present case antibodies against the a polypeptide of the inventoin may be employed and mimotope peptides from such panels may be identified.
C. Vectors
Polynucleotides of the invention can be incorporated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described below in connection with expression vectors.
D. Expression Vectors
Preferably, a polynucleotide of the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Such vectors may be transformed into a suitable host cell as described above to provide for expression of a polypeptide of the invention. Thus, in a further aspect the invention provides a process for preparing polypeptides according to the invention which comprises cultivating a host cell transformed or transfected with an expression vector as described above, under conditions to provide for expression by the vector of a coding sequence encoding the polypeptides, and recovering the expressed polypeptides.
A further embodiment of the invention provides vectors for the replication and expression of polynucleotides of the invention, or fragments thereof. The vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell. The vector may also be adapted to be used in vivo, for example in a method of naked DNA vaccination or gene therapy. A further embodiment of the invention provides host cells transformed or transfected with the vectors for the replication and expression of polynucleotides of the invention, including the DNA of GS, the open reading frames thereof and other corresponding ORFs particularly ORFs B, C, B and F from Mtb. The cells will be chosen to be compatible with the said vector and may for example be bacterial, yeast, insect or mammalian.
Expression vectors are widely available in the art and can be obtained commercially. Mammalian expression vectors may comprise a mammalian or viral promoter. Mammalian promoters include the metallothionien promoter. Viral promoters include promoters from adenovirus, the SV40 large T promoter and retroviral LTR promoters. Promoters compatible with insect cells include the polyhedrin promoter. Yeast promoters include the alcohol dehydrogenase promoter. Bacterial promoters include the .beta.-galactosidase promoter.
The expression vectors may also comprise enhancers, and in the case of eukaryotic vectors polyadenylation signal sequence downstream of the coding sequence being expressed.
Polypeptides of the invention may be expressed in suitable host cells, for example bacterial, yeast, plant, insect and mammalian cells, and recovered using standard purification techniques including, for example affinity chromatography, HPLC or other chromatographic separation techniques.
Polynucleotides according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense polynucleotides or ligands may also be produced by synthetic means. Such antisense polynucleotides may be used in a method of controlling the levels of the proteins encoded by the ORFs of the invention in a mycobacterial cell.
Polynucleotides of the invention may also be carried by vectors suitable for gene therapy methods. Such gene therapy methods include those designed to provide vaccination against diseases caused by pathogenic mycobacteria or to boost the immune response of a human or animal infected with a pathogenic mycobacteria.
For example, Ziegner et al, AIDS, 1995, 9;43-50 describes the use of a replication defective recombinant amphotropic retrovirus to boost the immune response in patients with HIV infection. Such a retrovirus may be modified to carry a polynucleotide encoding a polypeptide or fragment thereof of the invention and the retrovirus delivered to the cells of a human or animal subject in order to provide an immune response against said polypeptide. The retrovirus may be delivered directly to the patient or may be used to infecte cells ex-vivo, e.g. fibroblast cells, which are then introduced into the patient, optionally after being inactivated. The cells are desirably autologous or HLA-matched cells from the human or animal subject.
Gene therapy methods including methods for boosting an immune response to a particluar pathogen are disclosed generally in for example WO95/14091, the disclosure of which is incoporated herein by reference. Recombinant viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia virus vectors, herpes virus vectors and alphavirus vectors. Alpha virus vectors are described in, for example, WO95/07994, the disclosure of which is incorporated herein by reference.
Where direct administration of the recombinant viral vector is contemplated, either in the form of naked nucleic acid or in the form of packaged particles carrying the nucleic acid this may be done by any suitable means, for example oral administration or intravenous injection. From 10.sup.5 to 10.sup.8 c.f.u of virus represents a typical dose, which may be repeated for example weekly over a period of a few months. Administration of autologous or HLA-matched cells infected with the virus may be more convenient in some cases. This will generally be achieved by administering doses, for example from 10.sup.5 to 10.sup.8 cells per dose which may be repeated as described above.
The recombinant viral vector may further comprise nucleic acid capable of expressing an accessory molecule of the immune system designed to increase the immune response. Such a moleclue may be for example and interferon, particularly interferon gamma, an interleukin, for example IL-1.alpha., IL-1.beta. or IL-2, or an HLA class I or II moleclue. This may be particularly desirable where the vector is intended for use in the treatment of humans or animals already infected with a mycobacteria and it is desired to boost the immune response.
E. Antibodies
The invention also provides monoclonal or polyclonal antibodies to polypeptides of the invention or fragments thereof. The invention further provides a process for the production of monoclonal or polyclonal antibodies to polypeptides of the invention. Monoclonal antibodies may be prepared by conventional hybridoma technology using the polypeptides of the invention or peptide fragments thereof, as immunogens. Polyclonal antibodies may also be prepared by conventional means which comprise inoculating a host animal, for example a rat or a rabbit, with a polypeptide of the invention or peptide fragment thereof and recovering immune serum.
In order that such antibodies may be made, the invention also provides polypeptides of the invention or fragments thereof haptenised to another polypeptide for use as immunogens in animals or humans.
For the purposes of this invention, the term "antibody", unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a polypeptide of the invention. Such fragments include Fv, F(ab') and F(ab').sub.2 fragments, as well as single chain antibodies. Furthermore, the antibodies and fragments thereof may be humanised antibodies, e.g. as described in EP-A-239400.
Antibodies may be used in methods of detecting polypeptides of the invention present in biological samples (where such samples include the human or animal body samples, and environmental samples, mentioned above) by a method which comprises:
(a) providing an antibody of the invention;
(b) incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and
(c) determining whether antibody-antigen complex comprising said antibody is formed.
Antibodies of the invention may be bound to a solid support for example an immunoassay well, microparticle, dipstick or biosensor and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.
Antibodies of the invention may be used in the detection, diagnosis and prognosis of diseases as descirbed above in relation to polypeptides of the invention.
F. Compositions
The present invention also provides compositions comprising a polynucleotide or polypeptide of the invention together with a carrier or diluent. Compositions of the invention also include compositions comprising a nucleic acid, particularly and expression vector, of the invention. Compositions further include those carrying a recombinant virus of the invention. Such compositions include pharmaceutical compositions in which case the carrier or diluent will be pharmaceutically acceptable.
Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for inhalation as well as oral, parenteral (e.g. intramuscular or intravenous or transcutaneous) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
For example, formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the polynucleotide or the polypeptide of the invention to blood components or one or more organs, or to target cells such as M cells of the intestine after oral administration.
G. Vaccines
In another aspect, the invention provides novel vaccines for the prevention and treatment of infections caused by Mptb, Mavs, other GS-containing pathogenic mycobacteria and Mtb in animals and humans. The term "vaccine" as used herein means an agent used to stimulate the immune system of a vertebrate, particularly a warm blooded vertebrate including humans, so as to provide protection against future harm by an organism to which the vaccine is directed or to assist in the eradication of an organism in the treatment of established infection. The immune system will be stimulated by the production of cellular immunity antibodies, desirably neutralizing antibodies, directed to epitopes found on or in a pathogenic mycobacterium which expresses any one of the ORFs of the invention. The antibody so produced may be any of the immunological classes, such as the immunoglobulins A, D, E, G or M. Vaccines which stimulate the production of IgA are interest since this is the principle immunoglobulin produced by the secretory system of warm-blooded animals, and the production of such antibodies will help prevent infection or colonization of the intestinal tract. However an IgM and IgG response will also be desirable for systemic infections such as Crohn's disease or tuberculosis.
Vaccines of the invention include polynucleotides of the invention or fragments thereof in suitable vectors and administered by injection of naked DNA using standard protocols. Polynucleotides of the invention or fragments thereof in suitable vectors for the expression of the polypeptides of the invention may be given by injection, inhalation or by mouth. Suitable vectors include M.bovis BCG, M.smegmatis or other mycobacteria, Corynebacteria, Salmonella or other agents according to established protocols.
Polypeptides of the invention or fragments thereof in substantially isolated form may be used as vaccines by injection, inhalation, oral administration or by transcutaneous application according to standard protocols. Adjuvants (such as Iscoms or polylactide-coglycolide encapsulation), cytokines such as IL-12 and other immunomodulators may be used for the selective enhancement of the cell mediated or humoral immunological responses. Vaccination with polynucleotides and/or polypeptides of the invention may be undertaken to increase the susceptibility of pathogenic mycobacteria to antimicrobial agents in vivo.
In instances wherein the polypeptide is correctly configured so as to provide the correct epitope, but is too small to be immunogenic, the polypeptide may be linked to a suitable carrier.
A number of techniques for obtaining such linkage are known in the art, including the formation of disulfide linkages using N-succinimidyl-3-(2-pyridylthio) propionate (SPDP) and succinimidyl 4-(N-maleimido-methyl)cyclohexane-l-carboxylate (SMCC) obtained from Pierce Company, Rockford, Ill., (if the peptide lacks a sulfhydryl group, this can be provided by addition of a cysteine residue). These reagents create a disulfide linkage between themselves and peptide cysteine residues on one protein and an amide linkage through the epsilon-amino on a lysine, or other free amino group in the other. A variety of such disulfide/amide-forming agents are known. See, for example, Immun Rev (1982) 62:185. Other bifunctional coupling agents form a thioether rather than a disulfide linkage. Many of these thioether-forming agents are commercially available and include reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid, and the like. The carboxyl group can be activated by combining them with succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt. Additional methods of coupling antigens employs the rotavirus/"binding peptide" system described in EPO Pub. No. 259,149, the disclosure of which is incorporated herein by reference. The foregoing list is not meant to be exhaustive, and modifications of the named compounds can clearly be used.
Any carrier may be used which does not itself induce the production of antibodies harmful to the host. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins; polysaccharides, such as latex functionalized Sepharose.RTM., agarose, cellulose, cellulose beads and the like; polymeric amino acids, such as polyglutamic acid, polylysine, polylactide-coglycolide and the like; amino acid copolymers; and inactive virus particles. Especially useful protein substrates are serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well known to those skilled in the art.
The immunogenicity of the epitopes may also be enhanced by preparing them in mammalian or yeast systems fused with or assembled with particle-forming proteins such as, for example, that associated with hepatitis B surface antigen. See, e.g., U.S. Pat. No. 4,722,840. Constructs wherein the epitope is linked directly to the particle-forming protein coding sequences produce hybrids which are immunogenic with respect to the epitope. In addition, all of the vectors prepared include epitopes specific to HBV, having various degrees of immunogenicity, such as, for example, the pre-S peptide.
In addition, portions of the particle-forming protein coding sequence may be replaced with codons encoding an epitope of the invention. In this replacement, regions which are not required to mediate the aggregation of the units to form immunogenic particles in yeast or mammals can be deleted, thus eliminating additional HBV antigenic sites from competition with the epitope of the invention.
Vaccines may be prepared from one or more immunogenic polypeptides of the invention. These polypeptides may be expressed in various host cells (e.g., bacteria, yeast, insect, or mammalian cells), or alternatively may be isolated from viral preparations or made synthetically.
In addition to the above, it is also possible to prepare live vaccines of attenuated microorganisms which express one or more recombinant polypeptides of the invention. Suitable attenuated microorganisms are known in the art and include, for example, viruses (e.g., vaccinia virus), as well as bacteria.
The preparation of vaccines which contain an immunogenic polypeptide(s) as active ingredients, is known to one skilled in the art. Typically, such vaccines are prepared as injectables, or as suitably encapsulated oral preparations and either liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injestion or injection may also be prepared. The preparation may also be emulsified, or the protein encapsulated in liposomes. The active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween.RTM. 80 emulsion. The effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against an immunogenic polypeptide containing an antigenic sequence resulting from administration of this polypeptide in vaccines which are also comprised of the various adjuvants.
The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories, oral formulations or as enemas. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25%-70%.
The proteins may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective. The quantity to be administered, which is generally in the range of 5 .mu.g to 250 .mu.g, of antigen per dose, depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, mode of administration and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgement of the practitioner and may be peculiar to each subject.
The vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reenforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. The dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgement of the practitioner.
In a further aspect of the invention, there is provided an attenuated vaccine comprising a normally pathogenic mycobacteria which harbours an attenuating mutation in any one of the genes encoding a polypeptide of the invention. The gene is selected from the group of ORFs A, B, C, D, E, F, G and H, including the homologous ORFs B, C, E and F in Mtb.
The mycobacteria may be used in the form of killed bacteria or as a live attenuated vaccine. There are advantages to a live attenuated vaccine. The whole live organism is used, rather than dead cells or selected cell components which may exhibit modified or denatured antigens. Protein antigens in the outer membrane will maintain their tertiary and quaternary structures. Therefore the potential to elicit a good protective long term immunity should be higher.
The term "mutation" and the like refers to a genetic lesion in a gene which renders the gene non-functional. This may be at either the level of transcription or translation. The term thus envisages deletion of the entire gene or substantial portions thereof, and also point mutations in the coding sequence which result in truncated gene products unable to carry out the normal function of the gene.
A mutation introduced into a bacterium of the invention will generally be a non-reverting attenuating mutation. Non-reverting means that for practical purposes the probability of the mutated gene being restored to its normal function is small, for example less than 1 in 10.sup.6 such as less than 1 in 10.sup.9 or even less than 1 in 10.sup.12.
An attenuated mycobacteria of the invention may be in isolated form. This is usually desirable when the bacterium is to be used for the purposes of vaccination. The term "isolated" means that the bacterium is in a form in which it can be cultured, processed or otherwise used in a form in which it can be readily identified and in which it is substantially uncontaminated by other bacterial strains, for example non-attenuated parent strains or unrelated bacterial strains. The term "isolated bacterium" thus encompasses cultures of a bacterial mutant of the invention, for example in the form of colonies on a solid medium or in the form of a liquid culture, as well as frozen or dried preparations of the strains.
In a preferred aspect, the attenuated mycobacterium further comprises at least one additional mutation. This may be a mutation in a gene responsible for the production of products essential to bacterial growth which are absent in a human or animal host. For example, mutations to the gene for aspartate semi-aldehyde dehydrogenase (asd) have been proposed for the production of attenuated strains of Salmonella. The asd gene is described further in Gene (1993) 129; 123-128. A lesion in the asd gene, encoding the enzyme aspartate .beta.-semialdehyde dehydrogenase would render the organism auxotrophic for the essential nutrient diaminopelic acid (DAP), which can be provided exogenously during bulk culture of the vaccine strain. Since this compound is an essential constituent of the cell wall for gram-negative and some gram-positive organisms and is absent from mammalian or other vertebrate tissues, mutants would undergo lysis after about three rounds of division in such tissues. Analogous mutations may be made to the attenuated mycobacteria of the invention.
In addition or in the alternative, the attenuated mycobacteria may carry a recA mutation. The recA mutation knocks out homologous recombination--the process which is exploited for the construction of the mutations. Once the recA mutation has been incorporated the strain will be unable to repair the constructed deletion mutations. Such a mutation will provide attenuated strains in which the possibility of homologous recombination to with DNA from wild-type strains has been minimized. RecA genes have been widely studied in the art and their sequences are available. Further modifications may be made for additional safety.
The invention further provides a process for preparing a vaccine composition comprising an attenuated bacterium according to the invention process comprises (a) inoculating a culture vessel containing a nutrient medium suitable for growth of said bacterium; (b) culturing said bacterium; (c) recovering said bacteria and (d) mixing said bacteria with a pharmaceutically acceptable diluent or carrier.
Attenuated bacterial strains according to the invention may be constructed using recombinant DNA methodology which is known per se. In general, bacterial genes may be mutated by a process of targeted homologous recombination in which a DNA construct containing a mutated form of the gene is introduced into a host bacterium which it is desired to attenuate. The construct will recombine with the wild-type gene carried by the host and thus the mutated gene may be incorporated into the host genome to provide a bacterium of the present invention which may then be isolated.
The mutated gene may be obtained by introducing deletions into the gene, e.g by digesting with a restriction enzyme which cuts the coding sequence twice to excise a portion of the gene and then religating under conditions in which the excised portion is not reintroduced into the cut gene. Alternatively frame shift mutations may be introduced by cutting with a restriction enzyme which leaves overhanging 5' and 3' termini, filling in and/or trimming back the overhangs, and religating. Similar mutations may be made by site directed mutagenesis. These are only examples of the types of techniques which will readily be at the disposal of those of skill in the art.
Various assays are available to detect successful recombination. In the case of attenuations which mutate a target gene necessary for the production of an essential metabolite or catabolite compound, selection may be carried out by screening for bacteria unable to grow in the absence of such a compound. Bacteria may also be screened with antibodies or nucleic acids of the invention to determine the absence of production of a mutated gene product of the invention or to confirm that the genetic lesion introduced--e.g. a deletion--has been incorporated into the genome of the attenuated strain.
The concentration of the attenuated strain in the vaccine will be formulated to allow convenient unit dosage forms to be prepared. Concentrations of from about 10.sup.4 to 10.sup.9 bacteria per ml will generally be suitable, e.g. from about 10.sup.5 to 10.sup.8 such as about 10.sup.6 per ml. Live attenuated organisms may be administered subcutaneously or intramuscularly at up to 10.sup.8 organisms in one or more doses, e.g from around 10.sup.5 to 10.sup.8, e.g about 10.sup.6 or 10.sup.7 organisms in a single dose.
The vaccines of the invention may be administered to recipients to treat established disease or in order to protect them against diseases caused by the corresponding wild type mycobacteria, such as inflammatory diseases such as Crohn's disease or sarcoidosis in humans or Johne's disease in animals. The vaccine may be administered by any suitable route. In general, subcutaneous or intramuscular injection is most convenient, but oral, intranasal and colorectal administration may also be used.
The following Examples illustrates aspects of the invention.
EXAMPLE 1
Tests for the presence of the GS identifier sequence were performed on 5 .mu.l bacterial DNA extracts (25 .mu.g/ml to 500 .mu.g/ml) using polymerase chain reaction based on the oligonucleotide primers 5'-GATGCCGTGAGGAGGTAAAGCTGC-3' (Seq ID No. 40) and 5'-G ATACGGCTCTTGAATCCTGCACG-3' (Seq ID No. 41) from within the identifier DNA sequences (Seq. ID Nos 1 and 2). PCR was performed for 40 cycles in the presence of 1.5 mM magnesium and an annealing temperature of 58.degree. C. The presence or absence of the correct amplification product indicated the presence or absence of GS identifier sequence in the corresponding bacterium. GS identifier sequence is shown to be present in all the laboratory and field strains of Mptb and Mavs tested. This includes Mptb isolates 0025 (bovine CVL Weybridge), 0021 (caprine, Moredun), 0022 (bovine, Moredun), 0139 (human, Chiodini 1984), 0209, 0208, 0211, 0210, 0212, 0207, 0204, 0206 (bovine, Whipple 1990). All Mptb strains were IS900 positive. The Mavs strains include 0010 and 0012 (woodpigeon, Thorel) 0018 (armadillo, Portaels) and 0034, 0037, 0038, 0040 (AIDS, Hoffner). All Mavs strains were IS902 positive. One pathogenic M.avium strain 0033 (AIDS, Hoffner) also contained GS identifier sequence. GS identifier sequence is absent from other mycobacteria including other M.avium, M.malmoense, M.szulgai, M.gordonae, M.chelonei, M.fortuitum, M.phlei, as well as E.coli, S.areus, Nocardia sp, Streptococcus sp. Shigella sp. Pseudomonas sp.
EXAMPLE 2
To obtain the full sequence of GS in Mavs and Mptb we generated a genomic library of Mavs using the restriction endonuclease EcoRI and cloning into the vector pUC18. This achieved a representative library which was screened with .sup.32 P-labelled identifier sequence yielding a positive clone containing a 17 kbp insert. We constructed a restriction map of this insert and identified GS as fragments unique to Mavs and Mptb and not occurring in laboratory strains of M.avium. These fragments were sub-cloned into pUC18 and pGEM4Z. We identified GS contained within an 8 kb region. The full nucleotide sequence was determined for GS on both DNA strands using primer walking and automated DNA sequencing. DNA sequence for GS in Mptb was obtained using overlapping PCR products generated using PwoDNA polymerase, a proofreading thermostable enzyme. The final DNA sequences were derived using the University of Wisconsin GCG gel assembly software package.
EXAMPLE 3
The DNA sequence of GS in Mavs and Mptb was found to be more than 99% homologous. The ORFs encoded in GS were identified using GeneRunner and DNAStar computer programmes. Eight ORFs were identified and designated GSA, GSB, GSC, GSD, GSE, GSF, GSG and GSH. Database comparisons were carried out against the GenEMBL Database release version 48.0 (9/96), using the BLAST and BLIXEM programmes. GSA and GSB encoded proteins of 13.5 kDa and 30.7 kDa respectively, both of unknown functions. GSC encoded a protein of 38.4 kDa with a 65% homology to the amino acid sequence of rfbD of V.cholerae, a 62% amino acid sequence homology to gmd of E. coli and a 58% homology to gca of Ps.aeruginosa which are all GDP-D-mannose dehydratases. Equivalent gene products in H.influenzae, S.dysenteriae, Y.enterocolitica, N.gonorrhoea, K.pneumoniae and rfbD in Salmonella enterica are all involved in `O`-antigen processing known to be linked to pathogenicity. GSD encoded a protein of 37.1 kDa which showed 58% homology at the DNA level to wcaG from E.coli, a gene involved in the synthesis and regulation of capsular polysaccharides, also related to pathogenicity. GSE was found to have a >30% amino acid homology to rfbT of V. cholerae, involved in the transport of specific LPS components across the cell membrane. In V. cholerae the gene product causes a seroconversion from the Inaba to the Ogawa `epidemic` strain. GSF encoded a protein of 30.2 kDa which was homologous in the range 25-40% at the amino acid level to several glucosyl transferases such as rfpA of K.pneumoniae, rfbB of K.pneumoniae, lgtD of H.influenzae, lsi of N. gonorrhoae. In E. coli an equivalent gene galE adds .beta.-1-3 N-acetylglucosamine to galactose, the latter only found in `O` and `M` antigens which are also related to pathogenicity. GSH comprising the ORFs GSH.sub.1 and GSH.sub.2 encodes a protein totalling about 60 kDa which is a putative transposase with a 40-43% homology at the amino acid level to the equivalent gene product of IS21 in E.coli. This family of insertion sequences is broadly distributed amongst gram negative bacteria and is responsible for mobility and transposition of genetic elements. An IS21- like element in B.fragilis is split either side of the .beta.-lactamase gene controlling its activation and expression. We programmed an E.coli S30 cell-free extract with plasmid DNA containing the ORF GSH under the control of a lac promoter in the presence of a .sup.35 S-methionine, and demonstrated the translation of an abundant 60 kDa protein. The proteins homologous to GS encoded in other organisms are in general highly antigenic. Thus the proteins encoded by the ORFs in GS may be used in immunoassays of antibody or cell mediated immuno-reactivity for diagnosing infections caused by mycobacteria, particularly Mptb, Mavs and Mtb. Enhancement of host immune recognition of GS encoded proteins by vaccination using naked specific DNA or recombinant GS proteins, may be used in the prevention and treatment of infections caused by Mptb, Mavs and Mtb in humans and animals. Mutation or deletion of all or some of the ORFs A to H in GS may be used to generate attenuated strains of Mptb, Mavs or Mtb with lower pathogenicity for use as living or killed vaccines in humans and animals. Such vaccines are particularly relevant to Johne's disease in animals, to diseases caused by Mptb in humans such as Crohn's disease, and to the management of tuberculosis especially where the disease is caused by multiple drug-resistant organisms.
__________________________________________________________________________# SEQUENCE LISTING- <160> NUMBER OF SEQ ID NOS: 41- <210> SEQ ID NO 1<211> LENGTH: 674<212> TYPE: DNA<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 1- gatccaacta aacccgatgg aaccccgcgc aaactattgg acgtctccgc gc - #tacgcagt 60- tgggttggcg cccgcgaatc gcactgaaag agggcatcga tgcaacggtg tc - #gtggtacc 120- gcacaaatgc cgatgccgtg aggaggtaaa gctgcgggcc ggccgatgtt at - #ccctccgg 180- ccggacgggt agggcgacct gccatcgagt ggtacggcag tcgcctggcc gg - #cgaggcgc 240- atggcctatg tgagtatccc atagcctggc ttggctcgcc cctacgcatt at - #cagttgac 300- cgctttcgcg ccacgtcgca ggcttgcggc agcatcccgt tcaggtctcc tc - #atggtccg 360- gtgtggcacg accacgcaag ctcgaaccga ctcgtttccc aatttcgcat gc - #taatatcg 420- ctcgatggat tttttgcgca acgccggctt gatggctcgt aacgttagca cc - #gagatgct 480- gcgccactcc gaacgaaagc gcctattagt aaaccaagtc gaagcatacg ga - #gtcaacgt 540- tgttattgat gtcggtgcta actccggcca gttcggtagc gctttgcgtc gt - #gcaggatt 600- caagagccgt atcgtttcct ttgaacctct ttcggggcca tttgcgcaac ta - #acgcgcaa 660# 674- <210> SEQ ID NO 2<211> LENGTH: 674<212> TYPE: DNA<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 2- gatccgatgc cgacttgcgc gttagttgcg caaatggccc cgaaagaggt tc - #aaaggaaa 60- cgatacggct cttgaatcct gcacgacgca aagcgctacc gaactggccg ga - #gttagcac 120- cgacatcaat aacaacgttg actccgtatg cttcgacttg gtttactaat ag - #gcgctttc 180- gttcggagtg gcgcagcatc tcggtgctaa cgttacgagc catcaagccg gc - #gttgcgca 240- aaaaatccat cgagcgatat tagcatgcga aattgggaaa cgagtcggtt cg - #agcttgcg 300- tggtcgtgcc acaccggacc atgaggagac ctgaacggga tgctgccgca ag - #cctgcgac 360- gtggcgcgaa agcggtcaac tgataatgcg taggggcgag ccaagccagg ct - #atgggata 420- ctcacatagg ccatgcgcct cgccggccag gcgactgccg taccactcga tg - #gcaggtcg 480- ccctacccgt ccggccggag ggataacatc ggccggcccg cagctttacc tc - #ctcacggc 540- atcggcattt gtgcggtacc acgacaccgt tgcatcgatg ccctctttca gt - #gcgattcg 600- cgggcgccaa cccaactgcg tagcgcggag acgtccaata gtttgcgcgg gg - #ttccatcg 660# 674- <210> SEQ ID NO 3<211> LENGTH: 7995<212> TYPE: DNA<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 3- gaattctggg ttggagacga cgtcgaactc ctggtcggtc ttgcttcgaa tg - #atcgctgt 60- gatctggtcg gcggtgccga caggaaccgt cgacttgtcg acgatcacct tg - #taccggtc 120- gatgtatgac ccaatgtcgt ccgcaaccga gaagacgtac gtcaggtccg cc - #gccccgct 180- ttcacccatg ggcgtcggga cggcgatgaa aatgacgtcc gcgtgctcga tt - #ccgcgttg 240- ccggtcggtg gtgaagtcaa tcagcccgtt ctcacggttc ctcgcaatca ac - #tcccaacc 300- cgggctcgaa aatcgggaca ctgcctgcga ggagcaaatc gatcttggcc tg - #atcgatat 360- cgacacagac gacatcgttg ccgctatccg cgagacaggc gcccgtgacg ag - #gcctacat 420- agcctgatcc gaccaccgaa attttcaaga tgaccccttc aagtccccga tc - #ggtcgacg 480- accatactgc cgcaactctg taccctccgt gggtaattcg catgtcgcgt tc - #gtaaggag 540- cagccagcga gtcggggacg ttcggtgaga gagtcgcagg actacgaggt tg - #ccggtgcg 600- atacatcaca gtgttgcgtc tgtcggcaac gatgcagcaa gaacccacgg gg - #cagccctg 660- aactgcgcgc atgaccggtc cttgtcctgg cacctttgat cggccaccgc tt - #ccatgcga 720- acatgaccgg aatccatagc gcgtggtcaa gcagcgggga ggtagacgtc gg - #tgtcatct 780- gctccaaccg tgtcggtgat aacgatttcg ctgaacgatc tcgagggatt ga - #aaagcacc 840- gtggagagcg ttcgcgcgca gcgctatggg gggcgaatcg agcacatcgt ca - #tcgacggt 900- ggatcgggcg acgccgtcgt ggagtatctg tccggcgatc ctggctttgc at - #attggcaa 960- tctcagcccg acaacgggag atatgacgcg atgaatcagg gcattgccca tt - #cgtcgggc1020- gacctgttgt ggtttatgca ctccacggat cgtttctccg atccagatgc ag - #tcgcttcc1080- gtggtggagg cgctctcggg gcatggacca gtacgtgatt tgtggggtta cg - #ggaaaaac1140- aaccttgtcg gactcgacgg caaaccactt ttccctcggc cgtacggcta ta - #tgccgttt1200- aagatgcgga aatttctgct cggcgcgacg gttgcgcatc aggcgacatt ct - #tcggcgcg1260- tcgctggtag ccaagttggg cggttacgat cttgattttg gactcgaggc gg - #accagctg1320- ttcatctacc gtgccgcact aatacggcct cccgtcacga tcgaccgcgt gg - #tttgcgac1380- ttcgatgtca cgggacctgg ttcaacccag cccatccgtg agcactatcg ga - #ccctgcgg1440- cggctctggg acctgcatgg cgactacccg ctgggtgggc gcagagtgtc gt - #gggcttac1500- ttgcgtgtga aggagtactt gattcgggcc gacctggccg cattcaacgc gg - #taaagttc1560- ttgcgagcga agttcgccag agcttcgcgg aagcaaaatt catagaaacc aa - #cttctact1620- gcctgacctg agcagcgccg aggcgcgcag cgcgatcagt gcgacctgaa cg - #gccaggtg1680- gaaagcgcca ccgatcccgg caccgagtgc ctgacgcttc ggatcccttg ca - #ccacaacg1740- agagtgagag cgccatgatg aggaaatatc ggctgggcgg agtcaacgcc gg - #agtgacaa1800- aagtgagaac ccggtgaagc gagcgcttat aacagggatc acggggcagg at - #ggttccta1860- cctcgccgag ctactactga gcaagggata cgaggttcac gggctcgttc gt - #cgagcttc1920- gacgtttaac acgtcgcgga tcgatcacct ctacgttgac ccacaccaac cg - #ggcgcgcg1980- cttgttcttg cactatgcag acctcactga cggcacccgg ttggtgaccc tg - #ctcagcag2040- tatcgacccg gatgaggtct acaacctcgc agcgcagtcc catgtgcgcg tc - #agctttga2100- cgagccagtg cataccggag acaccaccgg catgggatcg atccgacttc tg - #gaagcagt2160- ccgcctttct cgggtggact gccggttcta tcaggcttcc tcgtcggaga tg - #ttcggcgc2220- atctccgcca ccgcagaacg aatcgacgcc gttctatccc cgttcgccat ac - #ggcgcggc2280- caaggtcttc tcgtactgga cgactcgcaa ctatcgagag gcgtacggat ta - #ttcgcagt2340- gaatggcatc ttgttcaacc atgagtcccc ccggcgcggc gagactttcg tg - #acccgaaa2400- gatcacgcgt gccgtggcgc gcatccgagc tggcgtccaa tcggaggtct at - #atgggcaa2460- cctcgatgcg atccgcgact ggggctacgc gcccgaatat gtcgagggga tg - #tggaggat2520- gttgcaagcg cctgaacctg atgactacgt cctggcgaca gggcgtggtt ac - #accgtacg2580- tgagttcgct caagctgctt ttgaccatgt cgggctcgac tggcaaaagc gc - #gtcaagtt2640- tgacgaccgc tatttgcgtc ccaccgaggt cgattcgcta gtaggagatg cc - #gacaaggc2700- ggcccagtca ctcggctgga aagcttcggt tcatactggt gaactcgcgc gc - #atcatggt2760- ggacgcggac atcgccgcgt tggagtgcga tggcacacca tggatcgaca cg - #ccgatgtt2820- gcctggttgg ggcagagtaa gttgacgact acacctgggc ctctggaccg cg - #caacgccc2880- gtgtatatcg ccggtcatcg ggggctggtc ggctcagcgc tcgtacgtag at - #ttgaggcc2940- gaggggttca ccaatctcat tgtgcgatca cgcgatgaga ttgatctgac gg - #accgagcc3000- gcaacgtttg attttgtgtc tgagacaaga ccacaggtga tcatcgatgc gg - #ccgcacgg3060- gtcggcggca tcatggcgaa taacacctat cccgcggact tcttgtccga aa - #acctccga3120- atccagacca atttgctcga cgcagctgtc gccgtgcgtg tgccgcggct cc - #ttttcctc3180- ggttcgtcat gcatctaccc gaagtacgct ccgcaaccta tccacgagag tg - #ctttattg3240- actggccctt tggagcccac caacgacgcg tatgcgatcg ccaagatcgc cg - #gtatcctg3300- caagttcagg cggttaggcg ccaatatggg ctggcgtgga tctctgcgat gc - #cgactaac3360- ctctacggac ccggcgacaa cttctccccg tccgggtcgc atctcttgcc gg - #cgctcatc3420- cgtcgatatg aggaagccaa agctggtggt gcagaagagg tgacgaattg gg - #ggaccggt3480- actccgcggc gcgaacttct gcatgtcgac gatctggcga gcgcatgcct gt - #tccttttg3540- gaacatttcg atggtccgaa ccacgtcaac gtgggcaccg gcgtcgatca ca - #gcattagc3600- gagatcgcag acatggtcgc tacagcggtg ggctacatcg gcgaaacacg tt - #gggatcca3660- actaaacccg atggaacccc gcgcaaacta ttggacgtct ccgcgctacg cg - #agttgggt3720- tggcgcccgc gaatcgcact gaaagacggc atcgatgcaa cggtgtcgtg gt - #accgcaca3780- aatgccgatg ccgtgaggag gtaaagctgc gggtcggccg atgttatccc tc - #cggccgga3840- cgggtggggc gacctgccgt cgagtggtac ggcagtcgcc tggccggcga gg - #cgcgtggc3900- ctatgggagt atccaatagc ctggcttggc tcgcccctac gcattatcag tt - #gaccgctt3960- tcgcgccagc tcgcaggctt gcggcagcat cccgttcagg tctcctcatg gt - #ccggtgtg4020- gcacgaccac gcaagctcga accgactcgt ttcccaattt cgcatgctaa ta - #tcgctcga4080- tggatttttt gcgcaacgcc ggcttgatgg ctcgtaacgt tagtaccgag at - #gctgcgcc4140- acttcgaacg aaagcgccta ttagtaaacc aattcaaagc atacggagtc aa - #cgttgtta4200- ttgatgtcgg tgctaactcc ggccagttcg gtagcgcttt gcgtcgtgca gg - #attcaaga4260- gccgtatcgt ttcctttgaa cctctttcgg ggccatttgc gcaactaacg cg - #caagtcgg4320- catcggatcc actatgggag tgtcaccagt atgccctagg cgacgccgat ga - #gacgatta4380- ccatcaatgt ggcaggcaat gcgggggcaa gtagttccgt gctgccgatg ct - #taaaagtc4440- atcaagatgc ctttcctccc gcgaattata ttggcaccga agacgttgca at - #acaccgcc4500- ttgattcggt tgcatcagaa tttctgaacc ctaccgatgt tactttcctg aa - #gatcgacg4560- tacagggttt cgagaagcag gttatcacgg gcagtaagtc aacgcttaac ga - #aagctgcg4620- tcggcatgca actcgaactt tcttttattc cgttgtacga aggtgacatg ct - #gattcatg4680- aagcgcttga acttgtctat tccctaggtt tcagactgac gggtttgttg cc - #cggcttta4740- cggatccgcg caatggtcga atgcttcaag ctgacggcat tttcttccgt gg - #ggacgatt4800- gacataaatg ctccgtcggc accctgccgg tatccaaacg ggcgatctgg tg - #agccggcc4860- tcccgggcac ctaatcgact atctaaattg aggcggccgc gacgtgcggc ac - #gaacaggt4920- ggccggctgc tagcgttaca cacgtcatga ctgcgccagt gttctcgata at - #tatcccta4980- ccttcaatgc agcggtgacg ctgcaagcct gcctcggaag catcgtcggg ca - #gacctacc5040- gggaagtgga agtggtcctt gtcgacggcg gttcgaccga tcggaccctc ga - #catcgcga5100- acagtttccg cccggaactc ggctcgcgac tggtcgttca cagcgggccc ga - #tgatggcc5160- cctacgacgc catgaaccgc ggcgtcggcg tggccacagg cgaatgggta ct - #ttttttag5220- gcgccgacga caccctctac gaaccaacca cgttggccca ggtagccgct tt - #tctcggcg5280- accatgcggc aagccatctt gtctatggcg atgttgtgat gcgttcgacg aa - #aagccggc5340- atgccggacc tttcgacctc gaccgcctcc tatttgagac gaatttgtgc ca - #ccaatcga5400- tcttttaccg ccgtgagctt ttcgacggca tcggccctta caacctgcgc ta - #ccgagtct5460- gggcggactg ggacttcaat attcgctgct tctccaaccc ggcgctgatt ac - #ccgctaca5520- tggacgtcgt gatttccgaa tacaacgaca tgaccggctt cagcatgagg ca - #ggggactg5580- ataaagagtt cagaaaacgg ctgccaatgt acttctgggt tgcagggtgg ga - #gacttgca5640- ggcgcatgct ggcgtttttg aaagacaagg agaatcgccg tctggccttg cg - #tacgcggt5700- tgataagggt taaggccgtc tccaaagaac gaagcgcaga accgtagtcg cg - #gatccaca5760- ttggacttct ttaacgcgtt tgcgtcctga tccacctttc aagcccgttc cg - #cgtaacgc5820- ggcgcgcaga gagtggtcgc atatcgcatc actgttctcg tgccagtgct tg - #gaaagcgt5880- cgagcactct ggttcgcgtt cttgacgttc gcgcccgctc ctagaggtag cg - #tgtcacgt5940- gactgaagcc aatgagtgca actcggcgtc gcgaaaggtt tcagtcgcgg tt - #gagcaaga6000- caccgcaaga ctactggagt gcgtgcacaa gcgcctccag ctcgcggctg aa - #agcggatg6060- caaagggatt cgaagcttga gcaacatgcg aaggggagaa cggcctatga gg - #ctgggaca6120- ggttttcgat ccgcgcgcga atgcactgtc aatggccaag tagaagtccc cg - #ctggtggc6180- cagcagaagt ccccactccg ctgcgggtgg ttggctaatt cttggcggct cc - #cttcttgt6240- ggtcggcgtg gcgcatccgg taggactcgc cggaggtgac gacgatgctg gc - #gtggtgca6300- gcagccgatc gaggatgctg gcggcggtgg tgtgctcggg caggaatcgc cc - #ccattgtt6360- cgaagggcca atgcgaggcg atggccaggg agcggcgctc gtagccggca gc - #cacgagcc6420- ggaacaacag ttgagtcccg gtgtcgtcga gcggggcgaa gccgatctcg tc - #caagatga6480- ccagatccgc gcggagcagg gtgtcgatga tcttgccgac ggtgttgtcg gc - #caggccgc6540- ggtagaggac ctcgatcagg tcggcggcgg tgaagtagcg gactttgaat cc - #ggcgtgga6600- cggcagcgtg cccgcagccg atgagcaggt gacttttgcc cgtaccaggt gg - #gccaatga6660- ccgccaggtt ctgttgtgcc cgaatccatt ccaggctcga caggtagtcg aa - #cgtggctg6720- cggtgatcga cgatccggtg acgtcgaacc cgtcgagggt cttggtgacc gg - #gaaggctg6780- cggccttgag acggttggcg gtgttggagg catcgcgggc agcgatctcg gc - #ctcaacca6840- acgtccgcag gatctcctcc ggtgtccagc gttgcgtctt ggcgacttgc aa - #cacctcgg6900- cggcgttgcg gcgcaccgtg gccagcttca accgccgcag cgccgcgtca ag - #gtcagcag6960- ccagcggtgc cgccgaggac ggtgccaccg gcttggcagc ggtggtcatg ag - #gccgtccc7020- gtcggtggtg ttgatcttgt aggcctccaa cgagcgggtc tcgacggtgg gc - #agatcgag7080- cacgagtgcg tcgccggcgg ggcggggttg tggggtgccg gcgccggcgg cc - #aggatcga7140- gcgcacgtcg gcagcgcgga accggcgaaa cgcaaccgcc cggcgcagcg cg - #tcaatcaa7200- agcctgttcg ccgtgggcgg cgccaaggcc gagcagaatg tcgagttcgg at - #ttcagtcg7260- ggtgttgccg atcgcagcag caccgacgag gaactgctgc gcttcggttc cc - #aatgcgca7320- gaatcgtttc tctgcttggg ttttcgggcg aggaccacgc gagggtgcgg gt - #ctgggtcc7380- gtcgtagtgt tcatcgagga tggacacctc acctgggctg acgagctcgt gc - #tcggccac7440- gatcacaccg gtcgcaggtt ccaacaggat cagggcgcca tgatcgacca cc - #accgccac7500- ggtggcaccg acgagccgct gaggcaccga gtaacgagct gagccgtaac gg - #atgcacga7560- gaggccgtcg accttacggc gcaccgaccc cgagccgatc gtcggccgca gc - #gagggcag7620- ctccctcaag acggtgcgct cgtcaaccaa gcgatcgttg ggcacggcgc ag - #atctccga7680- gtggaccgtg gcattgacct cggcgcacca tagttgcgcc tgggcgttga gg - #gcacgtag7740- gtcgacctgc tcaccggcta acgcagcttc ggtcagcagc ggcaccgcaa gg - #tcgtcctg7800- agcgtagcca cagaggttct ccacgatgcc cttcgattgc ggatccgcac cg - #tggcagaa7860- gtccggaacg aagccatagt gggacgcgaa tcgcacataa tccggtgttg ga - #acaacaac7920- attggcgacg acaccacctt tgaggcagcc catccggtcg gccaggatct tg - #gccggaac7980# 7995- <210> SEQ ID NO 4<211> LENGTH: 4435<212> TYPE: DNA<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 4- ttctactgcc tgacctgagc agcgccgagg cgcgcagcgc gatcactgcg ac - #ctgaatgg 60- ccaggtggaa agcgccaccg atcccggcac cgagtgcctg acgattcgga tc - #ccttgcac 120- cacaacgaga gtgagaccgc catgatgacg aaatatcggc tgggcggagt ca - #acgccgga 180- gtgacaaaag tgagaacccg gtgaagcgag cgcttataac agggatcacg gg - #gcaggatg 240- gttcctacct cgccgagcta ctactgagca agggatacga ggttcacggg ct - #cgttcgtc 300- gagcttcgac gtttaacacg tcgcggatcg atcacctcta cgttgaccca ca - #ccaaccgg 360- gcgcgcgctt gttcttgcac tatgcagacc tcactgacgg cacccggttg gt - #gaccctgc 420- tcagcagtat cgacccggat gaggtctaca acctcgcagc gcagtcccat gt - #gcgcgtca 480- gctttgacga gccagtgcat accggagaca ccaccggcat gggatcgatc cg - #acttctgg 540- aagcagtccg cctttctcgg gtggactgcc ggttctatca ggcttcctcg tc - #ggagatgt 600- tcggcgcatc tccgccaccg cagaacgaat cgacgccgtt ctatccccgt tc - #gccatacg 660- gcgcggccaa ggtcttctcg tactggacga ctcgcaacta tcgagaggcg ta - #cggattat 720- tcgcagtgaa tggcatcttg ttcaaccatg agtccccccg gcgcggcgag ac - #tttcgtga 780- cccgaaagat cacgcgtgcc gtggcgcgca tccgagctgg ctgccaatcg ga - #ggtctata 840- tgggcaacct cgatgcgatc cgcgactggg gctacgcgcc cgaatatgtc ga - #ggggatgt 900- ggaggatgtt gcaagcgcct gaacctgatg actacgtcct ggcgacaggg cg - #tggttaca 960- ccgtacgtga gttcgctcaa gctgcttttg accacgtcgg gctcgactgg ca - #aaagcacg1020- tcaagtttga cgaccgctat ttgcgcccca ccgaggtcga ttcgctagta gg - #agatgccg1080- acagggcggc ccagtcactc ggctggaaag cttcggttca tactggtgaa ct - #cgcgcgca1140- tcatggtgga cgcggacatc gccgcgtcgg agtgcgatgg cacaccatgg at - #cgacacgc1200- cgatgttgcc tggttggggc ggagtaagtt gacgactaca cctgggcctc tg - #gaccgcgc1260- aacgcccgtg tatatcgccg gtcatcgggg gctggtcggc tcagcgctcg ta - #cgtagatt1320- tgaggccgag gggttcacca atctcattgt gcgatcacgc gatgagattg at - #ctgacgga1380- ccgagccgca acgtttgatt ttgtgtctga gacaagacca caggtgatca tc - #gatgcggc1440- cgcacgggtc ggcggcatca tggcgaataa cacctatccc gcggacttct tg - #tccgaaaa1500- cctccgaatc cagaccaatt tgctcgacgc agctgtcgcc gtgcgtgtgc cg - #cggctcct1560- tttcctcggt tcgtcatgca tctacccgaa gtacgctccg caacctatcc ac - #gagagtgc1620- tttattgact ggccctttgg agcccaccaa cgacgcgtat gcgatcgcca ag - #atcgccgg1680- tatcctgcaa gttcaggcgg ttaggcgcca atatgggctg gcgtggatct ct - #gcgatgcc1740- gactaacctc tacggacccg gcgacaactt ctccccgtcc gggtcgcatc tc - #ttgccggc1800- gctcatccgt cgatatgagg aagccaaagc tggtggtgca gaagaggtga cg - #aattgggg1860- gaccggtact ccgcggcgcg aacttctgca tgtcgacgat ctggcgagcg ca - #tgcctgtt1920- ccttttggaa catttcgatg gtccgaacca cgtcaacgtg ggcaccggcg tc - #gatcacag1980- cattagcgag atcgcagaca tggtcgctac ggcggtgggc tacatcggcg aa - #acacgttg2040- ggatccaact aaacccgatg gaaccccgcg caaactattg gacgtctccg cg - #ctacgcga2100- gttgggttgg cgcccgcgaa tcgcactgaa agacggcatc gatgcaacgg tg - #tcgtggta2160- ccgcacaaat gccgatgccg tgaggaggta aagctgcggg ccggccgatg tt - #atccctcc2220- ggccggacgg gtagggcgac ctgccatcga gtggtacggc agtcgcctgg cc - #ggcgaggc2280- gcatggccta tgggagtatc ccatagcctg gcttggctcg cccctacgca tt - #atcagttg2340- accgctttcg cgccagctcg caggctcgcg gcagcatccc gttcaggtct cc - #tcatggtc2400- cggtgtggca cgaccacgca agctcgaacc gactcgtttc ccaatttcgc at - #gctaatat2460- cgctcgatgg attttttgcg caacgccggc ttgatggctc gtaacgttag ca - #ccgagatg2520- ctgcgccact tcgaacgaaa gcgcctatta gtaaaccaat tcaaagcata cg - #gagtcaac2580- gttgttattg atgtcggtgc taactccggc cagttcggta gcgctttgcg tc - #gtgcagga2640- ttcaagagcc gtatcgtttc ctttgaacct ctttcggggc catttgcgca ac - #taacgcgc2700- gagtcggcat cggatccact atgggagtgt caccagtatg ccctaggcga cg - #ccgatgag2760- acgattacca tcaatgtggc aggcaatgcg ggggcaagta gttccgtgct gc - #cgatgctt2820- aaaagtcatc aagatgcctt tcctcccgcg aattatattg gcaccgaaga cg - #ttgcaata2880- caccgccttg attcggttgc atcagaattt ctgaacccta ccgatgttac tt - #tcctgaag2940- atcgacgtac agggtttcga gaagcaggtt atcgcgggca gtaagtcaac gc - #ttaacgaa3000- agctgcgtcg gcatgcaact cgaactttct tttattccgt tgtacgaagg tg - #acatgctg3060- attcatgaag cgcttgaact tgtctattcc ctaggtttca gactgacggg tt - #tgttgccc3120- ggatttacgg atccgcgcaa tggtcgaatg cttcaagctg acggcatttt ct - #tccgtggg3180- gacgattgac ataaatgctt gcgtcggcac cctgccggta tccaaacggg cg - #atctggtg3240- agccggcctc ccgggcacct aatcgactat ctaaattgag gcggccgcga cg - #tgcggcac3300- gaacaggtgg ccggctgcta gcgttacaca cgtcatgact gcgccagtgt tc - #tcgataat3360- tatccctacc ttcaatgcag cggtgacgct gcaagcctgc ctcggaagca tc - #gtcgggca3420- gacctaccgg gaagtggaag tggtccttgt cgacggcggt tcgaccgatc gg - #accctcga3480- catcgcgaac agtttccgcc cggaactcgg ctcgcgactg gtcgttcaca gc - #gggcccga3540- tgatggcccc tacgacgcca tgaaccgcgg cgtcggcgta gccacaggcg aa - #tgggtact3600- ttttttaggc gccgacgaca ccctctacga accaaccacg ttggcccagg ta - #gccgcttt3660- tctcggcgac catgcggcaa gccatcttgt ctatggcgat gttgtgatgc gt - #tcgacgaa3720- aagccggcat gccggacctt tcgacctcga ccgcctccta tttgagacga at - #ttgtgcca3780- ccaatcgatc ttttaccgcc gtgagctttt cgacggcatc ggcccttaca ac - #ctgcgcta3840- ccgagtctgg gcggactggg acttcaatat tcgctgcttc tccaacccgg cg - #ctgattac3900- ccgctacatg gacgtcgtga tttccgaata caacgacatg accggcttca gc - #atgaggca3960- ggggactgat aaagagttca gaaaacggct gccaatgtac ttctgggttg ca - #gggtggga4020- gacttgcagg cgcatgctgg cgtttttgaa agacaaggag aatcgccgtc tg - #gccttgcg4080- tacgcggttg ataagggtta aggccgtctc caaagaacga agcgcagaac cg - #tagtcgcg4140- gatccacatt ggacttcttt aacgcgtttg cgtcctgatc cacctttcaa cc - #ccgttccg4200- cgtgacgcgg cgcgcagaga gtggtcgcat atcgcgtcac tgttctcgtg cc - #agtgcttg4260- gaaagcgtcg agcactctgg ttcgcgttct tgacgttcgc gcccgcccct ag - #aggtagcg4320- tgtcacgtga ctgaagccaa tgagtgcaac tcggcgtcgc gaaaggtttc ag - #tcgcggtt4380- gagcaagaca ccgcaagact actggagtgc gtgcacaagc gcctccagct ca - #cgg4435- <210> SEQ ID NO 5<211> LENGTH: 378<212> TYPE: DNA<213> ORGANISM: Mycobacterium<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)..(375)- <400> SEQUENCE: 5- atg atc gct gtg atc tgg tcg gcg gtg ccg ac - #a gga acc gtc gac ttg 48Met Ile Ala Val Ile Trp Ser Ala Val Pro Th - #r Gly Thr Val Asp Leu# 15- tcg acg atc acc ttg tac cgg tcg atg tat ga - #c cca atg tcg tcc gca 96Ser Thr Ile Thr Leu Tyr Arg Ser Met Tyr As - #p Pro Met Ser Ser Ala# 30- acc gag aag acg tac gtc agg tcc gcc gcc cc - #g ctt tca ccc atg ggc 144Thr Glu Lys Thr Tyr Val Arg Ser Ala Ala Pr - #o Leu Ser Pro Met Gly# 45- gtc ggg acg gcg atg aaa atg acg tcc gcg tg - #c tcg att ccg cgt tgc 192Val Gly Thr Ala Met Lys Met Thr Ser Ala Cy - #s Ser Ile Pro Arg Cys# 60- cgg tcg gtg gtg aag tca atc agc ccg ttc tc - #a cgg ttc ctc gca atc 240Arg Ser Val Val Lys Ser Ile Ser Pro Phe Se - #r Arg Phe Leu Ala Ile# 80- aac tcc caa ccc ggg ctc gaa aat cgg gac ac - #t gcc tgc gag gag caa 288Asn Ser Gln Pro Gly Leu Glu Asn Arg Asp Th - #r Ala Cys Glu Glu Gln# 95- atc gat ctt ggc ctg atc gat atc gac aca ga - #c gac atc gtt gcc gct 336Ile Asp Leu Gly Leu Ile Asp Ile Asp Thr As - #p Asp Ile Val Ala Ala# 110- atc cgc gag aca ggc gcc cgt gac gag gcc ta - #c ata gcc tga# 378Ile Arg Glu Thr Gly Ala Arg Asp Glu Ala Ty - #r Ile Ala# 125- <210> SEQ ID NO 6<211> LENGTH: 125<212> TYPE: PRT<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 6- Met Ile Ala Val Ile Trp Ser Ala Val Pro Th - #r Gly Thr Val Asp Leu# 15- Ser Thr Ile Thr Leu Tyr Arg Ser Met Tyr As - #p Pro Met Ser Ser Ala# 30- Thr Glu Lys Thr Tyr Val Arg Ser Ala Ala Pr - #o Leu Ser Pro Met Gly# 45- Val Gly Thr Ala Met Lys Met Thr Ser Ala Cy - #s Ser Ile Pro Arg Cys# 60- Arg Ser Val Val Lys Ser Ile Ser Pro Phe Se - #r Arg Phe Leu Ala Ile# 80- Asn Ser Gln Pro Gly Leu Glu Asn Arg Asp Th - #r Ala Cys Glu Glu Gln# 95- Ile Asp Leu Gly Leu Ile Asp Ile Asp Thr As - #p Asp Ile Val Ala Ala# 110- Ile Arg Glu Thr Gly Ala Arg Asp Glu Ala Ty - #r Ile Ala# 125- <210> SEQ ID NO 7<211> LENGTH: 834<212> TYPE: DNA<213> ORGANISM: Mycobacterium<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)..(831)- <400> SEQUENCE: 7- gtg tca tct gct cca acc gtg tcg gtg ata ac - #g att tcg ctg aac gat 48Val Ser Ser Ala Pro Thr Val Ser Val Ile Th - #r Ile Ser Leu Asn Asp# 15- ctc gag gga ttg aaa agc acc gtg gag agc gt - #t cgc gcg cag cgc tat 96Leu Glu Gly Leu Lys Ser Thr Val Glu Ser Va - #l Arg Ala Gln Arg Tyr# 30- ggg ggg cga atc gag cac atc gtc atc gac gg - #t gga tcg ggc gac gcc 144Gly Gly Arg Ile Glu His Ile Val Ile Asp Gl - #y Gly Ser Gly Asp Ala# 45- gtc gtg gag tat ctg tcc ggc gat cct ggc tt - #t gca tat tgg caa tct 192Val Val Glu Tyr Leu Ser Gly Asp Pro Gly Ph - #e Ala Tyr Trp Gln Ser# 60- cag ccc gac aac ggg aga tat gac gcg atg aa - #t cag ggc att gcc cat 240Gln Pro Asp Asn Gly Arg Tyr Asp Ala Met As - #n Gln Gly Ile Ala His# 80- tcg tcg ggc gac ctg ttg tgg ttt atg cac tc - #c acg gat cgt ttc tcc 288Ser Ser Gly Asp Leu Leu Trp Phe Met His Se - #r Thr Asp Arg Phe Ser# 95- gat cca gat gca gtc gct tcc gtg gtg gag gc - #g ctc tcg ggg cat gga 336Asp Pro Asp Ala Val Ala Ser Val Val Glu Al - #a Leu Ser Gly His Gly# 110- cca gta cgt gat ttg tgg ggt tac ggg aaa aa - #c aac ctt gtc gga ctc 384Pro Val Arg Asp Leu Trp Gly Tyr Gly Lys As - #n Asn Leu Val Gly Leu# 125- gac ggc aaa cca ctt ttc cct cgg ccg tac gg - #c tat atg ccg ttt aag 432Asp Gly Lys Pro Leu Phe Pro Arg Pro Tyr Gl - #y Tyr Met Pro Phe Lys# 140- atg cgg aaa ttt ctg ctc ggc gcg acg gtt gc - #g cat cag gcg aca ttc 480Met Arg Lys Phe Leu Leu Gly Ala Thr Val Al - #a His Gln Ala Thr Phe145 1 - #50 1 - #55 1 -#60- ttc ggc gcg tcg ctg gta gcc aag ttg ggc gg - #t tac gat ctt gat ttt 528Phe Gly Ala Ser Leu Val Ala Lys Leu Gly Gl - #y Tyr Asp Leu Asp Phe# 175- gga ctc gag gcg gac cag ctg ttc atc tac cg - #t gcc gca cta ata cgg 576Gly Leu Glu Ala Asp Gln Leu Phe Ile Tyr Ar - #g Ala Ala Leu Ile Arg# 190- cct ccc gtc acg atc gac cgc gtg gtt tgc ga - #c ttc gat gtc acg gga 624Pro Pro Val Thr Ile Asp Arg Val Val Cys As - #p Phe Asp Val Thr Gly# 205- cct ggt tca acc cag ccc atc cgt gag cac ta - #t cgg acc ctg cgg cgg 672Pro Gly Ser Thr Gln Pro Ile Arg Glu His Ty - #r Arg Thr Leu Arg Arg# 220- ctc tgg gac ctg cat ggc gac tac ccg ctg gg - #t ggg cgc aga gtg tcg 720Leu Trp Asp Leu His Gly Asp Tyr Pro Leu Gl - #y Gly Arg Arg Val Ser225 2 - #30 2 - #35 2 -#40- tgg gct tac ttg cgt gtg aag gag tac ttg at - #t cgg gcc gac ctg gcc 768Trp Ala Tyr Leu Arg Val Lys Glu Tyr Leu Il - #e Arg Ala Asp Leu Ala# 255- gca ttc aac gcg gta aag ttc ttg cga gcg aa - #g ttc gcc aga gct tcg 816Ala Phe Asn Ala Val Lys Phe Leu Arg Ala Ly - #s Phe Ala Arg Ala Ser# 270# 834 ca tagArg Lys Gln Asn Ser 275- <210> SEQ ID NO 8<211> LENGTH: 277<212> TYPE: PRT<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 8- Val Ser Ser Ala Pro Thr Val Ser Val Ile Th - #r Ile Ser Leu Asn Asp# 15- Leu Glu Gly Leu Lys Ser Thr Val Glu Ser Va - #l Arg Ala Gln Arg Tyr# 30- Gly Gly Arg Ile Glu His Ile Val Ile Asp Gl - #y Gly Ser Gly Asp Ala# 45- Val Val Glu Tyr Leu Ser Gly Asp Pro Gly Ph - #e Ala Tyr Trp Gln Ser# 60- Gln Pro Asp Asn Gly Arg Tyr Asp Ala Met As - #n Gln Gly Ile Ala His# 80- Ser Ser Gly Asp Leu Leu Trp Phe Met His Se - #r Thr Asp Arg Phe Ser# 95- Asp Pro Asp Ala Val Ala Ser Val Val Glu Al - #a Leu Ser Gly His Gly# 110- Pro Val Arg Asp Leu Trp Gly Tyr Gly Lys As - #n Asn Leu Val Gly Leu# 125- Asp Gly Lys Pro Leu Phe Pro Arg Pro Tyr Gl - #y Tyr Met Pro Phe Lys# 140- Met Arg Lys Phe Leu Leu Gly Ala Thr Val Al - #a His Gln Ala Thr Phe145 1 - #50 1 - #55 1 -#60- Phe Gly Ala Ser Leu Val Ala Lys Leu Gly Gl - #y Tyr Asp Leu Asp Phe# 175- Gly Leu Glu Ala Asp Gln Leu Phe Ile Tyr Ar - #g Ala Ala Leu Ile Arg# 190- Pro Pro Val Thr Ile Asp Arg Val Val Cys As - #p Phe Asp Val Thr Gly# 205- Pro Gly Ser Thr Gln Pro Ile Arg Glu His Ty - #r Arg Thr Leu Arg Arg# 220- Leu Trp Asp Leu His Gly Asp Tyr Pro Leu Gl - #y Gly Arg Arg Val Ser225 2 - #30 2 - #35 2 -#40- Trp Ala Tyr Leu Arg Val Lys Glu Tyr Leu Il - #e Arg Ala Asp Leu Ala# 255- Ala Phe Asn Ala Val Lys Phe Leu Arg Ala Ly - #s Phe Ala Arg Ala Ser# 270- Arg Lys Gln Asn Ser 275- <210> SEQ ID NO 9<211> LENGTH: 1032<212> TYPE: DNA<213> ORGANISM: Mycobacterium<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)..(1029)- <400> SEQUENCE: 9- gtg aag cga gcg ctt ata aca ggg atc acg gg - #g cag gat ggt tcc tac 48Val Lys Arg Ala Leu Ile Thr Gly Ile Thr Gl - #y Gln Asp Gly Ser Tyr# 15- ctc gcc gag cta cta ctg agc aag gga tac ga - #g gtt cac ggg ctc gtt 96Leu Ala Glu Leu Leu Leu Ser Lys Gly Tyr Gl - #u Val His Gly Leu Val# 30- cgt cga gct tcg acg ttt aac acg tcg cgg at - #c gat cac ctc tac gtt 144Arg Arg Ala Ser Thr Phe Asn Thr Ser Arg Il - #e Asp His Leu Tyr Val# 45- gac cca cac caa ccg ggc gcg cgc ttg ttc tt - #g cac tat gca gac ctc 192Asp Pro His Gln Pro Gly Ala Arg Leu Phe Le - #u His Tyr Ala Asp Leu# 60- act gac ggc acc cgg ttg gtg acc ctg ctc ag - #c agt atc gac ccg gat 240Thr Asp Gly Thr Arg Leu Val Thr Leu Leu Se - #r Ser Ile Asp Pro Asp# 80- gag gtc tac aac ctc gca gcg cag tcc cat gt - #g cgc gtc agc ttt gac 288Glu Val Tyr Asn Leu Ala Ala Gln Ser His Va - #l Arg Val Ser Phe Asp# 95- gag cca gtg cat acc gga gac acc acc ggc at - #g gga tcg atc cga ctt 336Glu Pro Val His Thr Gly Asp Thr Thr Gly Me - #t Gly Ser Ile Arg Leu# 110- ctg gaa gca gtc cgc ctt tct cgg gtg gac tg - #c cgg ttc tat cag gct 384Leu Glu Ala Val Arg Leu Ser Arg Val Asp Cy - #s Arg Phe Tyr Gln Ala# 125- tcc tcg tcg gag atg ttc ggc gca tct ccg cc - #a ccg cag aac gaa tcg 432Ser Ser Ser Glu Met Phe Gly Ala Ser Pro Pr - #o Pro Gln Asn Glu Ser# 140- acg ccg ttc tat ccc cgt tcg cca tac ggc gc - #g gcc aag gtc ttc tcg 480Thr Pro Phe Tyr Pro Arg Ser Pro Tyr Gly Al - #a Ala Lys Val Phe Ser145 1 - #50 1 - #55 1 -#60- tac tgg acg act cgc aac tat cga gag gcg ta - #c gga tta ttc gca gtg 528Tyr Trp Thr Thr Arg Asn Tyr Arg Glu Ala Ty - #r Gly Leu Phe Ala Val# 175- aat ggc atc ttg ttc aac cat gag tcc ccc cg - #g cgc ggc gag act ttc 576Asn Gly Ile Leu Phe Asn His Glu Ser Pro Ar - #g Arg Gly Glu Thr Phe# 190- gtg acc cga aag atc acg cgt gcc gtg gcg cg - #c atc cga gct ggc gtc 624Val Thr Arg Lys Ile Thr Arg Ala Val Ala Ar - #g Ile Arg Ala Gly Val# 205- caa tcg gag gtc tat atg ggc aac ctc gat gc - #g atc cgc gac tgg ggc 672Gln Ser Glu Val Tyr Met Gly Asn Leu Asp Al - #a Ile Arg Asp Trp Gly# 220- tac gcg ccc gaa tat gtc gag ggg atg tgg ag - #g atg ttg caa gcg cct 720Tyr Ala Pro Glu Tyr Val Glu Gly Met Trp Ar - #g Met Leu Gln Ala Pro225 2 - #30 2 - #35 2 -#40- gaa cct gat gac tac gtc ctg gcg aca ggg cg - #t ggt tac acc gta cgt 768Glu Pro Asp Asp Tyr Val Leu Ala Thr Gly Ar - #g Gly Tyr Thr Val Arg# 255- gag ttc gct caa gct gct ttt gac cat gtc gg - #g ctc gac tgg caa aag 816Glu Phe Ala Gln Ala Ala Phe Asp His Val Gl - #y Leu Asp Trp Gln Lys# 270- cgc gtc aag ttt gac gac cgc tat ttg cgt cc - #c acc gag gtc gat tcg 864Arg Val Lys Phe Asp Asp Arg Tyr Leu Arg Pr - #o Thr Glu Val Asp Ser# 285- cta gta gga gat gcc gac aag gcg gcc cag tc - #a ctc ggc tgg aaa gct 912Leu Val Gly Asp Ala Asp Lys Ala Ala Gln Se - #r Leu Gly Trp Lys Ala# 300- tcg gtt cat act ggt gaa ctc gcg cgc atc at - #g gtg gac gcg gac atc 960Ser Val His Thr Gly Glu Leu Ala Arg Ile Me - #t Val Asp Ala Asp Ile305 3 - #10 3 - #15 3 -#20- gcc gcg ttg gag tgc gat ggc aca cca tgg at - #c gac acg ccg atg ttg1008Ala Ala Leu Glu Cys Asp Gly Thr Pro Trp Il - #e Asp Thr Pro Met Leu# 335# 1032ga gta agt tgaPro Gly Trp Gly Arg Val Ser 340- <210> SEQ ID NO 10<211> LENGTH: 343<212> TYPE: PRT<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 10- Val Lys Arg Ala Leu Ile Thr Gly Ile Thr Gl - #y Gln Asp Gly Ser Tyr# 15- Leu Ala Glu Leu Leu Leu Ser Lys Gly Tyr Gl - #u Val His Gly Leu Val# 30- Arg Arg Ala Ser Thr Phe Asn Thr Ser Arg Il - #e Asp His Leu Tyr Val# 45- Asp Pro His Gln Pro Gly Ala Arg Leu Phe Le - #u His Tyr Ala Asp Leu# 60- Thr Asp Gly Thr Arg Leu Val Thr Leu Leu Se - #r Ser Ile Asp Pro Asp# 80- Glu Val Tyr Asn Leu Ala Ala Gln Ser His Va - #l Arg Val Ser Phe Asp# 95- Glu Pro Val His Thr Gly Asp Thr Thr Gly Me - #t Gly Ser Ile Arg Leu# 110- Leu Glu Ala Val Arg Leu Ser Arg Val Asp Cy - #s Arg Phe Tyr Gln Ala# 125- Ser Ser Ser Glu Met Phe Gly Ala Ser Pro Pr - #o Pro Gln Asn Glu Ser# 140- Thr Pro Phe Tyr Pro Arg Ser Pro Tyr Gly Al - #a Ala Lys Val Phe Ser145 1 - #50 1 - #55 1 -#60- Tyr Trp Thr Thr Arg Asn Tyr Arg Glu Ala Ty - #r Gly Leu Phe Ala Val# 175- Asn Gly Ile Leu Phe Asn His Glu Ser Pro Ar - #g Arg Gly Glu Thr Phe# 190- Val Thr Arg Lys Ile Thr Arg Ala Val Ala Ar - #g Ile Arg Ala Gly Val# 205- Gln Ser Glu Val Tyr Met Gly Asn Leu Asp Al - #a Ile Arg Asp Trp Gly# 220- Tyr Ala Pro Glu Tyr Val Glu Gly Met Trp Ar - #g Met Leu Gln Ala Pro225 2 - #30 2 - #35 2 -#40- Glu Pro Asp Asp Tyr Val Leu Ala Thr Gly Ar - #g Gly Tyr Thr Val Arg# 255- Glu Phe Ala Gln Ala Ala Phe Asp His Val Gl - #y Leu Asp Trp Gln Lys# 270- Arg Val Lys Phe Asp Asp Arg Tyr Leu Arg Pr - #o Thr Glu Val Asp Ser# 285- Leu Val Gly Asp Ala Asp Lys Ala Ala Gln Se - #r Leu Gly Trp Lys Ala# 300- Ser Val His Thr Gly Glu Leu Ala Arg Ile Me - #t Val Asp Ala Asp Ile305 3 - #10 3 - #15 3 -#20- Ala Ala Leu Glu Cys Asp Gly Thr Pro Trp Il - #e Asp Thr Pro Met Leu# 335- Pro Gly Trp Gly Arg Val Ser 340- <210> SEQ ID NO 11<211> LENGTH: 1032<212> TYPE: DNA<213> ORGANISM: Mycobacterium<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)..(1029)- <400> SEQUENCE: 11- gtg aag cga gcg ctt ata aca ggg atc acg gg - #g cag gat ggt tcc tac 48Val Lys Arg Ala Leu Ile Thr Gly Ile Thr Gl - #y Gln Asp Gly Ser Tyr# 15- ctc gcc gag cta cta ctg agc aag gga tac ga - #g gtt cac ggg ctc gtt 96Leu Ala Glu Leu Leu Leu Ser Lys Gly Tyr Gl - #u Val His Gly Leu Val# 30- cgt cga gct tcg acg ttt aac acg tcg cgg at - #c gat cac ctc tac gtt 144Arg Arg Ala Ser Thr Phe Asn Thr Ser Arg Il - #e Asp His Leu Tyr Val# 45- gac cca cac caa ccg ggc gcg cgc ttg ttc tt - #g cac tat gca gac ctc 192Asp Pro His Gln Pro Gly Ala Arg Leu Phe Le - #u His Tyr Ala Asp Leu# 60- act gac ggc acc cgg ttg gtg acc ctg ctc ag - #c agt atc gac ccg gat 240Thr Asp Gly Thr Arg Leu Val Thr Leu Leu Se - #r Ser Ile Asp Pro Asp# 80- gag gtc tac aac ctc gca gcg cag tcc cat gt - #g cgc gtc agc ttt gac 288Glu Val Tyr Asn Leu Ala Ala Gln Ser His Va - #l Arg Val Ser Phe Asp# 95- gag cca gtg cat acc gga gac acc acc ggc at - #g gga tcg atc cga ctt 336Glu Pro Val His Thr Gly Asp Thr Thr Gly Me - #t Gly Ser Ile Arg Leu# 110- ctg gaa gca gtc cgc ctt tct cgg gtg gac tg - #c cgg ttc tat cag gct 384Leu Glu Ala Val Arg Leu Ser Arg Val Asp Cy - #s Arg Phe Tyr Gln Ala# 125- tcc tcg tcg gag atg ttc ggc gca tct ccg cc - #a ccg cag aac gaa tcg 432Ser Ser Ser Glu Met Phe Gly Ala Ser Pro Pr - #o Pro Gln Asn Glu Ser# 140- acg ccg ttc tat ccc cgt tcg cca tac ggc gc - #g gcc aag gtc ttc tcg 480Thr Pro Phe Tyr Pro Arg Ser Pro Tyr Gly Al - #a Ala Lys Val Phe Ser145 1 - #50 1 - #55 1 -#60- tac tgg acg act cgc aac tat cga gag gcg ta - #c gga tta ttc gca gtg 528Tyr Trp Thr Thr Arg Asn Tyr Arg Glu Ala Ty - #r Gly Leu Phe Ala Val# 175- aat ggc atc ttg ttc aac cat gag tcc ccc cg - #g cgc ggc gag act ttc 576Asn Gly Ile Leu Phe Asn His Glu Ser Pro Ar - #g Arg Gly Glu Thr Phe# 190- gtg acc cga aag atc acg cgt gcc gtg gcg cg - #c atc cga gct ggc gtc 624Val Thr Arg Lys Ile Thr Arg Ala Val Ala Ar - #g Ile Arg Ala Gly Val# 205- caa tcg gag gtc tat atg ggc aac ctc gat gc - #g atc cgc gac tgg ggc 672Gln Ser Glu Val Tyr Met Gly Asn Leu Asp Al - #a Ile Arg Asp Trp Gly# 220- tac gcg ccc gaa tat gtc gag ggg atg tgg ag - #g atg ttg caa gcg cct 720Tyr Ala Pro Glu Tyr Val Glu Gly Met Trp Ar - #g Met Leu Gln Ala Pro225 2 - #30 2 - #35 2 -#40- gaa cct gat gac tac gtc ctg gcg aca ggg cg - #t ggt tac acc gta cgt 768Glu Pro Asp Asp Tyr Val Leu Ala Thr Gly Ar - #g Gly Tyr Thr Val Arg# 255- gag ttc gct caa gct gct ttt gac cac gtc gg - #g ctc gac tgg caa aag 816Glu Phe Ala Gln Ala Ala Phe Asp His Val Gl - #y Leu Asp Trp Gln Lys# 270- cac gtc aag ttt gac gac cgc tat ttg cgc cc - #c acc gag gtc gat tcg 864His Val Lys Phe Asp Asp Arg Tyr Leu Arg Pr - #o Thr Glu Val Asp Ser# 285- cta gta gga gat gcc gac agg gcg gcc cag tc - #a ctc ggc tgg aaa gct 912Leu Val Gly Asp Ala Asp Arg Ala Ala Gln Se - #r Leu Gly Trp Lys Ala# 300- tcg gtt cat act ggt gaa ctc gcg cgc atc at - #g gtg gac gcg gac atc 960Ser Val His Thr Gly Glu Leu Ala Arg Ile Me - #t Val Asp Ala Asp Ile305 3 - #10 3 - #15 3 -#20- gcc gcg tcg gag tgc gat ggc aca cca tgg at - #c gac acg ccg atg ttg 1008Ala Ala Ser Glu Cys Asp Gly Thr Pro Trp Il - #e Asp Thr Pro Met Leu# 335# 1032a gta agt tgaPro Gly Trp Gly Gly Val Ser 340- <210> SEQ ID NO 12<211> LENGTH: 343<212> TYPE: PRT<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 12- Val Lys Arg Ala Leu Ile Thr Gly Ile Thr Gl - #y Gln Asp Gly Ser Tyr# 15- Leu Ala Glu Leu Leu Leu Ser Lys Gly Tyr Gl - #u Val His Gly Leu Val# 30- Arg Arg Ala Ser Thr Phe Asn Thr Ser Arg Il - #e Asp His Leu Tyr Val# 45- Asp Pro His Gln Pro Gly Ala Arg Leu Phe Le - #u His Tyr Ala Asp Leu# 60- Thr Asp Gly Thr Arg Leu Val Thr Leu Leu Se - #r Ser Ile Asp Pro Asp# 80- Glu Val Tyr Asn Leu Ala Ala Gln Ser His Va - #l Arg Val Ser Phe Asp# 95- Glu Pro Val His Thr Gly Asp Thr Thr Gly Me - #t Gly Ser Ile Arg Leu# 110- Leu Glu Ala Val Arg Leu Ser Arg Val Asp Cy - #s Arg Phe Tyr Gln Ala# 125- Ser Ser Ser Glu Met Phe Gly Ala Ser Pro Pr - #o Pro Gln Asn Glu Ser# 140- Thr Pro Phe Tyr Pro Arg Ser Pro Tyr Gly Al - #a Ala Lys Val Phe Ser145 1 - #50 1 - #55 1 -#60- Tyr Trp Thr Thr Arg Asn Tyr Arg Glu Ala Ty - #r Gly Leu Phe Ala Val# 175- Asn Gly Ile Leu Phe Asn His Glu Ser Pro Ar - #g Arg Gly Glu Thr Phe# 190- Val Thr Arg Lys Ile Thr Arg Ala Val Ala Ar - #g Ile Arg Ala Gly Val# 205- Gln Ser Glu Val Tyr Met Gly Asn Leu Asp Al - #a Ile Arg Asp Trp Gly# 220- Tyr Ala Pro Glu Tyr Val Glu Gly Met Trp Ar - #g Met Leu Gln Ala Pro225 2 - #30 2 - #35 2 -#40- Glu Pro Asp Asp Tyr Val Leu Ala Thr Gly Ar - #g Gly Tyr Thr Val Arg# 255- Glu Phe Ala Gln Ala Ala Phe Asp His Val Gl - #y Leu Asp Trp Gln Lys# 270- His Val Lys Phe Asp Asp Arg Tyr Leu Arg Pr - #o Thr Glu Val Asp Ser# 285- Leu Val Gly Asp Ala Asp Arg Ala Ala Gln Se - #r Leu Gly Trp Lys Ala# 300- Ser Val His Thr Gly Glu Leu Ala Arg Ile Me - #t Val Asp Ala Asp Ile305 3 - #10 3 - #15 3 -#20- Ala Ala Ser Glu Cys Asp Gly Thr Pro Trp Il - #e Asp Thr Pro Met Leu# 335- Pro Gly Trp Gly Gly Val Ser 340- <210> SEQ ID NO 13<211> LENGTH: 1020<212> TYPE: DNA<213> ORGANISM: Mycobacterium<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)..(1017)- <400> SEQUENCE: 13- gtg cga tgg cac acc atg gat cga cac gcc ga - #t gtt gcc tgg ttg ggg 48Val Arg Trp His Thr Met Asp Arg His Ala As - #p Val Ala Trp Leu Gly# 15- cag agt aag ttg acg act aca cct ggg cct ct - #g gac cgc gca acg ccc 96Gln Ser Lys Leu Thr Thr Thr Pro Gly Pro Le - #u Asp Arg Ala Thr Pro# 30- gtg tat atc gcc ggt cat cgg ggg ctg gtc gg - #c tca gcg ctc gta cgt 144Val Tyr Ile Ala Gly His Arg Gly Leu Val Gl - #y Ser Ala Leu Val Arg# 45- aga ttt gag gcc gag ggg ttc acc aat ctc at - #t gtg cga tca cgc gat 192Arg Phe Glu Ala Glu Gly Phe Thr Asn Leu Il - #e Val Arg Ser Arg Asp# 60- gag att gat ctg acg gac cga gcc gca acg tt - #t gat ttt gtg tct gag 240Glu Ile Asp Leu Thr Asp Arg Ala Ala Thr Ph - #e Asp Phe Val Ser Glu# 80- aca aga cca cag gtg atc atc gat gcg gcc gc - #a cgg gtc ggc ggc atc 288Thr Arg Pro Gln Val Ile Ile Asp Ala Ala Al - #a Arg Val Gly Gly Ile# 95- atg gcg aat aac acc tat ccc gcg gac ttc tt - #g tcc gaa aac ctc cga 336Met Ala Asn Asn Thr Tyr Pro Ala Asp Phe Le - #u Ser Glu Asn Leu Arg# 110- atc cag acc aat ttg ctc gac gca gct gtc gc - #c gtg cgt gtg ccg cgg 384Ile Gln Thr Asn Leu Leu Asp Ala Ala Val Al - #a Val Arg Val Pro Arg# 125- ctc ctt ttc ctc ggt tcg tca tgc atc tac cc - #g aag tac gct ccg caa 432Leu Leu Phe Leu Gly Ser Ser Cys Ile Tyr Pr - #o Lys Tyr Ala Pro Gln# 140- cct atc cac gag agt gct tta ttg act ggc cc - #t ttg gag ccc acc aac 480Pro Ile His Glu Ser Ala Leu Leu Thr Gly Pr - #o Leu Glu Pro Thr Asn145 1 - #50 1 - #55 1 -#60- gac gcg tat gcg atc gcc aag atc gcc ggt at - #c ctg caa gtt cag gcg 528Asp Ala Tyr Ala Ile Ala Lys Ile Ala Gly Il - #e Leu Gln Val Gln Ala# 175- gtt agg cgc caa tat ggg ctg gcg tgg atc tc - #t gcg atg ccg act aac 576Val Arg Arg Gln Tyr Gly Leu Ala Trp Ile Se - #r Ala Met Pro Thr Asn# 190- ctc tac gga ccc ggc gac aac ttc tcc ccg tc - #c ggg tcg cat ctc ttg 624Leu Tyr Gly Pro Gly Asp Asn Phe Ser Pro Se - #r Gly Ser His Leu Leu# 205- ccg gcg ctc atc cgt cga tat gag gaa gcc aa - #a gct ggt ggt gca gaa 672Pro Ala Leu Ile Arg Arg Tyr Glu Glu Ala Ly - #s Ala Gly Gly Ala Glu# 220- gag gtg acg aat tgg ggg acc ggt act ccg cg - #g cgc gaa ctt ctg cat 720Glu Val Thr Asn Trp Gly Thr Gly Thr Pro Ar - #g Arg Glu Leu Leu His225 2 - #30 2 - #35 2 -#40- gtc gac gat ctg gcg agc gca tgc ctg ttc ct - #t ttg gaa cat ttc gat 768Val Asp Asp Leu Ala Ser Ala Cys Leu Phe Le - #u Leu Glu His Phe Asp# 255- ggt ccg aac cac gtc aac gtg ggc acc ggc gt - #c gat cac agc att agc 816Gly Pro Asn His Val Asn Val Gly Thr Gly Va - #l Asp His Ser Ile Ser# 270- gag atc gca gac atg gtc gct aca gcg gtg gg - #c tac atc ggc gaa aca 864Glu Ile Ala Asp Met Val Ala Thr Ala Val Gl - #y Tyr Ile Gly Glu Thr# 285- cgt tgg gat cca act aaa ccc gat gga acc cc - #g cgc aaa cta ttg gac 912Arg Trp Asp Pro Thr Lys Pro Asp Gly Thr Pr - #o Arg Lys Leu Leu Asp# 300- gtc tcc gcg cta cgc gag ttg ggt tgg cgc cc - #g cga atc gca ctg aaa 960Val Ser Ala Leu Arg Glu Leu Gly Trp Arg Pr - #o Arg Ile Ala Leu Lys305 3 - #10 3 - #15 3 -#20- gac ggc atc gat gca acg gtg tcg tgg tac cg - #c aca aat gcc gat gcc1008Asp Gly Ile Asp Ala Thr Val Ser Trp Tyr Ar - #g Thr Asn Ala Asp Ala# 335# 1020Val Arg Arg- <210> SEQ ID NO 14<211> LENGTH: 339<212> TYPE: PRT<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 14- Val Arg Trp His Thr Met Asp Arg His Ala As - #p Val Ala Trp Leu Gly# 15- Gln Ser Lys Leu Thr Thr Thr Pro Gly Pro Le - #u Asp Arg Ala Thr Pro# 30- Val Tyr Ile Ala Gly His Arg Gly Leu Val Gl - #y Ser Ala Leu Val Arg# 45- Arg Phe Glu Ala Glu Gly Phe Thr Asn Leu Il - #e Val Arg Ser Arg Asp# 60- Glu Ile Asp Leu Thr Asp Arg Ala Ala Thr Ph - #e Asp Phe Val Ser Glu# 80- Thr Arg Pro Gln Val Ile Ile Asp Ala Ala Al - #a Arg Val Gly Gly Ile# 95- Met Ala Asn Asn Thr Tyr Pro Ala Asp Phe Le - #u Ser Glu Asn Leu Arg# 110- Ile Gln Thr Asn Leu Leu Asp Ala Ala Val Al - #a Val Arg Val Pro Arg# 125- Leu Leu Phe Leu Gly Ser Ser Cys Ile Tyr Pr - #o Lys Tyr Ala Pro Gln# 140- Pro Ile His Glu Ser Ala Leu Leu Thr Gly Pr - #o Leu Glu Pro Thr Asn145 1 - #50 1 - #55 1 -#60- Asp Ala Tyr Ala Ile Ala Lys Ile Ala Gly Il - #e Leu Gln Val Gln Ala# 175- Val Arg Arg Gln Tyr Gly Leu Ala Trp Ile Se - #r Ala Met Pro Thr Asn# 190- Leu Tyr Gly Pro Gly Asp Asn Phe Ser Pro Se - #r Gly Ser His Leu Leu# 205- Pro Ala Leu Ile Arg Arg Tyr Glu Glu Ala Ly - #s Ala Gly Gly Ala Glu# 220- Glu Val Thr Asn Trp Gly Thr Gly Thr Pro Ar - #g Arg Glu Leu Leu His225 2 - #30 2 - #35 2 -#40- Val Asp Asp Leu Ala Ser Ala Cys Leu Phe Le - #u Leu Glu His Phe Asp# 255- Gly Pro Asn His Val Asn Val Gly Thr Gly Va - #l Asp His Ser Ile Ser# 270- Glu Ile Ala Asp Met Val Ala Thr Ala Val Gl - #y Tyr Ile Gly Glu Thr# 285- Arg Trp Asp Pro Thr Lys Pro Asp Gly Thr Pr - #o Arg Lys Leu Leu Asp# 300- Val Ser Ala Leu Arg Glu Leu Gly Trp Arg Pr - #o Arg Ile Ala Leu Lys305 3 - #10 3 - #15 3 -#20- Asp Gly Ile Asp Ala Thr Val Ser Trp Tyr Ar - #g Thr Asn Ala Asp Ala# 335- Val Arg Arg- <210> SEQ ID NO 15<211> LENGTH: 1020<212> TYPE: DNA<213> ORGANISM: Mycobacterium<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)..(1017)- <400> SEQUENCE: 15- gtg cga tgg cac acc atg gat cga cac gcc ga - #t gtt gcc tgg ttg ggg 48Val Arg Trp His Thr Met Asp Arg His Ala As - #p Val Ala Trp Leu Gly# 15- cgg agt aag ttg acg act aca cct ggg cct ct - #g gac cgc gca acg ccc 96Arg Ser Lys Leu Thr Thr Thr Pro Gly Pro Le - #u Asp Arg Ala Thr Pro# 30- gtg tat atc gcc ggt cat cgg ggg ctg gtc gg - #c tca gcg ctc gta cgt 144Val Tyr Ile Ala Gly His Arg Gly Leu Val Gl - #y Ser Ala Leu Val Arg# 45- aga ttt gag gcc gag ggg ttc acc aat ctc at - #t gtg cga tca cgc gat 192Arg Phe Glu Ala Glu Gly Phe Thr Asn Leu Il - #e Val Arg Ser Arg Asp# 60- gag att gat ctg acg gac cga gcc gca acg tt - #t gat ttt gtg tct gag 240Glu Ile Asp Leu Thr Asp Arg Ala Ala Thr Ph - #e Asp Phe Val Ser Glu# 80- aca aga cca cag gtg atc atc gat gcg gcc gc - #a cgg gtc ggc ggc atc 288Thr Arg Pro Gln Val Ile Ile Asp Ala Ala Al - #a Arg Val Gly Gly Ile# 95- atg gcg aat aac acc tat ccc gcg gac ttc tt - #g tcc gaa aac ctc cga 336Met Ala Asn Asn Thr Tyr Pro Ala Asp Phe Le - #u Ser Glu Asn Leu Arg# 110- atc cag acc aat ttg ctc gac gca gct gtc gc - #c gtg cgt gtg ccg cgg 384Ile Gln Thr Asn Leu Leu Asp Ala Ala Val Al - #a Val Arg Val Pro Arg# 125- ctc ctt ttc ctc ggt tcg tca tgc atc tac cc - #g aag tac gct ccg caa 432Leu Leu Phe Leu Gly Ser Ser Cys Ile Tyr Pr - #o Lys Tyr Ala Pro Gln# 140- cct atc cac gag agt gct tta ttg act ggc cc - #t ttg gag ccc acc aac 480Pro Ile His Glu Ser Ala Leu Leu Thr Gly Pr - #o Leu Glu Pro Thr Asn145 1 - #50 1 - #55 1 -#60- gac gcg tat gcg atc gcc aag atc gcc ggt at - #c ctg caa gtt cag gcg 528Asp Ala Tyr Ala Ile Ala Lys Ile Ala Gly Il - #e Leu Gln Val Gln Ala# 175- gtt agg cgc caa tat ggg ctg gcg tgg atc tc - #t gcg atg ccg act aac 576Val Arg Arg Gln Tyr Gly Leu Ala Trp Ile Se - #r Ala Met Pro Thr Asn# 190- ctc tac gga ccc ggc gac aac ttc tcc ccg tc - #c ggg tcg cat ctc ttg 624Leu Tyr Gly Pro Gly Asp Asn Phe Ser Pro Se - #r Gly Ser His Leu Leu# 205- ccg gcg ctc atc cgt cga tat gag gaa gcc aa - #a gct ggt ggt gca gaa 672Pro Ala Leu Ile Arg Arg Tyr Glu Glu Ala Ly - #s Ala Gly Gly Ala Glu# 220- gag gtg acg aat tgg ggg acc ggt act ccg cg - #g cgc gaa ctt ctg cat 720Glu Val Thr Asn Trp Gly Thr Gly Thr Pro Ar - #g Arg Glu Leu Leu His225 2 - #30 2 - #35 2 -#40- gtc gac gat ctg gcg agc gca tgc ctg ttc ct - #t ttg gaa cat ttc gat 768Val Asp Asp Leu Ala Ser Ala Cys Leu Phe Le - #u Leu Glu His Phe Asp# 255- ggt ccg aac cac gtc aac gtg ggc acc ggc gt - #c gat cac agc att agc 816Gly Pro Asn His Val Asn Val Gly Thr Gly Va - #l Asp His Ser Ile Ser# 270- gag atc gca gac atg gtc gct acg gcg gtg gg - #c tac atc ggc gaa aca 864Glu Ile Ala Asp Met Val Ala Thr Ala Val Gl - #y Tyr Ile Gly Glu Thr# 285- cgt tgg gat cca act aaa ccc gat gga acc cc - #g cgc aaa cta ttg gac 912Arg Trp Asp Pro Thr Lys Pro Asp Gly Thr Pr - #o Arg Lys Leu Leu Asp# 300- gtc tcc gcg cta cgc gag ttg ggt tgg cgc cc - #g cga atc gca ctg aaa 960Val Ser Ala Leu Arg Glu Leu Gly Trp Arg Pr - #o Arg Ile Ala Leu Lys305 3 - #10 3 - #15 3 -#20- gac ggc atc gat gca acg gtg tcg tgg tac cg - #c aca aat gcc gat gcc1008Asp Gly Ile Asp Ala Thr Val Ser Trp Tyr Ar - #g Thr Asn Ala Asp Ala# 335# 1020Val Arg Arg- <210> SEQ ID NO 16<211> LENGTH: 339<212> TYPE: PRT<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 16- Val Arg Trp His Thr Met Asp Arg His Ala As - #p Val Ala Trp Leu Gly# 15- Arg Ser Lys Leu Thr Thr Thr Pro Gly Pro Le - #u Asp Arg Ala Thr Pro# 30- Val Tyr Ile Ala Gly His Arg Gly Leu Val Gl - #y Ser Ala Leu Val Arg# 45- Arg Phe Glu Ala Glu Gly Phe Thr Asn Leu Il - #e Val Arg Ser Arg Asp# 60- Glu Ile Asp Leu Thr Asp Arg Ala Ala Thr Ph - #e Asp Phe Val Ser Glu# 80- Thr Arg Pro Gln Val Ile Ile Asp Ala Ala Al - #a Arg Val Gly Gly Ile# 95- Met Ala Asn Asn Thr Tyr Pro Ala Asp Phe Le - #u Ser Glu Asn Leu Arg# 110- Ile Gln Thr Asn Leu Leu Asp Ala Ala Val Al - #a Val Arg Val Pro Arg# 125- Leu Leu Phe Leu Gly Ser Ser Cys Ile Tyr Pr - #o Lys Tyr Ala Pro Gln# 140- Pro Ile His Glu Ser Ala Leu Leu Thr Gly Pr - #o Leu Glu Pro Thr Asn145 1 - #50 1 - #55 1 -#60- Asp Ala Tyr Ala Ile Ala Lys Ile Ala Gly Il - #e Leu Gln Val Gln Ala# 175- Val Arg Arg Gln Tyr Gly Leu Ala Trp Ile Se - #r Ala Met Pro Thr Asn# 190- Leu Tyr Gly Pro Gly Asp Asn Phe Ser Pro Se - #r Gly Ser His Leu Leu# 205- Pro Ala Leu Ile Arg Arg Tyr Glu Glu Ala Ly - #s Ala Gly Gly Ala Glu# 220- Glu Val Thr Asn Trp Gly Thr Gly Thr Pro Ar - #g Arg Glu Leu Leu His225 2 - #30 2 - #35 2 -#40- Val Asp Asp Leu Ala Ser Ala Cys Leu Phe Le - #u Leu Glu His Phe Asp# 255- Gly Pro Asn His Val Asn Val Gly Thr Gly Va - #l Asp His Ser Ile Ser# 270- Glu Ile Ala Asp Met Val Ala Thr Ala Val Gl - #y Tyr Ile Gly Glu Thr# 285- Arg Trp Asp Pro Thr Lys Pro Asp Gly Thr Pr - #o Arg Lys Leu Leu Asp# 300- Val Ser Ala Leu Arg Glu Leu Gly Trp Arg Pr - #o Arg Ile Ala Leu Lys305 3 - #10 3 - #15 3 -#20- Asp Gly Ile Asp Ala Thr Val Ser Trp Tyr Ar - #g Thr Asn Ala Asp Ala# 335- Val Arg Arg- <210> SEQ ID NO 17<211> LENGTH: 723<212> TYPE: DNA<213> ORGANISM: Mycobacterium<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)..(720)- <400> SEQUENCE: 17- atg gat ttt ttg cgc aac gcc ggc ttg atg gc - #t cgt aac gtt agt acc 48Met Asp Phe Leu Arg Asn Ala Gly Leu Met Al - #a Arg Asn Val Ser Thr# 15- gag atg ctg cgc cac ttc gaa cga aag cgc ct - #a tta gta aac caa ttc 96Glu Met Leu Arg His Phe Glu Arg Lys Arg Le - #u Leu Val Asn Gln Phe# 30- aaa gca tac gga gtc aac gtt gtt att gat gt - #c ggt gct aac tcc ggc 144Lys Ala Tyr Gly Val Asn Val Val Ile Asp Va - #l Gly Ala Asn Ser Gly# 45- cag ttc ggt agc gct ttg cgt cgt gca gga tt - #c aag agc cgt atc gtt 192Gln Phe Gly Ser Ala Leu Arg Arg Ala Gly Ph - #e Lys Ser Arg Ile Val# 60- tcc ttt gaa cct ctt tcg ggg cca ttt gcg ca - #a cta acg cgc aag tcg 240Ser Phe Glu Pro Leu Ser Gly Pro Phe Ala Gl - #n Leu Thr Arg Lys Ser# 80- gca tcg gat cca cta tgg gag tgt cac cag ta - #t gcc cta ggc gac gcc 288Ala Ser Asp Pro Leu Trp Glu Cys His Gln Ty - #r Ala Leu Gly Asp Ala# 95- gat gag acg att acc atc aat gtg gca ggc aa - #t gcg ggg gca agt agt 336Asp Glu Thr Ile Thr Ile Asn Val Ala Gly As - #n Ala Gly Ala Ser Ser# 110- tcc gtg ctg ccg atg ctt aaa agt cat caa ga - #t gcc ttt cct ccc gcg 384Ser Val Leu Pro Met Leu Lys Ser His Gln As - #p Ala Phe Pro Pro Ala# 125- aat tat att ggc acc gaa gac gtt gca ata ca - #c cgc ctt gat tcg gtt 432Asn Tyr Ile Gly Thr Glu Asp Val Ala Ile Hi - #s Arg Leu Asp Ser Val# 140- gca tca gaa ttt ctg aac cct acc gat gtt ac - #t ttc ctg aag atc gac 480Ala Ser Glu Phe Leu Asn Pro Thr Asp Val Th - #r Phe Leu Lys Ile Asp145 1 - #50 1 - #55 1 -#60- gta cag ggt ttc gag aag cag gtt atc acg gg - #c agt aag tca acg ctt 528Val Gln Gly Phe Glu Lys Gln Val Ile Thr Gl - #y Ser Lys Ser Thr Leu# 175- aac gaa agc tgc gtc ggc atg caa ctc gaa ct - #t tct ttt att ccg ttg 576Asn Glu Ser Cys Val Gly Met Gln Leu Glu Le - #u Ser Phe Ile Pro Leu# 190- tac gaa ggt gac atg ctg att cat gaa gcg ct - #t gaa ctt gtc tat tcc 624Tyr Glu Gly Asp Met Leu Ile His Glu Ala Le - #u Glu Leu Val Tyr Ser# 205- cta ggt ttc aga ctg acg ggt ttg ttg ccc gg - #c ttt acg gat ccg cgc 672Leu Gly Phe Arg Leu Thr Gly Leu Leu Pro Gl - #y Phe Thr Asp Pro Arg# 220- aat ggt cga atg ctt caa gct gac ggc att tt - #c ttc cgt ggg gac gat 720Asn Gly Arg Met Leu Gln Ala Asp Gly Ile Ph - #e Phe Arg Gly Asp Asp225 2 - #30 2 - #35 2 -#40# 723- <210> SEQ ID NO 18<211> LENGTH: 240<212> TYPE: PRT<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 18- Met Asp Phe Leu Arg Asn Ala Gly Leu Met Al - #a Arg Asn Val Ser Thr# 15- Glu Met Leu Arg His Phe Glu Arg Lys Arg Le - #u Leu Val Asn Gln Phe# 30- Lys Ala Tyr Gly Val Asn Val Val Ile Asp Va - #l Gly Ala Asn Ser Gly# 45- Gln Phe Gly Ser Ala Leu Arg Arg Ala Gly Ph - #e Lys Ser Arg Ile Val# 60- Ser Phe Glu Pro Leu Ser Gly Pro Phe Ala Gl - #n Leu Thr Arg Lys Ser# 80- Ala Ser Asp Pro Leu Trp Glu Cys His Gln Ty - #r Ala Leu Gly Asp Ala# 95- Asp Glu Thr Ile Thr Ile Asn Val Ala Gly As - #n Ala Gly Ala Ser Ser# 110- Ser Val Leu Pro Met Leu Lys Ser His Gln As - #p Ala Phe Pro Pro Ala# 125- Asn Tyr Ile Gly Thr Glu Asp Val Ala Ile Hi - #s Arg Leu Asp Ser Val# 140- Ala Ser Glu Phe Leu Asn Pro Thr Asp Val Th - #r Phe Leu Lys Ile Asp145 1 - #50 1 - #55 1 -#60- Val Gln Gly Phe Glu Lys Gln Val Ile Thr Gl - #y Ser Lys Ser Thr Leu# 175- Asn Glu Ser Cys Val Gly Met Gln Leu Glu Le - #u Ser Phe Ile Pro Leu# 190- Tyr Glu Gly Asp Met Leu Ile His Glu Ala Le - #u Glu Leu Val Tyr Ser# 205- Leu Gly Phe Arg Leu Thr Gly Leu Leu Pro Gl - #y Phe Thr Asp Pro Arg# 220- Asn Gly Arg Met Leu Gln Ala Asp Gly Ile Ph - #e Phe Arg Gly Asp Asp225 2 - #30 2 - #35 2 -#40- <210> SEQ ID NO 19<211> LENGTH: 723<212> TYPE: DNA<213> ORGANISM: Mycobacterium<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)..(720)- <400> SEQUENCE: 19- atg gat ttt ttg cgc aac gcc ggc ttg atg gc - #t cgt aac gtt agc acc 48Met Asp Phe Leu Arg Asn Ala Gly Leu Met Al - #a Arg Asn Val Ser Thr# 15- gag atg ctg cgc cac ttc gaa cga aag cgc ct - #a tta gta aac caa ttc 96Glu Met Leu Arg His Phe Glu Arg Lys Arg Le - #u Leu Val Asn Gln Phe# 30- aaa gca tac gga gtc aac gtt gtt att gat gt - #c ggt gct aac tcc ggc 144Lys Ala Tyr Gly Val Asn Val Val Ile Asp Va - #l Gly Ala Asn Ser Gly# 45- cag ttc ggt agc gct ttg cgt cgt gca gga tt - #c aag agc cgt atc gtt 192Gln Phe Gly Ser Ala Leu Arg Arg Ala Gly Ph - #e Lys Ser Arg Ile Val# 60- tcc ttt gaa cct ctt tcg ggg cca ttt gcg ca - #a cta acg cgc gag tcg 240Ser Phe Glu Pro Leu Ser Gly Pro Phe Ala Gl - #n Leu Thr Arg Glu Ser# 80- gca tcg gat cca cta tgg gag tgt cac cag ta - #t gcc cta ggc gac gcc 288Ala Ser Asp Pro Leu Trp Glu Cys His Gln Ty - #r Ala Leu Gly Asp Ala# 95- gat gag acg att acc atc aat gtg gca ggc aa - #t gcg ggg gca agt agt 336Asp Glu Thr Ile Thr Ile Asn Val Ala Gly As - #n Ala Gly Ala Ser Ser# 110- tcc gtg ctg ccg atg ctt aaa agt cat caa ga - #t gcc ttt cct ccc gcg 384Ser Val Leu Pro Met Leu Lys Ser His Gln As - #p Ala Phe Pro Pro Ala# 125- aat tat att ggc acc gaa gac gtt gca ata ca - #c cgc ctt gat tcg gtt 432Asn Tyr Ile Gly Thr Glu Asp Val Ala Ile Hi - #s Arg Leu Asp Ser Val# 140- gca tca gaa ttt ctg aac cct acc gat gtt ac - #t ttc ctg aag atc gac 480Ala Ser Glu Phe Leu Asn Pro Thr Asp Val Th - #r Phe Leu Lys Ile Asp145 1 - #50 1 - #55 1 -#60- gta cag ggt ttc gag aag cag gtt atc gcg gg - #c agt aag tca acg ctt 528Val Gln Gly Phe Glu Lys Gln Val Ile Ala Gl - #y Ser Lys Ser Thr Leu# 175- aac gaa agc tgc gtc ggc atg caa ctc gaa ct - #t tct ttt att ccg ttg 576Asn Glu Ser Cys Val Gly Met Gln Leu Glu Le - #u Ser Phe Ile Pro Leu# 190- tac gaa ggt gac atg ctg att cat gaa gcg ct - #t gaa ctt gtc tat tcc 624Tyr Glu Gly Asp Met Leu Ile His Glu Ala Le - #u Glu Leu Val Tyr Ser# 205- cta ggt ttc aga ctg acg ggt ttg ttg ccc gg - #a ttt acg gat ccg cgc 672Leu Gly Phe Arg Leu Thr Gly Leu Leu Pro Gl - #y Phe Thr Asp Pro Arg# 220- aat ggt cga atg ctt caa gct gac ggc att tt - #c ttc cgt ggg gac gat 720Asn Gly Arg Met Leu Gln Ala Asp Gly Ile Ph - #e Phe Arg Gly Asp Asp225 2 - #30 2 - #35 2 -#40# 723- <210> SEQ ID NO 20<211> LENGTH: 240<212> TYPE: PRT<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 20- Met Asp Phe Leu Arg Asn Ala Gly Leu Met Al - #a Arg Asn Val Ser Thr# 15- Glu Met Leu Arg His Phe Glu Arg Lys Arg Le - #u Leu Val Asn Gln Phe# 30- Lys Ala Tyr Gly Val Asn Val Val Ile Asp Va - #l Gly Ala Asn Ser Gly# 45- Gln Phe Gly Ser Ala Leu Arg Arg Ala Gly Ph - #e Lys Ser Arg Ile Val# 60- Ser Phe Glu Pro Leu Ser Gly Pro Phe Ala Gl - #n Leu Thr Arg Glu Ser# 80- Ala Ser Asp Pro Leu Trp Glu Cys His Gln Ty - #r Ala Leu Gly Asp Ala# 95- Asp Glu Thr Ile Thr Ile Asn Val Ala Gly As - #n Ala Gly Ala Ser Ser# 110- Ser Val Leu Pro Met Leu Lys Ser His Gln As - #p Ala Phe Pro Pro Ala# 125- Asn Tyr Ile Gly Thr Glu Asp Val Ala Ile Hi - #s Arg Leu Asp Ser Val# 140- Ala Ser Glu Phe Leu Asn Pro Thr Asp Val Th - #r Phe Leu Lys Ile Asp145 1 - #50 1 - #55 1 -#60- Val Gln Gly Phe Glu Lys Gln Val Ile Ala Gl - #y Ser Lys Ser Thr Leu# 175- Asn Glu Ser Cys Val Gly Met Gln Leu Glu Le - #u Ser Phe Ile Pro Leu# 190- Tyr Glu Gly Asp Met Leu Ile His Glu Ala Le - #u Glu Leu Val Tyr Ser# 205- Leu Gly Phe Arg Leu Thr Gly Leu Leu Pro Gl - #y Phe Thr Asp Pro Arg# 220- Asn Gly Arg Met Leu Gln Ala Asp Gly Ile Ph - #e Phe Arg Gly Asp Asp225 2 - #30 2 - #35 2 -#40- <210> SEQ ID NO 21<211> LENGTH: 801<212> TYPE: DNA<213> ORGANISM: Mycobacterium<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)..(798)- <400> SEQUENCE: 21- atg act gcg cca gtg ttc tcg ata att atc cc - #t acc ttc aat gca gcg 48Met Thr Ala Pro Val Phe Ser Ile Ile Ile Pr - #o Thr Phe Asn Ala Ala# 15- gtg acg ctg caa gcc tgc ctc gga agc atc gt - #c ggg cag acc tac cgg 96Val Thr Leu Gln Ala Cys Leu Gly Ser Ile Va - #l Gly Gln Thr Tyr Arg# 30- gaa gtg gaa gtg gtc ctt gtc gac ggc ggt tc - #g acc gat cgg acc ctc 144Glu Val Glu Val Val Leu Val Asp Gly Gly Se - #r Thr Asp Arg Thr Leu# 45- gac atc gcg aac agt ttc cgc ccg gaa ctc gg - #c tcg cga ctg gtc gtt 192Asp Ile Ala Asn Ser Phe Arg Pro Glu Leu Gl - #y Ser Arg Leu Val Val# 60- cac agc ggg ccc gat gat ggc ccc tac gac gc - #c atg aac cgc ggc gtc 240His Ser Gly Pro Asp Asp Gly Pro Tyr Asp Al - #a Met Asn Arg Gly Val# 80- ggc gtg gcc aca ggc gaa tgg gta ctt ttt tt - #a ggc gcc gac gac acc 288Gly Val Ala Thr Gly Glu Trp Val Leu Phe Le - #u Gly Ala Asp Asp Thr# 95- ctc tac gaa cca acc acg ttg gcc cag gta gc - #c gct ttt ctc ggc gac 336Leu Tyr Glu Pro Thr Thr Leu Ala Gln Val Al - #a Ala Phe Leu Gly Asp# 110- cat gcg gca agc cat ctt gtc tat ggc gat gt - #t gtg atg cgt tcg acg 384His Ala Ala Ser His Leu Val Tyr Gly Asp Va - #l Val Met Arg Ser Thr# 125- aaa agc cgg cat gcc gga cct ttc gac ctc ga - #c cgc ctc cta ttt gag 432Lys Ser Arg His Ala Gly Pro Phe Asp Leu As - #p Arg Leu Leu Phe Glu# 140- acg aat ttg tgc cac caa tcg atc ttt tac cg - #c cgt gag ctt ttc gac 480Thr Asn Leu Cys His Gln Ser Ile Phe Tyr Ar - #g Arg Glu Leu Phe Asp145 1 - #50 1 - #55 1 -#60- ggc atc ggc cct tac aac ctg cgc tac cga gt - #c tgg gcg gac tgg gac 528Gly Ile Gly Pro Tyr Asn Leu Arg Tyr Arg Va - #l Trp Ala Asp Trp Asp# 175- ttc aat att cgc tgc ttc tcc aac ccg gcg ct - #g att acc cgc tac atg 576Phe Asn Ile Arg Cys Phe Ser Asn Pro Ala Le - #u Ile Thr Arg Tyr Met# 190- gac gtc gtg att tcc gaa tac aac gac atg ac - #c ggc ttc agc atg agg 624Asp Val Val Ile Ser Glu Tyr Asn Asp Met Th - #r Gly Phe Ser Met Arg# 205- cag ggg act gat aaa gag ttc aga aaa cgg ct - #g cca atg tac ttc tgg 672Gln Gly Thr Asp Lys Glu Phe Arg Lys Arg Le - #u Pro Met Tyr Phe Trp# 220- gtt gca ggg tgg gag act tgc agg cgc atg ct - #g gcg ttt ttg aaa gac 720Val Ala Gly Trp Glu Thr Cys Arg Arg Met Le - #u Ala Phe Leu Lys Asp225 2 - #30 2 - #35 2 -#40- aag gag aat cgc cgt ctg gcc ttg cgt acg cg - #g ttg ata agg gtt aag 768Lys Glu Asn Arg Arg Leu Ala Leu Arg Thr Ar - #g Leu Ile Arg Val Lys# 255# 801c aaa gaa cga agc gca gaa ccg ta - #gAla Val Ser Lys Glu Arg Ser Ala Glu Pro# 265- <210> SEQ ID NO 22<211> LENGTH: 266<212> TYPE: PRT<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 22- Met Thr Ala Pro Val Phe Ser Ile Ile Ile Pr - #o Thr Phe Asn Ala Ala# 15- Val Thr Leu Gln Ala Cys Leu Gly Ser Ile Va - #l Gly Gln Thr Tyr Arg# 30- Glu Val Glu Val Val Leu Val Asp Gly Gly Se - #r Thr Asp Arg Thr Leu# 45- Asp Ile Ala Asn Ser Phe Arg Pro Glu Leu Gl - #y Ser Arg Leu Val Val# 60- His Ser Gly Pro Asp Asp Gly Pro Tyr Asp Al - #a Met Asn Arg Gly Val# 80- Gly Val Ala Thr Gly Glu Trp Val Leu Phe Le - #u Gly Ala Asp Asp Thr# 95- Leu Tyr Glu Pro Thr Thr Leu Ala Gln Val Al - #a Ala Phe Leu Gly Asp# 110- His Ala Ala Ser His Leu Val Tyr Gly Asp Va - #l Val Met Arg Ser Thr# 125- Lys Ser Arg His Ala Gly Pro Phe Asp Leu As - #p Arg Leu Leu Phe Glu# 140- Thr Asn Leu Cys His Gln Ser Ile Phe Tyr Ar - #g Arg Glu Leu Phe Asp145 1 - #50 1 - #55 1 -#60- Gly Ile Gly Pro Tyr Asn Leu Arg Tyr Arg Va - #l Trp Ala Asp Trp Asp# 175- Phe Asn Ile Arg Cys Phe Ser Asn Pro Ala Le - #u Ile Thr Arg Tyr Met# 190- Asp Val Val Ile Ser Glu Tyr Asn Asp Met Th - #r Gly Phe Ser Met Arg# 205- Gln Gly Thr Asp Lys Glu Phe Arg Lys Arg Le - #u Pro Met Tyr Phe Trp# 220- Val Ala Gly Trp Glu Thr Cys Arg Arg Met Le - #u Ala Phe Leu Lys Asp225 2 - #30 2 - #35 2 -#40- Lys Glu Asn Arg Arg Leu Ala Leu Arg Thr Ar - #g Leu Ile Arg Val Lys# 255- Ala Val Ser Lys Glu Arg Ser Ala Glu Pro# 265- <210> SEQ ID NO 23<211> LENGTH: 801<212> TYPE: DNA<213> ORGANISM: Mycobacterium<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)..(798)- <400> SEQUENCE: 23- atg act gcg cca gtg ttc tcg ata att atc cc - #t acc ttc aat gca gcg 48Met Thr Ala Pro Val Phe Ser Ile Ile Ile Pr - #o Thr Phe Asn Ala Ala# 15- gtg acg ctg caa gcc tgc ctc gga agc atc gt - #c ggg cag acc tac cgg 96Val Thr Leu Gln Ala Cys Leu Gly Ser Ile Va - #l Gly Gln Thr Tyr Arg# 30- gaa gtg gaa gtg gtc ctt gtc gac ggc ggt tc - #g acc gat cgg acc ctc 144Glu Val Glu Val Val Leu Val Asp Gly Gly Se - #r Thr Asp Arg Thr Leu# 45- gac atc gcg aac agt ttc cgc ccg gaa ctc gg - #c tcg cga ctg gtc gtt 192Asp Ile Ala Asn Ser Phe Arg Pro Glu Leu Gl - #y Ser Arg Leu Val Val# 60- cac agc ggg ccc gat gat ggc ccc tac gac gc - #c atg aac cgc ggc gtc 240His Ser Gly Pro Asp Asp Gly Pro Tyr Asp Al - #a Met Asn Arg Gly Val# 80- ggc gta gcc aca ggc gaa tgg gta ctt ttt tt - #a ggc gcc gac gac acc 288Gly Val Ala Thr Gly Glu Trp Val Leu Phe Le - #u Gly Ala Asp Asp Thr# 95- ctc tac gaa cca acc acg ttg gcc cag gta gc - #c gct ttt ctc ggc gac 336Leu Tyr Glu Pro Thr Thr Leu Ala Gln Val Al - #a Ala Phe Leu Gly Asp# 110- cat gcg gca agc cat ctt gtc tat ggc gat gt - #t gtg atg cgt tcg acg 384His Ala Ala Ser His Leu Val Tyr Gly Asp Va - #l Val Met Arg Ser Thr# 125- aaa agc cgg cat gcc gga cct ttc gac ctc ga - #c cgc ctc cta ttt gag 432Lys Ser Arg His Ala Gly Pro Phe Asp Leu As - #p Arg Leu Leu Phe Glu# 140- acg aat ttg tgc cac caa tcg atc ttt tac cg - #c cgt gag ctt ttc gac 480Thr Asn Leu Cys His Gln Ser Ile Phe Tyr Ar - #g Arg Glu Leu Phe Asp145 1 - #50 1 - #55 1 -#60- ggc atc ggc cct tac aac ctg cgc tac cga gt - #c tgg gcg gac tgg gac 528Gly Ile Gly Pro Tyr Asn Leu Arg Tyr Arg Va - #l Trp Ala Asp Trp Asp# 175- ttc aat att cgc tgc ttc tcc aac ccg gcg ct - #g att acc cgc tac atg 576Phe Asn Ile Arg Cys Phe Ser Asn Pro Ala Le - #u Ile Thr Arg Tyr Met# 190- gac gtc gtg att tcc gaa tac aac gac atg ac - #c ggc ttc agc atg agg 624Asp Val Val Ile Ser Glu Tyr Asn Asp Met Th - #r Gly Phe Ser Met Arg# 205- cag ggg act gat aaa gag ttc aga aaa cgg ct - #g cca atg tac ttc tgg 672Gln Gly Thr Asp Lys Glu Phe Arg Lys Arg Le - #u Pro Met Tyr Phe Trp# 220- gtt gca ggg tgg gag act tgc agg cgc atg ct - #g gcg ttt ttg aaa gac 720Val Ala Gly Trp Glu Thr Cys Arg Arg Met Le - #u Ala Phe Leu Lys Asp225 2 - #30 2 - #35 2 -#40- aag gag aat cgc cgt ctg gcc ttg cgt acg cg - #g ttg ata agg gtt aag 768Lys Glu Asn Arg Arg Leu Ala Leu Arg Thr Ar - #g Leu Ile Arg Val Lys# 255# 801c aaa gaa cga agc gca gaa ccg ta - #gAla Val Ser Lys Glu Arg Ser Ala Glu Pro# 265- <210> SEQ ID NO 24<211> LENGTH: 266<212> TYPE: PRT<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 24- Met Thr Ala Pro Val Phe Ser Ile Ile Ile Pr - #o Thr Phe Asn Ala Ala# 15- Val Thr Leu Gln Ala Cys Leu Gly Ser Ile Va - #l Gly Gln Thr Tyr Arg# 30- Glu Val Glu Val Val Leu Val Asp Gly Gly Se - #r Thr Asp Arg Thr Leu# 45- Asp Ile Ala Asn Ser Phe Arg Pro Glu Leu Gl - #y Ser Arg Leu Val Val# 60- His Ser Gly Pro Asp Asp Gly Pro Tyr Asp Al - #a Met Asn Arg Gly Val# 80- Gly Val Ala Thr Gly Glu Trp Val Leu Phe Le - #u Gly Ala Asp Asp Thr# 95- Leu Tyr Glu Pro Thr Thr Leu Ala Gln Val Al - #a Ala Phe Leu Gly Asp# 110- His Ala Ala Ser His Leu Val Tyr Gly Asp Va - #l Val Met Arg Ser Thr# 125- Lys Ser Arg His Ala Gly Pro Phe Asp Leu As - #p Arg Leu Leu Phe Glu# 140- Thr Asn Leu Cys His Gln Ser Ile Phe Tyr Ar - #g Arg Glu Leu Phe Asp145 1 - #50 1 - #55 1 -#60- Gly Ile Gly Pro Tyr Asn Leu Arg Tyr Arg Va - #l Trp Ala Asp Trp Asp# 175- Phe Asn Ile Arg Cys Phe Ser Asn Pro Ala Le - #u Ile Thr Arg Tyr Met# 190- Asp Val Val Ile Ser Glu Tyr Asn Asp Met Th - #r Gly Phe Ser Met Arg# 205- Gln Gly Thr Asp Lys Glu Phe Arg Lys Arg Le - #u Pro Met Tyr Phe Trp# 220- Val Ala Gly Trp Glu Thr Cys Arg Arg Met Le - #u Ala Phe Leu Lys Asp225 2 - #30 2 - #35 2 -#40- Lys Glu Asn Arg Arg Leu Ala Leu Arg Thr Ar - #g Leu Ile Arg Val Lys# 255- Ala Val Ser Lys Glu Arg Ser Ala Glu Pro# 265- <210> SEQ ID NO 25<211> LENGTH: 867<212> TYPE: DNA<213> ORGANISM: Mycobacterium<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)..(864)- <400> SEQUENCE: 25- gtg gcc agc aga agt ccc cac tcc gct gcg gg - #t ggt tgg cta att ctt 48Val Ala Ser Arg Ser Pro His Ser Ala Ala Gl - #y Gly Trp Leu Ile Leu# 15- ggc ggc tcc ctt ctt gtg gtc ggc gtg gcg ca - #t ccg gta gga ctc gcc 96Gly Gly Ser Leu Leu Val Val Gly Val Ala Hi - #s Pro Val Gly Leu Ala# 30- gga ggt gac gac gat gct ggc gtg gtg cag ca - #g ccg atc gag gat gct 144Gly Gly Asp Asp Asp Ala Gly Val Val Gln Gl - #n Pro Ile Glu Asp Ala# 45- ggc ggc ggt ggt gtg ctc ggg cag gaa tcg cc - #c cca ttg ttc gaa ggg 192Gly Gly Gly Gly Val Leu Gly Gln Glu Ser Pr - #o Pro Leu Phe Glu Gly# 60- cca atg cga ggc gat ggc cag gga gcg gcg ct - #c gta gcc ggc agc cac 240Pro Met Arg Gly Asp Gly Gln Gly Ala Ala Le - #u Val Ala Gly Ser His# 80- gag ccg gaa caa cag ttg agt ccc ggt gtc gt - #c gag cgg ggc gaa gcc 288Glu Pro Glu Gln Gln Leu Ser Pro Gly Val Va - #l Glu Arg Gly Glu Ala# 95- gat ctc gtc caa gat gac cag atc cgc gcg ga - #g cag ggt gtc gat gat 336Asp Leu Val Gln Asp Asp Gln Ile Arg Ala Gl - #u Gln Gly Val Asp Asp# 110- ctt gcc gac ggt gtt gtc ggc cag gcc gcg gt - #a gag gac ctc gat cag 384Leu Ala Asp Gly Val Val Gly Gln Ala Ala Va - #l Glu Asp Leu Asp Gln# 125- gtc ggc ggc ggt gaa gta gcg gac ttt gaa tc - #c ggc gtg gac ggc agc 432Val Gly Gly Gly Glu Val Ala Asp Phe Glu Se - #r Gly Val Asp Gly Ser# 140- gtg ccc gca gcc gat gag cag gtg act ttt gc - #c cgt acc agg tgg gcc 480Val Pro Ala Ala Asp Glu Gln Val Thr Phe Al - #a Arg Thr Arg Trp Ala145 1 - #50 1 - #55 1 -#60- aat gac cgc cag gtt ctg ttg tgc ccg aat cc - #a ttc cag gct cga cag 528Asn Asp Arg Gln Val Leu Leu Cys Pro Asn Pr - #o Phe Gln Ala Arg Gln# 175- gta gtc gaa cgt ggc tgc ggt gat cga cga tc - #c ggt gac gtc gaa ccc 576Val Val Glu Arg Gly Cys Gly Asp Arg Arg Se - #r Gly Asp Val Glu Pro# 190- gtc gag ggt ctt ggt gac cgg gaa ggc tgc gg - #c ctt gag acg gtt ggc 624Val Glu Gly Leu Gly Asp Arg Glu Gly Cys Gl - #y Leu Glu Thr Val Gly# 205- ggt gtt gga ggc atc gcg ggc agc gat ctc gg - #c ctc aac caa cgt ccg 672Gly Val Gly Gly Ile Ala Gly Ser Asp Leu Gl - #y Leu Asn Gln Arg Pro# 220- cag gat ctc ctc cgg tgt cca gcg ttg cgt ct - #t ggc gac ttg caa cac 720Gln Asp Leu Leu Arg Cys Pro Ala Leu Arg Le - #u Gly Asp Leu Gln His225 2 - #30 2 - #35 2 -#40- ctc ggc ggc gtt gcg gcg cac cgt ggc cag ct - #t caa ccg ccg cag cgc 768Leu Gly Gly Val Ala Ala His Arg Gly Gln Le - #u Gln Pro Pro Gln Arg# 255- cgc gtc aag gtc agc agc cag cgg tgc cgc cg - #a gga cgg tgc cac cgg 816Arg Val Lys Val Ser Ser Gln Arg Cys Arg Ar - #g Gly Arg Cys His Arg# 270- ctt ggc agc ggt ggt cat gag gcc gtc ccg tc - #g gtg gtg ttg atc ttg 864Leu Gly Ser Gly Gly His Glu Ala Val Pro Se - #r Val Val Leu Ile Leu# 285# 867- <210> SEQ ID NO 26<211> LENGTH: 288<212> TYPE: PRT<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 26- Val Ala Ser Arg Ser Pro His Ser Ala Ala Gl - #y Gly Trp Leu Ile Leu# 15- Gly Gly Ser Leu Leu Val Val Gly Val Ala Hi - #s Pro Val Gly Leu Ala# 30- Gly Gly Asp Asp Asp Ala Gly Val Val Gln Gl - #n Pro Ile Glu Asp Ala# 45- Gly Gly Gly Gly Val Leu Gly Gln Glu Ser Pr - #o Pro Leu Phe Glu Gly# 60- Pro Met Arg Gly Asp Gly Gln Gly Ala Ala Le - #u Val Ala Gly Ser His# 80- Glu Pro Glu Gln Gln Leu Ser Pro Gly Val Va - #l Glu Arg Gly Glu Ala# 95- Asp Leu Val Gln Asp Asp Gln Ile Arg Ala Gl - #u Gln Gly Val Asp Asp# 110- Leu Ala Asp Gly Val Val Gly Gln Ala Ala Va - #l Glu Asp Leu Asp Gln# 125- Val Gly Gly Gly Glu Val Ala Asp Phe Glu Se - #r Gly Val Asp Gly Ser# 140- Val Pro Ala Ala Asp Glu Gln Val Thr Phe Al - #a Arg Thr Arg Trp Ala145 1 - #50 1 - #55 1 -#60- Asn Asp Arg Gln Val Leu Leu Cys Pro Asn Pr - #o Phe Gln Ala Arg Gln# 175- Val Val Glu Arg Gly Cys Gly Asp Arg Arg Se - #r Gly Asp Val Glu Pro# 190- Val Glu Gly Leu Gly Asp Arg Glu Gly Cys Gl - #y Leu Glu Thr Val Gly# 205- Gly Val Gly Gly Ile Ala Gly Ser Asp Leu Gl - #y Leu Asn Gln Arg Pro# 220- Gln Asp Leu Leu Arg Cys Pro Ala Leu Arg Le - #u Gly Asp Leu Gln His225 2 - #30 2 - #35 2 -#40- Leu Gly Gly Val Ala Ala His Arg Gly Gln Le - #u Gln Pro Pro Gln Arg# 255- Arg Val Lys Val Ser Ser Gln Arg Cys Arg Ar - #g Gly Arg Cys His Arg# 270- Leu Gly Ser Gly Gly His Glu Ala Val Pro Se - #r Val Val Leu Ile Leu# 285- <210> SEQ ID NO 27<211> LENGTH: 1739<212> TYPE: DNA<213> ORGANISM: Mycobacterium<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)..(945)- <400> SEQUENCE: 27- atg ggc tgc ctc aaa ggt ggt gtc gtc gcc aa - #t gtt gtt gtt cca aca 48Met Gly Cys Leu Lys Gly Gly Val Val Ala As - #n Val Val Val Pro Thr# 15- ccg gat tat gtg cga ttc gcg tcc cac tat gg - #c ttc gtt ccg gac ttc 96Pro Asp Tyr Val Arg Phe Ala Ser His Tyr Gl - #y Phe Val Pro Asp Phe# 30- tgc cac ggt gcg gat ccg caa tcg aag ggc at - #c gtg gag aac ctc tgt 144Cys His Gly Ala Asp Pro Gln Ser Lys Gly Il - #e Val Glu Asn Leu Cys# 45- ggc tac gct cag gac gac ctt gcg gtg ccg ct - #g ctg acc gaa gct gcg 192Gly Tyr Ala Gln Asp Asp Leu Ala Val Pro Le - #u Leu Thr Glu Ala Ala# 60- tta gcc ggt gag cag gtc gac cta cgt gcc ct - #c aac gcc cag gcg caa 240Leu Ala Gly Glu Gln Val Asp Leu Arg Ala Le - #u Asn Ala Gln Ala Gln# 80- cta tgg tgc gcc gag gtc aat gcc acg gtc ca - #c tcg gag atc tgc gcc 288Leu Trp Cys Ala Glu Val Asn Ala Thr Val Hi - #s Ser Glu Ile Cys Ala# 95- gtg ccc aac gat cgc ttg gtt gac gag cgc ac - #c gtc ttg agg gag ctg 336Val Pro Asn Asp Arg Leu Val Asp Glu Arg Th - #r Val Leu Arg Glu Leu# 110- ccc tcg ctg cgg ccg acg atc ggc tcg ggg tc - #g gtg cgc cgt aag gtc 384Pro Ser Leu Arg Pro Thr Ile Gly Ser Gly Se - #r Val Arg Arg Lys Val# 125- gac ggc ctc tcg tgc atc cgt tac ggc tca gc - #t cgt tac tcg gtg cct 432Asp Gly Leu Ser Cys Ile Arg Tyr Gly Ser Al - #a Arg Tyr Ser Val Pro# 140- cag cgg ctc gtc ggt gcc acc gtg gcg gtg gt - #g gtc gat cat ggc gcc 480Gln Arg Leu Val Gly Ala Thr Val Ala Val Va - #l Val Asp His Gly Ala145 1 - #50 1 - #55 1 -#60- ctg atc ctg ttg gaa cct gcg acc ggt gtg at - #c gtg gcc gag cac gag 528Leu Ile Leu Leu Glu Pro Ala Thr Gly Val Il - #e Val Ala Glu His Glu# 175- ctc gtc agc cca ggt gag gtg tcc atc ctc ga - #t gaa cac tac gac gga 576Leu Val Ser Pro Gly Glu Val Ser Ile Leu As - #p Glu His Tyr Asp Gly# 190- ccc aga ccc gca ccc tcg cgt ggt cct cgc cc - #g aaa acc caa gca gag 624Pro Arg Pro Ala Pro Ser Arg Gly Pro Arg Pr - #o Lys Thr Gln Ala Glu# 205- aaa cga ttc tgc gca ttg gga acc gaa gcg ca - #g cag ttc ctc gtc ggt 672Lys Arg Phe Cys Ala Leu Gly Thr Glu Ala Gl - #n Gln Phe Leu Val Gly# 220- gct gct gcg atc ggc aac acc cga ctg aaa tc - #c gaa ctc gac att ctg 720Ala Ala Ala Ile Gly Asn Thr Arg Leu Lys Se - #r Glu Leu Asp Ile Leu225 2 - #30 2 - #35 2 -#40- ctc ggc ctt ggc gcc gcc cac ggc gaa cag gc - #t ttg att gac gcg ctg 768Leu Gly Leu Gly Ala Ala His Gly Glu Gln Al - #a Leu Ile Asp Ala Leu# 255- cgc cgg gcg gtt gcg ttt cgc cgg ttc cgc gc - #t gcc gac gtg cgc tcg 816Arg Arg Ala Val Ala Phe Arg Arg Phe Arg Al - #a Ala Asp Val Arg Ser# 270- atc ctg gcc gcc ggc gcc ggc acc cca caa cc - #c cgc ccc gcc ggc gac 864Ile Leu Ala Ala Gly Ala Gly Thr Pro Gln Pr - #o Arg Pro Ala Gly Asp# 285- gca ctc gtg ctc gat ctg ccc acc gtc gag ac - #c cgc tcg ttg gag gcc 912Ala Leu Val Leu Asp Leu Pro Thr Val Glu Th - #r Arg Ser Leu Glu Ala# 300- tac aag atc aac acc acc gac ggg acg gcc tc - #a tgaccaccgc tgccaagccg 965Tyr Lys Ile Asn Thr Thr Asp Gly Thr Ala Se - #r305 3 - #10 3 - #15- gtggcaccgt cctcggcggc accgctggct gctgaccttg acgcggcgct gc - #ggcggttg1025- aagctggcca cggtgcgccg caacgccgcc gaggtgttgc aagtcgccaa ga - #cgcaacgc1085- tggacaccgg aggagatcct gcggacgttg gttgaggccg agatcgctgc cc - #gcgatgcc1145- tccaacaccg ccaaccgtct caaggccgca gccttcccgg tcaccaagac cc - #tcgacggg1205- ttcgacgtca ccggatcgtc gatcaccgca gccacgttcg actacctgtc ga - #gcctggaa1265- tggattcggg cacaacagaa cctggcggtc attggcccac ctggtacggg ca - #aaagtcac1325- ctgctcatcg gctgcgggca cgctgccgtc cacgccggat tcaaagtccg ct - #acttcacc1385- gccgccgacc tgatcgaggt cctctaccgc ggcctggccg acaacaccgt cg - #gcaagatc1445- atcgacaccc tgctccgcgc ggatctggtc atcttggacg agatcggctt cg - #ccccgctc1505- gacgacaccg ggactcaact gttgttccgg ctcgtggctg ccggctacga gc - #gccgctcc1565- ctggccatcg cctcgcattg gcccttcgaa caatgggggc gattcctgcc cg - #agcacacc1625- accgccgcca gcatcctcga tcggctgctg caccacgcca gcatcgtcgt ca - #cctccggc1685- gagtcctacc ggatgcgcca cgccgaccac aagaagggag ccgccaagaa tt - #ag1739- <210> SEQ ID NO 28<211> LENGTH: 315<212> TYPE: PRT<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 28- Met Gly Cys Leu Lys Gly Gly Val Val Ala As - #n Val Val Val Pro Thr# 15- Pro Asp Tyr Val Arg Phe Ala Ser His Tyr Gl - #y Phe Val Pro Asp Phe# 30- Cys His Gly Ala Asp Pro Gln Ser Lys Gly Il - #e Val Glu Asn Leu Cys# 45- Gly Tyr Ala Gln Asp Asp Leu Ala Val Pro Le - #u Leu Thr Glu Ala Ala# 60- Leu Ala Gly Glu Gln Val Asp Leu Arg Ala Le - #u Asn Ala Gln Ala Gln# 80- Leu Trp Cys Ala Glu Val Asn Ala Thr Val Hi - #s Ser Glu Ile Cys Ala# 95- Val Pro Asn Asp Arg Leu Val Asp Glu Arg Th - #r Val Leu Arg Glu Leu# 110- Pro Ser Leu Arg Pro Thr Ile Gly Ser Gly Se - #r Val Arg Arg Lys Val# 125- Asp Gly Leu Ser Cys Ile Arg Tyr Gly Ser Al - #a Arg Tyr Ser Val Pro# 140- Gln Arg Leu Val Gly Ala Thr Val Ala Val Va - #l Val Asp His Gly Ala145 1 - #50 1 - #55 1 -#60- Leu Ile Leu Leu Glu Pro Ala Thr Gly Val Il - #e Val Ala Glu His Glu# 175- Leu Val Ser Pro Gly Glu Val Ser Ile Leu As - #p Glu His Tyr Asp Gly# 190- Pro Arg Pro Ala Pro Ser Arg Gly Pro Arg Pr - #o Lys Thr Gln Ala Glu# 205- Lys Arg Phe Cys Ala Leu Gly Thr Glu Ala Gl - #n Gln Phe Leu Val Gly# 220- Ala Ala Ala Ile Gly Asn Thr Arg Leu Lys Se - #r Glu Leu Asp Ile Leu225 2 - #30 2 - #35 2 -#40- Leu Gly Leu Gly Ala Ala His Gly Glu Gln Al - #a Leu Ile Asp Ala Leu# 255- Arg Arg Ala Val Ala Phe Arg Arg Phe Arg Al - #a Ala Asp Val Arg Ser# 270- Ile Leu Ala Ala Gly Ala Gly Thr Pro Gln Pr - #o Arg Pro Ala Gly Asp# 285- Ala Leu Val Leu Asp Leu Pro Thr Val Glu Th - #r Arg Ser Leu Glu Ala# 300- Tyr Lys Ile Asn Thr Thr Asp Gly Thr Ala Se - #r305 3 - #10 3 - #15- <210> SEQ ID NO 29<211> LENGTH: 264<212> TYPE: PRT<213> ORGANISM: Mycobacteria<220> FEATURE:<221> NAME/KEY: DOMAIN<222> LOCATION: (1)..(264)#encoded by nucleotides: amino acid sequence is 945-1736 of SEQ ID NO:27- <400> SEQUENCE: 29- Met Thr Thr Ala Ala Lys Pro Val Ala Pro Se - #r Ser Ala Ala Pro Leu# 15- Ala Ala Asp Leu Asp Ala Ala Leu Arg Arg Le - #u Lys Leu Ala Thr Val# 30- Arg Arg Asn Ala Ala Glu Val Leu Gln Val Al - #a Lys Thr Gln Arg Trp# 45- Thr Pro Glu Glu Ile Leu Arg Thr Leu Val Gl - #u Ala Glu Ile Ala Ala# 60- Arg Asp Ala Ser Asn Thr Ala Asn Arg Leu Ly - #s Ala Ala Ala Phe Pro# 80- Val Thr Lys Thr Leu Asp Gly Phe Asp Val Th - #r Gly Ser Ser Ile Thr# 95- Ala Ala Thr Phe Asp Tyr Leu Ser Ser Leu Gl - #u Trp Ile Arg Ala Gln# 110- Gln Asn Leu Ala Val Ile Gly Pro Pro Gly Th - #r Gly Lys Ser His Leu# 125- Leu Ile Gly Cys Gly His Ala Ala Val His Al - #a Gly Phe Lys Val Arg# 140- Tyr Phe Thr Ala Ala Asp Leu Ile Glu Val Le - #u Tyr Arg Gly Leu Ala145 1 - #50 1 - #55 1 -#60- Asp Asn Thr Val Gly Lys Ile Ile Asp Thr Le - #u Leu Arg Ala Asp Leu# 175- Val Ile Leu Asp Glu Ile Gly Phe Ala Pro Le - #u Asp Asp Thr Gly Thr# 190- Gln Leu Leu Phe Arg Leu Val Ala Ala Gly Ty - #r Glu Arg Arg Ser Leu# 205- Ala Ile Ala Ser His Trp Pro Phe Glu Gln Tr - #p Gly Arg Phe Leu Pro# 220- Glu His Thr Thr Ala Ala Ser Ile Leu Asp Ar - #g Leu Leu His His Ala225 2 - #30 2 - #35 2 -#40- Ser Ile Val Val Thr Ser Gly Glu Ser Tyr Ar - #g Met Arg His Ala Asp# 255- His Lys Lys Gly Ala Ala Lys Asn 260- <210> SEQ ID NO 30<211> LENGTH: 789<212> TYPE: DNA<213> ORGANISM: Mycobacterium<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)..(786)- <400> SEQUENCE: 30- gtg acg tct gct ccg acc gtc tcg gtg ata ac - #g atc tcg ttc aac gac 48Met Thr Ser Ala Pro Thr Val Ser Val Ile Th - #r Ile Ser Phe Asn Asp# 15- ctc gac ggg ttg cag cgc acg gtg aaa agt gt - #g cgg gcg caa cgc tac 96Leu Asp Gly Leu Gln Arg Thr Val Lys Ser Va - #l Arg Ala Gln Arg Tyr# 30- cgg gga cgc atc gag cac atc gta atc gac gg - #t ggc agc ggc gac gac 144Arg Gly Arg Ile Glu His Ile Val Ile Asp Gl - #y Gly Ser Gly Asp Asp# 45- gtg gtg gca tac ctg tcc ggg tgt gaa cca gg - #c ttc gcg tat tgg cag 192Val Val Ala Tyr Leu Ser Gly Cys Glu Pro Gl - #y Phe Ala Tyr Trp Gln# 60- tcc gag ccc gac ggc ggg cgg tac gac gcg at - #g aac cag ggc atc gcg 240Ser Glu Pro Asp Gly Gly Arg Tyr Asp Ala Me - #t Asn Gln Gly Ile Ala# 80- cac gca tcg ggt gat ctg ttg tgg ttc ttg ca - #c tcc gcc gat cgt ttt 288His Ala Ser Gly Asp Leu Leu Trp Phe Leu Hi - #s Ser Ala Asp Arg Phe# 95- tcc ggg ccc gac gtg gta gcc cag gcc gtg ga - #g gcg cta tcc ggc aag 336Ser Gly Pro Asp Val Val Ala Gln Ala Val Gl - #u Ala Leu Ser Gly Lys# 110- gga ccg gtg tcc gaa ttg tgg ggc ttc ggg at - #g gat cgt ctc gtc ggg 384Gly Pro Val Ser Glu Leu Trp Gly Phe Gly Me - #t Asp Arg Leu Val Gly# 125- ctc gat cgg gtg cgc ggc ccg ata cct ttc ag - #c ctg cgc aaa ttc ctg 432Leu Asp Arg Val Arg Gly Pro Ile Pro Phe Se - #r Leu Arg Lys Phe Leu# 140- gcc ggc aag cag gtt gtt ccg cat caa gca tc - #g ttc ttc gga tca tcg 480Ala Gly Lys Gln Val Val Pro His Gln Ala Se - #r Phe Phe Gly Ser Ser145 1 - #50 1 - #55 1 -#60- ctg gtg gcc aag atc ggt ggc tac gac ctt ga - #t ttc ggg atc gcc gcc 528Leu Val Ala Lys Ile Gly Gly Tyr Asp Leu As - #p Phe Gly Ile Ala Ala# 175- gac cag gaa ttc ata ttg cgg gcc gcg ctg gt - #a tgc gag ccg gtc acg 576Asp Gln Glu Phe Ile Leu Arg Ala Ala Leu Va - #l Cys Glu Pro Val Thr# 190- att cgg tgt gtg ctg tgc gag ttc gac acc ac - #g ggc gtc ggc tcg cac 624Ile Arg Cys Val Leu Cys Glu Phe Asp Thr Th - #r Gly Val Gly Ser His# 205- cgg gaa cca agc gcg gtc ttc ggt gat ctg cg - #c cgc atg ggc gac ctt 672Arg Glu Pro Ser Ala Val Phe Gly Asp Leu Ar - #g Arg Met Gly Asp Leu# 220- cat cgc cgc tac ccg ttc ggg gga agg cga at - #a tca cat gcc tac cta 720His Arg Arg Tyr Pro Phe Gly Gly Arg Arg Il - #e Ser His Ala Tyr Leu225 2 - #30 2 - #35 2 -#40- cgc ggc cgg gag ttc tac gcc tac aac agt cg - #a ttc tgg gaa aac gtc 768Arg Gly Arg Glu Phe Tyr Ala Tyr Asn Ser Ar - #g Phe Trp Glu Asn Val# 255# 789 aaa tagPhe Thr Arg Met Ser Lys 260- <210> SEQ ID NO 31<211> LENGTH: 262<212> TYPE: PRT<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 31- Met Thr Ser Ala Pro Thr Val Ser Val Ile Th - #r Ile Ser Phe Asn Asp# 15- Leu Asp Gly Leu Gln Arg Thr Val Lys Ser Va - #l Arg Ala Gln Arg Tyr# 30- Arg Gly Arg Ile Glu His Ile Val Ile Asp Gl - #y Gly Ser Gly Asp Asp# 45- Val Val Ala Tyr Leu Ser Gly Cys Glu Pro Gl - #y Phe Ala Tyr Trp Gln# 60- Ser Glu Pro Asp Gly Gly Arg Tyr Asp Ala Me - #t Asn Gln Gly Ile Ala# 80- His Ala Ser Gly Asp Leu Leu Trp Phe Leu Hi - #s Ser Ala Asp Arg Phe# 95- Ser Gly Pro Asp Val Val Ala Gln Ala Val Gl - #u Ala Leu Ser Gly Lys# 110- Gly Pro Val Ser Glu Leu Trp Gly Phe Gly Me - #t Asp Arg Leu Val Gly# 125- Leu Asp Arg Val Arg Gly Pro Ile Pro Phe Se - #r Leu Arg Lys Phe Leu# 140- Ala Gly Lys Gln Val Val Pro His Gln Ala Se - #r Phe Phe Gly Ser Ser145 1 - #50 1 - #55 1 -#60- Leu Val Ala Lys Ile Gly Gly Tyr Asp Leu As - #p Phe Gly Ile Ala Ala# 175- Asp Gln Glu Phe Ile Leu Arg Ala Ala Leu Va - #l Cys Glu Pro Val Thr# 190- Ile Arg Cys Val Leu Cys Glu Phe Asp Thr Th - #r Gly Val Gly Ser His# 205- Arg Glu Pro Ser Ala Val Phe Gly Asp Leu Ar - #g Arg Met Gly Asp Leu# 220- His Arg Arg Tyr Pro Phe Gly Gly Arg Arg Il - #e Ser His Ala Tyr Leu225 2 - #30 2 - #35 2 -#40- Arg Gly Arg Glu Phe Tyr Ala Tyr Asn Ser Ar - #g Phe Trp Glu Asn Val# 255- Phe Thr Arg Met Ser Lys 260- <210> SEQ ID NO 32<211> LENGTH: 1023<212> TYPE: DNA<213> ORGANISM: Mycobacterium<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)..(1020)- <400> SEQUENCE: 32- gtg aag cga gcg ctc atc acc gga atc acc gg - #c cag gac ggc tcg tat 48Met Lys Arg Ala Leu Ile Thr Gly Ile Thr Gl - #y Gln Asp Gly Ser Tyr# 15- ctc gcc gaa ctg ctg ctg gcc aag ggg tat ga - #g gtt cac ggg ctc atc 96Leu Ala Glu Leu Leu Leu Ala Lys Gly Tyr Gl - #u Val His Gly Leu Ile# 30- cgg cgc gct tcg acg ttc aac acc tcg cgg at - #c gat cac ctc tac gtc 144Arg Arg Ala Ser Thr Phe Asn Thr Ser Arg Il - #e Asp His Leu Tyr Val# 45- gac ccg cac caa ccg ggc gcg cgg ctg ttt ct - #g cac tat ggt gac ctg 192Asp Pro His Gln Pro Gly Ala Arg Leu Phe Le - #u His Tyr Gly Asp Leu# 60- atc gac gga acc cgg ttg gtg acc ctg ctg ag - #c acc atc gaa ccc gac 240Ile Asp Gly Thr Arg Leu Val Thr Leu Leu Se - #r Thr Ile Glu Pro Asp# 80- gag gtg tac aac ctg gcg gcg cag tca cac gt - #g cgg gtg agc ttc gac 288Glu Val Tyr Asn Leu Ala Ala Gln Ser His Va - #l Arg Val Ser Phe Asp# 95- gaa ccc gtg cac acc ggt gac acc acc ggc at - #g gga tcc atg cga ctg 336Glu Pro Val His Thr Gly Asp Thr Thr Gly Me - #t Gly Ser Met Arg Leu# 110- ctg gaa gcc gtt cgg ctc tct cgg gtg cac tg - #c cgc ttc tat cag gcg 384Leu Glu Ala Val Arg Leu Ser Arg Val His Cy - #s Arg Phe Tyr Gln Ala# 125- tcc tcg tcg gag atg ttc ggc gcc tcg ccg cc - #a ccg cag aac gag ctg 432Ser Ser Ser Glu Met Phe Gly Ala Ser Pro Pr - #o Pro Gln Asn Glu Leu# 140- acg ccg ttc tac ccg cgg tca ccg tat ggc gc - #c gcc aag gtc tat tcg 480Thr Pro Phe Tyr Pro Arg Ser Pro Tyr Gly Al - #a Ala Lys Val Tyr Ser145 1 - #50 1 - #55 1 -#60- tac tgg gcg acc cgc aat tat cgc gaa gcg ta - #c gga ttg ttc gcc gtt 528Tyr Trp Ala Thr Arg Asn Tyr Arg Glu Ala Ty - #r Gly Leu Phe Ala Val# 175- aac ggc atc ttg ttc aat cac gaa tca ccg cg - #g cgc ggt gag acg ttc 576Asn Gly Ile Leu Phe Asn His Glu Ser Pro Ar - #g Arg Gly Glu Thr Phe# 190- gtg acc cga aag atc acc agg gcc gtg gca cg - #c atc aag gcc ggt atc 624Val Thr Arg Lys Ile Thr Arg Ala Val Ala Ar - #g Ile Lys Ala Gly Ile# 205- cag tcc gag gtc tat atg ggc aat ctg gat gc - #g gtc cgc gac tgg ggg 672Gln Ser Glu Val Tyr Met Gly Asn Leu Asp Al - #a Val Arg Asp Trp Gly# 220- tac gcg ccc gaa tac gtc gaa ggc atg tgg cg - #g atg ctg cag acc gac 720Tyr Ala Pro Glu Tyr Val Glu Gly Met Trp Ar - #g Met Leu Gln Thr Asp225 2 - #30 2 - #35 2 -#40- gag ccc gac gac ttc gtt ttg gcg acc ggg cg - #c ggt ttc acc gtg cgt 768Glu Pro Asp Asp Phe Val Leu Ala Thr Gly Ar - #g Gly Phe Thr Val Arg# 255- gag ttc gcg cgg gcc gcg ttc gag cat gcc gg - #t ttg gac tgg cag cag 816Glu Phe Ala Arg Ala Ala Phe Glu His Ala Gl - #y Leu Asp Trp Gln Gln# 270- tac gtg aaa ttc gac caa cgc tat ctg cgg cc - #c acc gag gtg gat tcg 864Tyr Val Lys Phe Asp Gln Arg Tyr Leu Arg Pr - #o Thr Glu Val Asp Ser# 285- ctg atc ggc gac gcg acc aag gct gcc gaa tt - #g ctg ggc tgg agg gct 912Leu Ile Gly Asp Ala Thr Lys Ala Ala Glu Le - #u Leu Gly Trp Arg Ala# 300- tcg gtg cac act gac gag ttg gct cgg atc at - #g gtc gac gcg gac atg 960Ser Val His Thr Asp Glu Leu Ala Arg Ile Me - #t Val Asp Ala Asp Met305 3 - #10 3 - #15 3 -#20- gcg gcg ctg gag tgc gaa ggc aag ccg tgg at - #c gac aag ccg atg atc1008Ala Ala Leu Glu Cys Glu Gly Lys Pro Trp Il - #e Asp Lys Pro Met Ile# 335# 1023 gaAla Gly Arg Thr 340- <210> SEQ ID NO 33<211> LENGTH: 340<212> TYPE: PRT<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 33- Met Lys Arg Ala Leu Ile Thr Gly Ile Thr Gl - #y Gln Asp Gly Ser Tyr# 15- Leu Ala Glu Leu Leu Leu Ala Lys Gly Tyr Gl - #u Val His Gly Leu Ile# 30- Arg Arg Ala Ser Thr Phe Asn Thr Ser Arg Il - #e Asp His Leu Tyr Val# 45- Asp Pro His Gln Pro Gly Ala Arg Leu Phe Le - #u His Tyr Gly Asp Leu# 60- Ile Asp Gly Thr Arg Leu Val Thr Leu Leu Se - #r Thr Ile Glu Pro Asp# 80- Glu Val Tyr Asn Leu Ala Ala Gln Ser His Va - #l Arg Val Ser Phe Asp# 95- Glu Pro Val His Thr Gly Asp Thr Thr Gly Me - #t Gly Ser Met Arg Leu# 110- Leu Glu Ala Val Arg Leu Ser Arg Val His Cy - #s Arg Phe Tyr Gln Ala# 125- Ser Ser Ser Glu Met Phe Gly Ala Ser Pro Pr - #o Pro Gln Asn Glu Leu# 140- Thr Pro Phe Tyr Pro Arg Ser Pro Tyr Gly Al - #a Ala Lys Val Tyr Ser145 1 - #50 1 - #55 1 -#60- Tyr Trp Ala Thr Arg Asn Tyr Arg Glu Ala Ty - #r Gly Leu Phe Ala Val# 175- Asn Gly Ile Leu Phe Asn His Glu Ser Pro Ar - #g Arg Gly Glu Thr Phe# 190- Val Thr Arg Lys Ile Thr Arg Ala Val Ala Ar - #g Ile Lys Ala Gly Ile# 205- Gln Ser Glu Val Tyr Met Gly Asn Leu Asp Al - #a Val Arg Asp Trp Gly# 220- Tyr Ala Pro Glu Tyr Val Glu Gly Met Trp Ar - #g Met Leu Gln Thr Asp225 2 - #30 2 - #35 2 -#40- Glu Pro Asp Asp Phe Val Leu Ala Thr Gly Ar - #g Gly Phe Thr Val Arg# 255- Glu Phe Ala Arg Ala Ala Phe Glu His Ala Gl - #y Leu Asp Trp Gln Gln# 270- Tyr Val Lys Phe Asp Gln Arg Tyr Leu Arg Pr - #o Thr Glu Val Asp Ser# 285- Leu Ile Gly Asp Ala Thr Lys Ala Ala Glu Le - #u Leu Gly Trp Arg Ala# 300- Ser Val His Thr Asp Glu Leu Ala Arg Ile Me - #t Val Asp Ala Asp Met305 3 - #10 3 - #15 3 -#20- Ala Ala Leu Glu Cys Glu Gly Lys Pro Trp Il - #e Asp Lys Pro Met Ile# 335- Ala Gly Arg Thr 340- <210> SEQ ID NO 34<211> LENGTH: 732<212> TYPE: DNA<213> ORGANISM: Mycobacterium<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)..(729)- <400> SEQUENCE: 34- atg agg ctg gcc cgt cgc gct cgg aac atc tt - #g cgt cgc aac ggc atc 48Met Arg Leu Ala Arg Arg Ala Arg Asn Ile Le - #u Arg Arg Asn Gly Ile# 15- gag gtg tcg cgc tac ttt gcc gaa ctg gac tg - #g gaa cgc aat ttc ttg 96Glu Val Ser Arg Tyr Phe Ala Glu Leu Asp Tr - #p Glu Arg Asn Phe Leu# 30- cgc caa ctg caa tcg cat cgg gtc agt gcc gt - #g ctc gat gtc ggg gcc 144Arg Gln Leu Gln Ser His Arg Val Ser Ala Va - #l Leu Asp Val Gly Ala# 45- aat tcg ggg cag tac gcc agg ggt ctg cgc gg - #c gcg ggc ttc gcg ggc 192Asn Ser Gly Gln Tyr Ala Arg Gly Leu Arg Gl - #y Ala Gly Phe Ala Gly# 60- cgc atc gtc tcg ttc gag ccg ctg ccc ggg cc - #c ttt gcc gtc ttg cag 240Arg Ile Val Ser Phe Glu Pro Leu Pro Gly Pr - #o Phe Ala Val Leu Gln# 80- cgc agc gcc tcc acg gac ccg ttg tgg gaa tg - #c cgg cgc tgt gcg ctg 288Arg Ser Ala Ser Thr Asp Pro Leu Trp Glu Cy - #s Arg Arg Cys Ala Leu# 95- ggc gat gtc gat gga acc atc tcg atc aac gt - #c gcc ggc aac gag ggc 336Gly Asp Val Asp Gly Thr Ile Ser Ile Asn Va - #l Ala Gly Asn Glu Gly# 110- gcc agc agt tcc gtc ttg ccg atg ttg aaa cg - #a cat cag gac gcc ttt 384Ala Ser Ser Ser Val Leu Pro Met Leu Lys Ar - #g His Gln Asp Ala Phe# 125- cca cca gcc aac tac gtg ggc gcc caa cgg gt - #g ccg ata cat cga ctc 432Pro Pro Ala Asn Tyr Val Gly Ala Gln Arg Va - #l Pro Ile His Arg Leu# 140- gat tcc gtg gct gca gac gtt ctg cgg ccc aa - #c gat att gcg ttc ttg 480Asp Ser Val Ala Ala Asp Val Leu Arg Pro As - #n Asp Ile Ala Phe Leu145 1 - #50 1 - #55 1 -#60- aag atc gac gtt caa gga ttc gag aag cag gt - #g atc gcg ggt ggc gat 528Lys Ile Asp Val Gln Gly Phe Glu Lys Gln Va - #l Ile Ala Gly Gly Asp# 175- tca acg gtg cac gac cga tgc gtc ggc atg ca - #g ctc gag ctg tct ttc 576Ser Thr Val His Asp Arg Cys Val Gly Met Gl - #n Leu Glu Leu Ser Phe# 190- cag ccg ttg tac gag ggt ggc atg ctc atc cg - #c gag gcg ctc gat ctc 624Gln Pro Leu Tyr Glu Gly Gly Met Leu Ile Ar - #g Glu Ala Leu Asp Leu# 205- gtg gat tcg ttg ggc ttt acg ctc tcg gga tt - #g caa ccc ggt ttc acc 672Val Asp Ser Leu Gly Phe Thr Leu Ser Gly Le - #u Gln Pro Gly Phe Thr# 220- gac ccc cgc aac ggt cga atg ctg cag gcc ga - #t ggc atc ttc ttc cgg 720Asp Pro Arg Asn Gly Arg Met Leu Gln Ala As - #p Gly Ile Phe Phe Arg225 2 - #30 2 - #35 2 -#40# 732Gly Ser Asp- <210> SEQ ID NO 35<211> LENGTH: 243<212> TYPE: PRT<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 35- Met Arg Leu Ala Arg Arg Ala Arg Asn Ile Le - #u Arg Arg Asn Gly Ile# 15- Glu Val Ser Arg Tyr Phe Ala Glu Leu Asp Tr - #p Glu Arg Asn Phe Leu# 30- Arg Gln Leu Gln Ser His Arg Val Ser Ala Va - #l Leu Asp Val Gly Ala# 45- Asn Ser Gly Gln Tyr Ala Arg Gly Leu Arg Gl - #y Ala Gly Phe Ala Gly# 60- Arg Ile Val Ser Phe Glu Pro Leu Pro Gly Pr - #o Phe Ala Val Leu Gln# 80- Arg Ser Ala Ser Thr Asp Pro Leu Trp Glu Cy - #s Arg Arg Cys Ala Leu# 95- Gly Asp Val Asp Gly Thr Ile Ser Ile Asn Va - #l Ala Gly Asn Glu Gly# 110- Ala Ser Ser Ser Val Leu Pro Met Leu Lys Ar - #g His Gln Asp Ala Phe# 125- Pro Pro Ala Asn Tyr Val Gly Ala Gln Arg Va - #l Pro Ile His Arg Leu# 140- Asp Ser Val Ala Ala Asp Val Leu Arg Pro As - #n Asp Ile Ala Phe Leu145 1 - #50 1 - #55 1 -#60- Lys Ile Asp Val Gln Gly Phe Glu Lys Gln Va - #l Ile Ala Gly Gly Asp# 175- Ser Thr Val His Asp Arg Cys Val Gly Met Gl - #n Leu Glu Leu Ser Phe# 190- Gln Pro Leu Tyr Glu Gly Gly Met Leu Ile Ar - #g Glu Ala Leu Asp Leu# 205- Val Asp Ser Leu Gly Phe Thr Leu Ser Gly Le - #u Gln Pro Gly Phe Thr# 220- Asp Pro Arg Asn Gly Arg Met Leu Gln Ala As - #p Gly Ile Phe Phe Arg225 2 - #30 2 - #35 2 -#40- Gly Ser Asp- <210> SEQ ID NO 36<211> LENGTH: 732<212> TYPE: DNA<213> ORGANISM: Mycobacterium<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)..(729)- <400> SEQUENCE: 36- gtg aaa tcg ttg aaa ctc gct cgt ttc atc gc - #g cgt agc gcc gcc ttc 48Met Lys Ser Leu Lys Leu Ala Arg Phe Ile Al - #a Arg Ser Ala Ala Phe# 15- gag gtt tcg cgc cgc tat tct gag cga gac ct - #g aag cac cag ttt gtg 96Glu Val Ser Arg Arg Tyr Ser Glu Arg Asp Le - #u Lys His Gln Phe Val# 30- aag caa ctc aaa tcg cgt cgg gta gat gtc gt - #t ttc gat gtc ggc gcc 144Lys Gln Leu Lys Ser Arg Arg Val Asp Val Va - #l Phe Asp Val Gly Ala# 45- aac tca gga caa tac gcc gcc ggc ctc cgc cg - #a gca gca tat aag ggc 192Asn Ser Gly Gln Tyr Ala Ala Gly Leu Arg Ar - #g Ala Ala Tyr Lys Gly# 60- cgc att gtc tcg ttc gaa ccg cta tcc gga cc - #g ttt acg atc ttg gaa 240Arg Ile Val Ser Phe Glu Pro Leu Ser Gly Pr - #o Phe Thr Ile Leu Glu# 80- agc aaa gcg tca acg gat cca ctt tgg gat tg - #c cgg cag cat gcg ttg 288Ser Lys Ala Ser Thr Asp Pro Leu Trp Asp Cy - #s Arg Gln His Ala Leu# 95- ggc gat tct gat gga acg gtt acg atc aat at - #c gca gga aac gcc ggt 336Gly Asp Ser Asp Gly Thr Val Thr Ile Asn Il - #e Ala Gly Asn Ala Gly# 110- cag agc agt tcc gtc ttg ccc atg ctg aaa ag - #t cat cag aac gct ttt 384Gln Ser Ser Ser Val Leu Pro Met Leu Lys Se - #r His Gln Asn Ala Phe# 125- ccc ccg gca aac tat gtc ggt acc caa gag gc - #g tcc ata cat cga ctt 432Pro Pro Ala Asn Tyr Val Gly Thr Gln Glu Al - #a Ser Ile His Arg Leu# 140- gat tcc gtg gcg cca gaa ttt cta ggc atg aa - #c ggt gtc gct ttt ctc 480Asp Ser Val Ala Pro Glu Phe Leu Gly Met As - #n Gly Val Ala Phe Leu145 1 - #50 1 - #55 1 -#60- aag gtc gac gtt caa ggc ttt gaa aag cag gt - #g ctc gcc ggg ggc aaa 528Lys Val Asp Val Gln Gly Phe Glu Lys Gln Va - #l Leu Ala Gly Gly Lys# 175- tca acc ata gat gac cat tgc gtc ggc atg ca - #a ctc gaa ctg tcc ttc 576Ser Thr Ile Asp Asp His Cys Val Gly Met Gl - #n Leu Glu Leu Ser Phe# 190- ctg ccg ttg tac gaa ggt ggc atg ctc att cc - #t gaa gcc ctc gat ctc 624Leu Pro Leu Tyr Glu Gly Gly Met Leu Ile Pr - #o Glu Ala Leu Asp Leu# 205- gtg tat tcc ttg ggc ttc acg ttg acg gga tt - #g ctg cct tgt ttc att 672Val Tyr Ser Leu Gly Phe Thr Leu Thr Gly Le - #u Leu Pro Cys Phe Ile# 220- gat gca aat aat ggt cga atg ttg cag gcc ga - #c ggc atc ttt ttc cgc 720Asp Ala Asn Asn Gly Arg Met Leu Gln Ala As - #p Gly Ile Phe Phe Arg225 2 - #30 2 - #35 2 -#40# 732Glu Asp Asp- <210> SEQ ID NO 37<211> LENGTH: 243<212> TYPE: PRT<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 37- Met Lys Ser Leu Lys Leu Ala Arg Phe Ile Al - #a Arg Ser Ala Ala Phe# 15- Glu Val Ser Arg Arg Tyr Ser Glu Arg Asp Le - #u Lys His Gln Phe Val# 30- Lys Gln Leu Lys Ser Arg Arg Val Asp Val Va - #l Phe Asp Val Gly Ala# 45- Asn Ser Gly Gln Tyr Ala Ala Gly Leu Arg Ar - #g Ala Ala Tyr Lys Gly# 60- Arg Ile Val Ser Phe Glu Pro Leu Ser Gly Pr - #o Phe Thr Ile Leu Glu# 80- Ser Lys Ala Ser Thr Asp Pro Leu Trp Asp Cy - #s Arg Gln His Ala Leu# 95- Gly Asp Ser Asp Gly Thr Val Thr Ile Asn Il - #e Ala Gly Asn Ala Gly# 110- Gln Ser Ser Ser Val Leu Pro Met Leu Lys Se - #r His Gln Asn Ala Phe# 125- Pro Pro Ala Asn Tyr Val Gly Thr Gln Glu Al - #a Ser Ile His Arg Leu# 140- Asp Ser Val Ala Pro Glu Phe Leu Gly Met As - #n Gly Val Ala Phe Leu145 1 - #50 1 - #55 1 -#60- Lys Val Asp Val Gln Gly Phe Glu Lys Gln Va - #l Leu Ala Gly Gly Lys# 175- Ser Thr Ile Asp Asp His Cys Val Gly Met Gl - #n Leu Glu Leu Ser Phe# 190- Leu Pro Leu Tyr Glu Gly Gly Met Leu Ile Pr - #o Glu Ala Leu Asp Leu# 205- Val Tyr Ser Leu Gly Phe Thr Leu Thr Gly Le - #u Leu Pro Cys Phe Ile# 220- Asp Ala Asn Asn Gly Arg Met Leu Gln Ala As - #p Gly Ile Phe Phe Arg225 2 - #30 2 - #35 2 -#40- Glu Asp Asp- <210> SEQ ID NO 38<211> LENGTH: 828<212> TYPE: DNA<213> ORGANISM: Mycobacterium<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)..(825)- <400> SEQUENCE: 38- atg gtg cag acg aaa cga tac gcc ggc ttg ac - #c gca gct aac aca aag 48Met Val Gln Thr Lys Arg Tyr Ala Gly Leu Th - #r Ala Ala Asn Thr Lys# 15- aaa gtc gcc atg gcc gca cca atg ttt tcg at - #c atc atc ccc acc ttg 96Lys Val Ala Met Ala Ala Pro Met Phe Ser Il - #e Ile Ile Pro Thr Leu# 30- aac gtg gct gcg gta ttg cct gcc tgc ctc ga - #c agc atc gcc cgt cag 144Asn Val Ala Ala Val Leu Pro Ala Cys Leu As - #p Ser Ile Ala Arg Gln# 45- acc tgc ggt gac ttc gag ctg gta ctg gtc ga - #c ggc ggc tcg acg gac 192Thr Cys Gly Asp Phe Glu Leu Val Leu Val As - #p Gly Gly Ser Thr Asp# 60- gaa acc ctc gac atc gcc aac att ttc gcc cc - #c aac ctc ggc gag cgg 240Glu Thr Leu Asp Ile Ala Asn Ile Phe Ala Pr - #o Asn Leu Gly Glu Arg# 80- ttg atc att cat cgc gac acc gac cag ggc gt - #c tac gac gcc atg aac 288Leu Ile Ile His Arg Asp Thr Asp Gln Gly Va - #l Tyr Asp Ala Met Asn# 95- cgc ggc gtg gac ctg gcc acc gga acg tgg tt - #g ctc ttt ctg ggc gcg 336Arg Gly Val Asp Leu Ala Thr Gly Thr Trp Le - #u Leu Phe Leu Gly Ala# 110- gac gac agc ctg tac gag gct gac acc ctg gc - #g cgg gtg gcc gcc ttc 384Asp Asp Ser Leu Tyr Glu Ala Asp Thr Leu Al - #a Arg Val Ala Ala Phe# 125- att ggc gaa cac gag ccc agc gat ctg gta ta - #t ggc gac gtg atc atg 432Ile Gly Glu His Glu Pro Ser Asp Leu Val Ty - #r Gly Asp Val Ile Met# 140- cgc tca acc aat ttc cgc tgg ggt ggc gcc tt - #c gac ctc gac cgt ctg 480Arg Ser Thr Asn Phe Arg Trp Gly Gly Ala Ph - #e Asp Leu Asp Arg Leu145 1 - #50 1 - #55 1 -#60- ttg ttc aag cgc aac atc tgc cat cag gcg at - #c ttc tac cgc cgc gga 528Leu Phe Lys Arg Asn Ile Cys His Gln Ala Il - #e Phe Tyr Arg Arg Gly# 175- ctc ttc ggc acc atc ggt ccc tac aac ctc cg - #c tac cgg gtc ctg gcc 576Leu Phe Gly Thr Ile Gly Pro Tyr Asn Leu Ar - #g Tyr Arg Val Leu Ala# 190- gac tgg gac ttc aat att cgc tgc ttt tcc aa - #c cca gcg ctc gtc acc 624Asp Trp Asp Phe Asn Ile Arg Cys Phe Ser As - #n Pro Ala Leu Val Thr# 205- cgc tac atg cac gtg gtc gtt gca agc tac aa - #c gaa ttc ggc ggg ctc 672Arg Tyr Met His Val Val Val Ala Ser Tyr As - #n Glu Phe Gly Gly Leu# 220- agc aat acg atc gtc gac aag gag ttt ttg aa - #g cgg ctg ccg atg tcc 720Ser Asn Thr Ile Val Asp Lys Glu Phe Leu Ly - #s Arg Leu Pro Met Ser225 2 - #30 2 - #35 2 -#40- acg aga ctc ggc ata agg ctg gtc ata gtt ct - #g gtg cgc agg tgg cca 768Thr Arg Leu Gly Ile Arg Leu Val Ile Val Le - #u Val Arg Arg Trp Pro# 255- aag gtg atc agc agg gcc atg gta atg cgc ac - #c gtc att tct tgg cgg 816Lys Val Ile Ser Arg Ala Met Val Met Arg Th - #r Val Ile Ser Trp Arg# 270# 828Arg Arg Arg 275- <210> SEQ ID NO 39<211> LENGTH: 275<212> TYPE: PRT<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 39- Met Val Gln Thr Lys Arg Tyr Ala Gly Leu Th - #r Ala Ala Asn Thr Lys# 15- Lys Val Ala Met Ala Ala Pro Met Phe Ser Il - #e Ile Ile Pro Thr Leu# 30- Asn Val Ala Ala Val Leu Pro Ala Cys Leu As - #p Ser Ile Ala Arg Gln# 45- Thr Cys Gly Asp Phe Glu Leu Val Leu Val As - #p Gly Gly Ser Thr Asp# 60- Glu Thr Leu Asp Ile Ala Asn Ile Phe Ala Pr - #o Asn Leu Gly Glu Arg# 80- Leu Ile Ile His Arg Asp Thr Asp Gln Gly Va - #l Tyr Asp Ala Met Asn# 95- Arg Gly Val Asp Leu Ala Thr Gly Thr Trp Le - #u Leu Phe Leu Gly Ala# 110- Asp Asp Ser Leu Tyr Glu Ala Asp Thr Leu Al - #a Arg Val Ala Ala Phe# 125- Ile Gly Glu His Glu Pro Ser Asp Leu Val Ty - #r Gly Asp Val Ile Met# 140- Arg Ser Thr Asn Phe Arg Trp Gly Gly Ala Ph - #e Asp Leu Asp Arg Leu145 1 - #50 1 - #55 1 -#60- Leu Phe Lys Arg Asn Ile Cys His Gln Ala Il - #e Phe Tyr Arg Arg Gly# 175- Leu Phe Gly Thr Ile Gly Pro Tyr Asn Leu Ar - #g Tyr Arg Val Leu Ala# 190- Asp Trp Asp Phe Asn Ile Arg Cys Phe Ser As - #n Pro Ala Leu Val Thr# 205- Arg Tyr Met His Val Val Val Ala Ser Tyr As - #n Glu Phe Gly Gly Leu# 220- Ser Asn Thr Ile Val Asp Lys Glu Phe Leu Ly - #s Arg Leu Pro Met Ser225 2 - #30 2 - #35 2 -#40- Thr Arg Leu Gly Ile Arg Leu Val Ile Val Le - #u Val Arg Arg Trp Pro# 255- Lys Val Ile Ser Arg Ala Met Val Met Arg Th - #r Val Ile Ser Trp Arg# 270- Arg Arg Arg 275- <210> SEQ ID NO 40<211> LENGTH: 24<212> TYPE: DNA<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 40# 24aaag ctgc- <210> SEQ ID NO 41<211> LENGTH: 24<212> TYPE: DNA<213> ORGANISM: Mycobacterium- <400> SEQUENCE: 41# 24cctg cacg__________________________________________________________________________

Number	Date	Country
WO 94 26312	Nov 1994	WOX
WO 95 01441	Jan 1995	WOX

Polynucleotides and polypeptides in pathogenic mycobacteria and their use as diagnostics, vaccines and targets for chemotherapy

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Date Filed

Date Issued

Inventors

Original Assignees

Examiners

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CPC

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Abstract

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Priority Claims (1)

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Foreign Referenced Citations (2)

Non-Patent Literature Citations (7)

Entry
Database EMBL, Entry MT024, Accession No. U00024, Jan. 5, 1995 nt. 15203-15934 100% homology with SeqID:36 nt. 14306-15133 100% homology with SeqID:38 XP002033471 cited in the application.
Database EMBL, Entry MTCY277, Accession No. Z79701, Sep. 18, 1996 nt:34705-36493 100% homology with SeqID:30 nt.31972-32994 100% homology with SeqID:32 nt.33956-34687 100% homology with SeqID:34 XP002033472 cited in the application.
Database EMBL, Entry MTAD1, Accession No. AD000001, Dec. 15, 1996 nt.6775-7562 100% homology with Seq.ID:30 nt.9273-10295 100% homology with Seq.ID:32 7580-8311 100% homology with Seq.ID:34 XP002033473.
Database EMBL, Entry MTCY349, Accession No. Z83018, Nov. 26, 1996 nt.34695-35426 100% homology with SeqID:36 nt.33797-34624 100% homology with SeqID:38 XP002033474.
Database EMBL, Entry MTAD9, Accession No. AD000009, Dec. 15, 1996 nt.15203-15934 100% homology with SeqID:36 nt.14306-15133 100% homology with SeqID:38 XP002033475.
Vaccine, vol. 12, No. 16, 1994, pp. 1537-1540, XP002026338 Lowrie D B et al: "Towards A DNA Vaccine Against Tuberculosis" see p. 1537-p. 1538.
Nature, vol. 351, No. 6326, Jun. 6, 1991, pp. 456-460, XP000605495 Stover C K et al: "New Use Of BCG For Recombinant Vaccines" see p. 456-p.457.