US20040260078A1

US20040260078A1 - Novel polynucleotides and polypeptides in pathogenic mycobacteria and their use as diagnostics, vaccines and targets for chemotherapy

Info

Publication number: US20040260078A1
Application number: US10/805,311
Authority: US
Inventors: John Hermon-Taylor; Tim Doran; Douglas Millar; Mark Tizard; Mark Loughlin; Nazira Sumar; John Ford
Original assignee: St Georges Hospital Medical School
Current assignee: St Georges Hospital Medical School
Priority date: 1995-12-21
Filing date: 2004-03-22
Publication date: 2004-12-23
Also published as: US6156322A; DE69634918T2; AU735112B2; ATE299182T1; WO1997023624A3; EP0870032B1; AU1202797A; US7541181B2; DE69634918D1; US20060204521A1; EP0870032A2; WO1997023624A2; GB9526178D0

Abstract

The invention provides a nucleotide sequence representing a pathogenicity island found in species of pathogenic mycobacteria. The islands are shown as SEQ ID NOS: 3 and 4 and comprises several open reading frames encoding polypeptides. These polypeptides and their use in diagnosis and therapy form a further aspect of the invention.

Description

This invention relates to the novel polynucleocide 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 ion 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

FIG. 1([0005] 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. [0006] No 1 and 2 where the Seq. ID No 2 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[0007] ₁H₂on 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. [0008] 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. [0009] 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 [0010]
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. [0011]
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. [0012]
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. [0013] 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. [0014]
ORF H: Sec. ID No 27 Nucleotides 7953 to 6215 from Mavs. [0015]
ORF H[0016] ₁: Seq. ID No 28 Amino acid sequence encoded by nucleotides 7953 to 7006 of Seq. ID No 27
ORF H[0017] 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: [0018]
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. [0019]
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. [0020]
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. [0021]
ORF E: Seq. ID No 36 Cosmid MT024 nucleotides 15934 to 15203 Seq. ID No 37 Amino acid sequence encoded by Seq. ID No 36. [0022]
ORF F: Seq. ID No 38 Cosmid MT024 nucleotides 15133 to 14306 Seq. ID No 39 Amino acid sequence encoded by Seq. ID No 38. [0023]
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. [0024]
The present invention thus provides a polynucleotide in substantially isolated form which is capable of selectively hybridising to sequence [0025] 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. [0026]
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 polynucleotide 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. [0027]
A further aspect of the invention is our discovery of homologies between the [0028] ORFs 3, 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 MT024 (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 [0029]
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 polynucleocide to a solid phase or to enhance the recognition, the in vivo activity, or the lifespan of polynucleotides of the invention. [0030]
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. [0031]
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. [0032]
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, 3 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. [0033]
Polynucleotides of the invention will be capable of selectively hybridizing to the corresponding portion of the GS region, or to the corresponding OR-s 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° C. to about 60° 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 [0034] 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. [0035]
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. [0036]
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. [0037]
An additional class of polynucleotides of the invention are those obtainable from cosmids MTCY277 and MT024 (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. [0038]
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. [0039]
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, [0040] 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. [0041]
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. [0042]
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. [0043]
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. [0044]
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. [0045]
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 EF., Maniatis T (1989). Molecular cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory. [0046]
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. [0047]
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 0.03M sodium chloride and 0.03M sodium citrate at from about 50° C. to about 60° C.) A particularly preferred group of pathogenic mycobacteria are isolates of [0048] 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. [0049]
The invention further provides double stranded polynucleotides comprising a polynucleotide of the invention and its complement. [0050]
Polynucleotides or primers of the invention may carry a revealing label. Suitable labels include radioisotopes such as [0051] ³²P or ³⁵S, 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. [0052]
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. [0053]
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. [0054]
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. [0055]
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. [0056]
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. [0057]
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. [0058]
B. Polypeptides. [0059]
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%. [0060]
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. [0061]
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. [0062]
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. [0063]
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 [0064] 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. [0065]
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. [0066]
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 α-factor signal sequence. [0067]
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. [0068] ¹²⁵I, ³⁵S 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. [0069]
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. [0070]
Immunoassay methods are well known in the art and will generally comprise: [0071]
(a) providing a polypeptide of the invention comprising an epitope bindable by an antibody against said mycobacterial polypeptide; [0072]
(b) incubating a biological sample with said polypeptide under conditions which allow for the formation of an antibody-antigen complex; and [0073]
(c) determining whether antibody-antigen complex comprising said polypeptide is formed. [0074]
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 [0075]
(a) providing a polypeptide of the invention comprising an epitope bindable by a lymphocyte or macrophage or other cell receptor; [0076]
(b) incubating a cell sample with said polypeptide under conditions which allow for a cellular immune response [0077]
such as release of cytokines or other mediator to occur; and [0078]
(c) detecting the presence o: said cytokine or mediator in the incubate. [0079]
Polypeptides of the invention may be made by standard synthetic means well known in the art or recombinantly, as described below. [0080]
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. [0081]
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. [0082]
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. [0083]
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. [0084]
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. [0085]
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 [0086] ³⁵S, ¹²⁵I, 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 inhibitors 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 β-1-3-N-acetylglucosamine may be used as substrates in assays. [0087]
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. [0088]
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. [0089]
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. [0090]
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. [0091]
C. Vectors. [0092]
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. [0093]
D. Expression Vectors. [0094]
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. [0095]
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, E 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. [0096]
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 β-galactosidase promoter. [0097]
The expression vectors may also comprise enhancers, and in the case of eukaryotic vectors polyadenylation signal sequence downstream of the coding sequence being expressed. [0098]
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. [0099]
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. [0100]
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. [0101]
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. [0102]
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. [0103]
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[0104] ⁵to 10⁸c.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 10⁵to 10⁸cells 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-1α, IL-1βor 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. [0105]
E. Antibodies. [0106]
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. [0107]
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. [0108]
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′)[0109] ₂fragments, 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: [0110]
(a) providing an antibody of the invention; [0111]
(b) incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and [0112]
(c) determining whether antibody-antigen complex comprising said antibody is formed. [0113]
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. [0114]
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. [0115]
F. Compositions. [0116]
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. [0117]
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. [0118]
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. [0119]
G. Vaccines. [0120]
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. [0121]
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 [0122] M.bovisBCG, 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. [0123]
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. [0124]
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-1-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, [0125] Immun Rev (1982) 62:185. Other bifunctional coupling agents form a thioether rather than a disulfide linkage. Many of these thio-ether-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 Seoharose®, 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. [0126]
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. [0127]
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. [0128]
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. [0129]
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. [0130]
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 no: 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® 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. [0131]
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%. [0132]
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. [0133]
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 μg to 250 μg, 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. [0134]
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 ocher 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. [0135]
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 [0136] homologous ORFs 3, 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. [0137]
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. [0138]
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[0139] ⁶such as less than 1 in 10⁹or even less than 1 in 10¹²
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. [0140]
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 [0141] Salmonella. The asd gene is described further in Gene (1993) 129; 123-128. A lesion in the asd gene, encoding the enzyme aspartate β-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. [0142]
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. [0143]
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. [0144]
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. [0145]
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. [0146]
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[0147] ⁴to 10⁹bacteria per ml will generally be suitable, e.g. from about 10⁵to 10⁸such as about 10⁶per ml. Live attenuated organisms may be administered subcutaneously or intramuscularly at up to 10⁸organisms in one or more doses, e.g from around 10⁵to 10⁸, e.g about 10⁶or 10⁷organisms 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. [0148]
The following Examples illustrates aspects of the invention. [0149]

EXAMPLE 1

Tests for the presence of the GS identifier sequence were performed on 5 μl bacterial DNA extracts (25 μg/ml to 500 μg/ml) using polymerase chain reaction based on the [0150] oligonucleotide primers 5′-GATGCCGTGAGGAGGTAAAGCTGC-3′ (Seq ID No. 40) and 5′-GATACGGCTCTTGAATCCTGCACG-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° 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.forcuiitum, 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 [0151] ³²P-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 [0152] 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.encerocolitica, 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 β-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, and GSH, 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 β-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 ³⁵S-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.
1 41 1 674 DNA Mycobacterium 1 gatccaacta aacccgatgg aaccccgcgc aaactattgg acgtctccgc gctacgcagt 60 tgggttggcg cccgcgaatc gcactgaaag agggcatcga tgcaacggtg tcgtggtacc 120 gcacaaatgc cgatgccgtg aggaggtaaa gctgcgggcc ggccgatgtt atccctccgg 180 ccggacgggt agggcgacct gccatcgagt ggtacggcag tcgcctggcc ggcgaggcgc 240 atggcctatg tgagtatccc atagcctggc ttggctcgcc cctacgcatt atcagttgac 300 cgctttcgcg ccacgtcgca ggcttgcggc agcatcccgt tcaggtctcc tcatggtccg 360 gtgtggcacg accacgcaag ctcgaaccga ctcgtttccc aatttcgcat gctaatatcg 420 ctcgatggat tttttgcgca acgccggctt gatggctcgt aacgttagca ccgagatgct 480 gcgccactcc gaacgaaagc gcctattagt aaaccaagtc gaagcatacg gagtcaacgt 540 tgttattgat gtcggtgcta actccggcca gttcggtagc gctttgcgtc gtgcaggatt 600 caagagccgt atcgtttcct ttgaacctct ttcggggcca tttgcgcaac taacgcgcaa 660 gtcggcatcg gatc 674 2 674 DNA Mycobacterium 2 gatccgatgc cgacttgcgc gttagttgcg caaatggccc cgaaagaggt tcaaaggaaa 60 cgatacggct cttgaatcct gcacgacgca aagcgctacc gaactggccg gagttagcac 120 cgacatcaat aacaacgttg actccgtatg cttcgacttg gtttactaat aggcgctttc 180 gttcggagtg gcgcagcatc tcggtgctaa cgttacgagc catcaagccg gcgttgcgca 240 aaaaatccat cgagcgatat tagcatgcga aattgggaaa cgagtcggtt cgagcttgcg 300 tggtcgtgcc acaccggacc atgaggagac ctgaacggga tgctgccgca agcctgcgac 360 gtggcgcgaa agcggtcaac tgataatgcg taggggcgag ccaagccagg ctatgggata 420 ctcacatagg ccatgcgcct cgccggccag gcgactgccg taccactcga tggcaggtcg 480 ccctacccgt ccggccggag ggataacatc ggccggcccg cagctttacc tcctcacggc 540 atcggcattt gtgcggtacc acgacaccgt tgcatcgatg ccctctttca gtgcgattcg 600 cgggcgccaa cccaactgcg tagcgcggag acgtccaata gtttgcgcgg ggttccatcg 660 ggtttagttg gatc 674 3 7995 DNA Mycobacterium 3 gaattctggg ttggagacga cgtcgaactc ctggtcggtc ttgcttcgaa tgatcgctgt 60 gatctggtcg gcggtgccga caggaaccgt cgacttgtcg acgatcacct tgtaccggtc 120 gatgtatgac ccaatgtcgt ccgcaaccga gaagacgtac gtcaggtccg ccgccccgct 180 ttcacccatg ggcgtcggga cggcgatgaa aatgacgtcc gcgtgctcga ttccgcgttg 240 ccggtcggtg gtgaagtcaa tcagcccgtt ctcacggttc ctcgcaatca actcccaacc 300 cgggctcgaa aatcgggaca ctgcctgcga ggagcaaatc gatcttggcc tgatcgatat 360 cgacacagac gacatcgttg ccgctatccg cgagacaggc gcccgtgacg aggcctacat 420 agcctgatcc gaccaccgaa attttcaaga tgaccccttc aagtccccga tcggtcgacg 480 accatactgc cgcaactctg taccctccgt gggtaattcg catgtcgcgt tcgtaaggag 540 cagccagcga gtcggggacg ttcggtgaga gagtcgcagg actacgaggt tgccggtgcg 600 atacatcaca gtgttgcgtc tgtcggcaac gatgcagcaa gaacccacgg ggcagccctg 660 aactgcgcgc atgaccggtc cttgtcctgg cacctttgat cggccaccgc ttccatgcga 720 acatgaccgg aatccatagc gcgtggtcaa gcagcgggga ggtagacgtc ggtgtcatct 780 gctccaaccg tgtcggtgat aacgatttcg ctgaacgatc tcgagggatt gaaaagcacc 840 gtggagagcg ttcgcgcgca gcgctatggg gggcgaatcg agcacatcgt catcgacggt 900 ggatcgggcg acgccgtcgt ggagtatctg tccggcgatc ctggctttgc atattggcaa 960 tctcagcccg acaacgggag atatgacgcg atgaatcagg gcattgccca ttcgtcgggc 1020 gacctgttgt ggtttatgca ctccacggat cgtttctccg atccagatgc agtcgcttcc 1080 gtggtggagg cgctctcggg gcatggacca gtacgtgatt tgtggggtta cgggaaaaac 1140 aaccttgtcg gactcgacgg caaaccactt ttccctcggc cgtacggcta tatgccgttt 1200 aagatgcgga aatttctgct cggcgcgacg gttgcgcatc aggcgacatt cttcggcgcg 1260 tcgctggtag ccaagttggg cggttacgat cttgattttg gactcgaggc ggaccagctg 1320 ttcatctacc gtgccgcact aatacggcct cccgtcacga tcgaccgcgt ggtttgcgac 1380 ttcgatgtca cgggacctgg ttcaacccag cccatccgtg agcactatcg gaccctgcgg 1440 cggctctggg acctgcatgg cgactacccg ctgggtgggc gcagagtgtc gtgggcttac 1500 ttgcgtgtga aggagtactt gattcgggcc gacctggccg cattcaacgc ggtaaagttc 1560 ttgcgagcga agttcgccag agcttcgcgg aagcaaaatt catagaaacc aacttctact 1620 gcctgacctg agcagcgccg aggcgcgcag cgcgatcagt gcgacctgaa cggccaggtg 1680 gaaagcgcca ccgatcccgg caccgagtgc ctgacgcttc ggatcccttg caccacaacg 1740 agagtgagag cgccatgatg aggaaatatc ggctgggcgg agtcaacgcc ggagtgacaa 1800 aagtgagaac ccggtgaagc gagcgcttat aacagggatc acggggcagg atggttccta 1860 cctcgccgag ctactactga gcaagggata cgaggttcac gggctcgttc gtcgagcttc 1920 gacgtttaac acgtcgcgga tcgatcacct ctacgttgac ccacaccaac cgggcgcgcg 1980 cttgttcttg cactatgcag acctcactga cggcacccgg ttggtgaccc tgctcagcag 2040 tatcgacccg gatgaggtct acaacctcgc agcgcagtcc catgtgcgcg tcagctttga 2100 cgagccagtg cataccggag acaccaccgg catgggatcg atccgacttc tggaagcagt 2160 ccgcctttct cgggtggact gccggttcta tcaggcttcc tcgtcggaga tgttcggcgc 2220 atctccgcca ccgcagaacg aatcgacgcc gttctatccc cgttcgccat acggcgcggc 2280 caaggtcttc tcgtactgga cgactcgcaa ctatcgagag gcgtacggat tattcgcagt 2340 gaatggcatc ttgttcaacc atgagtcccc ccggcgcggc gagactttcg tgacccgaaa 2400 gatcacgcgt gccgtggcgc gcatccgagc tggcgtccaa tcggaggtct atatgggcaa 2460 cctcgatgcg atccgcgact ggggctacgc gcccgaatat gtcgagggga tgtggaggat 2520 gttgcaagcg cctgaacctg atgactacgt cctggcgaca gggcgtggtt acaccgtacg 2580 tgagttcgct caagctgctt ttgaccatgt cgggctcgac tggcaaaagc gcgtcaagtt 2640 tgacgaccgc tatttgcgtc ccaccgaggt cgattcgcta gtaggagatg ccgacaaggc 2700 ggcccagtca ctcggctgga aagcttcggt tcatactggt gaactcgcgc gcatcatggt 2760 ggacgcggac atcgccgcgt tggagtgcga tggcacacca tggatcgaca cgccgatgtt 2820 gcctggttgg ggcagagtaa gttgacgact acacctgggc ctctggaccg cgcaacgccc 2880 gtgtatatcg ccggtcatcg ggggctggtc ggctcagcgc tcgtacgtag atttgaggcc 2940 gaggggttca ccaatctcat tgtgcgatca cgcgatgaga ttgatctgac ggaccgagcc 3000 gcaacgtttg attttgtgtc tgagacaaga ccacaggtga tcatcgatgc ggccgcacgg 3060 gtcggcggca tcatggcgaa taacacctat cccgcggact tcttgtccga aaacctccga 3120 atccagacca atttgctcga cgcagctgtc gccgtgcgtg tgccgcggct ccttttcctc 3180 ggttcgtcat gcatctaccc gaagtacgct ccgcaaccta tccacgagag tgctttattg 3240 actggccctt tggagcccac caacgacgcg tatgcgatcg ccaagatcgc cggtatcctg 3300 caagttcagg cggttaggcg ccaatatggg ctggcgtgga tctctgcgat gccgactaac 3360 ctctacggac ccggcgacaa cttctccccg tccgggtcgc atctcttgcc ggcgctcatc 3420 cgtcgatatg aggaagccaa agctggtggt gcagaagagg tgacgaattg ggggaccggt 3480 actccgcggc gcgaacttct gcatgtcgac gatctggcga gcgcatgcct gttccttttg 3540 gaacatttcg atggtccgaa ccacgtcaac gtgggcaccg gcgtcgatca cagcattagc 3600 gagatcgcag acatggtcgc tacagcggtg ggctacatcg gcgaaacacg ttgggatcca 3660 actaaacccg atggaacccc gcgcaaacta ttggacgtct ccgcgctacg cgagttgggt 3720 tggcgcccgc gaatcgcact gaaagacggc atcgatgcaa cggtgtcgtg gtaccgcaca 3780 aatgccgatg ccgtgaggag gtaaagctgc gggtcggccg atgttatccc tccggccgga 3840 cgggtggggc gacctgccgt cgagtggtac ggcagtcgcc tggccggcga ggcgcgtggc 3900 ctatgggagt atccaatagc ctggcttggc tcgcccctac gcattatcag ttgaccgctt 3960 tcgcgccagc tcgcaggctt gcggcagcat cccgttcagg tctcctcatg gtccggtgtg 4020 gcacgaccac gcaagctcga accgactcgt ttcccaattt cgcatgctaa tatcgctcga 4080 tggatttttt gcgcaacgcc ggcttgatgg ctcgtaacgt tagtaccgag atgctgcgcc 4140 acttcgaacg aaagcgccta ttagtaaacc aattcaaagc atacggagtc aacgttgtta 4200 ttgatgtcgg tgctaactcc ggccagttcg gtagcgcttt gcgtcgtgca ggattcaaga 4260 gccgtatcgt ttcctttgaa cctctttcgg ggccatttgc gcaactaacg cgcaagtcgg 4320 catcggatcc actatgggag tgtcaccagt atgccctagg cgacgccgat gagacgatta 4380 ccatcaatgt ggcaggcaat gcgggggcaa gtagttccgt gctgccgatg cttaaaagtc 4440 atcaagatgc ctttcctccc gcgaattata ttggcaccga agacgttgca atacaccgcc 4500 ttgattcggt tgcatcagaa tttctgaacc ctaccgatgt tactttcctg aagatcgacg 4560 tacagggttt cgagaagcag gttatcacgg gcagtaagtc aacgcttaac gaaagctgcg 4620 tcggcatgca actcgaactt tcttttattc cgttgtacga aggtgacatg ctgattcatg 4680 aagcgcttga acttgtctat tccctaggtt tcagactgac gggtttgttg cccggcttta 4740 cggatccgcg caatggtcga atgcttcaag ctgacggcat tttcttccgt ggggacgatt 4800 gacataaatg ctccgtcggc accctgccgg tatccaaacg ggcgatctgg tgagccggcc 4860 tcccgggcac ctaatcgact atctaaattg aggcggccgc gacgtgcggc acgaacaggt 4920 ggccggctgc tagcgttaca cacgtcatga ctgcgccagt gttctcgata attatcccta 4980 ccttcaatgc agcggtgacg ctgcaagcct gcctcggaag catcgtcggg cagacctacc 5040 gggaagtgga agtggtcctt gtcgacggcg gttcgaccga tcggaccctc gacatcgcga 5100 acagtttccg cccggaactc ggctcgcgac tggtcgttca cagcgggccc gatgatggcc 5160 cctacgacgc catgaaccgc ggcgtcggcg tggccacagg cgaatgggta ctttttttag 5220 gcgccgacga caccctctac gaaccaacca cgttggccca ggtagccgct tttctcggcg 5280 accatgcggc aagccatctt gtctatggcg atgttgtgat gcgttcgacg aaaagccggc 5340 atgccggacc tttcgacctc gaccgcctcc tatttgagac gaatttgtgc caccaatcga 5400 tcttttaccg ccgtgagctt ttcgacggca tcggccctta caacctgcgc taccgagtct 5460 gggcggactg ggacttcaat attcgctgct tctccaaccc ggcgctgatt acccgctaca 5520 tggacgtcgt gatttccgaa tacaacgaca tgaccggctt cagcatgagg caggggactg 5580 ataaagagtt cagaaaacgg ctgccaatgt acttctgggt tgcagggtgg gagacttgca 5640 ggcgcatgct ggcgtttttg aaagacaagg agaatcgccg tctggccttg cgtacgcggt 5700 tgataagggt taaggccgtc tccaaagaac gaagcgcaga accgtagtcg cggatccaca 5760 ttggacttct ttaacgcgtt tgcgtcctga tccacctttc aagcccgttc cgcgtaacgc 5820 ggcgcgcaga gagtggtcgc atatcgcatc actgttctcg tgccagtgct tggaaagcgt 5880 cgagcactct ggttcgcgtt cttgacgttc gcgcccgctc ctagaggtag cgtgtcacgt 5940 gactgaagcc aatgagtgca actcggcgtc gcgaaaggtt tcagtcgcgg ttgagcaaga 6000 caccgcaaga ctactggagt gcgtgcacaa gcgcctccag ctcgcggctg aaagcggatg 6060 caaagggatt cgaagcttga gcaacatgcg aaggggagaa cggcctatga ggctgggaca 6120 ggttttcgat ccgcgcgcga atgcactgtc aatggccaag tagaagtccc cgctggtggc 6180 cagcagaagt ccccactccg ctgcgggtgg ttggctaatt cttggcggct cccttcttgt 6240 ggtcggcgtg gcgcatccgg taggactcgc cggaggtgac gacgatgctg gcgtggtgca 6300 gcagccgatc gaggatgctg gcggcggtgg tgtgctcggg caggaatcgc ccccattgtt 6360 cgaagggcca atgcgaggcg atggccaggg agcggcgctc gtagccggca gccacgagcc 6420 ggaacaacag ttgagtcccg gtgtcgtcga gcggggcgaa gccgatctcg tccaagatga 6480 ccagatccgc gcggagcagg gtgtcgatga tcttgccgac ggtgttgtcg gccaggccgc 6540 ggtagaggac ctcgatcagg tcggcggcgg tgaagtagcg gactttgaat ccggcgtgga 6600 cggcagcgtg cccgcagccg atgagcaggt gacttttgcc cgtaccaggt gggccaatga 6660 ccgccaggtt ctgttgtgcc cgaatccatt ccaggctcga caggtagtcg aacgtggctg 6720 cggtgatcga cgatccggtg acgtcgaacc cgtcgagggt cttggtgacc gggaaggctg 6780 cggccttgag acggttggcg gtgttggagg catcgcgggc agcgatctcg gcctcaacca 6840 acgtccgcag gatctcctcc ggtgtccagc gttgcgtctt ggcgacttgc aacacctcgg 6900 cggcgttgcg gcgcaccgtg gccagcttca accgccgcag cgccgcgtca aggtcagcag 6960 ccagcggtgc cgccgaggac ggtgccaccg gcttggcagc ggtggtcatg aggccgtccc 7020 gtcggtggtg ttgatcttgt aggcctccaa cgagcgggtc tcgacggtgg gcagatcgag 7080 cacgagtgcg tcgccggcgg ggcggggttg tggggtgccg gcgccggcgg ccaggatcga 7140 gcgcacgtcg gcagcgcgga accggcgaaa cgcaaccgcc cggcgcagcg cgtcaatcaa 7200 agcctgttcg ccgtgggcgg cgccaaggcc gagcagaatg tcgagttcgg atttcagtcg 7260 ggtgttgccg atcgcagcag caccgacgag gaactgctgc gcttcggttc ccaatgcgca 7320 gaatcgtttc tctgcttggg ttttcgggcg aggaccacgc gagggtgcgg gtctgggtcc 7380 gtcgtagtgt tcatcgagga tggacacctc acctgggctg acgagctcgt gctcggccac 7440 gatcacaccg gtcgcaggtt ccaacaggat cagggcgcca tgatcgacca ccaccgccac 7500 ggtggcaccg acgagccgct gaggcaccga gtaacgagct gagccgtaac ggatgcacga 7560 gaggccgtcg accttacggc gcaccgaccc cgagccgatc gtcggccgca gcgagggcag 7620 ctccctcaag acggtgcgct cgtcaaccaa gcgatcgttg ggcacggcgc agatctccga 7680 gtggaccgtg gcattgacct cggcgcacca tagttgcgcc tgggcgttga gggcacgtag 7740 gtcgacctgc tcaccggcta acgcagcttc ggtcagcagc ggcaccgcaa ggtcgtcctg 7800 agcgtagcca cagaggttct ccacgatgcc cttcgattgc ggatccgcac cgtggcagaa 7860 gtccggaacg aagccatagt gggacgcgaa tcgcacataa tccggtgttg gaacaacaac 7920 attggcgacg acaccacctt tgaggcagcc catccggtcg gccaggatct tggccggaac 7980 cccaccgatc gcctc 7995 4 4435 DNA Mycobacterium 4 ttctactgcc tgacctgagc agcgccgagg cgcgcagcgc gatcactgcg acctgaatgg 60 ccaggtggaa agcgccaccg atcccggcac cgagtgcctg acgattcgga tcccttgcac 120 cacaacgaga gtgagaccgc catgatgacg aaatatcggc tgggcggagt caacgccgga 180 gtgacaaaag tgagaacccg gtgaagcgag cgcttataac agggatcacg gggcaggatg 240 gttcctacct cgccgagcta ctactgagca agggatacga ggttcacggg ctcgttcgtc 300 gagcttcgac gtttaacacg tcgcggatcg atcacctcta cgttgaccca caccaaccgg 360 gcgcgcgctt gttcttgcac tatgcagacc tcactgacgg cacccggttg gtgaccctgc 420 tcagcagtat cgacccggat gaggtctaca acctcgcagc gcagtcccat gtgcgcgtca 480 gctttgacga gccagtgcat accggagaca ccaccggcat gggatcgatc cgacttctgg 540 aagcagtccg cctttctcgg gtggactgcc ggttctatca ggcttcctcg tcggagatgt 600 tcggcgcatc tccgccaccg cagaacgaat cgacgccgtt ctatccccgt tcgccatacg 660 gcgcggccaa ggtcttctcg tactggacga ctcgcaacta tcgagaggcg tacggattat 720 tcgcagtgaa tggcatcttg ttcaaccatg agtccccccg gcgcggcgag actttcgtga 780 cccgaaagat cacgcgtgcc gtggcgcgca tccgagctgg ctgccaatcg gaggtctata 840 tgggcaacct cgatgcgatc cgcgactggg gctacgcgcc cgaatatgtc gaggggatgt 900 ggaggatgtt gcaagcgcct gaacctgatg actacgtcct ggcgacaggg cgtggttaca 960 ccgtacgtga gttcgctcaa gctgcttttg accacgtcgg gctcgactgg caaaagcacg 1020 tcaagtttga cgaccgctat ttgcgcccca ccgaggtcga ttcgctagta ggagatgccg 1080 acagggcggc ccagtcactc ggctggaaag cttcggttca tactggtgaa ctcgcgcgca 1140 tcatggtgga cgcggacatc gccgcgtcgg agtgcgatgg cacaccatgg atcgacacgc 1200 cgatgttgcc tggttggggc ggagtaagtt gacgactaca cctgggcctc tggaccgcgc 1260 aacgcccgtg tatatcgccg gtcatcgggg gctggtcggc tcagcgctcg tacgtagatt 1320 tgaggccgag gggttcacca atctcattgt gcgatcacgc gatgagattg atctgacgga 1380 ccgagccgca acgtttgatt ttgtgtctga gacaagacca caggtgatca tcgatgcggc 1440 cgcacgggtc ggcggcatca tggcgaataa cacctatccc gcggacttct tgtccgaaaa 1500 cctccgaatc cagaccaatt tgctcgacgc agctgtcgcc gtgcgtgtgc cgcggctcct 1560 tttcctcggt tcgtcatgca tctacccgaa gtacgctccg caacctatcc acgagagtgc 1620 tttattgact ggccctttgg agcccaccaa cgacgcgtat gcgatcgcca agatcgccgg 1680 tatcctgcaa gttcaggcgg ttaggcgcca atatgggctg gcgtggatct ctgcgatgcc 1740 gactaacctc tacggacccg gcgacaactt ctccccgtcc gggtcgcatc tcttgccggc 1800 gctcatccgt cgatatgagg aagccaaagc tggtggtgca gaagaggtga cgaattgggg 1860 gaccggtact ccgcggcgcg aacttctgca tgtcgacgat ctggcgagcg catgcctgtt 1920 ccttttggaa catttcgatg gtccgaacca cgtcaacgtg ggcaccggcg tcgatcacag 1980 cattagcgag atcgcagaca tggtcgctac ggcggtgggc tacatcggcg aaacacgttg 2040 ggatccaact aaacccgatg gaaccccgcg caaactattg gacgtctccg cgctacgcga 2100 gttgggttgg cgcccgcgaa tcgcactgaa agacggcatc gatgcaacgg tgtcgtggta 2160 ccgcacaaat gccgatgccg tgaggaggta aagctgcggg ccggccgatg ttatccctcc 2220 ggccggacgg gtagggcgac ctgccatcga gtggtacggc agtcgcctgg ccggcgaggc 2280 gcatggccta tgggagtatc ccatagcctg gcttggctcg cccctacgca ttatcagttg 2340 accgctttcg cgccagctcg caggctcgcg gcagcatccc gttcaggtct cctcatggtc 2400 cggtgtggca cgaccacgca agctcgaacc gactcgtttc ccaatttcgc atgctaatat 2460 cgctcgatgg attttttgcg caacgccggc ttgatggctc gtaacgttag caccgagatg 2520 ctgcgccact tcgaacgaaa gcgcctatta gtaaaccaat tcaaagcata cggagtcaac 2580 gttgttattg atgtcggtgc taactccggc cagttcggta gcgctttgcg tcgtgcagga 2640 ttcaagagcc gtatcgtttc ctttgaacct ctttcggggc catttgcgca actaacgcgc 2700 gagtcggcat cggatccact atgggagtgt caccagtatg ccctaggcga cgccgatgag 2760 acgattacca tcaatgtggc aggcaatgcg ggggcaagta gttccgtgct gccgatgctt 2820 aaaagtcatc aagatgcctt tcctcccgcg aattatattg gcaccgaaga cgttgcaata 2880 caccgccttg attcggttgc atcagaattt ctgaacccta ccgatgttac tttcctgaag 2940 atcgacgtac agggtttcga gaagcaggtt atcgcgggca gtaagtcaac gcttaacgaa 3000 agctgcgtcg gcatgcaact cgaactttct tttattccgt tgtacgaagg tgacatgctg 3060 attcatgaag cgcttgaact tgtctattcc ctaggtttca gactgacggg tttgttgccc 3120 ggatttacgg atccgcgcaa tggtcgaatg cttcaagctg acggcatttt cttccgtggg 3180 gacgattgac ataaatgctt gcgtcggcac cctgccggta tccaaacggg cgatctggtg 3240 agccggcctc ccgggcacct aatcgactat ctaaattgag gcggccgcga cgtgcggcac 3300 gaacaggtgg ccggctgcta gcgttacaca cgtcatgact gcgccagtgt tctcgataat 3360 tatccctacc ttcaatgcag cggtgacgct gcaagcctgc ctcggaagca tcgtcgggca 3420 gacctaccgg gaagtggaag tggtccttgt cgacggcggt tcgaccgatc ggaccctcga 3480 catcgcgaac agtttccgcc cggaactcgg ctcgcgactg gtcgttcaca gcgggcccga 3540 tgatggcccc tacgacgcca tgaaccgcgg cgtcggcgta gccacaggcg aatgggtact 3600 ttttttaggc gccgacgaca ccctctacga accaaccacg ttggcccagg tagccgcttt 3660 tctcggcgac catgcggcaa gccatcttgt ctatggcgat gttgtgatgc gttcgacgaa 3720 aagccggcat gccggacctt tcgacctcga ccgcctccta tttgagacga atttgtgcca 3780 ccaatcgatc ttttaccgcc gtgagctttt cgacggcatc ggcccttaca acctgcgcta 3840 ccgagtctgg gcggactggg acttcaatat tcgctgcttc tccaacccgg cgctgattac 3900 ccgctacatg gacgtcgtga tttccgaata caacgacatg accggcttca gcatgaggca 3960 ggggactgat aaagagttca gaaaacggct gccaatgtac ttctgggttg cagggtggga 4020 gacttgcagg cgcatgctgg cgtttttgaa agacaaggag aatcgccgtc tggccttgcg 4080 tacgcggttg ataagggtta aggccgtctc caaagaacga agcgcagaac cgtagtcgcg 4140 gatccacatt ggacttcttt aacgcgtttg cgtcctgatc cacctttcaa ccccgttccg 4200 cgtgacgcgg cgcgcagaga gtggtcgcat atcgcgtcac tgttctcgtg ccagtgcttg 4260 gaaagcgtcg agcactctgg ttcgcgttct tgacgttcgc gcccgcccct agaggtagcg 4320 tgtcacgtga ctgaagccaa tgagtgcaac tcggcgtcgc gaaaggtttc agtcgcggtt 4380 gagcaagaca ccgcaagact actggagtgc gtgcacaagc gcctccagct cacgg 4435 5 378 DNA Mycobacterium CDS (1)..(375) 5 atg atc gct gtg atc tgg tcg gcg gtg ccg aca gga acc gtc gac ttg 48 Met Ile Ala Val Ile Trp Ser Ala Val Pro Thr Gly Thr Val Asp Leu 1 5 10 15 tcg acg atc acc ttg tac cgg tcg atg tat gac cca atg tcg tcc gca 96 Ser Thr Ile Thr Leu Tyr Arg Ser Met Tyr Asp Pro Met Ser Ser Ala 20 25 30 acc gag aag acg tac gtc agg tcc gcc gcc ccg ctt tca ccc atg ggc 144 Thr Glu Lys Thr Tyr Val Arg Ser Ala Ala Pro Leu Ser Pro Met Gly 35 40 45 gtc ggg acg gcg atg aaa atg acg tcc gcg tgc tcg att ccg cgt tgc 192 Val Gly Thr Ala Met Lys Met Thr Ser Ala Cys Ser Ile Pro Arg Cys 50 55 60 cgg tcg gtg gtg aag tca atc agc ccg ttc tca cgg ttc ctc gca atc 240 Arg Ser Val Val Lys Ser Ile Ser Pro Phe Ser Arg Phe Leu Ala Ile 65 70 75 80 aac tcc caa ccc ggg ctc gaa aat cgg gac act gcc tgc gag gag caa 288 Asn Ser Gln Pro Gly Leu Glu Asn Arg Asp Thr Ala Cys Glu Glu Gln 85 90 95 atc gat ctt ggc ctg atc gat atc gac aca gac gac atc gtt gcc gct 336 Ile Asp Leu Gly Leu Ile Asp Ile Asp Thr Asp Asp Ile Val Ala Ala 100 105 110 atc cgc gag aca ggc gcc cgt gac gag gcc tac ata gcc tga 378 Ile Arg Glu Thr Gly Ala Arg Asp Glu Ala Tyr Ile Ala 115 120 125 6 125 PRT Mycobacterium 6 Met Ile Ala Val Ile Trp Ser Ala Val Pro Thr Gly Thr Val Asp Leu 1 5 10 15 Ser Thr Ile Thr Leu Tyr Arg Ser Met Tyr Asp Pro Met Ser Ser Ala 20 25 30 Thr Glu Lys Thr Tyr Val Arg Ser Ala Ala Pro Leu Ser Pro Met Gly 35 40 45 Val Gly Thr Ala Met Lys Met Thr Ser Ala Cys Ser Ile Pro Arg Cys 50 55 60 Arg Ser Val Val Lys Ser Ile Ser Pro Phe Ser Arg Phe Leu Ala Ile 65 70 75 80 Asn Ser Gln Pro Gly Leu Glu Asn Arg Asp Thr Ala Cys Glu Glu Gln 85 90 95 Ile Asp Leu Gly Leu Ile Asp Ile Asp Thr Asp Asp Ile Val Ala Ala 100 105 110 Ile Arg Glu Thr Gly Ala Arg Asp Glu Ala Tyr Ile Ala 115 120 125 7 834 DNA Mycobacterium CDS (1)..(831) 7 gtg tca tct gct cca acc gtg tcg gtg ata acg att tcg ctg aac gat 48 Val Ser Ser Ala Pro Thr Val Ser Val Ile Thr Ile Ser Leu Asn Asp 1 5 10 15 ctc gag gga ttg aaa agc acc gtg gag agc gtt cgc gcg cag cgc tat 96 Leu Glu Gly Leu Lys Ser Thr Val Glu Ser Val Arg Ala Gln Arg Tyr 20 25 30 ggg ggg cga atc gag cac atc gtc atc gac ggt gga tcg ggc gac gcc 144 Gly Gly Arg Ile Glu His Ile Val Ile Asp Gly Gly Ser Gly Asp Ala 35 40 45 gtc gtg gag tat ctg tcc ggc gat cct ggc ttt gca tat tgg caa tct 192 Val Val Glu Tyr Leu Ser Gly Asp Pro Gly Phe Ala Tyr Trp Gln Ser 50 55 60 cag ccc gac aac ggg aga tat gac gcg atg aat cag ggc att gcc cat 240 Gln Pro Asp Asn Gly Arg Tyr Asp Ala Met Asn Gln Gly Ile Ala His 65 70 75 80 tcg tcg ggc gac ctg ttg tgg ttt atg cac tcc acg gat cgt ttc tcc 288 Ser Ser Gly Asp Leu Leu Trp Phe Met His Ser Thr Asp Arg Phe Ser 85 90 95 gat cca gat gca gtc gct tcc gtg gtg gag gcg ctc tcg ggg cat gga 336 Asp Pro Asp Ala Val Ala Ser Val Val Glu Ala Leu Ser Gly His Gly 100 105 110 cca gta cgt gat ttg tgg ggt tac ggg aaa aac aac ctt gtc gga ctc 384 Pro Val Arg Asp Leu Trp Gly Tyr Gly Lys Asn Asn Leu Val Gly Leu 115 120 125 gac ggc aaa cca ctt ttc cct cgg ccg tac ggc tat atg ccg ttt aag 432 Asp Gly Lys Pro Leu Phe Pro Arg Pro Tyr Gly Tyr Met Pro Phe Lys 130 135 140 atg cgg aaa ttt ctg ctc ggc gcg acg gtt gcg cat cag gcg aca ttc 480 Met Arg Lys Phe Leu Leu Gly Ala Thr Val Ala His Gln Ala Thr Phe 145 150 155 160 ttc ggc gcg tcg ctg gta gcc aag ttg ggc ggt tac gat ctt gat ttt 528 Phe Gly Ala Ser Leu Val Ala Lys Leu Gly Gly Tyr Asp Leu Asp Phe 165 170 175 gga ctc gag gcg gac cag ctg ttc atc tac cgt gcc gca cta ata cgg 576 Gly Leu Glu Ala Asp Gln Leu Phe Ile Tyr Arg Ala Ala Leu Ile Arg 180 185 190 cct ccc gtc acg atc gac cgc gtg gtt tgc gac ttc gat gtc acg gga 624 Pro Pro Val Thr Ile Asp Arg Val Val Cys Asp Phe Asp Val Thr Gly 195 200 205 cct ggt tca acc cag ccc atc cgt gag cac tat cgg acc ctg cgg cgg 672 Pro Gly Ser Thr Gln Pro Ile Arg Glu His Tyr Arg Thr Leu Arg Arg 210 215 220 ctc tgg gac ctg cat ggc gac tac ccg ctg ggt ggg cgc aga gtg tcg 720 Leu Trp Asp Leu His Gly Asp Tyr Pro Leu Gly Gly Arg Arg Val Ser 225 230 235 240 tgg gct tac ttg cgt gtg aag gag tac ttg att cgg gcc gac ctg gcc 768 Trp Ala Tyr Leu Arg Val Lys Glu Tyr Leu Ile Arg Ala Asp Leu Ala 245 250 255 gca ttc aac gcg gta aag ttc ttg cga gcg aag ttc gcc aga gct tcg 816 Ala Phe Asn Ala Val Lys Phe Leu Arg Ala Lys Phe Ala Arg Ala Ser 260 265 270 cgg aag caa aat tca tag 834 Arg Lys Gln Asn Ser 275 8 277 PRT Mycobacterium 8 Val Ser Ser Ala Pro Thr Val Ser Val Ile Thr Ile Ser Leu Asn Asp 1 5 10 15 Leu Glu Gly Leu Lys Ser Thr Val Glu Ser Val Arg Ala Gln Arg Tyr 20 25 30 Gly Gly Arg Ile Glu His Ile Val Ile Asp Gly Gly Ser Gly Asp Ala 35 40 45 Val Val Glu Tyr Leu Ser Gly Asp Pro Gly Phe Ala Tyr Trp Gln Ser 50 55 60 Gln Pro Asp Asn Gly Arg Tyr Asp Ala Met Asn Gln Gly Ile Ala His 65 70 75 80 Ser Ser Gly Asp Leu Leu Trp Phe Met His Ser Thr Asp Arg Phe Ser 85 90 95 Asp Pro Asp Ala Val Ala Ser Val Val Glu Ala Leu Ser Gly His Gly 100 105 110 Pro Val Arg Asp Leu Trp Gly Tyr Gly Lys Asn Asn Leu Val Gly Leu 115 120 125 Asp Gly Lys Pro Leu Phe Pro Arg Pro Tyr Gly Tyr Met Pro Phe Lys 130 135 140 Met Arg Lys Phe Leu Leu Gly Ala Thr Val Ala His Gln Ala Thr Phe 145 150 155 160 Phe Gly Ala Ser Leu Val Ala Lys Leu Gly Gly Tyr Asp Leu Asp Phe 165 170 175 Gly Leu Glu Ala Asp Gln Leu Phe Ile Tyr Arg Ala Ala Leu Ile Arg 180 185 190 Pro Pro Val Thr Ile Asp Arg Val Val Cys Asp Phe Asp Val Thr Gly 195 200 205 Pro Gly Ser Thr Gln Pro Ile Arg Glu His Tyr Arg Thr Leu Arg Arg 210 215 220 Leu Trp Asp Leu His Gly Asp Tyr Pro Leu Gly Gly Arg Arg Val Ser 225 230 235 240 Trp Ala Tyr Leu Arg Val Lys Glu Tyr Leu Ile Arg Ala Asp Leu Ala 245 250 255 Ala Phe Asn Ala Val Lys Phe Leu Arg Ala Lys Phe Ala Arg Ala Ser 260 265 270 Arg Lys Gln Asn Ser 275 9 1032 DNA Mycobacterium CDS (1)..(1029) 9 gtg aag cga gcg ctt ata aca ggg atc acg ggg cag gat ggt tcc tac 48 Val Lys Arg Ala Leu Ile Thr Gly Ile Thr Gly Gln Asp Gly Ser Tyr 1 5 10 15 ctc gcc gag cta cta ctg agc aag gga tac gag gtt cac ggg ctc gtt 96 Leu Ala Glu Leu Leu Leu Ser Lys Gly Tyr Glu Val His Gly Leu Val 20 25 30 cgt cga gct tcg acg ttt aac acg tcg cgg atc gat cac ctc tac gtt 144 Arg Arg Ala Ser Thr Phe Asn Thr Ser Arg Ile Asp His Leu Tyr Val 35 40 45 gac cca cac caa ccg ggc gcg cgc ttg ttc ttg cac tat gca gac ctc 192 Asp Pro His Gln Pro Gly Ala Arg Leu Phe Leu His Tyr Ala Asp Leu 50 55 60 act gac ggc acc cgg ttg gtg acc ctg ctc agc agt atc gac ccg gat 240 Thr Asp Gly Thr Arg Leu Val Thr Leu Leu Ser Ser Ile Asp Pro Asp 65 70 75 80 gag gtc tac aac ctc gca gcg cag tcc cat gtg cgc gtc agc ttt gac 288 Glu Val Tyr Asn Leu Ala Ala Gln Ser His Val Arg Val Ser Phe Asp 85 90 95 gag cca gtg cat acc gga gac acc acc ggc atg gga tcg atc cga ctt 336 Glu Pro Val His Thr Gly Asp Thr Thr Gly Met Gly Ser Ile Arg Leu 100 105 110 ctg gaa gca gtc cgc ctt tct cgg gtg gac tgc cgg ttc tat cag gct 384 Leu Glu Ala Val Arg Leu Ser Arg Val Asp Cys Arg Phe Tyr Gln Ala 115 120 125 tcc tcg tcg gag atg ttc ggc gca tct ccg cca ccg cag aac gaa tcg 432 Ser Ser Ser Glu Met Phe Gly Ala Ser Pro Pro Pro Gln Asn Glu Ser 130 135 140 acg ccg ttc tat ccc cgt tcg cca tac ggc gcg gcc aag gtc ttc tcg 480 Thr Pro Phe Tyr Pro Arg Ser Pro Tyr Gly Ala Ala Lys Val Phe Ser 145 150 155 160 tac tgg acg act cgc aac tat cga gag gcg tac gga tta ttc gca gtg 528 Tyr Trp Thr Thr Arg Asn Tyr Arg Glu Ala Tyr Gly Leu Phe Ala Val 165 170 175 aat ggc atc ttg ttc aac cat gag tcc ccc cgg cgc ggc gag act ttc 576 Asn Gly Ile Leu Phe Asn His Glu Ser Pro Arg Arg Gly Glu Thr Phe 180 185 190 gtg acc cga aag atc acg cgt gcc gtg gcg cgc atc cga gct ggc gtc 624 Val Thr Arg Lys Ile Thr Arg Ala Val Ala Arg Ile Arg Ala Gly Val 195 200 205 caa tcg gag gtc tat atg ggc aac ctc gat gcg atc cgc gac tgg ggc 672 Gln Ser Glu Val Tyr Met Gly Asn Leu Asp Ala Ile Arg Asp Trp Gly 210 215 220 tac gcg ccc gaa tat gtc gag ggg atg tgg agg atg ttg caa gcg cct 720 Tyr Ala Pro Glu Tyr Val Glu Gly Met Trp Arg Met Leu Gln Ala Pro 225 230 235 240 gaa cct gat gac tac gtc ctg gcg aca ggg cgt ggt tac acc gta cgt 768 Glu Pro Asp Asp Tyr Val Leu Ala Thr Gly Arg Gly Tyr Thr Val Arg 245 250 255 gag ttc gct caa gct gct ttt gac cat gtc ggg ctc gac tgg caa aag 816 Glu Phe Ala Gln Ala Ala Phe Asp His Val Gly Leu Asp Trp Gln Lys 260 265 270 cgc gtc aag ttt gac gac cgc tat ttg cgt ccc acc gag gtc gat tcg 864 Arg Val Lys Phe Asp Asp Arg Tyr Leu Arg Pro Thr Glu Val Asp Ser 275 280 285 cta gta gga gat gcc gac aag gcg gcc cag tca ctc ggc tgg aaa gct 912 Leu Val Gly Asp Ala Asp Lys Ala Ala Gln Ser Leu Gly Trp Lys Ala 290 295 300 tcg gtt cat act ggt gaa ctc gcg cgc atc atg gtg gac gcg gac atc 960 Ser Val His Thr Gly Glu Leu Ala Arg Ile Met Val Asp Ala Asp Ile 305 310 315 320 gcc gcg ttg gag tgc gat ggc aca cca tgg atc gac acg ccg atg ttg 1008 Ala Ala Leu Glu Cys Asp Gly Thr Pro Trp Ile Asp Thr Pro Met Leu 325 330 335 cct ggt tgg ggc aga gta agt tga 1032 Pro Gly Trp Gly Arg Val Ser 340 10 343 PRT Mycobacterium 10 Val Lys Arg Ala Leu Ile Thr Gly Ile Thr Gly Gln Asp Gly Ser Tyr 1 5 10 15 Leu Ala Glu Leu Leu Leu Ser Lys Gly Tyr Glu Val His Gly Leu Val 20 25 30 Arg Arg Ala Ser Thr Phe Asn Thr Ser Arg Ile Asp His Leu Tyr Val 35 40 45 Asp Pro His Gln Pro Gly Ala Arg Leu Phe Leu His Tyr Ala Asp Leu 50 55 60 Thr Asp Gly Thr Arg Leu Val Thr Leu Leu Ser Ser Ile Asp Pro Asp 65 70 75 80 Glu Val Tyr Asn Leu Ala Ala Gln Ser His Val Arg Val Ser Phe Asp 85 90 95 Glu Pro Val His Thr Gly Asp Thr Thr Gly Met Gly Ser Ile Arg Leu 100 105 110 Leu Glu Ala Val Arg Leu Ser Arg Val Asp Cys Arg Phe Tyr Gln Ala 115 120 125 Ser Ser Ser Glu Met Phe Gly Ala Ser Pro Pro Pro Gln Asn Glu Ser 130 135 140 Thr Pro Phe Tyr Pro Arg Ser Pro Tyr Gly Ala Ala Lys Val Phe Ser 145 150 155 160 Tyr Trp Thr Thr Arg Asn Tyr Arg Glu Ala Tyr Gly Leu Phe Ala Val 165 170 175 Asn Gly Ile Leu Phe Asn His Glu Ser Pro Arg Arg Gly Glu Thr Phe 180 185 190 Val Thr Arg Lys Ile Thr Arg Ala Val Ala Arg Ile Arg Ala Gly Val 195 200 205 Gln Ser Glu Val Tyr Met Gly Asn Leu Asp Ala Ile Arg Asp Trp Gly 210 215 220 Tyr Ala Pro Glu Tyr Val Glu Gly Met Trp Arg Met Leu Gln Ala Pro 225 230 235 240 Glu Pro Asp Asp Tyr Val Leu Ala Thr Gly Arg Gly Tyr Thr Val Arg 245 250 255 Glu Phe Ala Gln Ala Ala Phe Asp His Val Gly Leu Asp Trp Gln Lys 260 265 270 Arg Val Lys Phe Asp Asp Arg Tyr Leu Arg Pro Thr Glu Val Asp Ser 275 280 285 Leu Val Gly Asp Ala Asp Lys Ala Ala Gln Ser Leu Gly Trp Lys Ala 290 295 300 Ser Val His Thr Gly Glu Leu Ala Arg Ile Met Val Asp Ala Asp Ile 305 310 315 320 Ala Ala Leu Glu Cys Asp Gly Thr Pro Trp Ile Asp Thr Pro Met Leu 325 330 335 Pro Gly Trp Gly Arg Val Ser 340 11 1032 DNA Mycobacterium CDS (1)..(1029) 11 gtg aag cga gcg ctt ata aca ggg atc acg ggg cag gat ggt tcc tac 48 Val Lys Arg Ala Leu Ile Thr Gly Ile Thr Gly Gln Asp Gly Ser Tyr 1 5 10 15 ctc gcc gag cta cta ctg agc aag gga tac gag gtt cac ggg ctc gtt 96 Leu Ala Glu Leu Leu Leu Ser Lys Gly Tyr Glu Val His Gly Leu Val 20 25 30 cgt cga gct tcg acg ttt aac acg tcg cgg atc gat cac ctc tac gtt 144 Arg Arg Ala Ser Thr Phe Asn Thr Ser Arg Ile Asp His Leu Tyr Val 35 40 45 gac cca cac caa ccg ggc gcg cgc ttg ttc ttg cac tat gca gac ctc 192 Asp Pro His Gln Pro Gly Ala Arg Leu Phe Leu His Tyr Ala Asp Leu 50 55 60 act gac ggc acc cgg ttg gtg acc ctg ctc agc agt atc gac ccg gat 240 Thr Asp Gly Thr Arg Leu Val Thr Leu Leu Ser Ser Ile Asp Pro Asp 65 70 75 80 gag gtc tac aac ctc gca gcg cag tcc cat gtg cgc gtc agc ttt gac 288 Glu Val Tyr Asn Leu Ala Ala Gln Ser His Val Arg Val Ser Phe Asp 85 90 95 gag cca gtg cat acc gga gac acc acc ggc atg gga tcg atc cga ctt 336 Glu Pro Val His Thr Gly Asp Thr Thr Gly Met Gly Ser Ile Arg Leu 100 105 110 ctg gaa gca gtc cgc ctt tct cgg gtg gac tgc cgg ttc tat cag gct 384 Leu Glu Ala Val Arg Leu Ser Arg Val Asp Cys Arg Phe Tyr Gln Ala 115 120 125 tcc tcg tcg gag atg ttc ggc gca tct ccg cca ccg cag aac gaa tcg 432 Ser Ser Ser Glu Met Phe Gly Ala Ser Pro Pro Pro Gln Asn Glu Ser 130 135 140 acg ccg ttc tat ccc cgt tcg cca tac ggc gcg gcc aag gtc ttc tcg 480 Thr Pro Phe Tyr Pro Arg Ser Pro Tyr Gly Ala Ala Lys Val Phe Ser 145 150 155 160 tac tgg acg act cgc aac tat cga gag gcg tac gga tta ttc gca gtg 528 Tyr Trp Thr Thr Arg Asn Tyr Arg Glu Ala Tyr Gly Leu Phe Ala Val 165 170 175 aat ggc atc ttg ttc aac cat gag tcc ccc cgg cgc ggc gag act ttc 576 Asn Gly Ile Leu Phe Asn His Glu Ser Pro Arg Arg Gly Glu Thr Phe 180 185 190 gtg acc cga aag atc acg cgt gcc gtg gcg cgc atc cga gct ggc gtc 624 Val Thr Arg Lys Ile Thr Arg Ala Val Ala Arg Ile Arg Ala Gly Val 195 200 205 caa tcg gag gtc tat atg ggc aac ctc gat gcg atc cgc gac tgg ggc 672 Gln Ser Glu Val Tyr Met Gly Asn Leu Asp Ala Ile Arg Asp Trp Gly 210 215 220 tac gcg ccc gaa tat gtc gag ggg atg tgg agg atg ttg caa gcg cct 720 Tyr Ala Pro Glu Tyr Val Glu Gly Met Trp Arg Met Leu Gln Ala Pro 225 230 235 240 gaa cct gat gac tac gtc ctg gcg aca ggg cgt ggt tac acc gta cgt 768 Glu Pro Asp Asp Tyr Val Leu Ala Thr Gly Arg Gly Tyr Thr Val Arg 245 250 255 gag ttc gct caa gct gct ttt gac cac gtc ggg ctc gac tgg caa aag 816 Glu Phe Ala Gln Ala Ala Phe Asp His Val Gly Leu Asp Trp Gln Lys 260 265 270 cac gtc aag ttt gac gac cgc tat ttg cgc ccc acc gag gtc gat tcg 864 His Val Lys Phe Asp Asp Arg Tyr Leu Arg Pro Thr Glu Val Asp Ser 275 280 285 cta gta gga gat gcc gac agg gcg gcc cag tca ctc ggc tgg aaa gct 912 Leu Val Gly Asp Ala Asp Arg Ala Ala Gln Ser Leu Gly Trp Lys Ala 290 295 300 tcg gtt cat act ggt gaa ctc gcg cgc atc atg gtg gac gcg gac atc 960 Ser Val His Thr Gly Glu Leu Ala Arg Ile Met Val Asp Ala Asp Ile 305 310 315 320 gcc gcg tcg gag tgc gat ggc aca cca tgg atc gac acg ccg atg ttg 1008 Ala Ala Ser Glu Cys Asp Gly Thr Pro Trp Ile Asp Thr Pro Met Leu 325 330 335 cct ggt tgg ggc gga gta agt tga 1032 Pro Gly Trp Gly Gly Val Ser 340 12 343 PRT Mycobacterium 12 Val Lys Arg Ala Leu Ile Thr Gly Ile Thr Gly Gln Asp Gly Ser Tyr 1 5 10 15 Leu Ala Glu Leu Leu Leu Ser Lys Gly Tyr Glu Val His Gly Leu Val 20 25 30 Arg Arg Ala Ser Thr Phe Asn Thr Ser Arg Ile Asp His Leu Tyr Val 35 40 45 Asp Pro His Gln Pro Gly Ala Arg Leu Phe Leu His Tyr Ala Asp Leu 50 55 60 Thr Asp Gly Thr Arg Leu Val Thr Leu Leu Ser Ser Ile Asp Pro Asp 65 70 75 80 Glu Val Tyr Asn Leu Ala Ala Gln Ser His Val Arg Val Ser Phe Asp 85 90 95 Glu Pro Val His Thr Gly Asp Thr Thr Gly Met Gly Ser Ile Arg Leu 100 105 110 Leu Glu Ala Val Arg Leu Ser Arg Val Asp Cys Arg Phe Tyr Gln Ala 115 120 125 Ser Ser Ser Glu Met Phe Gly Ala Ser Pro Pro Pro Gln Asn Glu Ser 130 135 140 Thr Pro Phe Tyr Pro Arg Ser Pro Tyr Gly Ala Ala Lys Val Phe Ser 145 150 155 160 Tyr Trp Thr Thr Arg Asn Tyr Arg Glu Ala Tyr Gly Leu Phe Ala Val 165 170 175 Asn Gly Ile Leu Phe Asn His Glu Ser Pro Arg Arg Gly Glu Thr Phe 180 185 190 Val Thr Arg Lys Ile Thr Arg Ala Val Ala Arg Ile Arg Ala Gly Val 195 200 205 Gln Ser Glu Val Tyr Met Gly Asn Leu Asp Ala Ile Arg Asp Trp Gly 210 215 220 Tyr Ala Pro Glu Tyr Val Glu Gly Met Trp Arg Met Leu Gln Ala Pro 225 230 235 240 Glu Pro Asp Asp Tyr Val Leu Ala Thr Gly Arg Gly Tyr Thr Val Arg 245 250 255 Glu Phe Ala Gln Ala Ala Phe Asp His Val Gly Leu Asp Trp Gln Lys 260 265 270 His Val Lys Phe Asp Asp Arg Tyr Leu Arg Pro Thr Glu Val Asp Ser 275 280 285 Leu Val Gly Asp Ala Asp Arg Ala Ala Gln Ser Leu Gly Trp Lys Ala 290 295 300 Ser Val His Thr Gly Glu Leu Ala Arg Ile Met Val Asp Ala Asp Ile 305 310 315 320 Ala Ala Ser Glu Cys Asp Gly Thr Pro Trp Ile Asp Thr Pro Met Leu 325 330 335 Pro Gly Trp Gly Gly Val Ser 340 13 1020 DNA Mycobacterium CDS (1)..(1017) 13 gtg cga tgg cac acc atg gat cga cac gcc gat gtt gcc tgg ttg ggg 48 Val Arg Trp His Thr Met Asp Arg His Ala Asp Val Ala Trp Leu Gly 1 5 10 15 cag agt aag ttg acg act aca cct ggg cct ctg gac cgc gca acg ccc 96 Gln Ser Lys Leu Thr Thr Thr Pro Gly Pro Leu Asp Arg Ala Thr Pro 20 25 30 gtg tat atc gcc ggt cat cgg ggg ctg gtc ggc tca gcg ctc gta cgt 144 Val Tyr Ile Ala Gly His Arg Gly Leu Val Gly Ser Ala Leu Val Arg 35 40 45 aga ttt gag gcc gag ggg ttc acc aat ctc att gtg cga tca cgc gat 192 Arg Phe Glu Ala Glu Gly Phe Thr Asn Leu Ile Val Arg Ser Arg Asp 50 55 60 gag att gat ctg acg gac cga gcc gca acg ttt gat ttt gtg tct gag 240 Glu Ile Asp Leu Thr Asp Arg Ala Ala Thr Phe Asp Phe Val Ser Glu 65 70 75 80 aca aga cca cag gtg atc atc gat gcg gcc gca cgg gtc ggc ggc atc 288 Thr Arg Pro Gln Val Ile Ile Asp Ala Ala Ala Arg Val Gly Gly Ile 85 90 95 atg gcg aat aac acc tat ccc gcg gac ttc ttg tcc gaa aac ctc cga 336 Met Ala Asn Asn Thr Tyr Pro Ala Asp Phe Leu Ser Glu Asn Leu Arg 100 105 110 atc cag acc aat ttg ctc gac gca gct gtc gcc gtg cgt gtg ccg cgg 384 Ile Gln Thr Asn Leu Leu Asp Ala Ala Val Ala Val Arg Val Pro Arg 115 120 125 ctc ctt ttc ctc ggt tcg tca tgc atc tac ccg aag tac gct ccg caa 432 Leu Leu Phe Leu Gly Ser Ser Cys Ile Tyr Pro Lys Tyr Ala Pro Gln 130 135 140 cct atc cac gag agt gct tta ttg act ggc cct ttg gag ccc acc aac 480 Pro Ile His Glu Ser Ala Leu Leu Thr Gly Pro Leu Glu Pro Thr Asn 145 150 155 160 gac gcg tat gcg atc gcc aag atc gcc ggt atc ctg caa gtt cag gcg 528 Asp Ala Tyr Ala Ile Ala Lys Ile Ala Gly Ile Leu Gln Val Gln Ala 165 170 175 gtt agg cgc caa tat ggg ctg gcg tgg atc tct gcg atg ccg act aac 576 Val Arg Arg Gln Tyr Gly Leu Ala Trp Ile Ser Ala Met Pro Thr Asn 180 185 190 ctc tac gga ccc ggc gac aac ttc tcc ccg tcc ggg tcg cat ctc ttg 624 Leu Tyr Gly Pro Gly Asp Asn Phe Ser Pro Ser Gly Ser His Leu Leu 195 200 205 ccg gcg ctc atc cgt cga tat gag gaa gcc aaa gct ggt ggt gca gaa 672 Pro Ala Leu Ile Arg Arg Tyr Glu Glu Ala Lys Ala Gly Gly Ala Glu 210 215 220 gag gtg acg aat tgg ggg acc ggt act ccg cgg cgc gaa ctt ctg cat 720 Glu Val Thr Asn Trp Gly Thr Gly Thr Pro Arg Arg Glu Leu Leu His 225 230 235 240 gtc gac gat ctg gcg agc gca tgc ctg ttc ctt ttg gaa cat ttc gat 768 Val Asp Asp Leu Ala Ser Ala Cys Leu Phe Leu Leu Glu His Phe Asp 245 250 255 ggt ccg aac cac gtc aac gtg ggc acc ggc gtc gat cac agc att agc 816 Gly Pro Asn His Val Asn Val Gly Thr Gly Val Asp His Ser Ile Ser 260 265 270 gag atc gca gac atg gtc gct aca gcg gtg ggc tac atc ggc gaa aca 864 Glu Ile Ala Asp Met Val Ala Thr Ala Val Gly Tyr Ile Gly Glu Thr 275 280 285 cgt tgg gat cca act aaa ccc gat gga acc ccg cgc aaa cta ttg gac 912 Arg Trp Asp Pro Thr Lys Pro Asp Gly Thr Pro Arg Lys Leu Leu Asp 290 295 300 gtc tcc gcg cta cgc gag ttg ggt tgg cgc ccg cga atc gca ctg aaa 960 Val Ser Ala Leu Arg Glu Leu Gly Trp Arg Pro Arg Ile Ala Leu Lys 305 310 315 320 gac ggc atc gat gca acg gtg tcg tgg tac cgc aca aat gcc gat gcc 1008 Asp Gly Ile Asp Ala Thr Val Ser Trp Tyr Arg Thr Asn Ala Asp Ala 325 330 335 gtg agg agg taa 1020 Val Arg Arg 14 339 PRT Mycobacterium 14 Val Arg Trp His Thr Met Asp Arg His Ala Asp Val Ala Trp Leu Gly 1 5 10 15 Gln Ser Lys Leu Thr Thr Thr Pro Gly Pro Leu Asp Arg Ala Thr Pro 20 25 30 Val Tyr Ile Ala Gly His Arg Gly Leu Val Gly Ser Ala Leu Val Arg 35 40 45 Arg Phe Glu Ala Glu Gly Phe Thr Asn Leu Ile Val Arg Ser Arg Asp 50 55 60 Glu Ile Asp Leu Thr Asp Arg Ala Ala Thr Phe Asp Phe Val Ser Glu 65 70 75 80 Thr Arg Pro Gln Val Ile Ile Asp Ala Ala Ala Arg Val Gly Gly Ile 85 90 95 Met Ala Asn Asn Thr Tyr Pro Ala Asp Phe Leu Ser Glu Asn Leu Arg 100 105 110 Ile Gln Thr Asn Leu Leu Asp Ala Ala Val Ala Val Arg Val Pro Arg 115 120 125 Leu Leu Phe Leu Gly Ser Ser Cys Ile Tyr Pro Lys Tyr Ala Pro Gln 130 135 140 Pro Ile His Glu Ser Ala Leu Leu Thr Gly Pro Leu Glu Pro Thr Asn 145 150 155 160 Asp Ala Tyr Ala Ile Ala Lys Ile Ala Gly Ile Leu Gln Val Gln Ala 165 170 175 Val Arg Arg Gln Tyr Gly Leu Ala Trp Ile Ser Ala Met Pro Thr Asn 180 185 190 Leu Tyr Gly Pro Gly Asp Asn Phe Ser Pro Ser Gly Ser His Leu Leu 195 200 205 Pro Ala Leu Ile Arg Arg Tyr Glu Glu Ala Lys Ala Gly Gly Ala Glu 210 215 220 Glu Val Thr Asn Trp Gly Thr Gly Thr Pro Arg Arg Glu Leu Leu His 225 230 235 240 Val Asp Asp Leu Ala Ser Ala Cys Leu Phe Leu Leu Glu His Phe Asp 245 250 255 Gly Pro Asn His Val Asn Val Gly Thr Gly Val Asp His Ser Ile Ser 260 265 270 Glu Ile Ala Asp Met Val Ala Thr Ala Val Gly Tyr Ile Gly Glu Thr 275 280 285 Arg Trp Asp Pro Thr Lys Pro Asp Gly Thr Pro Arg Lys Leu Leu Asp 290 295 300 Val Ser Ala Leu Arg Glu Leu Gly Trp Arg Pro Arg Ile Ala Leu Lys 305 310 315 320 Asp Gly Ile Asp Ala Thr Val Ser Trp Tyr Arg Thr Asn Ala Asp Ala 325 330 335 Val Arg Arg 15 1020 DNA Mycobacterium CDS (1)..(1017) 15 gtg cga tgg cac acc atg gat cga cac gcc gat gtt gcc tgg ttg ggg 48 Val Arg Trp His Thr Met Asp Arg His Ala Asp Val Ala Trp Leu Gly 1 5 10 15 cgg agt aag ttg acg act aca cct ggg cct ctg gac cgc gca acg ccc 96 Arg Ser Lys Leu Thr Thr Thr Pro Gly Pro Leu Asp Arg Ala Thr Pro 20 25 30 gtg tat atc gcc ggt cat cgg ggg ctg gtc ggc tca gcg ctc gta cgt 144 Val Tyr Ile Ala Gly His Arg Gly Leu Val Gly Ser Ala Leu Val Arg 35 40 45 aga ttt gag gcc gag ggg ttc acc aat ctc att gtg cga tca cgc gat 192 Arg Phe Glu Ala Glu Gly Phe Thr Asn Leu Ile Val Arg Ser Arg Asp 50 55 60 gag att gat ctg acg gac cga gcc gca acg ttt gat ttt gtg tct gag 240 Glu Ile Asp Leu Thr Asp Arg Ala Ala Thr Phe Asp Phe Val Ser Glu 65 70 75 80 aca aga cca cag gtg atc atc gat gcg gcc gca cgg gtc ggc ggc atc 288 Thr Arg Pro Gln Val Ile Ile Asp Ala Ala Ala Arg Val Gly Gly Ile 85 90 95 atg gcg aat aac acc tat ccc gcg gac ttc ttg tcc gaa aac ctc cga 336 Met Ala Asn Asn Thr Tyr Pro Ala Asp Phe Leu Ser Glu Asn Leu Arg 100 105 110 atc cag acc aat ttg ctc gac gca gct gtc gcc gtg cgt gtg ccg cgg 384 Ile Gln Thr Asn Leu Leu Asp Ala Ala Val Ala Val Arg Val Pro Arg 115 120 125 ctc ctt ttc ctc ggt tcg tca tgc atc tac ccg aag tac gct ccg caa 432 Leu Leu Phe Leu Gly Ser Ser Cys Ile Tyr Pro Lys Tyr Ala Pro Gln 130 135 140 cct atc cac gag agt gct tta ttg act ggc cct ttg gag ccc acc aac 480 Pro Ile His Glu Ser Ala Leu Leu Thr Gly Pro Leu Glu Pro Thr Asn 145 150 155 160 gac gcg tat gcg atc gcc aag atc gcc ggt atc ctg caa gtt cag gcg 528 Asp Ala Tyr Ala Ile Ala Lys Ile Ala Gly Ile Leu Gln Val Gln Ala 165 170 175 gtt agg cgc caa tat ggg ctg gcg tgg atc tct gcg atg ccg act aac 576 Val Arg Arg Gln Tyr Gly Leu Ala Trp Ile Ser Ala Met Pro Thr Asn 180 185 190 ctc tac gga ccc ggc gac aac ttc tcc ccg tcc ggg tcg cat ctc ttg 624 Leu Tyr Gly Pro Gly Asp Asn Phe Ser Pro Ser Gly Ser His Leu Leu 195 200 205 ccg gcg ctc atc cgt cga tat gag gaa gcc aaa gct ggt ggt gca gaa 672 Pro Ala Leu Ile Arg Arg Tyr Glu Glu Ala Lys Ala Gly Gly Ala Glu 210 215 220 gag gtg acg aat tgg ggg acc ggt act ccg cgg cgc gaa ctt ctg cat 720 Glu Val Thr Asn Trp Gly Thr Gly Thr Pro Arg Arg Glu Leu Leu His 225 230 235 240 gtc gac gat ctg gcg agc gca tgc ctg ttc ctt ttg gaa cat ttc gat 768 Val Asp Asp Leu Ala Ser Ala Cys Leu Phe Leu Leu Glu His Phe Asp 245 250 255 ggt ccg aac cac gtc aac gtg ggc acc ggc gtc gat cac agc att agc 816 Gly Pro Asn His Val Asn Val Gly Thr Gly Val Asp His Ser Ile Ser 260 265 270 gag atc gca gac atg gtc gct acg gcg gtg ggc tac atc ggc gaa aca 864 Glu Ile Ala Asp Met Val Ala Thr Ala Val Gly Tyr Ile Gly Glu Thr 275 280 285 cgt tgg gat cca act aaa ccc gat gga acc ccg cgc aaa cta ttg gac 912 Arg Trp Asp Pro Thr Lys Pro Asp Gly Thr Pro Arg Lys Leu Leu Asp 290 295 300 gtc tcc gcg cta cgc gag ttg ggt tgg cgc ccg cga atc gca ctg aaa 960 Val Ser Ala Leu Arg Glu Leu Gly Trp Arg Pro Arg Ile Ala Leu Lys 305 310 315 320 gac ggc atc gat gca acg gtg tcg tgg tac cgc aca aat gcc gat gcc 1008 Asp Gly Ile Asp Ala Thr Val Ser Trp Tyr Arg Thr Asn Ala Asp Ala 325 330 335 gtg agg agg taa 1020 Val Arg Arg 16 339 PRT Mycobacterium 16 Val Arg Trp His Thr Met Asp Arg His Ala Asp Val Ala Trp Leu Gly 1 5 10 15 Arg Ser Lys Leu Thr Thr Thr Pro Gly Pro Leu Asp Arg Ala Thr Pro 20 25 30 Val Tyr Ile Ala Gly His Arg Gly Leu Val Gly Ser Ala Leu Val Arg 35 40 45 Arg Phe Glu Ala Glu Gly Phe Thr Asn Leu Ile Val Arg Ser Arg Asp 50 55 60 Glu Ile Asp Leu Thr Asp Arg Ala Ala Thr Phe Asp Phe Val Ser Glu 65 70 75 80 Thr Arg Pro Gln Val Ile Ile Asp Ala Ala Ala Arg Val Gly Gly Ile 85 90 95 Met Ala Asn Asn Thr Tyr Pro Ala Asp Phe Leu Ser Glu Asn Leu Arg 100 105 110 Ile Gln Thr Asn Leu Leu Asp Ala Ala Val Ala Val Arg Val Pro Arg 115 120 125 Leu Leu Phe Leu Gly Ser Ser Cys Ile Tyr Pro Lys Tyr Ala Pro Gln 130 135 140 Pro Ile His Glu Ser Ala Leu Leu Thr Gly Pro Leu Glu Pro Thr Asn 145 150 155 160 Asp Ala Tyr Ala Ile Ala Lys Ile Ala Gly Ile Leu Gln Val Gln Ala 165 170 175 Val Arg Arg Gln Tyr Gly Leu Ala Trp Ile Ser Ala Met Pro Thr Asn 180 185 190 Leu Tyr Gly Pro Gly Asp Asn Phe Ser Pro Ser Gly Ser His Leu Leu 195 200 205 Pro Ala Leu Ile Arg Arg Tyr Glu Glu Ala Lys Ala Gly Gly Ala Glu 210 215 220 Glu Val Thr Asn Trp Gly Thr Gly Thr Pro Arg Arg Glu Leu Leu His 225 230 235 240 Val Asp Asp Leu Ala Ser Ala Cys Leu Phe Leu Leu Glu His Phe Asp 245 250 255 Gly Pro Asn His Val Asn Val Gly Thr Gly Val Asp His Ser Ile Ser 260 265 270 Glu Ile Ala Asp Met Val Ala Thr Ala Val Gly Tyr Ile Gly Glu Thr 275 280 285 Arg Trp Asp Pro Thr Lys Pro Asp Gly Thr Pro Arg Lys Leu Leu Asp 290 295 300 Val Ser Ala Leu Arg Glu Leu Gly Trp Arg Pro Arg Ile Ala Leu Lys 305 310 315 320 Asp Gly Ile Asp Ala Thr Val Ser Trp Tyr Arg Thr Asn Ala Asp Ala 325 330 335 Val Arg Arg 17 723 DNA Mycobacterium CDS (1)..(720) 17 atg gat ttt ttg cgc aac gcc ggc ttg atg gct cgt aac gtt agt acc 48 Met Asp Phe Leu Arg Asn Ala Gly Leu Met Ala Arg Asn Val Ser Thr 1 5 10 15 gag atg ctg cgc cac ttc gaa cga aag cgc cta tta gta aac caa ttc 96 Glu Met Leu Arg His Phe Glu Arg Lys Arg Leu Leu Val Asn Gln Phe 20 25 30 aaa gca tac gga gtc aac gtt gtt att gat gtc ggt gct aac tcc ggc 144 Lys Ala Tyr Gly Val Asn Val Val Ile Asp Val Gly Ala Asn Ser Gly 35 40 45 cag ttc ggt agc gct ttg cgt cgt gca gga ttc aag agc cgt atc gtt 192 Gln Phe Gly Ser Ala Leu Arg Arg Ala Gly Phe Lys Ser Arg Ile Val 50 55 60 tcc ttt gaa cct ctt tcg ggg cca ttt gcg caa cta acg cgc aag tcg 240 Ser Phe Glu Pro Leu Ser Gly Pro Phe Ala Gln Leu Thr Arg Lys Ser 65 70 75 80 gca tcg gat cca cta tgg gag tgt cac cag tat gcc cta ggc gac gcc 288 Ala Ser Asp Pro Leu Trp Glu Cys His Gln Tyr Ala Leu Gly Asp Ala 85 90 95 gat gag acg att acc atc aat gtg gca ggc aat gcg ggg gca agt agt 336 Asp Glu Thr Ile Thr Ile Asn Val Ala Gly Asn Ala Gly Ala Ser Ser 100 105 110 tcc gtg ctg ccg atg ctt aaa agt cat caa gat gcc ttt cct ccc gcg 384 Ser Val Leu Pro Met Leu Lys Ser His Gln Asp Ala Phe Pro Pro Ala 115 120 125 aat tat att ggc acc gaa gac gtt gca ata cac cgc ctt gat tcg gtt 432 Asn Tyr Ile Gly Thr Glu Asp Val Ala Ile His Arg Leu Asp Ser Val 130 135 140 gca tca gaa ttt ctg aac cct acc gat gtt act ttc ctg aag atc gac 480 Ala Ser Glu Phe Leu Asn Pro Thr Asp Val Thr Phe Leu Lys Ile Asp 145 150 155 160 gta cag ggt ttc gag aag cag gtt atc acg ggc agt aag tca acg ctt 528 Val Gln Gly Phe Glu Lys Gln Val Ile Thr Gly Ser Lys Ser Thr Leu 165 170 175 aac gaa agc tgc gtc ggc atg caa ctc gaa ctt tct ttt att ccg ttg 576 Asn Glu Ser Cys Val Gly Met Gln Leu Glu Leu Ser Phe Ile Pro Leu 180 185 190 tac gaa ggt gac atg ctg att cat gaa gcg ctt gaa ctt gtc tat tcc 624 Tyr Glu Gly Asp Met Leu Ile His Glu Ala Leu Glu Leu Val Tyr Ser 195 200 205 cta ggt ttc aga ctg acg ggt ttg ttg ccc ggc ttt acg gat ccg cgc 672 Leu Gly Phe Arg Leu Thr Gly Leu Leu Pro Gly Phe Thr Asp Pro Arg 210 215 220 aat ggt cga atg ctt caa gct gac ggc att ttc ttc cgt ggg gac gat 720 Asn Gly Arg Met Leu Gln Ala Asp Gly Ile Phe Phe Arg Gly Asp Asp 225 230 235 240 tga 723 18 240 PRT Mycobacterium 18 Met Asp Phe Leu Arg Asn Ala Gly Leu Met Ala Arg Asn Val Ser Thr 1 5 10 15 Glu Met Leu Arg His Phe Glu Arg Lys Arg Leu Leu Val Asn Gln Phe 20 25 30 Lys Ala Tyr Gly Val Asn Val Val Ile Asp Val Gly Ala Asn Ser Gly 35 40 45 Gln Phe Gly Ser Ala Leu Arg Arg Ala Gly Phe Lys Ser Arg Ile Val 50 55 60 Ser Phe Glu Pro Leu Ser Gly Pro Phe Ala Gln Leu Thr Arg Lys Ser 65 70 75 80 Ala Ser Asp Pro Leu Trp Glu Cys His Gln Tyr Ala Leu Gly Asp Ala 85 90 95 Asp Glu Thr Ile Thr Ile Asn Val Ala Gly Asn Ala Gly Ala Ser Ser 100 105 110 Ser Val Leu Pro Met Leu Lys Ser His Gln Asp Ala Phe Pro Pro Ala 115 120 125 Asn Tyr Ile Gly Thr Glu Asp Val Ala Ile His Arg Leu Asp Ser Val 130 135 140 Ala Ser Glu Phe Leu Asn Pro Thr Asp Val Thr Phe Leu Lys Ile Asp 145 150 155 160 Val Gln Gly Phe Glu Lys Gln Val Ile Thr Gly Ser Lys Ser Thr Leu 165 170 175 Asn Glu Ser Cys Val Gly Met Gln Leu Glu Leu Ser Phe Ile Pro Leu 180 185 190 Tyr Glu Gly Asp Met Leu Ile His Glu Ala Leu Glu Leu Val Tyr Ser 195 200 205 Leu Gly Phe Arg Leu Thr Gly Leu Leu Pro Gly Phe Thr Asp Pro Arg 210 215 220 Asn Gly Arg Met Leu Gln Ala Asp Gly Ile Phe Phe Arg Gly Asp Asp 225 230 235 240 19 723 DNA Mycobacterium CDS (1)..(720) 19 atg gat ttt ttg cgc aac gcc ggc ttg atg gct cgt aac gtt agc acc 48 Met Asp Phe Leu Arg Asn Ala Gly Leu Met Ala Arg Asn Val Ser Thr 1 5 10 15 gag atg ctg cgc cac ttc gaa cga aag cgc cta tta gta aac caa ttc 96 Glu Met Leu Arg His Phe Glu Arg Lys Arg Leu Leu Val Asn Gln Phe 20 25 30 aaa gca tac gga gtc aac gtt gtt att gat gtc ggt gct aac tcc ggc 144 Lys Ala Tyr Gly Val Asn Val Val Ile Asp Val Gly Ala Asn Ser Gly 35 40 45 cag ttc ggt agc gct ttg cgt cgt gca gga ttc aag agc cgt atc gtt 192 Gln Phe Gly Ser Ala Leu Arg Arg Ala Gly Phe Lys Ser Arg Ile Val 50 55 60 tcc ttt gaa cct ctt tcg ggg cca ttt gcg caa cta acg cgc gag tcg 240 Ser Phe Glu Pro Leu Ser Gly Pro Phe Ala Gln Leu Thr Arg Glu Ser 65 70 75 80 gca tcg gat cca cta tgg gag tgt cac cag tat gcc cta ggc gac gcc 288 Ala Ser Asp Pro Leu Trp Glu Cys His Gln Tyr Ala Leu Gly Asp Ala 85 90 95 gat gag acg att acc atc aat gtg gca ggc aat gcg ggg gca agt agt 336 Asp Glu Thr Ile Thr Ile Asn Val Ala Gly Asn Ala Gly Ala Ser Ser 100 105 110 tcc gtg ctg ccg atg ctt aaa agt cat caa gat gcc ttt cct ccc gcg 384 Ser Val Leu Pro Met Leu Lys Ser His Gln Asp Ala Phe Pro Pro Ala 115 120 125 aat tat att ggc acc gaa gac gtt gca ata cac cgc ctt gat tcg gtt 432 Asn Tyr Ile Gly Thr Glu Asp Val Ala Ile His Arg Leu Asp Ser Val 130 135 140 gca tca gaa ttt ctg aac cct acc gat gtt act ttc ctg aag atc gac 480 Ala Ser Glu Phe Leu Asn Pro Thr Asp Val Thr Phe Leu Lys Ile Asp 145 150 155 160 gta cag ggt ttc gag aag cag gtt atc gcg ggc agt aag tca acg ctt 528 Val Gln Gly Phe Glu Lys Gln Val Ile Ala Gly Ser Lys Ser Thr Leu 165 170 175 aac gaa agc tgc gtc ggc atg caa ctc gaa ctt tct ttt att ccg ttg 576 Asn Glu Ser Cys Val Gly Met Gln Leu Glu Leu Ser Phe Ile Pro Leu 180 185 190 tac gaa ggt gac atg ctg att cat gaa gcg ctt gaa ctt gtc tat tcc 624 Tyr Glu Gly Asp Met Leu Ile His Glu Ala Leu Glu Leu Val Tyr Ser 195 200 205 cta ggt ttc aga ctg acg ggt ttg ttg ccc gga ttt acg gat ccg cgc 672 Leu Gly Phe Arg Leu Thr Gly Leu Leu Pro Gly Phe Thr Asp Pro Arg 210 215 220 aat ggt cga atg ctt caa gct gac ggc att ttc ttc cgt ggg gac gat 720 Asn Gly Arg Met Leu Gln Ala Asp Gly Ile Phe Phe Arg Gly Asp Asp 225 230 235 240 tga 723 20 240 PRT Mycobacterium 20 Met Asp Phe Leu Arg Asn Ala Gly Leu Met Ala Arg Asn Val Ser Thr 1 5 10 15 Glu Met Leu Arg His Phe Glu Arg Lys Arg Leu Leu Val Asn Gln Phe 20 25 30 Lys Ala Tyr Gly Val Asn Val Val Ile Asp Val Gly Ala Asn Ser Gly 35 40 45 Gln Phe Gly Ser Ala Leu Arg Arg Ala Gly Phe Lys Ser Arg Ile Val 50 55 60 Ser Phe Glu Pro Leu Ser Gly Pro Phe Ala Gln Leu Thr Arg Glu Ser 65 70 75 80 Ala Ser Asp Pro Leu Trp Glu Cys His Gln Tyr Ala Leu Gly Asp Ala 85 90 95 Asp Glu Thr Ile Thr Ile Asn Val Ala Gly Asn Ala Gly Ala Ser Ser 100 105 110 Ser Val Leu Pro Met Leu Lys Ser His Gln Asp Ala Phe Pro Pro Ala 115 120 125 Asn Tyr Ile Gly Thr Glu Asp Val Ala Ile His Arg Leu Asp Ser Val 130 135 140 Ala Ser Glu Phe Leu Asn Pro Thr Asp Val Thr Phe Leu Lys Ile Asp 145 150 155 160 Val Gln Gly Phe Glu Lys Gln Val Ile Ala Gly Ser Lys Ser Thr Leu 165 170 175 Asn Glu Ser Cys Val Gly Met Gln Leu Glu Leu Ser Phe Ile Pro Leu 180 185 190 Tyr Glu Gly Asp Met Leu Ile His Glu Ala Leu Glu Leu Val Tyr Ser 195 200 205 Leu Gly Phe Arg Leu Thr Gly Leu Leu Pro Gly Phe Thr Asp Pro Arg 210 215 220 Asn Gly Arg Met Leu Gln Ala Asp Gly Ile Phe Phe Arg Gly Asp Asp 225 230 235 240 21 801 DNA Mycobacterium CDS (1)..(798) 21 atg act gcg cca gtg ttc tcg ata att atc cct acc ttc aat gca gcg 48 Met Thr Ala Pro Val Phe Ser Ile Ile Ile Pro Thr Phe Asn Ala Ala 1 5 10 15 gtg acg ctg caa gcc tgc ctc gga agc atc gtc ggg cag acc tac cgg 96 Val Thr Leu Gln Ala Cys Leu Gly Ser Ile Val Gly Gln Thr Tyr Arg 20 25 30 gaa gtg gaa gtg gtc ctt gtc gac ggc ggt tcg acc gat cgg acc ctc 144 Glu Val Glu Val Val Leu Val Asp Gly Gly Ser Thr Asp Arg Thr Leu 35 40 45 gac atc gcg aac agt ttc cgc ccg gaa ctc ggc tcg cga ctg gtc gtt 192 Asp Ile Ala Asn Ser Phe Arg Pro Glu Leu Gly Ser Arg Leu Val Val 50 55 60 cac agc ggg ccc gat gat ggc ccc tac gac gcc atg aac cgc ggc gtc 240 His Ser Gly Pro Asp Asp Gly Pro Tyr Asp Ala Met Asn Arg Gly Val 65 70 75 80 ggc gtg gcc aca ggc gaa tgg gta ctt ttt tta ggc gcc gac gac acc 288 Gly Val Ala Thr Gly Glu Trp Val Leu Phe Leu Gly Ala Asp Asp Thr 85 90 95 ctc tac gaa cca acc acg ttg gcc cag gta gcc gct ttt ctc ggc gac 336 Leu Tyr Glu Pro Thr Thr Leu Ala Gln Val Ala Ala Phe Leu Gly Asp 100 105 110 cat gcg gca agc cat ctt gtc tat ggc gat gtt gtg atg cgt tcg acg 384 His Ala Ala Ser His Leu Val Tyr Gly Asp Val Val Met Arg Ser Thr 115 120 125 aaa agc cgg cat gcc gga cct ttc gac ctc gac cgc ctc cta ttt gag 432 Lys Ser Arg His Ala Gly Pro Phe Asp Leu Asp Arg Leu Leu Phe Glu 130 135 140 acg aat ttg tgc cac caa tcg atc ttt tac cgc cgt gag ctt ttc gac 480 Thr Asn Leu Cys His Gln Ser Ile Phe Tyr Arg Arg Glu Leu Phe Asp 145 150 155 160 ggc atc ggc cct tac aac ctg cgc tac cga gtc tgg gcg gac tgg gac 528 Gly Ile Gly Pro Tyr Asn Leu Arg Tyr Arg Val Trp Ala Asp Trp Asp 165 170 175 ttc aat att cgc tgc ttc tcc aac ccg gcg ctg att acc cgc tac atg 576 Phe Asn Ile Arg Cys Phe Ser Asn Pro Ala Leu Ile Thr Arg Tyr Met 180 185 190 gac gtc gtg att tcc gaa tac aac gac atg acc ggc ttc agc atg agg 624 Asp Val Val Ile Ser Glu Tyr Asn Asp Met Thr Gly Phe Ser Met Arg 195 200 205 cag ggg act gat aaa gag ttc aga aaa cgg ctg cca atg tac ttc tgg 672 Gln Gly Thr Asp Lys Glu Phe Arg Lys Arg Leu Pro Met Tyr Phe Trp 210 215 220 gtt gca ggg tgg gag act tgc agg cgc atg ctg gcg ttt ttg aaa gac 720 Val Ala Gly Trp Glu Thr Cys Arg Arg Met Leu Ala Phe Leu Lys Asp 225 230 235 240 aag gag aat cgc cgt ctg gcc ttg cgt acg cgg ttg ata agg gtt aag 768 Lys Glu Asn Arg Arg Leu Ala Leu Arg Thr Arg Leu Ile Arg Val Lys 245 250 255 gcc gtc tcc aaa gaa cga agc gca gaa ccg tag 801 Ala Val Ser Lys Glu Arg Ser Ala Glu Pro 260 265 22 266 PRT Mycobacterium 22 Met Thr Ala Pro Val Phe Ser Ile Ile Ile Pro Thr Phe Asn Ala Ala 1 5 10 15 Val Thr Leu Gln Ala Cys Leu Gly Ser Ile Val Gly Gln Thr Tyr Arg 20 25 30 Glu Val Glu Val Val Leu Val Asp Gly Gly Ser Thr Asp Arg Thr Leu 35 40 45 Asp Ile Ala Asn Ser Phe Arg Pro Glu Leu Gly Ser Arg Leu Val Val 50 55 60 His Ser Gly Pro Asp Asp Gly Pro Tyr Asp Ala Met Asn Arg Gly Val 65 70 75 80 Gly Val Ala Thr Gly Glu Trp Val Leu Phe Leu Gly Ala Asp Asp Thr 85 90 95 Leu Tyr Glu Pro Thr Thr Leu Ala Gln Val Ala Ala Phe Leu Gly Asp 100 105 110 His Ala Ala Ser His Leu Val Tyr Gly Asp Val Val Met Arg Ser Thr 115 120 125 Lys Ser Arg His Ala Gly Pro Phe Asp Leu Asp Arg Leu Leu Phe Glu 130 135 140 Thr Asn Leu Cys His Gln Ser Ile Phe Tyr Arg Arg Glu Leu Phe Asp 145 150 155 160 Gly Ile Gly Pro Tyr Asn Leu Arg Tyr Arg Val Trp Ala Asp Trp Asp 165 170 175 Phe Asn Ile Arg Cys Phe Ser Asn Pro Ala Leu Ile Thr Arg Tyr Met 180 185 190 Asp Val Val Ile Ser Glu Tyr Asn Asp Met Thr Gly Phe Ser Met Arg 195 200 205 Gln Gly Thr Asp Lys Glu Phe Arg Lys Arg Leu Pro Met Tyr Phe Trp 210 215 220 Val Ala Gly Trp Glu Thr Cys Arg Arg Met Leu Ala Phe Leu Lys Asp 225 230 235 240 Lys Glu Asn Arg Arg Leu Ala Leu Arg Thr Arg Leu Ile Arg Val Lys 245 250 255 Ala Val Ser Lys Glu Arg Ser Ala Glu Pro 260 265 23 801 DNA Mycobacterium CDS (1)..(798) 23 atg act gcg cca gtg ttc tcg ata att atc cct acc ttc aat gca gcg 48 Met Thr Ala Pro Val Phe Ser Ile Ile Ile Pro Thr Phe Asn Ala Ala 1 5 10 15 gtg acg ctg caa gcc tgc ctc gga agc atc gtc ggg cag acc tac cgg 96 Val Thr Leu Gln Ala Cys Leu Gly Ser Ile Val Gly Gln Thr Tyr Arg 20 25 30 gaa gtg gaa gtg gtc ctt gtc gac ggc ggt tcg acc gat cgg acc ctc 144 Glu Val Glu Val Val Leu Val Asp Gly Gly Ser Thr Asp Arg Thr Leu 35 40 45 gac atc gcg aac agt ttc cgc ccg gaa ctc ggc tcg cga ctg gtc gtt 192 Asp Ile Ala Asn Ser Phe Arg Pro Glu Leu Gly Ser Arg Leu Val Val 50 55 60 cac agc ggg ccc gat gat ggc ccc tac gac gcc atg aac cgc ggc gtc 240 His Ser Gly Pro Asp Asp Gly Pro Tyr Asp Ala Met Asn Arg Gly Val 65 70 75 80 ggc gta gcc aca ggc gaa tgg gta ctt ttt tta ggc gcc gac gac acc 288 Gly Val Ala Thr Gly Glu Trp Val Leu Phe Leu Gly Ala Asp Asp Thr 85 90 95 ctc tac gaa cca acc acg ttg gcc cag gta gcc gct ttt ctc ggc gac 336 Leu Tyr Glu Pro Thr Thr Leu Ala Gln Val Ala Ala Phe Leu Gly Asp 100 105 110 cat gcg gca agc cat ctt gtc tat ggc gat gtt gtg atg cgt tcg acg 384 His Ala Ala Ser His Leu Val Tyr Gly Asp Val Val Met Arg Ser Thr 115 120 125 aaa agc cgg cat gcc gga cct ttc gac ctc gac cgc ctc cta ttt gag 432 Lys Ser Arg His Ala Gly Pro Phe Asp Leu Asp Arg Leu Leu Phe Glu 130 135 140 acg aat ttg tgc cac caa tcg atc ttt tac cgc cgt gag ctt ttc gac 480 Thr Asn Leu Cys His Gln Ser Ile Phe Tyr Arg Arg Glu Leu Phe Asp 145 150 155 160 ggc atc ggc cct tac aac ctg cgc tac cga gtc tgg gcg gac tgg gac 528 Gly Ile Gly Pro Tyr Asn Leu Arg Tyr Arg Val Trp Ala Asp Trp Asp 165 170 175 ttc aat att cgc tgc ttc tcc aac ccg gcg ctg att acc cgc tac atg 576 Phe Asn Ile Arg Cys Phe Ser Asn Pro Ala Leu Ile Thr Arg Tyr Met 180 185 190 gac gtc gtg att tcc gaa tac aac gac atg acc ggc ttc agc atg agg 624 Asp Val Val Ile Ser Glu Tyr Asn Asp Met Thr Gly Phe Ser Met Arg 195 200 205 cag ggg act gat aaa gag ttc aga aaa cgg ctg cca atg tac ttc tgg 672 Gln Gly Thr Asp Lys Glu Phe Arg Lys Arg Leu Pro Met Tyr Phe Trp 210 215 220 gtt gca ggg tgg gag act tgc agg cgc atg ctg gcg ttt ttg aaa gac 720 Val Ala Gly Trp Glu Thr Cys Arg Arg Met Leu Ala Phe Leu Lys Asp 225 230 235 240 aag gag aat cgc cgt ctg gcc ttg cgt acg cgg ttg ata agg gtt aag 768 Lys Glu Asn Arg Arg Leu Ala Leu Arg Thr Arg Leu Ile Arg Val Lys 245 250 255 gcc gtc tcc aaa gaa cga agc gca gaa ccg tag 801 Ala Val Ser Lys Glu Arg Ser Ala Glu Pro 260 265 24 266 PRT Mycobacterium 24 Met Thr Ala Pro Val Phe Ser Ile Ile Ile Pro Thr Phe Asn Ala Ala 1 5 10 15 Val Thr Leu Gln Ala Cys Leu Gly Ser Ile Val Gly Gln Thr Tyr Arg 20 25 30 Glu Val Glu Val Val Leu Val Asp Gly Gly Ser Thr Asp Arg Thr Leu 35 40 45 Asp Ile Ala Asn Ser Phe Arg Pro Glu Leu Gly Ser Arg Leu Val Val 50 55 60 His Ser Gly Pro Asp Asp Gly Pro Tyr Asp Ala Met Asn Arg Gly Val 65 70 75 80 Gly Val Ala Thr Gly Glu Trp Val Leu Phe Leu Gly Ala Asp Asp Thr 85 90 95 Leu Tyr Glu Pro Thr Thr Leu Ala Gln Val Ala Ala Phe Leu Gly Asp 100 105 110 His Ala Ala Ser His Leu Val Tyr Gly Asp Val Val Met Arg Ser Thr 115 120 125 Lys Ser Arg His Ala Gly Pro Phe Asp Leu Asp Arg Leu Leu Phe Glu 130 135 140 Thr Asn Leu Cys His Gln Ser Ile Phe Tyr Arg Arg Glu Leu Phe Asp 145 150 155 160 Gly Ile Gly Pro Tyr Asn Leu Arg Tyr Arg Val Trp Ala Asp Trp Asp 165 170 175 Phe Asn Ile Arg Cys Phe Ser Asn Pro Ala Leu Ile Thr Arg Tyr Met 180 185 190 Asp Val Val Ile Ser Glu Tyr Asn Asp Met Thr Gly Phe Ser Met Arg 195 200 205 Gln Gly Thr Asp Lys Glu Phe Arg Lys Arg Leu Pro Met Tyr Phe Trp 210 215 220 Val Ala Gly Trp Glu Thr Cys Arg Arg Met Leu Ala Phe Leu Lys Asp 225 230 235 240 Lys Glu Asn Arg Arg Leu Ala Leu Arg Thr Arg Leu Ile Arg Val Lys 245 250 255 Ala Val Ser Lys Glu Arg Ser Ala Glu Pro 260 265 25 867 DNA Mycobacterium CDS (1)..(864) 25 gtg gcc agc aga agt ccc cac tcc gct gcg ggt ggt tgg cta att ctt 48 Val Ala Ser Arg Ser Pro His Ser Ala Ala Gly Gly Trp Leu Ile Leu 1 5 10 15 ggc ggc tcc ctt ctt gtg gtc ggc gtg gcg cat ccg gta gga ctc gcc 96 Gly Gly Ser Leu Leu Val Val Gly Val Ala His Pro Val Gly Leu Ala 20 25 30 gga ggt gac gac gat gct ggc gtg gtg cag cag ccg atc gag gat gct 144 Gly Gly Asp Asp Asp Ala Gly Val Val Gln Gln Pro Ile Glu Asp Ala 35 40 45 ggc ggc ggt ggt gtg ctc ggg cag gaa tcg ccc cca ttg ttc gaa ggg 192 Gly Gly Gly Gly Val Leu Gly Gln Glu Ser Pro Pro Leu Phe Glu Gly 50 55 60 cca atg cga ggc gat ggc cag gga gcg gcg ctc gta gcc ggc agc cac 240 Pro Met Arg Gly Asp Gly Gln Gly Ala Ala Leu Val Ala Gly Ser His 65 70 75 80 gag ccg gaa caa cag ttg agt ccc ggt gtc gtc gag cgg ggc gaa gcc 288 Glu Pro Glu Gln Gln Leu Ser Pro Gly Val Val Glu Arg Gly Glu Ala 85 90 95 gat ctc gtc caa gat gac cag atc cgc gcg gag cag ggt gtc gat gat 336 Asp Leu Val Gln Asp Asp Gln Ile Arg Ala Glu Gln Gly Val Asp Asp 100 105 110 ctt gcc gac ggt gtt gtc ggc cag gcc gcg gta gag gac ctc gat cag 384 Leu Ala Asp Gly Val Val Gly Gln Ala Ala Val Glu Asp Leu Asp Gln 115 120 125 gtc ggc ggc ggt gaa gta gcg gac ttt gaa tcc ggc gtg gac ggc agc 432 Val Gly Gly Gly Glu Val Ala Asp Phe Glu Ser Gly Val Asp Gly Ser 130 135 140 gtg ccc gca gcc gat gag cag gtg act ttt gcc cgt acc agg tgg gcc 480 Val Pro Ala Ala Asp Glu Gln Val Thr Phe Ala Arg Thr Arg Trp Ala 145 150 155 160 aat gac cgc cag gtt ctg ttg tgc ccg aat cca ttc cag gct cga cag 528 Asn Asp Arg Gln Val Leu Leu Cys Pro Asn Pro Phe Gln Ala Arg Gln 165 170 175 gta gtc gaa cgt ggc tgc ggt gat cga cga tcc ggt gac gtc gaa ccc 576 Val Val Glu Arg Gly Cys Gly Asp Arg Arg Ser Gly Asp Val Glu Pro 180 185 190 gtc gag ggt ctt ggt gac cgg gaa ggc tgc ggc ctt gag acg gtt ggc 624 Val Glu Gly Leu Gly Asp Arg Glu Gly Cys Gly Leu Glu Thr Val Gly 195 200 205 ggt gtt gga ggc atc gcg ggc agc gat ctc ggc ctc aac caa cgt ccg 672 Gly Val Gly Gly Ile Ala Gly Ser Asp Leu Gly Leu Asn Gln Arg Pro 210 215 220 cag gat ctc ctc cgg tgt cca gcg ttg cgt ctt ggc gac ttg caa cac 720 Gln Asp Leu Leu Arg Cys Pro Ala Leu Arg Leu Gly Asp Leu Gln His 225 230 235 240 ctc ggc ggc gtt gcg gcg cac cgt ggc cag ctt caa ccg ccg cag cgc 768 Leu Gly Gly Val Ala Ala His Arg Gly Gln Leu Gln Pro Pro Gln Arg 245 250 255 cgc gtc aag gtc agc agc cag cgg tgc cgc cga gga cgg tgc cac cgg 816 Arg Val Lys Val Ser Ser Gln Arg Cys Arg Arg Gly Arg Cys His Arg 260 265 270 ctt ggc agc ggt ggt cat gag gcc gtc ccg tcg gtg gtg ttg atc ttg 864 Leu Gly Ser Gly Gly His Glu Ala Val Pro Ser Val Val Leu Ile Leu 275 280 285 tag 867 26 288 PRT Mycobacterium 26 Val Ala Ser Arg Ser Pro His Ser Ala Ala Gly Gly Trp Leu Ile Leu 1 5 10 15 Gly Gly Ser Leu Leu Val Val Gly Val Ala His Pro Val Gly Leu Ala 20 25 30 Gly Gly Asp Asp Asp Ala Gly Val Val Gln Gln Pro Ile Glu Asp Ala 35 40 45 Gly Gly Gly Gly Val Leu Gly Gln Glu Ser Pro Pro Leu Phe Glu Gly 50 55 60 Pro Met Arg Gly Asp Gly Gln Gly Ala Ala Leu Val Ala Gly Ser His 65 70 75 80 Glu Pro Glu Gln Gln Leu Ser Pro Gly Val Val Glu Arg Gly Glu Ala 85 90 95 Asp Leu Val Gln Asp Asp Gln Ile Arg Ala Glu Gln Gly Val Asp Asp 100 105 110 Leu Ala Asp Gly Val Val Gly Gln Ala Ala Val Glu Asp Leu Asp Gln 115 120 125 Val Gly Gly Gly Glu Val Ala Asp Phe Glu Ser Gly Val Asp Gly Ser 130 135 140 Val Pro Ala Ala Asp Glu Gln Val Thr Phe Ala Arg Thr Arg Trp Ala 145 150 155 160 Asn Asp Arg Gln Val Leu Leu Cys Pro Asn Pro Phe Gln Ala Arg Gln 165 170 175 Val Val Glu Arg Gly Cys Gly Asp Arg Arg Ser Gly Asp Val Glu Pro 180 185 190 Val Glu Gly Leu Gly Asp Arg Glu Gly Cys Gly Leu Glu Thr Val Gly 195 200 205 Gly Val Gly Gly Ile Ala Gly Ser Asp Leu Gly Leu Asn Gln Arg Pro 210 215 220 Gln Asp Leu Leu Arg Cys Pro Ala Leu Arg Leu Gly Asp Leu Gln His 225 230 235 240 Leu Gly Gly Val Ala Ala His Arg Gly Gln Leu Gln Pro Pro Gln Arg 245 250 255 Arg Val Lys Val Ser Ser Gln Arg Cys Arg Arg Gly Arg Cys His Arg 260 265 270 Leu Gly Ser Gly Gly His Glu Ala Val Pro Ser Val Val Leu Ile Leu 275 280 285 27 1739 DNA Mycobacterium CDS (1)..(945) 27 atg ggc tgc ctc aaa ggt ggt gtc gtc gcc aat gtt gtt gtt cca aca 48 Met Gly Cys Leu Lys Gly Gly Val Val Ala Asn Val Val Val Pro Thr 1 5 10 15 ccg gat tat gtg cga ttc gcg tcc cac tat ggc ttc gtt ccg gac ttc 96 Pro Asp Tyr Val Arg Phe Ala Ser His Tyr Gly Phe Val Pro Asp Phe 20 25 30 tgc cac ggt gcg gat ccg caa tcg aag ggc atc gtg gag aac ctc tgt 144 Cys His Gly Ala Asp Pro Gln Ser Lys Gly Ile Val Glu Asn Leu Cys 35 40 45 ggc tac gct cag gac gac ctt gcg gtg ccg ctg ctg acc gaa gct gcg 192 Gly Tyr Ala Gln Asp Asp Leu Ala Val Pro Leu Leu Thr Glu Ala Ala 50 55 60 tta gcc ggt gag cag gtc gac cta cgt gcc ctc aac gcc cag gcg caa 240 Leu Ala Gly Glu Gln Val Asp Leu Arg Ala Leu Asn Ala Gln Ala Gln 65 70 75 80 cta tgg tgc gcc gag gtc aat gcc acg gtc cac tcg gag atc tgc gcc 288 Leu Trp Cys Ala Glu Val Asn Ala Thr Val His Ser Glu Ile Cys Ala 85 90 95 gtg ccc aac gat cgc ttg gtt gac gag cgc acc gtc ttg agg gag ctg 336 Val Pro Asn Asp Arg Leu Val Asp Glu Arg Thr Val Leu Arg Glu Leu 100 105 110 ccc tcg ctg cgg ccg acg atc ggc tcg ggg tcg gtg cgc cgt aag gtc 384 Pro Ser Leu Arg Pro Thr Ile Gly Ser Gly Ser Val Arg Arg Lys Val 115 120 125 gac ggc ctc tcg tgc atc cgt tac ggc tca gct cgt tac tcg gtg cct 432 Asp Gly Leu Ser Cys Ile Arg Tyr Gly Ser Ala Arg Tyr Ser Val Pro 130 135 140 cag cgg ctc gtc ggt gcc acc gtg gcg gtg gtg gtc gat cat ggc gcc 480 Gln Arg Leu Val Gly Ala Thr Val Ala Val Val Val Asp His Gly Ala 145 150 155 160 ctg atc ctg ttg gaa cct gcg acc ggt gtg atc gtg gcc gag cac gag 528 Leu Ile Leu Leu Glu Pro Ala Thr Gly Val Ile Val Ala Glu His Glu 165 170 175 ctc gtc agc cca ggt gag gtg tcc atc ctc gat gaa cac tac gac gga 576 Leu Val Ser Pro Gly Glu Val Ser Ile Leu Asp Glu His Tyr Asp Gly 180 185 190 ccc aga ccc gca ccc tcg cgt ggt cct cgc ccg aaa acc caa gca gag 624 Pro Arg Pro Ala Pro Ser Arg Gly Pro Arg Pro Lys Thr Gln Ala Glu 195 200 205 aaa cga ttc tgc gca ttg gga acc gaa gcg cag cag ttc ctc gtc ggt 672 Lys Arg Phe Cys Ala Leu Gly Thr Glu Ala Gln Gln Phe Leu Val Gly 210 215 220 gct gct gcg atc ggc aac acc cga ctg aaa tcc gaa ctc gac att ctg 720 Ala Ala Ala Ile Gly Asn Thr Arg Leu Lys Ser Glu Leu Asp Ile Leu 225 230 235 240 ctc ggc ctt ggc gcc gcc cac ggc gaa cag gct ttg att gac gcg ctg 768 Leu Gly Leu Gly Ala Ala His Gly Glu Gln Ala Leu Ile Asp Ala Leu 245 250 255 cgc cgg gcg gtt gcg ttt cgc cgg ttc cgc gct gcc gac gtg cgc tcg 816 Arg Arg Ala Val Ala Phe Arg Arg Phe Arg Ala Ala Asp Val Arg Ser 260 265 270 atc ctg gcc gcc ggc gcc ggc acc cca caa ccc cgc ccc gcc ggc gac 864 Ile Leu Ala Ala Gly Ala Gly Thr Pro Gln Pro Arg Pro Ala Gly Asp 275 280 285 gca ctc gtg ctc gat ctg ccc acc gtc gag acc cgc tcg ttg gag gcc 912 Ala Leu Val Leu Asp Leu Pro Thr Val Glu Thr Arg Ser Leu Glu Ala 290 295 300 tac aag atc aac acc acc gac ggg acg gcc tca tgaccaccgc tgccaagccg 965 Tyr Lys Ile Asn Thr Thr Asp Gly Thr Ala Ser 305 310 315 gtggcaccgt cctcggcggc accgctggct gctgaccttg acgcggcgct gcggcggttg 1025 aagctggcca cggtgcgccg caacgccgcc gaggtgttgc aagtcgccaa gacgcaacgc 1085 tggacaccgg aggagatcct gcggacgttg gttgaggccg agatcgctgc ccgcgatgcc 1145 tccaacaccg ccaaccgtct caaggccgca gccttcccgg tcaccaagac cctcgacggg 1205 ttcgacgtca ccggatcgtc gatcaccgca gccacgttcg actacctgtc gagcctggaa 1265 tggattcggg cacaacagaa cctggcggtc attggcccac ctggtacggg caaaagtcac 1325 ctgctcatcg gctgcgggca cgctgccgtc cacgccggat tcaaagtccg ctacttcacc 1385 gccgccgacc tgatcgaggt cctctaccgc ggcctggccg acaacaccgt cggcaagatc 1445 atcgacaccc tgctccgcgc ggatctggtc atcttggacg agatcggctt cgccccgctc 1505 gacgacaccg ggactcaact gttgttccgg ctcgtggctg ccggctacga gcgccgctcc 1565 ctggccatcg cctcgcattg gcccttcgaa caatgggggc gattcctgcc cgagcacacc 1625 accgccgcca gcatcctcga tcggctgctg caccacgcca gcatcgtcgt cacctccggc 1685 gagtcctacc ggatgcgcca cgccgaccac aagaagggag ccgccaagaa ttag 1739 28 315 PRT Mycobacterium 28 Met Gly Cys Leu Lys Gly Gly Val Val Ala Asn Val Val Val Pro Thr 1 5 10 15 Pro Asp Tyr Val Arg Phe Ala Ser His Tyr Gly Phe Val Pro Asp Phe 20 25 30 Cys His Gly Ala Asp Pro Gln Ser Lys Gly Ile Val Glu Asn Leu Cys 35 40 45 Gly Tyr Ala Gln Asp Asp Leu Ala Val Pro Leu Leu Thr Glu Ala Ala 50 55 60 Leu Ala Gly Glu Gln Val Asp Leu Arg Ala Leu Asn Ala Gln Ala Gln 65 70 75 80 Leu Trp Cys Ala Glu Val Asn Ala Thr Val His Ser Glu Ile Cys Ala 85 90 95 Val Pro Asn Asp Arg Leu Val Asp Glu Arg Thr Val Leu Arg Glu Leu 100 105 110 Pro Ser Leu Arg Pro Thr Ile Gly Ser Gly Ser Val Arg Arg Lys Val 115 120 125 Asp Gly Leu Ser Cys Ile Arg Tyr Gly Ser Ala Arg Tyr Ser Val Pro 130 135 140 Gln Arg Leu Val Gly Ala Thr Val Ala Val Val Val Asp His Gly Ala 145 150 155 160 Leu Ile Leu Leu Glu Pro Ala Thr Gly Val Ile Val Ala Glu His Glu 165 170 175 Leu Val Ser Pro Gly Glu Val Ser Ile Leu Asp Glu His Tyr Asp Gly 180 185 190 Pro Arg Pro Ala Pro Ser Arg Gly Pro Arg Pro Lys Thr Gln Ala Glu 195 200 205 Lys Arg Phe Cys Ala Leu Gly Thr Glu Ala Gln Gln Phe Leu Val Gly 210 215 220 Ala Ala Ala Ile Gly Asn Thr Arg Leu Lys Ser Glu Leu Asp Ile Leu 225 230 235 240 Leu Gly Leu Gly Ala Ala His Gly Glu Gln Ala Leu Ile Asp Ala Leu 245 250 255 Arg Arg Ala Val Ala Phe Arg Arg Phe Arg Ala Ala Asp Val Arg Ser 260 265 270 Ile Leu Ala Ala Gly Ala Gly Thr Pro Gln Pro Arg Pro Ala Gly Asp 275 280 285 Ala Leu Val Leu Asp Leu Pro Thr Val Glu Thr Arg Ser Leu Glu Ala 290 295 300 Tyr Lys Ile Asn Thr Thr Asp Gly Thr Ala Ser 305 310 315 29 264 PRT Mycobacteria DOMAIN (1)..(264) amino acid sequence is encoded by nucleotides 945-1736 of SEQ ID NO27 29 Met Thr Thr Ala Ala Lys Pro Val Ala Pro Ser Ser Ala Ala Pro Leu 1 5 10 15 Ala Ala Asp Leu Asp Ala Ala Leu Arg Arg Leu Lys Leu Ala Thr Val 20 25 30 Arg Arg Asn Ala Ala Glu Val Leu Gln Val Ala Lys Thr Gln Arg Trp 35 40 45 Thr Pro Glu Glu Ile Leu Arg Thr Leu Val Glu Ala Glu Ile Ala Ala 50 55 60 Arg Asp Ala Ser Asn Thr Ala Asn Arg Leu Lys Ala Ala Ala Phe Pro 65 70 75 80 Val Thr Lys Thr Leu Asp Gly Phe Asp Val Thr Gly Ser Ser Ile Thr 85 90 95 Ala Ala Thr Phe Asp Tyr Leu Ser Ser Leu Glu Trp Ile Arg Ala Gln 100 105 110 Gln Asn Leu Ala Val Ile Gly Pro Pro Gly Thr Gly Lys Ser His Leu 115 120 125 Leu Ile Gly Cys Gly His Ala Ala Val His Ala Gly Phe Lys Val Arg 130 135 140 Tyr Phe Thr Ala Ala Asp Leu Ile Glu Val Leu Tyr Arg Gly Leu Ala 145 150 155 160 Asp Asn Thr Val Gly Lys Ile Ile Asp Thr Leu Leu Arg Ala Asp Leu 165 170 175 Val Ile Leu Asp Glu Ile Gly Phe Ala Pro Leu Asp Asp Thr Gly Thr 180 185 190 Gln Leu Leu Phe Arg Leu Val Ala Ala Gly Tyr Glu Arg Arg Ser Leu 195 200 205 Ala Ile Ala Ser His Trp Pro Phe Glu Gln Trp Gly Arg Phe Leu Pro 210 215 220 Glu His Thr Thr Ala Ala Ser Ile Leu Asp Arg Leu Leu His His Ala 225 230 235 240 Ser Ile Val Val Thr Ser Gly Glu Ser Tyr Arg Met Arg His Ala Asp 245 250 255 His Lys Lys Gly Ala Ala Lys Asn 260 30 789 DNA Mycobacterium CDS (1)..(786) 30 gtg acg tct gct ccg acc gtc tcg gtg ata acg atc tcg ttc aac gac 48 Met Thr Ser Ala Pro Thr Val Ser Val Ile Thr Ile Ser Phe Asn Asp 1 5 10 15 ctc gac ggg ttg cag cgc acg gtg aaa agt gtg cgg gcg caa cgc tac 96 Leu Asp Gly Leu Gln Arg Thr Val Lys Ser Val Arg Ala Gln Arg Tyr 20 25 30 cgg gga cgc atc gag cac atc gta atc gac ggt ggc agc ggc gac gac 144 Arg Gly Arg Ile Glu His Ile Val Ile Asp Gly Gly Ser Gly Asp Asp 35 40 45 gtg gtg gca tac ctg tcc ggg tgt gaa cca ggc ttc gcg tat tgg cag 192 Val Val Ala Tyr Leu Ser Gly Cys Glu Pro Gly Phe Ala Tyr Trp Gln 50 55 60 tcc gag ccc gac ggc ggg cgg tac gac gcg atg aac cag ggc atc gcg 240 Ser Glu Pro Asp Gly Gly Arg Tyr Asp Ala Met Asn Gln Gly Ile Ala 65 70 75 80 cac gca tcg ggt gat ctg ttg tgg ttc ttg cac tcc gcc gat cgt ttt 288 His Ala Ser Gly Asp Leu Leu Trp Phe Leu His Ser Ala Asp Arg Phe 85 90 95 tcc ggg ccc gac gtg gta gcc cag gcc gtg gag gcg cta tcc ggc aag 336 Ser Gly Pro Asp Val Val Ala Gln Ala Val Glu Ala Leu Ser Gly Lys 100 105 110 gga ccg gtg tcc gaa ttg tgg ggc ttc ggg atg gat cgt ctc gtc ggg 384 Gly Pro Val Ser Glu Leu Trp Gly Phe Gly Met Asp Arg Leu Val Gly 115 120 125 ctc gat cgg gtg cgc ggc ccg ata cct ttc agc ctg cgc aaa ttc ctg 432 Leu Asp Arg Val Arg Gly Pro Ile Pro Phe Ser Leu Arg Lys Phe Leu 130 135 140 gcc ggc aag cag gtt gtt ccg cat caa gca tcg ttc ttc gga tca tcg 480 Ala Gly Lys Gln Val Val Pro His Gln Ala Ser Phe Phe Gly Ser Ser 145 150 155 160 ctg gtg gcc aag atc ggt ggc tac gac ctt gat ttc ggg atc gcc gcc 528 Leu Val Ala Lys Ile Gly Gly Tyr Asp Leu Asp Phe Gly Ile Ala Ala 165 170 175 gac cag gaa ttc ata ttg cgg gcc gcg ctg gta tgc gag ccg gtc acg 576 Asp Gln Glu Phe Ile Leu Arg Ala Ala Leu Val Cys Glu Pro Val Thr 180 185 190 att cgg tgt gtg ctg tgc gag ttc gac acc acg ggc gtc ggc tcg cac 624 Ile Arg Cys Val Leu Cys Glu Phe Asp Thr Thr Gly Val Gly Ser His 195 200 205 cgg gaa cca agc gcg gtc ttc ggt gat ctg cgc cgc atg ggc gac ctt 672 Arg Glu Pro Ser Ala Val Phe Gly Asp Leu Arg Arg Met Gly Asp Leu 210 215 220 cat cgc cgc tac ccg ttc ggg gga agg cga ata tca cat gcc tac cta 720 His Arg Arg Tyr Pro Phe Gly Gly Arg Arg Ile Ser His Ala Tyr Leu 225 230 235 240 cgc ggc cgg gag ttc tac gcc tac aac agt cga ttc tgg gaa aac gtc 768 Arg Gly Arg Glu Phe Tyr Ala Tyr Asn Ser Arg Phe Trp Glu Asn Val 245 250 255 ttc acg cga atg tcg aaa tag 789 Phe Thr Arg Met Ser Lys 260 31 262 PRT Mycobacterium 31 Met Thr Ser Ala Pro Thr Val Ser Val Ile Thr Ile Ser Phe Asn Asp 1 5 10 15 Leu Asp Gly Leu Gln Arg Thr Val Lys Ser Val Arg Ala Gln Arg Tyr 20 25 30 Arg Gly Arg Ile Glu His Ile Val Ile Asp Gly Gly Ser Gly Asp Asp 35 40 45 Val Val Ala Tyr Leu Ser Gly Cys Glu Pro Gly Phe Ala Tyr Trp Gln 50 55 60 Ser Glu Pro Asp Gly Gly Arg Tyr Asp Ala Met Asn Gln Gly Ile Ala 65 70 75 80 His Ala Ser Gly Asp Leu Leu Trp Phe Leu His Ser Ala Asp Arg Phe 85 90 95 Ser Gly Pro Asp Val Val Ala Gln Ala Val Glu Ala Leu Ser Gly Lys 100 105 110 Gly Pro Val Ser Glu Leu Trp Gly Phe Gly Met Asp Arg Leu Val Gly 115 120 125 Leu Asp Arg Val Arg Gly Pro Ile Pro Phe Ser Leu Arg Lys Phe Leu 130 135 140 Ala Gly Lys Gln Val Val Pro His Gln Ala Ser Phe Phe Gly Ser Ser 145 150 155 160 Leu Val Ala Lys Ile Gly Gly Tyr Asp Leu Asp Phe Gly Ile Ala Ala 165 170 175 Asp Gln Glu Phe Ile Leu Arg Ala Ala Leu Val Cys Glu Pro Val Thr 180 185 190 Ile Arg Cys Val Leu Cys Glu Phe Asp Thr Thr Gly Val Gly Ser His 195 200 205 Arg Glu Pro Ser Ala Val Phe Gly Asp Leu Arg Arg Met Gly Asp Leu 210 215 220 His Arg Arg Tyr Pro Phe Gly Gly Arg Arg Ile Ser His Ala Tyr Leu 225 230 235 240 Arg Gly Arg Glu Phe Tyr Ala Tyr Asn Ser Arg Phe Trp Glu Asn Val 245 250 255 Phe Thr Arg Met Ser Lys 260 32 1023 DNA Mycobacterium CDS (1)..(1020) 32 gtg aag cga gcg ctc atc acc gga atc acc ggc cag gac ggc tcg tat 48 Met Lys Arg Ala Leu Ile Thr Gly Ile Thr Gly Gln Asp Gly Ser Tyr 1 5 10 15 ctc gcc gaa ctg ctg ctg gcc aag ggg tat gag gtt cac ggg ctc atc 96 Leu Ala Glu Leu Leu Leu Ala Lys Gly Tyr Glu Val His Gly Leu Ile 20 25 30 cgg cgc gct tcg acg ttc aac acc tcg cgg atc gat cac ctc tac gtc 144 Arg Arg Ala Ser Thr Phe Asn Thr Ser Arg Ile Asp His Leu Tyr Val 35 40 45 gac ccg cac caa ccg ggc gcg cgg ctg ttt ctg cac tat ggt gac ctg 192 Asp Pro His Gln Pro Gly Ala Arg Leu Phe Leu His Tyr Gly Asp Leu 50 55 60 atc gac gga acc cgg ttg gtg acc ctg ctg agc acc atc gaa ccc gac 240 Ile Asp Gly Thr Arg Leu Val Thr Leu Leu Ser Thr Ile Glu Pro Asp 65 70 75 80 gag gtg tac aac ctg gcg gcg cag tca cac gtg cgg gtg agc ttc gac 288 Glu Val Tyr Asn Leu Ala Ala Gln Ser His Val Arg Val Ser Phe Asp 85 90 95 gaa ccc gtg cac acc ggt gac acc acc ggc atg gga tcc atg cga ctg 336 Glu Pro Val His Thr Gly Asp Thr Thr Gly Met Gly Ser Met Arg Leu 100 105 110 ctg gaa gcc gtt cgg ctc tct cgg gtg cac tgc cgc ttc tat cag gcg 384 Leu Glu Ala Val Arg Leu Ser Arg Val His Cys Arg Phe Tyr Gln Ala 115 120 125 tcc tcg tcg gag atg ttc ggc gcc tcg ccg cca ccg cag aac gag ctg 432 Ser Ser Ser Glu Met Phe Gly Ala Ser Pro Pro Pro Gln Asn Glu Leu 130 135 140 acg ccg ttc tac ccg cgg tca ccg tat ggc gcc gcc aag gtc tat tcg 480 Thr Pro Phe Tyr Pro Arg Ser Pro Tyr Gly Ala Ala Lys Val Tyr Ser 145 150 155 160 tac tgg gcg acc cgc aat tat cgc gaa gcg tac gga ttg ttc gcc gtt 528 Tyr Trp Ala Thr Arg Asn Tyr Arg Glu Ala Tyr Gly Leu Phe Ala Val 165 170 175 aac ggc atc ttg ttc aat cac gaa tca ccg cgg cgc ggt gag acg ttc 576 Asn Gly Ile Leu Phe Asn His Glu Ser Pro Arg Arg Gly Glu Thr Phe 180 185 190 gtg acc cga aag atc acc agg gcc gtg gca cgc atc aag gcc ggt atc 624 Val Thr Arg Lys Ile Thr Arg Ala Val Ala Arg Ile Lys Ala Gly Ile 195 200 205 cag tcc gag gtc tat atg ggc aat ctg gat gcg gtc cgc gac tgg ggg 672 Gln Ser Glu Val Tyr Met Gly Asn Leu Asp Ala Val Arg Asp Trp Gly 210 215 220 tac gcg ccc gaa tac gtc gaa ggc atg tgg cgg atg ctg cag acc gac 720 Tyr Ala Pro Glu Tyr Val Glu Gly Met Trp Arg Met Leu Gln Thr Asp 225 230 235 240 gag ccc gac gac ttc gtt ttg gcg acc ggg cgc ggt ttc acc gtg cgt 768 Glu Pro Asp Asp Phe Val Leu Ala Thr Gly Arg Gly Phe Thr Val Arg 245 250 255 gag ttc gcg cgg gcc gcg ttc gag cat gcc ggt ttg gac tgg cag cag 816 Glu Phe Ala Arg Ala Ala Phe Glu His Ala Gly Leu Asp Trp Gln Gln 260 265 270 tac gtg aaa ttc gac caa cgc tat ctg cgg ccc acc gag gtg gat tcg 864 Tyr Val Lys Phe Asp Gln Arg Tyr Leu Arg Pro Thr Glu Val Asp Ser 275 280 285 ctg atc ggc gac gcg acc aag gct gcc gaa ttg ctg ggc tgg agg gct 912 Leu Ile Gly Asp Ala Thr Lys Ala Ala Glu Leu Leu Gly Trp Arg Ala 290 295 300 tcg gtg cac act gac gag ttg gct cgg atc atg gtc gac gcg gac atg 960 Ser Val His Thr Asp Glu Leu Ala Arg Ile Met Val Asp Ala Asp Met 305 310 315 320 gcg gcg ctg gag tgc gaa ggc aag ccg tgg atc gac aag ccg atg atc 1008 Ala Ala Leu Glu Cys Glu Gly Lys Pro Trp Ile Asp Lys Pro Met Ile 325 330 335 gcc ggc cgg aca tga 1023 Ala Gly Arg Thr 340 33 340 PRT Mycobacterium 33 Met Lys Arg Ala Leu Ile Thr Gly Ile Thr Gly Gln Asp Gly Ser Tyr 1 5 10 15 Leu Ala Glu Leu Leu Leu Ala Lys Gly Tyr Glu Val His Gly Leu Ile 20 25 30 Arg Arg Ala Ser Thr Phe Asn Thr Ser Arg Ile Asp His Leu Tyr Val 35 40 45 Asp Pro His Gln Pro Gly Ala Arg Leu Phe Leu His Tyr Gly Asp Leu 50 55 60 Ile Asp Gly Thr Arg Leu Val Thr Leu Leu Ser Thr Ile Glu Pro Asp 65 70 75 80 Glu Val Tyr Asn Leu Ala Ala Gln Ser His Val Arg Val Ser Phe Asp 85 90 95 Glu Pro Val His Thr Gly Asp Thr Thr Gly Met Gly Ser Met Arg Leu 100 105 110 Leu Glu Ala Val Arg Leu Ser Arg Val His Cys Arg Phe Tyr Gln Ala 115 120 125 Ser Ser Ser Glu Met Phe Gly Ala Ser Pro Pro Pro Gln Asn Glu Leu 130 135 140 Thr Pro Phe Tyr Pro Arg Ser Pro Tyr Gly Ala Ala Lys Val Tyr Ser 145 150 155 160 Tyr Trp Ala Thr Arg Asn Tyr Arg Glu Ala Tyr Gly Leu Phe Ala Val 165 170 175 Asn Gly Ile Leu Phe Asn His Glu Ser Pro Arg Arg Gly Glu Thr Phe 180 185 190 Val Thr Arg Lys Ile Thr Arg Ala Val Ala Arg Ile Lys Ala Gly Ile 195 200 205 Gln Ser Glu Val Tyr Met Gly Asn Leu Asp Ala Val Arg Asp Trp Gly 210 215 220 Tyr Ala Pro Glu Tyr Val Glu Gly Met Trp Arg Met Leu Gln Thr Asp 225 230 235 240 Glu Pro Asp Asp Phe Val Leu Ala Thr Gly Arg Gly Phe Thr Val Arg 245 250 255 Glu Phe Ala Arg Ala Ala Phe Glu His Ala Gly Leu Asp Trp Gln Gln 260 265 270 Tyr Val Lys Phe Asp Gln Arg Tyr Leu Arg Pro Thr Glu Val Asp Ser 275 280 285 Leu Ile Gly Asp Ala Thr Lys Ala Ala Glu Leu Leu Gly Trp Arg Ala 290 295 300 Ser Val His Thr Asp Glu Leu Ala Arg Ile Met Val Asp Ala Asp Met 305 310 315 320 Ala Ala Leu Glu Cys Glu Gly Lys Pro Trp Ile Asp Lys Pro Met Ile 325 330 335 Ala Gly Arg Thr 340 34 732 DNA Mycobacterium CDS (1)..(729) 34 atg agg ctg gcc cgt cgc gct cgg aac atc ttg cgt cgc aac ggc atc 48 Met Arg Leu Ala Arg Arg Ala Arg Asn Ile Leu Arg Arg Asn Gly Ile 1 5 10 15 gag gtg tcg cgc tac ttt gcc gaa ctg gac tgg gaa cgc aat ttc ttg 96 Glu Val Ser Arg Tyr Phe Ala Glu Leu Asp Trp Glu Arg Asn Phe Leu 20 25 30 cgc caa ctg caa tcg cat cgg gtc agt gcc gtg ctc gat gtc ggg gcc 144 Arg Gln Leu Gln Ser His Arg Val Ser Ala Val Leu Asp Val Gly Ala 35 40 45 aat tcg ggg cag tac gcc agg ggt ctg cgc ggc gcg ggc ttc gcg ggc 192 Asn Ser Gly Gln Tyr Ala Arg Gly Leu Arg Gly Ala Gly Phe Ala Gly 50 55 60 cgc atc gtc tcg ttc gag ccg ctg ccc ggg ccc ttt gcc gtc ttg cag 240 Arg Ile Val Ser Phe Glu Pro Leu Pro Gly Pro Phe Ala Val Leu Gln 65 70 75 80 cgc agc gcc tcc acg gac ccg ttg tgg gaa tgc cgg cgc tgt gcg ctg 288 Arg Ser Ala Ser Thr Asp Pro Leu Trp Glu Cys Arg Arg Cys Ala Leu 85 90 95 ggc gat gtc gat gga acc atc tcg atc aac gtc gcc ggc aac gag ggc 336 Gly Asp Val Asp Gly Thr Ile Ser Ile Asn Val Ala Gly Asn Glu Gly 100 105 110 gcc agc agt tcc gtc ttg ccg atg ttg aaa cga cat cag gac gcc ttt 384 Ala Ser Ser Ser Val Leu Pro Met Leu Lys Arg His Gln Asp Ala Phe 115 120 125 cca cca gcc aac tac gtg ggc gcc caa cgg gtg ccg ata cat cga ctc 432 Pro Pro Ala Asn Tyr Val Gly Ala Gln Arg Val Pro Ile His Arg Leu 130 135 140 gat tcc gtg gct gca gac gtt ctg cgg ccc aac gat att gcg ttc ttg 480 Asp Ser Val Ala Ala Asp Val Leu Arg Pro Asn Asp Ile Ala Phe Leu 145 150 155 160 aag atc gac gtt caa gga ttc gag aag cag gtg atc gcg ggt ggc gat 528 Lys Ile Asp Val Gln Gly Phe Glu Lys Gln Val Ile Ala Gly Gly Asp 165 170 175 tca acg gtg cac gac cga tgc gtc ggc atg cag ctc gag ctg tct ttc 576 Ser Thr Val His Asp Arg Cys Val Gly Met Gln Leu Glu Leu Ser Phe 180 185 190 cag ccg ttg tac gag ggt ggc atg ctc atc cgc gag gcg ctc gat ctc 624 Gln Pro Leu Tyr Glu Gly Gly Met Leu Ile Arg Glu Ala Leu Asp Leu 195 200 205 gtg gat tcg ttg ggc ttt acg ctc tcg gga ttg caa ccc ggt ttc acc 672 Val Asp Ser Leu Gly Phe Thr Leu Ser Gly Leu Gln Pro Gly Phe Thr 210 215 220 gac ccc cgc aac ggt cga atg ctg cag gcc gat ggc atc ttc ttc cgg 720 Asp Pro Arg Asn Gly Arg Met Leu Gln Ala Asp Gly Ile Phe Phe Arg 225 230 235 240 ggc agc gat tga 732 Gly Ser Asp 35 243 PRT Mycobacterium 35 Met Arg Leu Ala Arg Arg Ala Arg Asn Ile Leu Arg Arg Asn Gly Ile 1 5 10 15 Glu Val Ser Arg Tyr Phe Ala Glu Leu Asp Trp Glu Arg Asn Phe Leu 20 25 30 Arg Gln Leu Gln Ser His Arg Val Ser Ala Val Leu Asp Val Gly Ala 35 40 45 Asn Ser Gly Gln Tyr Ala Arg Gly Leu Arg Gly Ala Gly Phe Ala Gly 50 55 60 Arg Ile Val Ser Phe Glu Pro Leu Pro Gly Pro Phe Ala Val Leu Gln 65 70 75 80 Arg Ser Ala Ser Thr Asp Pro Leu Trp Glu Cys Arg Arg Cys Ala Leu 85 90 95 Gly Asp Val Asp Gly Thr Ile Ser Ile Asn Val Ala Gly Asn Glu Gly 100 105 110 Ala Ser Ser Ser Val Leu Pro Met Leu Lys Arg His Gln Asp Ala Phe 115 120 125 Pro Pro Ala Asn Tyr Val Gly Ala Gln Arg Val Pro Ile His Arg Leu 130 135 140 Asp Ser Val Ala Ala Asp Val Leu Arg Pro Asn Asp Ile Ala Phe Leu 145 150 155 160 Lys Ile Asp Val Gln Gly Phe Glu Lys Gln Val Ile Ala Gly Gly Asp 165 170 175 Ser Thr Val His Asp Arg Cys Val Gly Met Gln Leu Glu Leu Ser Phe 180 185 190 Gln Pro Leu Tyr Glu Gly Gly Met Leu Ile Arg Glu Ala Leu Asp Leu 195 200 205 Val Asp Ser Leu Gly Phe Thr Leu Ser Gly Leu Gln Pro Gly Phe Thr 210 215 220 Asp Pro Arg Asn Gly Arg Met Leu Gln Ala Asp Gly Ile Phe Phe Arg 225 230 235 240 Gly Ser Asp 36 732 DNA Mycobacterium CDS (1)..(729) 36 gtg aaa tcg ttg aaa ctc gct cgt ttc atc gcg cgt agc gcc gcc ttc 48 Met Lys Ser Leu Lys Leu Ala Arg Phe Ile Ala Arg Ser Ala Ala Phe 1 5 10 15 gag gtt tcg cgc cgc tat tct gag cga gac ctg aag cac cag ttt gtg 96 Glu Val Ser Arg Arg Tyr Ser Glu Arg Asp Leu Lys His Gln Phe Val 20 25 30 aag caa ctc aaa tcg cgt cgg gta gat gtc gtt ttc gat gtc ggc gcc 144 Lys Gln Leu Lys Ser Arg Arg Val Asp Val Val Phe Asp Val Gly Ala 35 40 45 aac tca gga caa tac gcc gcc ggc ctc cgc cga gca gca tat aag ggc 192 Asn Ser Gly Gln Tyr Ala Ala Gly Leu Arg Arg Ala Ala Tyr Lys Gly 50 55 60 cgc att gtc tcg ttc gaa ccg cta tcc gga ccg ttt acg atc ttg gaa 240 Arg Ile Val Ser Phe Glu Pro Leu Ser Gly Pro Phe Thr Ile Leu Glu 65 70 75 80 agc aaa gcg tca acg gat cca ctt tgg gat tgc cgg cag cat gcg ttg 288 Ser Lys Ala Ser Thr Asp Pro Leu Trp Asp Cys Arg Gln His Ala Leu 85 90 95 ggc gat tct gat gga acg gtt acg atc aat atc gca gga aac gcc ggt 336 Gly Asp Ser Asp Gly Thr Val Thr Ile Asn Ile Ala Gly Asn Ala Gly 100 105 110 cag agc agt tcc gtc ttg ccc atg ctg aaa agt cat cag aac gct ttt 384 Gln Ser Ser Ser Val Leu Pro Met Leu Lys Ser His Gln Asn Ala Phe 115 120 125 ccc ccg gca aac tat gtc ggt acc caa gag gcg tcc ata cat cga ctt 432 Pro Pro Ala Asn Tyr Val Gly Thr Gln Glu Ala Ser Ile His Arg Leu 130 135 140 gat tcc gtg gcg cca gaa ttt cta ggc atg aac ggt gtc gct ttt ctc 480 Asp Ser Val Ala Pro Glu Phe Leu Gly Met Asn Gly Val Ala Phe Leu 145 150 155 160 aag gtc gac gtt caa ggc ttt gaa aag cag gtg ctc gcc ggg ggc aaa 528 Lys Val Asp Val Gln Gly Phe Glu Lys Gln Val Leu Ala Gly Gly Lys 165 170 175 tca acc ata gat gac cat tgc gtc ggc atg caa ctc gaa ctg tcc ttc 576 Ser Thr Ile Asp Asp His Cys Val Gly Met Gln Leu Glu Leu Ser Phe 180 185 190 ctg ccg ttg tac gaa ggt ggc atg ctc att cct gaa gcc ctc gat ctc 624 Leu Pro Leu Tyr Glu Gly Gly Met Leu Ile Pro Glu Ala Leu Asp Leu 195 200 205 gtg tat tcc ttg ggc ttc acg ttg acg gga ttg ctg cct tgt ttc att 672 Val Tyr Ser Leu Gly Phe Thr Leu Thr Gly Leu Leu Pro Cys Phe Ile 210 215 220 gat gca aat aat ggt cga atg ttg cag gcc gac ggc atc ttt ttc cgc 720 Asp Ala Asn Asn Gly Arg Met Leu Gln Ala Asp Gly Ile Phe Phe Arg 225 230 235 240 gag gac gat tga 732 Glu Asp Asp 37 243 PRT Mycobacterium 37 Met Lys Ser Leu Lys Leu Ala Arg Phe Ile Ala Arg Ser Ala Ala Phe 1 5 10 15 Glu Val Ser Arg Arg Tyr Ser Glu Arg Asp Leu Lys His Gln Phe Val 20 25 30 Lys Gln Leu Lys Ser Arg Arg Val Asp Val Val Phe Asp Val Gly Ala 35 40 45 Asn Ser Gly Gln Tyr Ala Ala Gly Leu Arg Arg Ala Ala Tyr Lys Gly 50 55 60 Arg Ile Val Ser Phe Glu Pro Leu Ser Gly Pro Phe Thr Ile Leu Glu 65 70 75 80 Ser Lys Ala Ser Thr Asp Pro Leu Trp Asp Cys Arg Gln His Ala Leu 85 90 95 Gly Asp Ser Asp Gly Thr Val Thr Ile Asn Ile Ala Gly Asn Ala Gly 100 105 110 Gln Ser Ser Ser Val Leu Pro Met Leu Lys Ser His Gln Asn Ala Phe 115 120 125 Pro Pro Ala Asn Tyr Val Gly Thr Gln Glu Ala Ser Ile His Arg Leu 130 135 140 Asp Ser Val Ala Pro Glu Phe Leu Gly Met Asn Gly Val Ala Phe Leu 145 150 155 160 Lys Val Asp Val Gln Gly Phe Glu Lys Gln Val Leu Ala Gly Gly Lys 165 170 175 Ser Thr Ile Asp Asp His Cys Val Gly Met Gln Leu Glu Leu Ser Phe 180 185 190 Leu Pro Leu Tyr Glu Gly Gly Met Leu Ile Pro Glu Ala Leu Asp Leu 195 200 205 Val Tyr Ser Leu Gly Phe Thr Leu Thr Gly Leu Leu Pro Cys Phe Ile 210 215 220 Asp Ala Asn Asn Gly Arg Met Leu Gln Ala Asp Gly Ile Phe Phe Arg 225 230 235 240 Glu Asp Asp 38 828 DNA Mycobacterium CDS (1)..(825) 38 atg gtg cag acg aaa cga tac gcc ggc ttg acc gca gct aac aca aag 48 Met Val Gln Thr Lys Arg Tyr Ala Gly Leu Thr Ala Ala Asn Thr Lys 1 5 10 15 aaa gtc gcc atg gcc gca cca atg ttt tcg atc atc atc ccc acc ttg 96 Lys Val Ala Met Ala Ala Pro Met Phe Ser Ile Ile Ile Pro Thr Leu 20 25 30 aac gtg gct gcg gta ttg cct gcc tgc ctc gac agc atc gcc cgt cag 144 Asn Val Ala Ala Val Leu Pro Ala Cys Leu Asp Ser Ile Ala Arg Gln 35 40 45 acc tgc ggt gac ttc gag ctg gta ctg gtc gac ggc ggc tcg acg gac 192 Thr Cys Gly Asp Phe Glu Leu Val Leu Val Asp Gly Gly Ser Thr Asp 50 55 60 gaa acc ctc gac atc gcc aac att ttc gcc ccc aac ctc ggc gag cgg 240 Glu Thr Leu Asp Ile Ala Asn Ile Phe Ala Pro Asn Leu Gly Glu Arg 65 70 75 80 ttg atc att cat cgc gac acc gac cag ggc gtc tac gac gcc atg aac 288 Leu Ile Ile His Arg Asp Thr Asp Gln Gly Val Tyr Asp Ala Met Asn 85 90 95 cgc ggc gtg gac ctg gcc acc gga acg tgg ttg ctc ttt ctg ggc gcg 336 Arg Gly Val Asp Leu Ala Thr Gly Thr Trp Leu Leu Phe Leu Gly Ala 100 105 110 gac gac agc ctg tac gag gct gac acc ctg gcg cgg gtg gcc gcc ttc 384 Asp Asp Ser Leu Tyr Glu Ala Asp Thr Leu Ala Arg Val Ala Ala Phe 115 120 125 att ggc gaa cac gag ccc agc gat ctg gta tat ggc gac gtg atc atg 432 Ile Gly Glu His Glu Pro Ser Asp Leu Val Tyr Gly Asp Val Ile Met 130 135 140 cgc tca acc aat ttc cgc tgg ggt ggc gcc ttc gac ctc gac cgt ctg 480 Arg Ser Thr Asn Phe Arg Trp Gly Gly Ala Phe Asp Leu Asp Arg Leu 145 150 155 160 ttg ttc aag cgc aac atc tgc cat cag gcg atc ttc tac cgc cgc gga 528 Leu Phe Lys Arg Asn Ile Cys His Gln Ala Ile Phe Tyr Arg Arg Gly 165 170 175 ctc ttc ggc acc atc ggt ccc tac aac ctc cgc tac cgg gtc ctg gcc 576 Leu Phe Gly Thr Ile Gly Pro Tyr Asn Leu Arg Tyr Arg Val Leu Ala 180 185 190 gac tgg gac ttc aat att cgc tgc ttt tcc aac cca gcg ctc gtc acc 624 Asp Trp Asp Phe Asn Ile Arg Cys Phe Ser Asn Pro Ala Leu Val Thr 195 200 205 cgc tac atg cac gtg gtc gtt gca agc tac aac gaa ttc ggc ggg ctc 672 Arg Tyr Met His Val Val Val Ala Ser Tyr Asn Glu Phe Gly Gly Leu 210 215 220 agc aat acg atc gtc gac aag gag ttt ttg aag cgg ctg ccg atg tcc 720 Ser Asn Thr Ile Val Asp Lys Glu Phe Leu Lys Arg Leu Pro Met Ser 225 230 235 240 acg aga ctc ggc ata agg ctg gtc ata gtt ctg gtg cgc agg tgg cca 768 Thr Arg Leu Gly Ile Arg Leu Val Ile Val Leu Val Arg Arg Trp Pro 245 250 255 aag gtg atc agc agg gcc atg gta atg cgc acc gtc att tct tgg cgg 816 Lys Val Ile Ser Arg Ala Met Val Met Arg Thr Val Ile Ser Trp Arg 260 265 270 cgc cga cgt tag 828 Arg Arg Arg 275 39 275 PRT Mycobacterium 39 Met Val Gln Thr Lys Arg Tyr Ala Gly Leu Thr Ala Ala Asn Thr Lys 1 5 10 15 Lys Val Ala Met Ala Ala Pro Met Phe Ser Ile Ile Ile Pro Thr Leu 20 25 30 Asn Val Ala Ala Val Leu Pro Ala Cys Leu Asp Ser Ile Ala Arg Gln 35 40 45 Thr Cys Gly Asp Phe Glu Leu Val Leu Val Asp Gly Gly Ser Thr Asp 50 55 60 Glu Thr Leu Asp Ile Ala Asn Ile Phe Ala Pro Asn Leu Gly Glu Arg 65 70 75 80 Leu Ile Ile His Arg Asp Thr Asp Gln Gly Val Tyr Asp Ala Met Asn 85 90 95 Arg Gly Val Asp Leu Ala Thr Gly Thr Trp Leu Leu Phe Leu Gly Ala 100 105 110 Asp Asp Ser Leu Tyr Glu Ala Asp Thr Leu Ala Arg Val Ala Ala Phe 115 120 125 Ile Gly Glu His Glu Pro Ser Asp Leu Val Tyr Gly Asp Val Ile Met 130 135 140 Arg Ser Thr Asn Phe Arg Trp Gly Gly Ala Phe Asp Leu Asp Arg Leu 145 150 155 160 Leu Phe Lys Arg Asn Ile Cys His Gln Ala Ile Phe Tyr Arg Arg Gly 165 170 175 Leu Phe Gly Thr Ile Gly Pro Tyr Asn Leu Arg Tyr Arg Val Leu Ala 180 185 190 Asp Trp Asp Phe Asn Ile Arg Cys Phe Ser Asn Pro Ala Leu Val Thr 195 200 205 Arg Tyr Met His Val Val Val Ala Ser Tyr Asn Glu Phe Gly Gly Leu 210 215 220 Ser Asn Thr Ile Val Asp Lys Glu Phe Leu Lys Arg Leu Pro Met Ser 225 230 235 240 Thr Arg Leu Gly Ile Arg Leu Val Ile Val Leu Val Arg Arg Trp Pro 245 250 255 Lys Val Ile Ser Arg Ala Met Val Met Arg Thr Val Ile Ser Trp Arg 260 265 270 Arg Arg Arg 275 40 24 DNA Mycobacterium 40 gatgccgtga ggaggtaaag ctgc 24 41 24 DNA Mycobacterium 41 gatacggctc ttgaatcctg cacg 24

Claims

1-23. (Canceled).

24. A substantially isolated polynucleotide comprising:

(a) a polynucleotide sequence according to any one of SEQ ID NOS: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27;

(b) a polynucleotide capable of selectively hybridising to a polynucleotide of (a);

(c) a polynucleotide having at least 80% sequence homology to a polynucleotide of (a) over 30 contiguous amino acids;

(d) a polynucleotide encoding a polypeptide of any one of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28; or

(e) a polynucleotide encoding a fragment of at least 10 amino acids of a polypeptide of any one of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28;

wherein said polynucleotide encodes a polypeptide having the ability to stimulate an immune response against a mycobacterium.

25. A substantially isolated polynucleotide according to claim 24 comprising:

(a) a polynucleotide sequence according to SEQ ID NO: 23;

(b) a polynucleotide capable of selectively hybridising to a polynucleotide of SEQ ID NO: 23;

(c) a polynucleotide having at least 80% sequence homology to the polynucleotide of SEQ ID NO: 23 over 30 contiguous amino acids;

(d) a polynucleotide encoding the polypeptide of SEQ ID NO: 24; or

(e) a polynucleotide encoding a fragment of at least 10 amino acids of the polypeptide of SEQ ID NO: 24;

26. A polynucleotide according to claim 24 which further comprises a label.

27. A vector carrying a polynucleotide according to claim 24.

28. A vector according to claim 27 which is an expression vector.

29. A vector according to claim 28 wherein said polynucleotide is operably linked to a control sequence which is capable of providing for the expression of the coding sequence of the polynucleotide.

30. A vector according to claim 27 which comprises one or more components selected from the group consisting of an origin of replication, a promoter for expression of the polypeptide encoded by said polynucleotide, a regulator of a promoter for expression of the polypeptide encoded by said polypeptide, an enhancer and a selectable marker gene.

31. A vector according to claim 30 wherein said promoter is a mammalian, viral, yeast or bacterial promoter.

32. A vector according to claim 31 wherein said promoter is selected from the group consisting of: a metallothionien promoter, an adenovirus promoter, the SV40 large T promoter, a retroviral LTR promoter, the polyhedrin promoter, an alcohol dehydrogenase promoter and a β-galactosidase promoter.

33. A vector according to claim 27 which is adapted for use in vivo.

34. A vector according to claim 27 which is a plasmid, virus or phage vector.

35. A vector according to claim 34 wherein said viral vector is selected from the group consisting of retroviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia virus vectors, herpes virus vector and alpha virus vectors.

36. A host cell comprising, transformed with or transfected by a vector according to claim 27.

37. A host cell according to claim 36 which is a bacterial, yeast, insect or mammalian cell.

38. A host cell according to claim 37 which is selected from the group consisting of M. bovis BCG, M. smegmatis, a mycobacterium, Corynebacteria and Salmonella.

39. A pharmaceutical composition comprising a polynucleotide according to claim 24 and a pharmaceutically acceptable carrier or diluent.

40. A pharmaceutical composition comprising a vector according to claim 27 and a pharmaceutically acceptable carrier or diluent.

41. A pharmaceutical composition comprising a host cell according to claim 36 and a pharmaceutically acceptable carrier or diluent.

42. A method of raising an immune response in an animal or human against a mycobacterium, which method comprises administering an effective amount of a polynucleotide capable of expressing a polypeptide selected from:

(i) a polypeptide according to any one of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 29;

(ii) a polypeptide comprising a polypeptide according to (i);

(iii) a polypeptide having at least 70% amino acid identity to a polypeptide of (i) over 30 or more contiguous amino acids, which retains the ability to stimulate an immune response against said mycobacterium; or

(iv) a fragment of a polypeptide of (i) comprising at least 10 amino acids which retains the ability to stimulate an immune response against said mycobacterium to said human or animal and allowing said polypeptide to be expressed.

43. A method according to claim 19 wherein said polynucleotide is selected from:

(e) a polynucleotide encoding a fragment of at least 10 amino acids of a polypeptide of any one of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28.

44. A method of raising an immune response in an animal or human against a mycobacterium, which method comprises administering an effective amount of a polynucleotide capable of expressing a polypeptide selected from:

(ii) a polypeptide according to SEQ ID NO: 24;

(ii) a polypeptide comprising a polypeptide according to (i);

45. A method according to claim 19 wherein said polynucleotide is selected from:

(a) a polynucleotide sequence according to SEQ ID NO: 23;

(d) a polynucleotide encoding the polypeptide of SEQ ID NO: 24; or

(e) a polynucleotide encoding a fragment of at least 10 amino acids of the polypeptide of SEQ ID NO: 24.

46. A method according to claim 42 wherein said polynucleotide is provided in a vector, operably linked to a control sequence which is capable of providing for the expression of said polypeptide from said vector.

47. A method according to claim 43 wherein said vector is a plasmid, virus or phage vector.

48. A method of enhancing the response of an animal or human infected with a mycobacterium to treatment with an antimycobacterial drug, which comprises raising an immune response in said animal or human according to claim 42.