WO1998043479A1 - 76 kDa, 32 kDa, AND 50 kDa HELICOBACTER POLYPEPTIDES AND CORRESPONDING POLYNUCLEOTIDE MOLECULES - Google Patents

76 kDa, 32 kDa, AND 50 kDa HELICOBACTER POLYPEPTIDES AND CORRESPONDING POLYNUCLEOTIDE MOLECULES Download PDF

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WO1998043479A1
WO1998043479A1 PCT/US1998/006421 US9806421W WO9843479A1 WO 1998043479 A1 WO1998043479 A1 WO 1998043479A1 US 9806421 W US9806421 W US 9806421W WO 9843479 A1 WO9843479 A1 WO 9843479A1
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ala
leu
ser
gly
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PCT/US1998/006421
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French (fr)
Inventor
Harold Kleanthous
Ling Lissolo
Jean-François TOMB
Charles Miller
Amal Al-Garawi
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Merieux Oravax
Human Genome Sciences, Inc.
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Priority claimed from US08/834,666 external-priority patent/US20020044949A1/en
Priority claimed from US08/831,310 external-priority patent/US20020026035A1/en
Application filed by Merieux Oravax, Human Genome Sciences, Inc. filed Critical Merieux Oravax
Priority to CA002286893A priority Critical patent/CA2286893A1/en
Priority to AU68757/98A priority patent/AU750792B2/en
Priority to JP54196298A priority patent/JP2001523954A/en
Priority to EP98914395A priority patent/EP0980204A4/en
Publication of WO1998043479A1 publication Critical patent/WO1998043479A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to Helicobacter polypeptides and corresponding polynucleotide molecules that can be used in methods to prevent or treat Helicobacter infection in mammals, such as humans.
  • Helicobacter is a genus of spiral, gram-negative bacteria that colonize the gastrointestinal tracts of mammals. Several species colonize the stomach, most notably H. pylori, H. heilmanii, H. felis, and H. mustelae. Although H. pylori is the species most commonly associated with human infection, H. heilmanii and H. felis have also been isolated from humans, but at lower frequencies than H. pylori. Helicobacter infects over 50% of adult populations in developed countries and nearly 100% in developing countries and some Pacific rim countries, making it one of the most prevalent infections worldwide.
  • H. pylori is now recognized as an important pathogen of humans, in that the chronic gastritis it causes is a risk factor for the development of peptic ulcer diseases and gastric carcinoma. It is thus highly desirable to develop safe and effective vaccines for preventing and treating Helicobacter infection.
  • Helicobacter antigens have been characterized or isolated. These include urease, which is composed of two structural subunits of approximately 30 and 67 kDa ( ⁇ u et al, Infect. Immun. 58:992, 1990; Dunn et al, J. Biol. Chem. 265:9464, 1990; Evans et al, Microbial Pathogenesis 10: 15, 1991 ; Labigne et al, J. Bact., 173: 1920, 1991); the 87 kDa vacuolar cytotoxin (VacA) (Cover et al, J. Biol. Chem. 267: 10570, 1992; Phadnis et al, Infect. Immun.
  • urease which is composed of two structural subunits of approximately 30 and 67 kDa ( ⁇ u et al, Infect. Immun. 58:992, 1990; Dunn et al, J. Biol. Chem. 265:9464, 1990; Evans et al, Microbial Pathogenesis 10
  • urease is believed to be a vaccine candidate (WO 94/9823; WO 95/22987; WO 95/3824; Michetti et al, Gastroenterology 107: 1002, 1994). Nevertheless, it is thought that several antigens may ultimately be necessary in a vaccine.
  • the invention provides polynucleotide molecules that encode a family of 76 kDa Helicobacter polypeptides, designated GHPO 386, GHPO 789, GHPO 1516, GHPO 1197, GHPO 1180, GHPO 896, GHPO 711, GHPO 190, GHPO 185, GHPO 1417, and GHPO 1414, a 32 kDa polypeptide, designated GHPO 1360, and a 50 kDa polypeptide, designated GHPO 750, which can be used, e.g., in methods to prevent, treat, or diagnose Helicobacter infection.
  • polypeptides include those having the amino acid sequences shown in SEQ ID NOs:2-22 (even numbers), 66, and 68.
  • polypeptides include those having the amino acid sequences shown in SEQ ID NOs:2-22 (even numbers), 66, and 68.
  • the invention also includes polynucleotide molecules that encode mutants and derivatives of these polypeptides, which can result from the addition, deletion, or substitution of non-essential amino acids, as is described further below.
  • the invention includes the corresponding polypeptides (i.e., polypeptides encoded by the polynucleotide molecules of the invention, or fragments thereof), and monospecific antibodies that specifically bind to these polypeptides.
  • the present invention has many applications and includes expression cassettes, vectors, and cells transformed or transfected with the polynucleotides of the invention.
  • the present invention provides (i) methods for producing polypeptides of the invention in recombinant host systems and related expression cassettes, vectors, and transformed or transfected cells; (ii) live vaccine vectors, such as pox virus, Salmonella typhimurium, and Vibrio cholerae vectors, that contain polynucleotides of the invention (such vaccine vectors being useful in, e.g., methods for preventing or treating Helicobacter infection) in combination with a diluent or carrier, and related pharmaceutical compositions and associated therapeutic and/or prophylactic methods; (iii) therapeutic and/or prophylactic methods involving administration of polynucleotide molecules, either in a naked form or formulated with a delivery vehicle, polypeptides or mixtures of polypeptides, or monospecific antibodies of the invention, and related pharmaceutical compositions; (iv) methods for detecting the presence of Helicobacter in biological samples, which can involve the use of polynucleotide molecules, monospecific antibodies, or polypeptide
  • Figure 1 is an alignment of the predicted amino acid sequences of GHPO 386 (SEQ ID NO:2), GHPO 789 (SEQ ID NO:4), and GHPO 1516 (SEQ ID NO:6), as well as a consensus sequence for the 76 kDa protein family.
  • Figure 2 is an alignment of the predicted amino acid sequences of
  • GHPO 1197 (SEQ ID NO: 8), GHPO 1180 (SEQ ID NO: 10), GHPO 896 (SEQ ID NO: 12), GHPO 711 (SEQ ID NO: 14), GHPO 190 (SEQ ID NO: 16), GHPO 185 (SEQ ID NO: 18), GHPO 1417 (SEQ ID NO:20), and GHPO 1414 (SEQ ID NO:22), as well as a consensus sequence for the 76 kDa protein family.
  • Open reading frames encoding a family of new, full length, membrane-associated 76 kDa polypeptides, designated GHPO 386, GHPO 789, GHPO 1516, GHPO 1197, GHPO 1180, GHPO 896, GHPO 711, GHPO 190, GHPO 185, GHPO 1417, and GHPO 1414, a 32 kDa polypeptide, designated GHPO 1360, and a 50 kDa polypeptide, designated GHPO 750, have been identified in the H. pylori genome.
  • the amino acid sequences of the 76 kDa polypeptides are aligned in Figures 1 and 2.
  • the 76 kDa, 32 kDa, and 50 kDa polypeptides can be used, for example, in vaccination methods for preventing or treating Helicobacter infection.
  • GHPO 750, GHPO 1360, GHPO 190, and GHPO 1516 have been shown to be protective antigens.
  • protective antigen is meant an antigen that is capable of reducing the infection level after challenge, relative to a positive control. Absolute protection from infection, although included in the invention, is not required.
  • polypeptides of the invention are secreted polypeptides that can be produced in their mature forms (i.e., as polypeptides that have been exported through class II or class III secretion pathways) or as precursors that include a signal peptide, which can be removed in the course of excretion/secretion by cleavage at the N-terminal end of the mature form.
  • the cleavage site is located at the C-terminal end of the signal peptide, adjacent to the mature form.
  • isolated polynucleotides that encode the precursor and mature forms of Helicobacter GHPO 386, GHPO 789, GHPO 1516, GHPO 1 197, GHPO 1180, GHPO 896, GHPO 711, GHPO 190, GHPO 185, GHPO 1417, GHPO 1414, GHPO 1360, and GHPO 750.
  • An isolated polynucleotide of the invention encodes: (i) a polypeptide having an amino acid sequence that is homologous to a Helicobacter amino acid sequence of a polypeptide associated with the Helicobacter membrane, the Helicobacter amino acid sequence being selected from the group consisting of the amino acid sequences shown:
  • -in SEQ ID NO: 22 beginning with an amino acid in any one of positions -18 to 5, preferably in position -18 or position 1 , and ending with an amino acid in position 673 (GHPO 1414); -in SEQ ID NO:66, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 279 (GHPO 1360); and -in SEQ ID NO:68, beginning with an amino acid in position 1 and ending with an amino acid in position 399 (GHPO 750); or (ii) a derivative of the polypeptide.
  • isolated polynucleotide is defined as a polynucleotide that is removed from the environment in which it naturally occurs.
  • a naturally- occurring DNA molecule present in the genome of a living bacteria or as part of a gene bank is not isolated, but the same molecule, separated from the remaining part of the bacterial genome, as a result of, e.g., a cloning event (amplification), is “isolated.”
  • an isolated DNA molecule is free from DNA regions (e.g., coding regions) with which it is immediately contiguous, at the 5' or 3' ends, in the naturally occurring genome.
  • Such isolated polynucleotides can be part of a vector or a composition and still be isolated, as such a vector or composition is not part of its natural environment.
  • a polynucleotide of the invention can consist of RNA or DNA (e.g., cDNA, genomic DNA, or synthetic DNA), or modifications or combinations of RNA or DNA.
  • the polynucleotide can be double-stranded or single-stranded and, if single-stranded, can be the coding (sense) strand or the non-coding (antisense) strand.
  • sequences that encode polypeptides of the invention can be (a) the coding sequence as shown in SEQ ID NOs: 1-21 (odd numbers), 65, and 67; (b) a ribonucleotide sequence derived by transcription of (a); or (c) a different coding sequence that, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptides as the polynucleotide molecules having the sequences illustrated in any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67.
  • polypeptides of the invention can be ones that are naturally secreted or excreted by, e.g., H. felis, H. mustelae, H. heilmanii, or H. pylori.
  • polypeptide or protein is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Both terms are used interchangeably in the present application.
  • homologous amino acid sequence is meant an amino acid sequence that differs from an amino acid sequence shown in any of SEQ ID NOs:2-22 (even numbers), 66, and 68, or an amino acid sequence encoded by the nucleotide sequence of any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67, by one or more non-conservative amino acid substitutions, deletions, or additions located at positions at which they do not destroy the specific antigenicity of the polypeptide.
  • such a sequence is at least 75%, more preferably at least 80%, and most preferably at least 90% identical to an amino acid sequence shown in any of SEQ ID NOs:2-22 (even numbers), 66, and 68.
  • homologous amino acid sequences include sequences that are identical or substantially identical to an amino acid sequence as shown in any of SEQ ID NOs:2-22 (even numbers), 66, and 68.
  • amino acid sequence that is substantially identical is meant a sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, and most preferably at least 99% identical to an amino acid sequence of reference and that differs from the sequence of reference, if at all, by a majority of conservative amino acid substitutions.
  • Conservative amino acid substitutions typically include substitutions among amino acids of the same class. These classes include, for example, amino acids having uncharged polar side chains, such as asparagine, glutamine, serine, threonine, and tyrosine; amino acids having basic side chains, such as lysine, arginine, and histidine; amino acids having acidic side chains, snch as aspartic acid and glutamic acid; and amino acids having nonpolar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine.
  • amino acids having uncharged polar side chains such as asparagine, glutamine, serine, threonine, and tyrosine
  • amino acids having basic side chains such as lysine, arginine, and histidine
  • amino acids having acidic side chains snch as aspartic acid and glutamic acid
  • sequence analysis software e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705. Similar amino acid sequences are aligned to obtain the maximum degree of homology (i.e., identity). To this end, it may be necessary to artificially introduce gaps into the sequence. Once the optimal alignment has been set up, the degree of homology (i.e., identity) is established by recording all of the positions in which the amino acids of both sequences are identical, relative to the total number of positions.
  • homologous polynucleotide sequences are defined in a similar way.
  • a homologous sequence is one that is at least 45%, more preferably at least 60%, and most preferably at least 85% identical to a coding sequence of any of SEQ ID NOs: l-21 (odd numbers), 65, and 67.
  • Polypeptides having a sequence homologous to one of the sequences shown in SEQ ID NOs:2-22 (even numbers), 66, and 68 include naturally- occurring allelic variants, as well as mutants or any other non-naturally occurring variants that are analogous in terms of antigenicity, to a polypeptide having a sequence as shown in SEQ ID NOs:2-22 (even numbers), 66, and 68.
  • an allelic variant is an alternate form of a polypeptide that is characterized as having a substitution, deletion, or addition of one or more amino acids that does not alter the biological function of the polypeptide.
  • biological function is meant a function of the polypeptide in the cells in which it naturally occurs, even if the function is not necessary for the growth or survival of the cells.
  • the biological function of a porin is to allow the entry into cells of compounds present in the extracellular medium.
  • the biological function is distinct from the antigenic function.
  • a polypeptide can have more than one biological function.
  • allelic variants are very common in nature.
  • a bacterial species e.g., H. pylori
  • H. pylori is usually represented by a variety of strains that differ from each other by minor allelic variations.
  • a polypeptide that fulfills the same biological function in different strains can have an amino acid sequence that is not identical in each of the strains.
  • Such an allelic variation can be equally reflected at the polynucleotide level. Support for the use of allelic variants of polypeptide antigens comes from, e.g., studies of the Helicobacter urease antigen.
  • the amino acid sequence of Helicobacter urease varies widely from species to species, yet cross-species protection occurs, indicating that the urease molecule, when used as an immunogen, is highly tolerant of amino acid variations. Even among different strains of the single species H. pylori, there are amino acid sequence variations.
  • UreA + UreB apoenzyme expressed from pORV214 (UreA and UreB sequences differ from H. pylori strain CPM630 by one and two amino acid changes, respectively; Lee et al, supra, 1995); a UreA-glutathione-S-transferase fusion protein (UreA sequence from H. pylori strain ATCC 43504; Thomas et al, Acta Gastro-Enterologica Belgica 56:54, 1993); UreA + UreB holoenzyme purified from H.
  • pylori strain NCTC11637 (Marchetti et al, Science 267: 1655, 1995); a UreA-MBP fusion protein (UreA from H. pylori strain 85P; Ferrero et al, Infection and Immunity 62:4981 , 1994); a UreB-MBP fusion protein (UreB from H. pylori strain 85P; Ferrero et al, supra); a UreA-MBP fusion protein (UreA from H. felis strain ATCC 49179; Ferrero et al, supra); a UreB-MBP fusion protein (UreB from H.
  • Polynucleotides e.g., DNA molecules, encoding allelic variants can easily be obtained by polymerase chain reaction (PCR) amplification of genomic bacterial DNA extracted by conventional methods.
  • PCR polymerase chain reaction
  • Suitable primers can be designed based on the nucleotide sequence information provided in SEQ ID NOs: 1-21 (odd numbers), 65, and 67.
  • a primer consists of 10 to 40, preferably 15 to 25 nucleotides.
  • primers containing C and G nucleotides in proportions sufficient to ensure efficient hybridization, e.g., an amount of C and G nucleotides of at least 40%, preferably 50%, of the total nucleotide amount.
  • an amount of C and G nucleotides of at least 40%, preferably 50%, of the total nucleotide amount can readily design primers that can be used to isolate the polynucleotides of the invention from different Helicobacter strains.
  • primers useful for cloning a polynucleotide molecule encoding a polypeptide having the amino acid sequence of unprocessed GHPO 386 are shown in SEQ ID NO:23 (matching at the 5' end) and in SEQ ID NO:25 (matching at the 3' end).
  • Primers useful for cloning a DNA molecule encoding a polypeptide having the amino acid sequence of mature GHPO 386 are shown in SEQ ID NO:24 (matching at the 5' end) and in SEQ ID NO:25 (matching at the 3' end).
  • Primers useful for cloning a DNA molecule encoding a polypeptide having the amino acid sequence of GHPO 1360 are shown in SEQ ID NO:78 (matching at the 5' end) and in SEQ ID NO: 79 (matching at the 3' end). Use of these primers enables amplification of the entire gene encoding GHPO 1360.
  • the first aspect of the invention includes: (i) isolated polynucleotide molecules (e.g., DNA molecules) that can be amplified and/or cloned using the polymerase chain reaction from a Helicobacter, e.g., H. pylori, genome using either:
  • isolated polynucleotide molecules e.g., DNA molecules
  • Helicobacter e.g., H. pylori
  • the 5' ends of the primers described above can advantageously include a restriction endonuclease recognition site that contains, typically, 4 to 6 nucleotides.
  • a restriction endonuclease recognition site that contains, typically, 4 to 6 nucleotides.
  • the sequences 5'-GGATCC-3' (BamHT) or 5'- CTCGAG-3' (Xhol) can be used. Restriction sites can be selected by those skilled in the art so that the amplified DNA, when digested, if necessary, can be conveniently cloned into an appropriately digested vector, such as a plasmid vector.
  • a 5' clamp e.g., GCC
  • GCC restriction endonuclease recognition site
  • Useful homologs that do not occur naturally can be designed using known methods for identifying regions of an antigen that are likely to be tolerant of amino acid sequence changes and/or deletions. For example, sequences of the antigen from different species can be compared to identify conserved sequences.
  • Polypeptide derivatives that are encoded by polynucleotides of the invention include, e.g., fragments, polypeptides having large internal deletions derived from full-length polypeptides, and fusion proteins.
  • Polypeptide fragments of the invention can be derived from a polypeptide having a sequence homologous to the sequences of any of SEQ ID NOs:2-22 (even numbers), 66, and 68, to the extent that the fragments retain the substantial antigenicity of the parent polypeptide (specific antigenicity).
  • Polypeptide derivatives can also be constructed by large internal deletions that remove a substantial part of the parent polypeptide, while retaining specific antigenicity. Generally, polypeptide derivatives should be about at least 12 amino acids in length to maintain antigenicity.
  • polypeptides can be at least 20 amino acids, preferably at least 50 amino acids, more preferably at least 75 amino acids, and most preferably at least 100 amino acids in length.
  • Useflil polypeptide derivatives e.g., polypeptide fragments
  • the Laser Gene Program from DNA Star can be used to obtain hydrophilicity, antigenic index, and intensity index plots for the polypeptides of the invention. This program can also be used to obtain information about homologies of the polypeptides with known protein motifs.
  • fragments spanning regions of the plots in which the antigenic index is relatively high can be selected.
  • fragments spanning regions in which both the antigenic index and the intensity plots are relatively high can also be selected.
  • Fragments containing conserved sequences, particularly hydrophilic conserved sequences can also be selected.
  • Polypeptide fragments and polypeptides having large internal deletions can be used for revealing epitopes that are otherwise masked in the parent polypeptide and that may be of importance for inducing a protective T cell-dependent immune response. Deletions can also remove immunodominant regions of high variability among strains.
  • polypeptide fragments and polypeptides having large internal deletions can be constructed using standard methods (see, e.g., Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994), for example, by PCR, including inverse PCR, by restriction enzyme treatment of the cloned DNA molecules, or by the method of Kunkel et al (Proc. Natl. Acad. Sci. USA 82:448, 1985; biological material available at Stratagene).
  • a polypeptide derivative can also be produced as a fusion polypeptide that contains a polypeptide or a polypeptide derivative of the invention fused, e.g., at the - or C-terminal end, to any other polypeptide (hereinafter referred to as a peptide tail).
  • a product can be easily obtained by translation of a genetic fusion, i.e., a hybrid gene.
  • Vectors for expressing fusion polypeptides are commercially available, and include the pMal-c2 or pMal-p2 systems of New England Biolabs, in which the peptide tail is a maltose binding protein, the glutathione-S-transferase system of Pharmacia, or the His-Tag system available from Novagen. These and other expression systems provide convenient means for further purification of polypeptides and derivatives of the invention.
  • fusion polypeptides included in invention includes a polypeptide or polypeptide derivative of the invention fused to a polypeptide having adjuvant activity, such as, e.g., subunit B of either cholera toxin or E. coli heat-labile toxin.
  • a polypeptide having adjuvant activity such as, e.g., subunit B of either cholera toxin or E. coli heat-labile toxin.
  • a polypeptide fragment of the invention can be fused within the amino acid sequence of the polypeptide having adjuvant activity. Spacer sequences can also be included, if desired.
  • the polynucleotides of the invention encode Helicobacter polypeptides in precursor or mature form. They can also encode hybrid precursors containing heterologous signal peptides, which can mature into polypeptides of the invention.
  • heterologous signal peptide is meant a signal peptide that is not found in the naturally-occurring precursor of a polypeptide of the invention.
  • a polynucleotide of the invention hybridizes, preferably under stringent conditions, to a polynucleotide having a sequence as shown in any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67.
  • Hybridization procedures are, e.g., described by Ausubel et al. (supra); Silhavy et al. (Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1984); and Davis et al. (A Manual for Genetic Engineering: Advanced Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1980).
  • Tm melting temperature
  • Thi 81.5 + 0.5 -x (% G+C) + 1.6 log (positive ion concentration) - 0.6 x (% formamide).
  • hybridization temperature (Th) is approximately 20 to 40°C, 20 to 25 °C, or, preferably, 30 to 40°C below the calculated Tm.
  • stringent conditions can be achieved, both for pre-hybridizing and hybridizing incubations, (i) within 4-16 hours at 42 °C, in 6 x SSC containing
  • Tm 4 x (G+C) + 2 (A+T).
  • Tm 4 x (G+C) + 2 (A+T).
  • a polynucleotide molecule of the invention can have various applications.
  • a polynucleotide molecule can be used (i) in a process for producing the encoded polypeptide in a recombinant host system, (ii) in the construction of vaccine vectors such as poxviruses, which are further used in methods and compositions for preventing and/or treating Helicobacter infection, (iii) as a vaccine agent, in a naked form or formulated with a delivery vehicle, and (iv) in the construction of attenuated Helicobacter strains that can over-express a polynucleotide of the invention or express it in a non-toxic, mutated form.
  • vaccine vectors such as poxviruses
  • elements e.g., a promoter
  • a recombinant expression system can be selected from procaryotic and eucaryotic hosts.
  • Eucaryotic hosts include, for example, yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris), mammalian cells (e.g., COS1, NIH3T3, or JEG3 cells), arthropods cells (e.g., Spodoptera frugiperda (SF9) cells), and plant cells.
  • yeast cells e.g., Saccharomyces cerevisiae or Pichia pastoris
  • mammalian cells e.g., COS1, NIH3T3, or JEG3 cells
  • arthropods cells e.g., Spodoptera frugiperda (SF9) cells
  • plant cells e.g., a procaryotic host such as E. coli is used.
  • Bacterial and eucaryotic cells are available from a number of different sources that are known to those skilled in the art,
  • an expression cassette includes a constitutive or inducible promoter that is functional in the selected host system; a ribosome binding site; a start codon (ATG); if necessary, a region encoding a signal peptide, e.g., a lipidation signal peptide; a polynucleotide molecule of the invention; a stop codon; and, optionally, a 3' terminal region (translation and/or transcription terminator).
  • the signal pepti de-encoding region is adjacent to the polynucleotide of the invention and is placed in the proper reading frame.
  • the signal pepti de-encoding region can be homologous or heterologous to the polynucleotide molecule encoding the mature polypeptide and it can be specific to the secretion apparatus of the host used for expression.
  • the open reading frame constituted by the polynucleotide molecule of the invention, alone or together with the signal peptide, is placed under the control of the promoter so that transcription and translation occur in the host system.
  • Promoters and signal peptide-encoding regions are widely known and available to those skilled in the art and include, for example, the promoter of Salmonella typhimurium (and derivatives) that is inducible by arabinose (promoter araB) and is functional in Gram-negative bacteria such as E. coli (U.S. Patent No. 5,028,530; Cagnon et al, Protein Engineering 4(7): 843, 1991); the promoter of the bacteriophage T7 RNA polymerase gene, which is functional in a number of E. coli strains expressing T7 polymerase (U.S. Patent No. 4,952,496); the OspA lipidation signal peptide; and RlpB lipidation signal peptide (Takase et al, J. Bact. 169:5692, 1987).
  • the expression cassette is typically part of an expression vector, which is selected for its ability to replicate in the chosen expression system.
  • Expression vectors e.g., plasmids or viral vectors
  • plasmids or viral vectors can be chosen from, for example, those described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985, Supp. 1987) and can purchased from various commercial sources. Methods for transforming or transfecting host cells with expression vectors are well known in the art and will depend on the host system selected, as described in Ausubel et al (supra).
  • a recombinant polypeptide of the invention (or a polypeptide derivative) is produced and remains in the intracellular compartment, is secreted/excreted in the extracellular medium or in the periplasmic space, or is embedded in the cellular membrane.
  • the polypeptide can then be recovered in a substantially purified form from the cell extract or from the supernatant after centrifugation of the cell culture.
  • the recombinant polypeptide can be purified by antibody-based affinity purification or by any other method known to a person skilled in the art, such as by genetic fusion to a small affinity-binding domain.
  • Antibody-based affinity purification methods are also available for purifying a polypeptide of the invention extracted from a Helicobacter strain.
  • Antibodies useful for immunoaffinity purification of the polypeptides of the invention can be obtained using methods described below.
  • Polynucleotides of the invention can also be used in DNA vaccination methods, using either a viral or bacterial host as gene delivery vehicle (live vaccine vector) or administering the gene in a free form, e.g., inserted into a plasmid.
  • Therapeutic or prophylactic efficacy of a polynucleotide of the invention can be evaluated as is described below.
  • a vaccine vector such as a poxvirus, containing a polynucleotide molecule of the invention placed under the control of elements required for expression; (ii) a composition of matter containing a vaccine vector of the invention, together with a diluent or carrier; (iii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a vaccine vector of the invention; (iv) a method for inducing an immune response against Helicobacter in a mammal (e.g., a human; alternatively, the method can be used in veterinary applications for treating or preventing Helicobacter infection of animals, e.g., cats or birds), which involves administering to the mammal an immunogenically effective amount of a vaccine vector of the invention to elicit an immune response, e.g., a protective or therapeutic immune response to Helicobacter; and (v) a method for preventing and/or treating a Helicobacter (
  • the third aspect of the invention encompasses the use of a vaccine vector of the invention in the preparation of a medicament for preventing and/or treating Helicobacter infection.
  • a vaccine vector of the invention can express one or several polypeptides or derivatives of the invention, as well as at least one additional Helicobacter antigen such as a urease apoenzyme or a subunit, fragment, homolog, mutant, or derivative thereof.
  • it can express a cytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12), that enhances the immune response.
  • a vaccine vector can include an additional polynucleotide molecules encoding, e.g., urease subunit A, B, or both, or a cytokine, placed under the control of elements required for expression in a mammalian cell.
  • composition of the invention can include several vaccine vectors, each of which being capable of expressing a polypeptide or derivative of the invention.
  • a composition can also contain a vaccine vector capable of expressing an additional Helicobacter antigen such as urease apoenzyme, a subunit, fragment, homolog, mutant, or derivative thereof, or a cytokine such as IL-2 or IL-12.
  • a vaccine vector of the invention can be administered by any conventional route in use in the vaccine field, for example, to a mucosal (e.g., ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract) surface or via a parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route.
  • a mucosal e.g., ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract
  • parenteral e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal
  • Prefened routes depend upon the choice of the vaccine vector.
  • the administration can be achieved in a single dose or repeated at intervals.
  • the appropriate dosage depends on various parameters that are understood by those skilled in the art, such as the nature of the vaccine vector itself, the route of administration, and the condition of the mammal to be vaccinated (e.g., the weight, age, and general health of the mammal).
  • Live vaccine vectors that can be used in the invention include viral vectors, such as adenoviruses and poxviruses, as well as bacterial vectors, e.g., Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bilie de Calmette- Guerin (BCG), and Streptococcus.
  • viral vectors such as adenoviruses and poxviruses
  • bacterial vectors e.g., Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bilie de Calmette- Guerin (BCG), and Streptococcus.
  • An example of an adenovirus vector, as well as a method for constructing an adenovirus vector capable of expressing a polynucleotide molecule of the invention, is described in U.S. Patent No. 4,920,209.
  • Poxvirus vectors that can be used in the invention include, e.g.,
  • Patent No. 4,722,848 and U.S. Patent No. 5,364,773, respectively also see, e.g., Tartaglia et al, Virology 188:217, 1992, for a description of a vaccinia virus vector, and Taylor et al, Vaccine 13:539, 1995, for a description of a canary poxvirus vector.
  • Poxvirus vectors capable of expressing a polynucleotide of the invention can be obtained by homologous recombination, as described in Kieny et al (Nature 312: 163, 1984) so that the polynucleotide of the invention is inserted in the viral genome under appropriate conditions for expression in mammalian cells.
  • the dose of viral vector vaccine for therapeutic or prophylactic use, can be from about lxl 0 4 to about 1x10", advantageously from about lxlO 7 to about lxlO 10 , or, preferably, from about lxlO 7 to about lxl 0 9 plaque-forming units per kilogram.
  • viral vectors are administered parenterally, for example, in 3 doses that are 4 weeks apart.
  • Non-toxicogenic Vibrio cholerae mutant strains that can be used in live oral vaccines are described by Mekalanos et al. (Nature 306:551, 1983) and in U.S. Patent No. 4,882,278 (strain in which a substantial amount of the coding sequence of each of the two ctxA alleles has been deleted so that no functional cholerae toxin is produced); WO 92/11354 (strain in which the z ' rgA locus is inactivated by mutation; this mutation can be combined in a single strain with ctxA mutations); and WO 94/1533 (deletion mutant lacking functional ctxA and attRSl DNA sequences).
  • An effective vaccine dose of a V. cholerae strain capable of expressing a polypeptide or polypeptide derivative encoded by a polynucleotide molecule of the invention can contain, e.g., about lxlO 5 to about lxlO 9 , preferably about lxlO 6 to about lxl 0 8 viable bacteria in an appropriate volume for the selected route of administration.
  • Preferred routes of administration include all mucosal routes, but, most preferably, these vectors are administered intranasally or orally.
  • Attenuated Salmonella typhimurium strains genetically engineered for recombinant expression of heterologous antigens, and their use as oral vaccines, are described by Nakayama et al. (Bio/Technology 6:693, 1988) and in WO 92/11361.
  • Preferred routes of administration for these vectors include all mucosal routes. Most preferably, the vectors are administered intranasally or orally. Others bacterial strains useful as vaccine vectors are described by
  • a polynucleotide of the invention can be inserted into the bacterial genome or it can remain in a free state, for example, carried on a plasmid.
  • An adjuvant can also be added to a composition containing a bacterial vector vaccine.
  • a number of adjuvants that can be used are known to those skilled in the art.
  • preferred adjuvants can be selected from the list provided below.
  • a composition of matter containing a polynucleotide of the invention, together with a diluent or carrier e.g., a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a polynucleotide of the invention
  • a method for inducing an immune response against Helicobacter, in a mammal by administering to the mammal an immunogenically effective amount of a polynucleotide of the invention to elicit an immune response, e.g., a protective immune response to Helicobacter
  • a method for preventing and or treating a Helicobacter e.g., H. pylori, H. felis, H.
  • the fourth aspect of the invention encompasses the use of a polynucleotide of the invention in the preparation of a medicament for preventing and/or treating Helicobacter infection.
  • the fourth aspect of the invention preferably includes the use of a polynucleotide molecule placed under conditions for expression in a mammalian cell, e.g., in a plasmid that is unable to replicate in mammalian cells and to substantially integrate into a mammalian genome.
  • Polynucleotides (for example, DNA or RNA molecules) of the invention can also be administered as such to a mammal as a vaccine.
  • a DNA molecule of the invention can be in the form of a plasmid that is unable to replicate in a mammalian cell and unable to integrate into the mammalian genome.
  • a DNA molecule is placed under the control of a promoter suitable for expression in a mammalian cell.
  • the promoter can function ubiquitously or tissue-specifically. Examples of non-tissue specific promoters include the early Cytomegalovirus (CMV) promoter (U.S. Patent No.
  • the desmin promoter (Li et al, Gene 78:243, 1989; Li et al, J. Biol. Chem. 266:6562, 1991 ; Li et al, J. Biol. Chem. 268: 10403, 1993) is tissue-specific and drives expression in muscle cells. More generally, useful promoters and vectors are described, e.g., in WO 94/21797 and by Hartikka et al. (Human Gene Therapy 7: 1205, 1996).
  • the polynucleotide of the invention can encode a precursor or a mature form of a polypeptide of the invention.
  • the precursor sequence can be homologous or heterologous.
  • a eucaryotic leader sequence can be used, such as the leader sequence of the tissue-type plasminogen factor (tPA).
  • a composition of the invention can contain one or several polynucleotides of the invention. It can also contain at least one additional polynucleotide encoding another Helicobacter antigen, such as urease subunit A, B, or both, or a fragment, derivative, mutant, or analog thereof.
  • a polynucleotide encoding a cytokine such as interleukin-2 (IL-2) or interleukin- 12 (IL-12), can also be added to the composition so that the immune response is enhanced.
  • additional polynucleotides are placed under appropriate control for expression.
  • DNA molecules of the invention and/or additional DNA molecules to be included in the same composition are earned in the same plasmid.
  • Standard methods can be used in the preparation of therapeutic polynucleotides of the invention.
  • a polynucleotide can be used in a naked form, free of any delivery vehicles, such as anionic liposomes, cationic lipids, microparticles, e.g., gold microparticles, precipitating agents, e.g., calcium phosphate, or any other transfection-facilitating agent.
  • the polynucleotide can be simply diluted in a physiologically acceptable solution, such as sterile saline or sterile buffered saline, with or without a carrier.
  • the ca ⁇ ier preferably is isotonic, hypotonic, or weakly hypertonic, and has a relatively low ionic strength, such as provided by a sucrose solution, e.g., a solution containing 20% sucrose.
  • a polynucleotide can be associated with agents that assist in cellular uptake. It can be, e.g., (i) complemented with a chemical agent that modifies cellular permeability, such as bupivacaine (see, e.g.,
  • Anionic and neutral liposomes are well-known in the art (see, e.g.,
  • Liposomes A Practical Approach, RPC New Ed, IRL Press, 1990, for a detailed description of methods for making liposomes) and are useful for delivering a large range of products, including polynucleotides.
  • Cationic lipids can also be used for gene delivery.
  • Such lipids include, for example, LipofectinTM, which is also known as DOTMA (N-[l-DOTMA) (N-[l-DOTMA) (N-[l-DOTMA) (N-[l-DOTMA)
  • Cationic lipids for gene delivery are preferably used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine; WO 90/11092).
  • DOPE dioleyl phosphatidylethanolamine
  • Other transfection- facilitating compounds can be added to a formulation containing cationic liposomes.
  • WO 93/18759 WO 93/19768
  • WO 94/25608 WO 95/2397
  • They include, e.g., spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane (see, for example, WO 93/18759) and membrane-permeabilizing compounds such as GALA, Gramicidine S, and cationic bile salts (see, for example, WO 93/19768).
  • Gold or tungsten microparticles can also be used for gene delivery, as described in WO 91/359, WO 93/17706, and by Tang et al. (Nature 356: 152, 1992).
  • the microparticle-coated polynucleotides can be injected via intradermal or intraepidermal routes using a needleless injection device
  • the amount of DNA to be used in a vaccine recipient depends, e.g., on the strength of the promoter used in the DNA construct, the immunogenicity of the expressed gene product, the condition of the mammal intended for administration (e.g., the weight, age, and general health of the mammal), the mode of administration, and the type of formulation.
  • a therapeutically or prophylactically effective dose from about 1 ⁇ g to about 1 mg, preferably, from about 10 ⁇ g to about 800 ⁇ g, and, more preferably, from about 25 ⁇ g to about 250 ⁇ g, can be administered to human adults.
  • the administration can be achieved in a single dose or repeated at intervals.
  • the route of administration can be any conventional route used in the vaccine field.
  • a polynucleotide of the invention can be administered via a mucosal surface, e.g., an ocular, intranasal, pulmonary, oral, intestinal, rectal, vaginal, or urinary tract surface, or via a parenteral route, e.g., by an intravenous, subcutaneous, intraperitoneal, intradermal, intraepidermal, or intramuscular route.
  • a parenteral route e.g., by an intravenous, subcutaneous, intraperitoneal, intradermal, intraepidermal, or intramuscular route.
  • the choice of administration route will depend on, e.g., the formulation that is selected.
  • a polynucleotide formulated in association with bupivacaine is advantageously administered into muscle.
  • the formulation can be advantageously injected via intravenous, intranasal (for example, by aerosolization), intramuscular, intradermal, and subcutaneous routes.
  • a polynucleotide in a naked form can advantageously be administered via the intramuscular, intradermal, or subcutaneous routes.
  • such a composition can also contain an adjuvant.
  • a systemic adjuvant that does not require concomitant administration in order to exhibit an adjuvant effect is preferable.
  • nucleotide probe or primer having a sequence found in, or derived by degeneracy of the genetic code from, a sequence shown in any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67, or a complementary sequence thereof.
  • probe refers to DNA (preferably single stranded) or RNA molecules (or modifications or combinations thereof) that hybridize under the stringent conditions, as defined above, to polynucleotide molecules having sequences homologous to those shown in any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67, or to a complementary or anti-sense sequence of any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67.
  • probes are significantly shorter than the full- length sequences shown in any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67.
  • they can contain from about 5 to about 100, preferably from about 10 to about 80 nucleotides.
  • probes have sequences that are at least 75%, preferably at least 85%, more preferably 95% homologous to a portion of a sequence as shown in any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67, or a sequence complementary to such sequences.
  • Probes can contain modified bases, such as inosine, methyl-5- deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, or diamino-2, 6- purine.
  • Sugar or phosphate residues can also be modified or substituted.
  • a deoxyribose residue can be replaced by a poly amide (Nielsen et al, Science 254: 1497, 1991) and phosphate residues can be replaced by ester groups such as diphosphate, alkyl, arylphosphonate, and phosphorothioate esters.
  • the 2'-hydroxyl group on ribonucleotides can be modified by addition of, e.g., alkyl groups.
  • Probes of the invention can be used in diagnostic tests, or as capture or detection probes. Such capture probes can be immobilized on solid supports, directly or indirectly, by covalent means or by passive adso ⁇ tion.
  • a detection probe can be labeled by a detectable label, for example a label selected from radioactive isotopes; enzymes, such as peroxidase and alkaline phosphatase; enzymes that are able to hydrolyze a chromogenic, fluorogenic, or luminescent substrate; compounds that are chromogenic, fluorogenic, or luminescent; nucleotide base analogs; and biotin.
  • Probes of the invention can be used in any conventional hybridization method, such as in dot blot methods (Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1982), Southern blot methods (Southern, J. Mol. Biol. 98:503, 1975), northern blot methods (identical to Southern blot to the exception that RNA is used as a target), or a sandwich method (Dunn et al, Cell 12:23, 1977). As is known in the art, the latter technique involves the use of a specific capture probe and a specific detection probe that have nucleotide sequences that are at least partially different from each other.
  • Primers used in the invention usually contain about 10 to 40 nucleotides and are used to initiate enzymatic polymerization of DNA in an amplification process (e.g., PCR), an elongation process, or a reverse transcription method. In a diagnostic method involving PCR, the primers can be labeled.
  • the invention also encompasses (i) a reagent containing a probe of the invention for detecting and/or identifying the presence of Helicobacter in a biological material; (ii) a method for detecting and/or identifying the presence of Helicobacter in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA or RNA is extracted from the material and denatured, and (c) the sample is exposed to a probe of the invention, for example, a capture probe, a detection probe, or both, under stringent hybridization conditions, so that hybridization is detected; and (iii) a method for detecting and/or identifying the presence of Helicobacter in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA is extracted therefrom, (c) the extracted DNA is contacted with at least one, or, preferably two, primers of the invention, and amplified by the polymerase chain reaction, and (d) an amplified DNA
  • polypeptides that can be produced by expression of the polynucleotides of the invention can be used as vaccine antigens.
  • a sixth aspect of the invention features a substantially purified polypeptide or polypeptide derivative having an amino acid sequence encoded by a polynucleotide of the invention.
  • a "substantially purified polypeptide” is defined as a polypeptide that is separated from the environment in which it naturally occurs and/or a polypeptide that is free of most of the other polypeptides that are present in the environment in which it was synthesized.
  • the polypeptides of the invention can be purified from a natural source, such as a Helicobacter strain, or can be produced using recombinant methods.
  • Homologous polypeptides or polypeptide derivatives encoded by polynucleotides of the invention can be screened for specific antigenicity by testing cross-reactivity with an antiserum raised against a polypeptide having an amino acid sequence as shown in any of SEQ ID NOs:2-22 (even numbers), 66, and 68.
  • a monospecific hyperimmune antiserum can be raised against a purified reference polypeptide as such or as a fusion polypeptide, for example, an expression product of MBP, GST, or His-tag systems, or a synthetic peptide predicted to be antigenic.
  • the homologous polypeptide or derivative that is screened for specific antigenicity can be produced as such or as a fusion polypeptide. In the latter case, and if the antiserum is also raised against a fusion polypeptide, two different fusion systems are employed.
  • Specific antigenicity can be determined using a number of methods, including Western blot (Towbin et al, Proc. Natl. Acad. Sci. USA 76:4350, 1979), dot blot, and ELISA methods, as described below.
  • the product to be screened is fractionated by SDS-PAGE, as described, for example, by Laemmli (Nature 227:680, 1970).
  • a filter such as a nitrocellulose membrane
  • the material is incubated with the monospecific hyperimmune antiserum, which is diluted in a range of dilutions from about 1 :50 to about 1 :5000, preferably from about 1 : 100 to about 1 :500.
  • Specific antigenicity is shown once a band co ⁇ esponding to the product exhibits reactivity at any of the dilutions in the range.
  • the product to be screened can be used as the coating antigen.
  • a purified preparation is preferred, but a whole cell extract can also be used. Briefly, about 100 ⁇ l of a preparation of about 10 ⁇ g protein/ml is distributed into wells of a 96-well ELISA plate. The plate is incubated for about 2 hours at 37°C, then overnight at 4°C. The plate is washed with phosphate buffered saline (PBS) containing 0.05% Tween 20 (PBS/Tween buffer) and the wells are saturated with 250 ⁇ l PBS containing 1% bovine serum albumin (BSA), to prevent non-specific antibody binding.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • the plate After 1 hour of incubation at 37 °C, the plate is washed with PBS/Tween buffer. The antiserum is serially diluted in PBS/Tween buffer containing 0.5% BSA, and 100 ⁇ l dilutions are added to each well. The plate is incubated for 90 minutes at 37 °C, washed, and evaluated using standard methods. For example, a goat anti-rabbit peroxidase conjugate can be added to the wells when the specific antibodies used were raised in rabbits. Incubation is carried out for about 90 minutes at 37° C and the plate is washed. The reaction is developed with the appropriate substrate and the reaction is measured by colorimetry (absorbance measured spectrophotometrically). Under these experimental conditions, a positive reaction is shown once an O.D. value of 1.0 is detected with a dilution of at least about 1 :50, preferably of at least about 1 :500.
  • a purified product is prefened, although a whole cell extract can be used.
  • a solution of the product at a concentration of about 100 ⁇ g/ml is serially diluted two-fold with 50 mM Tris-HCl (pH 7.5).
  • a filter such as a 0.45 ⁇ m nitrocellulose membrane, set in a 96- well dot blot apparatus (Biorad).
  • the buffer is removed by applying vacuum to the system.
  • Wells are washed by addition of 50 mM Tris-HCl (pH 7.5) and the membrane is air-dried.
  • the membrane is saturated in blocking buffer (50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 10 g/L skim milk) and incubated with an antiserum diluted from about 1 : 50 to about 1 : 5000, preferably about 1 :500.
  • the reaction is detected using standard methods. For example, a goat anti -rabbit peroxidase conjugate can be added to the wells when rabbit antibodies are used. Incubation is carried out for about 90 minutes at 37 °C and the blot is washed. The reaction is developed with the appropriate substrate and stopped. The reaction is then measured visually by the appearance of a colored spot, e.g., by colorimetry.
  • a positive reaction is associated with detection of a colored spot for reactions carried out with a dilution of at least about 1 :50, preferably, of at least about 1 :500.
  • Therapeutic or prophylactic efficacy of a polypeptide or polypeptide derivative of the invention can be evaluated as is described below.
  • a composition of matter containing a polypeptide of the invention together with a diluent or carrier containing a therapeutically or prophylactically effective amount of a polypeptide of the invention
  • a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a polypeptide of the invention containing a therapeutically or prophylactically effective amount of a polypeptide of the invention
  • a method for inducing an immune response against Helicobacter in a mammal by administering to the mammal an immunogenically effective amount of a polypeptide of the invention to elicit an immune response, e.g., a protective immune response to Helicobacter
  • a method for preventing and/or treating a Helicobacter e.g., H. pylori, H. felis, H.
  • this aspect of the invention includes the use of a polypeptide of the invention in the preparation of a medicament for preventing and/or treating Helicobacter infection.
  • the immunogenic compositions of the invention can be administered by any conventional route in use in the vaccine field, for example, to a mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface or via a parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route.
  • a mucosal e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract
  • a parenteral e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal
  • the choice of the administration route depends upon a number of parameters, such as the adjuvant used. For example, if a mucosal adjuvant is used, the intranasal or oral route will be prefened, and if a lipid formulation or an aluminum compound is
  • the subcutaneous or intramuscular route is most preferred.
  • the choice of administration route can also depend upon the nature of the vaccine agent.
  • a polypeptide of the invention fused to CTB or to LTB will be best administered to a mucosal surface.
  • a composition of the invention can contain one or several polypeptides or derivatives of the invention. It can also contain at least one additional Helicobacter antigen, such as the urease apoenzyme, or a subunit, fragment, homolog, mutant, or derivative thereof.
  • Helicobacter antigen such as the urease apoenzyme, or a subunit, fragment, homolog, mutant, or derivative thereof.
  • a polypeptide or polypeptide derivative can be formulated into or with liposomes, such as neutral or anionic liposomes, microspheres, ISCOMS, or virus-like particles (VLPs), to facilitate delivery and/or enhance the immune response.
  • liposomes such as neutral or anionic liposomes, microspheres, ISCOMS, or virus-like particles (VLPs)
  • VLPs virus-like particles
  • Adjuvants other than liposomes can also be used in the invention and are well known in the art (see, for example, the list provided below).
  • Administration can be achieved in a single dose or repeated as necessary at intervals that can be determined by one skilled in the art. For example, a priming dose can be followed by three booster doses at weekly or monthly intervals.
  • An appropriate dose depends on various parameters, including the nature of the recipient (e.g., whether the recipient is an adult or an infant), the particular vaccine antigen, the route and frequency of administration, the presence/absence or type of adjuvant, and the desired effect (e.g., protection and/or treatment), and can be readily determined by one skilled in the art.
  • a vaccine antigen of the invention can be administered mucosally in an amount ranging from about 10 ⁇ g to about 500 mg, preferably from about 1 mg to about 200 mg.
  • the dose usually should not exceed about 1 mg, and is, preferably, about 100 ⁇ g.
  • the polynucleotides and polypeptides of the invention can be used sequentially as part of a multi-step immunization process.
  • a mammal can be initially primed with a vaccine vector of the invention, such as a pox virus, e.g., via a parenteral route, and then boosted twice with a polypeptide encoded by the vaccine vector, e.g., via the mucosal route.
  • liposomes associated with a polypeptide or polypeptide derivative of the invention can be used for priming, with boosting being earned out mucosally using a soluble polypeptide or polypeptide derivative of the invention, in combination with a mucosal - adjuvant (e.g., LT).
  • a mucosal - adjuvant e.g., LT
  • Polypeptides and polypeptide derivatives of the invention can also be used as diagnostic reagents for detecting the presence of anti-Helicobacter antibodies, e.g., in blood samples.
  • Such polypeptides can be about 5 to about 80, preferably, about 10 to about 50 amino acids in length and can be labeled or unlabeled, depending upon the diagnostic method. Diagnostic methods involving such a reagent are described below.
  • a polypeptide or polypeptide derivative is produced and can be purified using known methods.
  • the polypeptide or polypeptide derivative can be produced as a fusion protein containing a fused tail that facilitates purification.
  • the fusion product can be used to immunize a small mammal, e.g., a mouse or a rabbit, in order to raise monospecific antibodies against the polypeptide or polypeptide derivative.
  • the eighth aspect of the invention thus provides a monospecific antibody that binds to a polypeptide or polypeptide derivative of the invention.
  • monospecific antibody an antibody that is capable of reacting with a unique, naturally-occurring Helicobacter polypeptide.
  • An antibody of the invention can be polyclonal or monoclonal.
  • Monospecific antibodies can be recombinant, e.g., chimeric (e.g., consisting of a variable region of murine origin and a human constant region), humanized (e.g., a human immunoglobulin constant region and a variable region of animal, e.g., murine, origin), and/or single chain.
  • Both polyclonal and monospecific antibodies can also be in the form of immunoglobulin fragments, e.g., F(ab)'2 or Fab fragments.
  • the antibodies of the invention can be of any isotype, e.g., IgG or IgA, and polyclonal antibodies can be of a single isotype or can contain a mixture of isotypes.
  • the antibodies of the invention which can be raised to a polypeptide or polypeptide derivative of the invention, can be produced and identified using standard immunological assays, e.g., Western blot assays, dot blot assays, or ELISA (see, e.g., Coligan et al, Current Protocols in Immunology, John Wiley & Sons, Inc., New York, NY, 1994).
  • the antibodies can be used in diagnostic methods to detect the presence of Helicobacter antigens in a sample, such as a biological sample.
  • the antibodies can also be used in affinity chromatography methods for purifying a polypeptide or polypeptide derivative of the invention. As is discussed further below, the antibodies can also be used in prophylactic and therapeutic passive immunization methods.
  • a ninth aspect of the invention provides (i) a reagent for detecting the presence of Helicobacter in a biological sample that contains an antibody, polypeptide, or polypeptide derivative of the invention; and (ii) a diagnostic method for detecting the presence of Helicobacter in a biological sample, by contacting the biological sample with an antibody, a polypeptide, or a polypeptide derivative of the invention, so that an immune complex is formed, and detecting the complex as an indication of the presence of Helicobacter in the sample or the organism from which the sample was derived.
  • the immune complex is formed between a component of the sample and the antibody, polypeptide, or polypeptide derivative, and that any unbound material can be removed prior to detecting the complex.
  • a polypeptide reagent can be used for detecting the presence of anti-Helicobacter antibodies in a sample, e.g., a blood sample, while an antibody of the invention can be used for screening a sample, such as a gastric extract or biopsy sample, for the presence of Helicobacter polypeptides.
  • the reagent e.g., the antibody, polypeptide, or polypeptide derivative of the invention
  • the reagent can be in a free ⁇ tate or can be immobilized on a solid support, such as, for example, on the interior surface of a tube or on the surface, or within pores, of a bead. Immobilization can be achieved using direct or indirect means. Direct means include passive adso ⁇ tion (i.e., non-covalent binding) or covalent binding between the support and the reagent. By “indirect means” is meant that an anti-reagent compound that interacts with the reagent is first attached to the solid support.
  • an antibody that binds to it can serve as an anti-reagent, provided that it binds to an epitope that is not involved in recognition of antibodies in biological samples.
  • Indirect means can also employ a ligand-receptor system, for example, a molecule, such as a vitamin, can be grafted onto the polypeptide reagent and the corresponding receptor can be immobilized on the solid phase. This concept is illustrated by the well known biotin-streptavidin system.
  • indirect means can be used, e.g., by adding to the reagent a peptide tail, chemically or by genetic engineering, and immobilizing the grafted or fused product by passive adsorption or covalent linkage of the peptide tail.
  • a process for purifying, from a biological sample, a polypeptide or polypeptide derivative of the invention which involves carrying out antibody-based affinity chromatography with the biological sample, wherein the antibody is a monospecific antibody of the invention.
  • the antibody can be polyclonal or monospecific, and preferably is of the IgG type.
  • Purified IgGs can be prepared from an antiserum using standard methods (see, e.g., Coligan et al, supra). Conventional chromatography supports, as well as standard methods for grafting antibodies, are described, for example, by Harlow et al (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988).
  • a biological sample such as an H. pylori extract, preferably in a buffer solution
  • a chromatography material which is, preferably, equilibrated with the buffer used to dilute the biological sample, so that the polypeptide or polypeptide derivative of the invention (i.e., the antigen) is allowed to adsorb onto the material.
  • the chromatography material such as a gel or a resin coupled to an antibody of the invention, can be in batch form or in a column.
  • the unbound components are washed off and the antigen is eluted with an appropriate elution buffer, such as a glycine buffer, a buffer containing a chaotropic agent, e.g., guanidine HC1, or a buffer having high salt concentration (e.g., 3 M MgCl 2 ).
  • an appropriate elution buffer such as a glycine buffer, a buffer containing a chaotropic agent, e.g., guanidine HC1, or a buffer having high salt concentration (e.g., 3 M MgCl 2 ).
  • Eluted fractions are recovered and the presence of the antigen is detected, e.g., by measuring the absorbance at 280 nm.
  • an antibody of the invention can be screened for therapeutic efficacy as follows.
  • a composition of matter containing a monospecific antibody of the invention together with a diluent or carrier;
  • a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a monospecific antibody of the invention and
  • a method for treating or preventing Helicobacter e.g., H. pylori, H. felis, H. mustelae, or H. heilmanii
  • the eleventh aspect of the invention includes the use of a monospecific antibody of the invention in the preparation of a medicament for treating or preventing Helicobacter infection.
  • the monospecific antibody can be polyclonal or monoclonal, and is, preferably, predominantly of the IgA isotype.
  • the antibody is administered to a mucosal surface of a mammal, e.g., the gastric mucosa, e.g., orally or intragastrically, optionally, in the presence of a bicarbonate buffer.
  • systemic administration not requiring a bicarbonate buffer, can be carried out.
  • a monospecific antibody of the invention can be administered as a single active agent or as a mixture with at least one additional monospecific antibody specific for a different Helicobacter polypeptide.
  • the amount of antibody and the particular regimen used can be readily determined by one skilled in the art. For example, daily administration of about 100 to 1,000 mg of antibody over one week, or three doses per day of about 100 to 1,000 mg of antibody over two or three days, can be effective regimens for most p poses.
  • Therapeutic or prophylactic efficacy can be evaluated using standard methods in the art, e.g., by measuring induction of a mucosal immune response or induction of protective and/or therapeutic immunity, using, e.g., the H. felis mouse model and the procedures described by Lee et al. (Eur. J. Gastroenterology & Hepatology 7:303, 1995) or Lee et al. (J. Infect. Dis. 172: 161, 1995).
  • the H. felis strain of the model can be replaced with another Helicobacter strain.
  • the efficacy of polynucleotide molecules and polypeptides from H. pylori is, preferably, evaluated in a mouse model using an H.
  • Protection can be determined by comparing the degree of Helicobacter infection in the gastric tissue assessed by, for example, urease activity, bacterial counts, or gastritis, to that of a control group. Protection is shown when infection is reduced by comparison to the control group. Such an evaluation can be made for polynucleotides, vaccine vectors, polypeptides, and polypeptide derivatives, as well as for antibodies of the invention.
  • an antibody of the invention can be administered to the gastric mucosa of mice previously challenged with an H. pylori strain, as described, e.g., by Lee et al. (supra). Then, after an appropriate period of time, the bacterial load of the mucosa can be estimated by assessing urease activity, as compared to a control. Reduced urease activity indicates that the antibody is therapeutically effective.
  • Adjuvants that can be used in any of the vaccine compositions described above are described as follows.
  • Adjuvants for parenteral administration include, for example, aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate.
  • the antigen can be precipitated with, or adsorbed onto, the aluminum compound using standard methods.
  • Other adjuvants such as RIBI (ImmunoChem, Hamilton, MT), can also be used in parenteral administration.
  • Adjuvants that can be used for mucosal administration include, for example, bacterial toxins, e.g., the cholera toxin (CT), the E.
  • CT cholera toxin
  • coli heat-labile toxin LT
  • Clostridium difficile toxin A the Clostridium difficile toxin A
  • PT pertussis toxin
  • CTB native cholera toxin subunit B
  • Fragments, homologs, derivatives, and fusions to any of these toxins can also be used, provided that they retain adjuvant activity.
  • a mutant having reduced toxicity is used.
  • Suitable mutants are described, e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant).
  • Additional LT mutants that can be used in the methods and compositions of the invention include, e.g., Ser-63-Lys, Ala-69-Gly, Glu-110-Asp, and Glu-112-Asp mutants.
  • Other adjuvants such as the bacterial monophosphoryl lipid A (MPLA) of, e.g., E.
  • MPLA bacterial monophosphoryl lipid A
  • coli Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri
  • saponins and polylactide glycolide (PLGA) microspheres
  • PLGA polylactide glycolide
  • Adjuvants useful for both mucosal and parenteral administrations such as polyphosphazene (WO 95/2415), can also be used.
  • Any pharmaceutical composition of the invention, containing a polynucleotide, polypeptide, polypeptide derivative, or antibody of the invention, can be manufactured using standard methods.
  • a pharmaceutically acceptable diluent or carrier e.g., water or a saline solution, such as PBS, optionally, including a bicarbonate salt, such as sodium bicarbonate, e.g., 0.1 to 0.5 M.
  • Bicarbonate can advantageously be added to compositions intended for oral or intragastric administration.
  • a diluent or ca ⁇ ier can be selected on the basis of the mode and route of administration, and standard pharmaceutical practice.
  • Suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use in pharmaceutical formulations, are described in Remington 's Pharmaceutical Sciences, a standard reference text in this field and in the USP/NF.
  • the invention also includes methods in which gastroduodenal infections, such as Helicobacter infection, are treated by oral administration of a Helicobacter polypeptide of the invention and a mucosal adjuvant, in combination with an antibiotic, an antisecretory agent, a bismuth salt, an antacid, sucralfate, or a combination thereof.
  • antibiotics including, e.g., macrolides, tetracyclines, ⁇ -lactams, aminoglycosides, quinolones, penicillins, and derivatives thereof
  • antibiotics include, e.g., amoxicillin, clarithromycin, tetracycline, metronidizole, erythromycin, cefuroxime, and erythromycin
  • antisecretory agents including, e.g., H 2 - receptor antagonists (e.g., cimetidine, ranitidine, famotidine, nizatidine and roxatidine), proton pump inhibitors (e.g., omeprazole, lansoprazole, and pantoprazole), prostaglandin analogs (e.g., misoprostil and enprostil), and anticholinergic agents (e.g., pirenzepine
  • H 2 - receptor antagonists e.g., cimetidine, ranitidine, fa
  • compositions for carrying out these methods i.e., compositions containing a Helicobacter antigen (or antigens) of the invention, an adjuvant, and one or more of the above-listed compounds, in a pharmaceutically acceptable carrier or diluent.
  • Amounts of the above-listed compounds used in the methods and compositions of the invention can readily be determined by one skilled in the art.
  • one skilled in the art can readily design treatment/immunization schedules.
  • the non- vaccine components can be administered on days 1-14, and the vaccine antigen + adjuvant can be administered on days 7, 14, 21, and 28.
  • Methods and pharmaceutical compositions of the invention can be used to treat or to prevent Helicobacter infections and, accordingly, gastroduodenal diseases associated with these infections, including acute, chronic, and atrophic gastritis, and peptic ulcer diseases, e.g., gastric and duodenal ulcers.
  • a 76 kDa protein band containing GHPO 386, GHPO 789, and GHPO 1516 (hereinafter the "purified 76 kDa proteins"), GHPO 1360,- and GHPO 750 were purified from Helicobacter pylori strain ATCC number 43579 (American Type Culture Collection, Rockville, Maryland) by immunoaffinity- based chromatography using the methods described below in Example 1, and were shown to be effective vaccine antigens as follows.
  • mice Groups of 10 mice each were orally immunized with 1, 5, or 25 ⁇ g of the purified 76 kDa proteins, purified GHPO 1360, or purified GHPO 750 in combination with 5 ⁇ g of the heat-labile enterotoxin (LT) of E. coli. Twenty five ⁇ g of recombinant urease, in combination with 5 ⁇ g LT, was used as a positive control, and 5 ⁇ g of LT in PBS was used as a negative control. The immunizations were carried out four times each, on days 0, 7, 14, and 21 of the experiment. On day 33, blood samples were collected from the mice and, on day 34, saliva samples were collected.
  • LT heat-labile enterotoxin
  • mice On day 35, all of the mice were challenged by intragastric administration of 1 x 10 7 streptomycin-resistant, mouse-adapted H. pylori. On day 49, additional saliva samples were collected and, about two weeks after challenge, on days 52-53, the mice were sacrificed. Stomachs were removed from the mice and were analyzed for Helicobacter infection by measuring urease activity in the intact stomach tissue and by a quantitative culture study (Table 1).
  • Example 1 describes purification of GHPO 1516 (76 kDa), GHPO 1360 (32 kDa), and GHPO 750 (50 kDa) from Helicobacter cultures.
  • Example 2 describes identification of genes, e.g., genes encoding 76 kDa proteins, such as GHPO 386, GHPO 789, GHPO 1516, GHPO 1197, GHPO 1180, GHPO 896, GHPO 711, GHPO 190, GHPO 185, GHPO 1417, and GHPO 1414, a 32 kDa protein (GHPO 1360), and a 50 kDa protein (GHPO 750) in the Helicobacter genome, as well as identification of signal sequences, and primer design for amplification of genes lacking signal sequences.
  • Example 3 describes cloning of DNA encoding GHPO 386, GHPO 789, GHPO 1516, GHPO 896, GHPO 1360, and GHPO 750 into a vector that provides a histidine tag, and production and purification of the resulting his-tagged fusion proteins.
  • Example 4 describes methods for cloning DNA encoding the polypeptides of the invention so that they can be produced without His-tags,
  • Example 5 describes methods for purifying recombinant polypeptides of the invention, and
  • Example 6 describes use of the GHPO 1360 polypeptide as a serodiagnostic tool for H. pylori infection
  • EXAMPLE 1 Purification and partial sequence analysis of GHPO 1516 (76 kDa), GHPO 1360 (32 kDa), and GHPO 750 (50 kDa) protein from Helicobacter pylori
  • Frozen seeds from H. pylori strain ATCC 43579 are used to seed a 75 cm 2 flask containing a biphasic medium (a solid phase made of Colombia gelose containing 6% fresh sheep blood and a liquid phase made of triptcase soja containing 20% fetal calf serum). After 24 hours of culturing under microaerophilic conditions, the liquid phase is used for seeding several 75 cm 2 flasks containing biphasic medium lacking sheep blood. After 24 hours of culture, the liquid phase is used to seed a 2 L biofermentor in triptcase soja liquid phase containing 10 g/L beta-cyclodextrine. At OD 1.5-1.8, this culture is diluted in a 10 L biofermentor containing the liquid medium. After 24 hours, the bacteria are spun in a centrifuge at 4,000 x g for 30 minutes at 4°C. A 10 L culture contains about 20 to 30 g (wet weight) bacteria.
  • the pellet obtained using the method described above is washed with 500 ml PBS (7.650 g NaCl, 0.724 g disodium phosphate, and 0.210 g monopotassium phosphate for one liter (pH 7.2)) for a one liter culture.
  • the bacteria are then spun in a centrifuge again under the same conditions.
  • the pellet (Cl) is suspended in 1% N-octyl-D-glucopyranoside (NOG; 30 ml/L; Sigma). The bacterial suspension is incubated for 1 hour at room temperature while stirring, spun in a centrifuge at 17,600 x g for 30 minutes at 4°C, and the pellet (C2) is recovered.
  • NOG N-octyl-D-glucopyranoside
  • the supernatant (S2) is dialyzed against PBS overnight at 4°C while stirring.
  • the precipitate is recovered by centrifugation at 2,600 x g for 30 minutes at 4°C.
  • the supernatant (S2d) is discarded and the pellet (Cs2d) is recovered and stored at -20°C.
  • the pellet (C2) is resuspended in 20 mM Tris-HCl buffer (pH 7.5) and
  • Pefabloc (Buffer A), and is homogenized with an ultra-turrax (3821, Janke and Kungel). Lysozyme and EDTA are added at 0.1 mg/ml and 1 mM, respectively.
  • the homogenate is sonicated three times for 2 minutes each at 4°C, and then is spun in an ultracentrifuge at 210,000 x g for 30 minutes at 4°C.
  • the supernatant (S3) which contains the cytoplasmic and periplasmic proteins, is eliminated, while the pellet is recovered, washed with buffer A, and spun in an ultracentrifuge at 210,000 x g for 30 minutes at 4°C.
  • the supernatant (S4) is eliminated and the pellet (C4) is stored at -20°C. This pellet (C4) contains membrane proteins.
  • the pellet (C4) is washed in 50 mM NaC0 3 (pH 9.5) and 100 ⁇ M Pefabloc (buffer B). The suspension is spun in an ultracentrifuge at
  • SDS-PAGE is carried out according to the method of Laemmli (supra), using a biphasic gel consisting of a 5% polyacrylamide concentrating gel and a 10% polyacrylamide separating gel.
  • the membrane fraction C4 is resuspended in buffer A, diluted in an equal volume of 2x sample buffer, and heated for 5 minutes at 95°C.
  • About 19 mg of protein is applied to the gel (16 x 12 cm; 5 mm thick).
  • Pre-migration is carried out for 2 hours at 50 V, and is followed by migration overnight at 65 V. After Coomassie blue staining, five major bands are revealed that have apparent molecular weights of 87, 76, 54, 50, and 32 kDa. Bands at 50 and 32 kDa appear to be slightly contaminated with bands at 47 and 35 kDa, respectively.
  • a band corresponding to the purified 76 kDa proteins, 32 kDa protein (GHPO 1360), or 50 kDa protein (GHPO 750) is cut out from the gel and is pounded with an ultra-turrax in 10-20 ml extraction buffer (25 mM Tris- HCl (pH 8.8), 8 M urea, 10% SDS, 100 ⁇ M phenyl methyl sulfonyl fluoride (PMSF), and 10 ⁇ M Pefabloc (buffer C)).
  • 10-20 ml extraction buffer 25 mM Tris- HCl (pH 8.8), 8 M urea, 10% SDS, 100 ⁇ M phenyl methyl sulfonyl fluoride (PMSF), and 10 ⁇ M Pefabloc (buffer C)
  • Each homogenate is filtered through a Millipore AP20 filter under 7 bars at room temperature, washed with 5-10 ml buffer C, and then filtered again. Each filtrate is precipitated with three volumes of a 50/50 mixture of 75% methanol and 75% isopropanol, and then is spun in a centrifuge at 240,000 x g for 16 hours at 10°C.
  • Each pellet is resuspended in 2 ml of 10 mM NaP0 4 (pH 7.0) containing 1 M NaCl, 0.1% Sarkosyl, 100 ⁇ M PMSF, and 6 M urea (buffer D).
  • the solubilized sample is dialyzed, in order, against 100 ml buffer D containing 4 M urea, 100 ml buffer D containing 2 M urea and 0.5% Sarkosyl, and twice against 100 ml buffer D that does not contain urea or Sarkosyl.
  • the dialyses are earned out for 1 hour each while stirring at room temperature.
  • the last dialysate is incubated for 30 minutes in an ice bath, and then is spun in a centrifuge at low speed for 10 minutes at 4°C.
  • the supernatant is recovered, filtered through a Millipore filter (0.45 ⁇ m), and stored at -20°C.
  • Specific polyclonal serum against the purified 76 kDa proteins, the 32 kDa protein (GHPO 1360), or the 50 kDa protein (GHPO 750), which are purified by preparative SDS-PAGE, is prepared by hyperimmunizing rabbits as follows. On day 0, a preparation containing 50 ⁇ g of the protein mixed with complete Freund's adjuvant is administered subcutaneously to the rabbits at multiple sites. The rabbits are boosted at days 21 and 42 with 25 ⁇ g of the protein in incomplete Freund's adjuvant, and are sacrificed at day 60. Complement is removed from the serum by heating for 30 minutes at 56°C. The hyperimmune serum is then sterilized by filtration through a Millipore membrane (0.22 ⁇ m).
  • the hyperimmune serum prepared as described above is applied to a Protein A Sepharose Fast Flow column (Pharmacia) that is equilibrated with
  • CNBr-activated Sepharose 4B gel (Pharmacia; reference: 17-0430-01) is suspended in 1 mM NaCl buffer (1 g dry gel provides for 3.5 ml hydrated gel; 5 to 10 mg IgGs can be retained per ml of hydrated gel). The gel is then washed using a buchner by adding small quantities of 1 mM HC1. The total volume of 1 mM HC1 that is used amounts to 200 ml/g of gel.
  • IgGs Purified IgGs are dialyzed for 4 hours at room temperature against 50 volumes of 500 mM sodium phosphate buffer (pH 7.5). The IgGs are then diluted to 3 mg/ml with the same buffer. IgGs are incubated with the gel overnight at 5+3 °C while stirring. The gel is packed in a chromatography column and is washed with 2 column volumes of 500 mM phosphate buffer (pH 7.5). The gel is then transferred to a tube and is incubated with 100 mM ethanolamine (pH 7.5), and then it is washed with 2 column volumes of PBS. The gel can be stored in PBS/merthiolate, 1/10,000. I.C.4. Adsorption and elution
  • the 76 kDa protein is adsorbed and eluted as follows.
  • the membrane fraction Cs2d is suspended in 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, and then is filtered through a 0.45 ⁇ m membrane.
  • the supernatant is applied to the column, which is equilibrated with 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, at a flow rate of about 10 ml/hour.
  • the column is washed with 20 column volumes of 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, and then with 2 to 6 volumes 10 mM phosphate buffer (pH 6.8).
  • the antigen is eluted with 100 mM glycine buffer (pH 2.5).
  • the eluate is collected in 3 ml fractions, to each of which is added 150 ⁇ l 1 M phosphate buffer (pH 8.0).
  • the optical density of each fraction is measured at 280 nm, fractions containing the 76 kDa protein are pooled, and stored at -70°C.
  • the 32 kDa protein (GHPO 1360) or the 50 kDa protein (GHPO 750) is purified by immunoaffinity-based chromatography as follows.
  • membrane fraction C4 is solubilized in 50 mM NaC0 3 (pH 9.5) for 30 minutes at room temperature under stirring and the preparation is centrifuged for 30 minutes at 200,000 x g at 4°C.
  • the 47 and 35 kDa proteins are insoluble in the NaC0 3 buffer and are eliminated in the pellet.
  • the supernatant is dialyzed against 50 mM Tris-HCL (pH 8.0), 2 mM EDTA, and then is filtered through a 0.45 ⁇ m membrane.
  • the filtered supernatant is applied to the column, which is equilibrated with 50 mM Tris- HCL (pH 8.0), 2 mM EDTA, at a flow rate of about 10 ml/hour.
  • the column is washed with 20 column volumes of 50 mM Tris-HCL (pH 8.0), 2 mM EDTA, and then with 2 to 6 volumes of 10 mM phosphate buffer (pH 6.8).
  • the antigen is eluted with 100 mM glycine buffer (pH 2.5).
  • the eluate is collected in 3 ml fractions, to each of which is added 150 ⁇ l 1 M phosphate buffer (pH 8.0).
  • the optical density of each fraction is measured at 280 nm, and fractions containing the 50 or 32 kDa protein are pooled and stored at -70°C.
  • N-terminal sequencing is carried out with the purified 50 kDa protein preparation. The sequence found is as follows: MKEKFNRTKPHVNIGTIGHVDH (SEQ ID NO:73). Similarly, N-terminal and internal sequencing is carried out with the purified 32 kDa preparation. The sequences found are as follows: AHNANNATHNTKK (SEQ ID NO: 74) and KPAHNA (SEQ ID NO:75) (N-terminal), and IDKQPKAKK (SEQ ID NO:76) and FWAKKQAE (SEQ ID NO:77) (internal).
  • the 76 kDa protein can also be purified as follows.
  • a 40 ml Q- Sepharose column (diameter: 2.5 cm; height: 8 cm) is prepared according to the manufacturer's instructions (Pharmacia). The column is washed and equilibrated with buffer B, containing 50 mM NaCO, (pH 9.5), 100 ⁇ M Pefabloc, and 0.1% Zwittergent 3-14. The chromatography is monitored by measuring absorbance at 280 nm at the column exit.
  • One hundred and forty mg of protein from the membrane fraction Cs2d resuspended in buffer B are applied to the column.
  • the column is washed with 0.1 M NaCl in buffer B, and then a 0.1-0.5 M NaCl gradient is applied to the column.
  • the fraction eluted between 0.35 and 0.45 M NaCl is further purified on a 10 ml S-Sepharose column (diameter: 1.5 cm; height: 5 cm; up to 10 mg protein/ml of gel), which is prepared according to the manufacturer's instructions (Pharmacia).
  • the fraction obtained is dialyzed against 50 mM acetate (pH 5.0) containing 100 ⁇ M Pefabloc and 0.1% Zwittergent 3-14, and then is applied to the column, which is equilibrated with the acetate buffer.
  • 0.15 M NaCl is enriched with the 76 kDa protein.
  • the 32 kDa protein (GHPO 1360) can also be purified as follows.
  • Membrane fraction C4 is solubilized in 50 mM NaC0 3 buffer (pH 9.5) at room temperature for 30 minutes under stirring. The suspension is then centrifuged at 200,000 x g for 30 minutes at 4°C. This allows the 32 and 35 kDa proteins to be separated, since the 35 kDa protein is insoluble in the NaC0 3 buffer. The supernatant is dialyzed against 50 mM NaP0 4 buffer (pH 7.0), and then is applied to an SP-Sepharose column, which is equilibrated with the NaP0 4 buffer. The column is washed with the NaP0 4 buffer, and then an 0-0.5 M
  • 0.31 M contains the 32 kDa protein.
  • the 50 kDa protein can also be purified as follows.
  • a 40 ml Q-Sepharose column (diameter: 2.5 cm; height: 8 cm) is prepared according to the manufacturer's instructions (Pharmacia), washed, and equilibrated with buffer B (pH 9.5) (50 mM NaC0 3 , 100 ⁇ M Pefabloc, and
  • the chromatography is monitored by UV detection at 280 nm at the column exit.
  • One hundred and forty mg of protein solubilized as is described above are applied to the column, which is then washed with buffer B until the absorbance at 280 nm is stabilized.
  • the proteins are eluted with a 0.1-
  • Fraction 9 which conesponds to the beginning of the washing at 1 M NaCl and contains acidic proteins, is further purified as follows.
  • a 10 ml DEAE Sepharose column (diameter: 1.5 cm, height: 5 cm) is prepared according to the manufacturer's instructions (Pharmacia) (up to 10 mg protein/ml of gel). The column is washed and equilibrated with buffer B. Chromatography is monitored as is described above. Fraction 9 is dialyzed against buffer B and contains about 10 mg protein.
  • Fraction 9 is applied to the DEAE-Sepharose column. The column is washed with buffer B until the absorbance at 280 nm is stabilized. The proteins are eluted with a 0-0.5 M NaCl gradient in buffer B (10 fold V ⁇ ), followed by washing in buffer B, containing 1 M NaCl (2 fold V ⁇ ). Fractions are recovered and analyzed by SDS-PAGE. The 50 kDa protein is found in the fractions eluted at 0.3-0.4 M NaCl.
  • EXAMPLE 2 Identification of genes in the H. pylori genome, such as genes encoding the 76 kDa proteins, the 32 kDa protein (GHPO 1360), and the 50 kDa protein (GHPO 750) identification of signal sequences, and primer design for amplification of genes lacking signal sequences
  • the H. pylori genome was provided as a text file containing a single contiguous string of nucleotides that had been determined to be 1.76 Megabases in length.
  • the complete genome was split into 17 separate files using the program SPLIT (Creativity in Action), giving rise to 16 contigs, each containing 100,000 nucleotides, and a 17 th contig containing the remaining 76,000 nucleotides.
  • a header was added to each of the 17 files using the format: >hpg0.txt (representing contig 1), .hpgl .txt (representing contig 2), etc.
  • the resulting 17 files were then copied together to form one file that represented the plus strand of the complete H. pylori genome.
  • the constructed database was given the designation " ⁇ .”
  • a negative strand database of the H. pylori genome was created similarly by first creating a reverse complement of the positive strand using the program SeqPup (D.G. Gilbert, Indiana University Biology Department) and then performing the same procedure as described above for the plus strand. This database was given the designation "N.”
  • ORFs open reading frames
  • FASTA Pearson et al, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988.
  • FASTA was used for searching either a DNA sequence against either of the gene databases (" ⁇ " and/or "N"), or a peptide sequence against the ORF library ("O").
  • TFASTX was used to search a peptide sequence against all possible reading frames of a DNA database (" ⁇ " and/or "N” libraries).
  • ⁇ " and/or "N” libraries - Potential frameshifts also being resolved, FASTX was used for searching the translated reading frames of a DNA sequence against either a DNA database, or a peptide sequence against the protein database.
  • the deduced protein encoded by a target gene sequence is analyzed using the PROTEAN software package (DNAStar, Inc.). This analysis predicts those areas of the protein that are hydrophobic by using the Kyte-Doolittle algorithm, and identifies any potential polar residues preceding the hydrophobic core region, which is typical for many signal sequences. For confirmation, the target protein is then searched against a PROSITE database (DNAStar, Inc.) consisting of motifs and signatures. Characteristic of many signal sequences and hydrophobic regions in general, is the identification of predicted prokaryotic lipid attachment sites. Where confirmation between the two approaches is apparent at the N-terminus of any protein, putative cleavage sites are sought.
  • this includes the presence of either an Alanine (A), Serine (S), or Glycine (G) residue immediately after the core hydrophobic region.
  • A Alanine
  • S Serine
  • G Glycine
  • C Cysteine
  • EXAMPLE 3 Preparation of isolated DNA encoding GHPO 386, GHPO 789, GHPO 1516, GHPO 896, GHPO 1360, and GHPO 750, and production of these proteins as a histidine-tagged fusion proteins
  • Helicobacter pylori strain ORV2001 stored in LB medium containing 50%) glycerol at -70°C, is grown on Colombia agar containing 7% sheep blood for 48 hours under microaerophilic conditions (8-10% C0 2 , 5-7% 0 2 , and 85- 87% N 2 ). Cells are harvested, washed with PBS (pH 7.2), and DNA is then extracted from the cells using the Rapid Prep Genomic DNA Isolation kit (Pharmacia Biotech). 3.B. PCR amplification
  • DNA encoding GHPO 386, GHPO 789, GHPO 1516, GHPO 896, GHPO 1360, and GHPO odd numbers), 65, and 67 is amplified from genomic DNA, as can be prepared as is described above, by the Polymerase Chain Reaction (PCR) using the following primers: GHPO 386:
  • N-terminal primer 5'-CTGAATTCGATTTCAAGGAGAAAACATGAAA-3' (SEQ ID NO:59); and C-terminal primer:
  • N-terminal primer 5'-CGCGGATCCGAATCCAATTTAATCCAAAAAGG-3' (SEQ ID NO:61);
  • N-terminal primer 5'-CGCGGATCCGAATCCAATTTAATCCAAAAAGG-3' (SEQ ID NO:56);
  • C-terminal primer 5'-CCGCTCGAGTTAAGTAAGCGAACACATATTCAA-3' (SEQ ID NO:57).
  • GHPO 896 N-terminal primer:
  • GHPO 1360 5'-CCGCTCGAGTTAGTAAGCAAACACATAATTGTG-3' (SEQ ID NO: 64).
  • N-terminal primer 5'-CGCGGATCCGAATGAAAAAAAATATCTTAAAT-3' (SEQ ID NO:69);
  • GHPO 750 5*-CCGCTCGAGTTACTTGTTGATAACAATTTT-3' (SEQ ID NO:70).
  • N-terminal primer 5*-CGCGGATCCGAATGGCAAAAGAAAAGTTTAAC-3' (SEQ ID NO:71);
  • C-terminal primer 5'-CCGCTCGAGTTATTCAATAATATTGCTCAC-3* (SEQ ID NO:72).
  • GHPO 711 N-terminal primer:
  • C-terminal primer 5'-CCCCTCGAGTTAATAGGCAAACAC-3' (SEQ ID NO:84).
  • the N-terminal and C-terminal primers for each clone both include a 5' clamp and a restriction enzyme recognition sequence for cloning pu ⁇ oses (BamHl (GGATCC) andXhol (CTCGAG) recognition sequences).
  • Amplification of gene-specific DNA is carried out using a heat-stable DNA Polymerase (e.g., Thermalase DNA Polymerase (Amresco)) according to the manufacturer's instructions.
  • the reaction mixture which is brought to a final volume of 100 ⁇ l with distilled water, is as follows: dNTPs mix 200 ⁇ M
  • amplification reaction conditions can readily be determined by one skilled in the art. In the present case, the following conditions were used.
  • GHPO 386 and GHPO 789 in a reaction containing Taq DNA polymerase (Appligene), a denaturing step was canied out at 95 °C for 30 seconds, followed by an annealing step at 50 °C for one minute, and an extension step at 72 °C for 2 minutes and 30 seconds. Twenty five cycles were canied out.
  • GHPO 896 in a reaction containing Taq DNA polymerase, a denaturing step was carried out at 97 °C for 30 seconds, followed by an annealing step at 50°C for one minute, and an extension step at 72°C for 2 minutes and 30 seconds.
  • GHPO 1516 was clone GHPO 1516, instead of Taq DNA polymerase, and the annealing temperature was 55 °C.
  • Vent DNA polymerase was used for clone GHPO 1516, instead of Taq DNA polymerase, and the annealing temperature was 55 °C.
  • GHPO 1360 and GHPO 750 Thermalase DNA polymerase was used for GHPO 1360 and GHPO 750.
  • a denaturing step was canied out at 95 °C for 30 seconds, followed by an annealing step at 55 °C for one minute, and an extension step at 72 °C for 2 minutes. Thirty cycles were carried out.
  • GHPO 711 Vent DNA polymerase was used for GHPO 711
  • a denaturing step was canied out at 94 °C for 30 seconds, followed by an annealing step at 50°C for 30 seconds, and an extension step at 72°C for 1 minute. Twenty five cycles were canied out. 3.C. Transformation and selection of transformants
  • a single PCR product is thus amplified and is then digested at 37 °C for 2 hours with BamHl and_A7r ⁇ I concunently in a 20 ⁇ l reaction volume.
  • the digested product is ligated to similarly cleaved pET28a (Novagen) that is dephosphorylated prior to the ligation by treatment with Calf Intestinal Alkaline Phosphatase (CIP).
  • CIP Calf Intestinal Alkaline Phosphatase
  • the ligation reaction (20 ⁇ l) is canied out at 14 °C overnight and then is used to transform 100 ⁇ l fresh E. coli XL 1 -blue competent cells (Novagen).
  • the cells are incubated on ice for 2 hours, then heat-shocked at 42 °C for 30 seconds, and returned to ice for 90 seconds.
  • the samples are then added to 1 ml LB broth in the absence of selection and grown at 37 °C for 2 hours.
  • the cells are then plated out on LB agar containing kanamycin (50 ⁇ g/ml) at a lOx and neat dilution and incubated overnight at 37 °C. The following day, 50 colonies are picked onto secondary plates and incubated at 37 °C overnight.
  • Plasmid DNA is extracted using the Quiagen mini -prep, method and is quantitated by agarose gel electrophoresis.
  • PCR is performed with the gene-specific primers under the conditions stated above and transformant DNA is confirmed to contain the desired insert. If PCR-positive, one of the five plasmid DNA samples (500 ng) extracted from the E. coli XL 1 -blue cells is used to transform competent BL21 ( ⁇ DE3) E. coli competent cells (Novagen; as described previously). Transformants (10) are picked onto selective kanamycin (50 ⁇ g/ml) containing LB agar plates and stored as a research stock in LB containing 50% glycerol.
  • One ml of frozen glycerol stock prepared as described in 3. C. is used to inoculate 50 ml of LB medium containing 25 ⁇ g/ml of kanamycin in a 250 ml Erlenmeyer flask.
  • the flask is incubated at 37°C for 2 hours or until the absorbance at 600 nm (OD 600 ) reaches 0.4-1.0.
  • the culture is stopped from growing by placing the flask at 4°C overnight.
  • 10 ml of the overnight culture are used to inoculate 240 ml LB medium containing kanamycin (25 ⁇ g/ml), with the initial OD 600 about 0.02-0.04.
  • Four flasks are inoculated for each ORF.
  • the cells are grown to an OD 600 of 1.0 (about 2 hours at 37°C), a 1 ml sample is harvested by centrifugation, and the sample is analyzed by SDS- PAGE to detect any leaky expression.
  • the remaining culture is induced with 1 mM IPTG and the induced cultures are grown for an additional 2 hours at 37°C.
  • the final OD 600 is taken and the cells are harvested by centrifugation at 5,000 x g for 15 minutes at 4°C. The supernatant is discarded and the pellets are resuspended in 50 mM Tris-HCl (pH 8.0), 2 mM EDTA. Two hundred and fifty ml of buffer are used for a 1 L culture and the cells are recovered by centrifugation at 12,000 x g for 20 minutes. The supernatant is discarded and the pellets are stored at -45°C.
  • Pellets obtained from 3.D. are thawed and resuspended in 95 ml of 50 mM Tris-HCl (pH 8.0). Pefabloc and lysozyme are added to final concentrations of 100 ⁇ M and 100 ⁇ g/ml, respectively.
  • the mixture is homogenized with magnetic stining at 5°C for 30 minutes.
  • Benzonase (Merck) is added at a 1 U/ml final concentration, in the presence of 10 mM MgCl2, to ensure total digestion of the DNA.
  • the suspension is sonicated (Branson Sonifier 450) for 3 cycles of 2 minutes each at maximum output.
  • the target protein is produced in a soluble form (i.e., in the supernatant obtained in 3.E.)
  • NaCl and imidazole are added to the supernatant to final concentrations of 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, and 10 mM imidazole (buffer A).
  • the mixture is filtered through a 0.45 ⁇ m membrane and loaded onto an IMAC column (Pharmacia HiTrap chelating Sepharose; 1 ml) that has been charged with nickel ions according to the manufacturer's recommendations.
  • the column is washed with 50 column volumes of buffer A and the recombinant target protein is eluted with 5 ml of buffer B (50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 500 mM imidazole).
  • buffer B 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 500 mM imidazole.
  • the elution profile is monitored by measuring the absorbance of the fractions at 280 nm. Fractions conesponding to the protein peak are pooled, dialyzed against PBS containing 0.5 M arginine, filtered through a 0.22 ⁇ m membrane, and stored at -45°C. 3.E.2. Insoluble fraction If the target protein is expressed in the insoluble fraction (pellets obtained from 3.E.), purification is conducted under denaturing conditions.
  • NaCl, imidazole, and urea are added to the resuspended pellet to final concentrations of 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 10 mM imidazole, and 6 M urea (buffer C). After complete solubilization, the mixture is filtered through a 0.45 ⁇ m membrane and loaded onto an IMAC column.
  • the purification procedures on the IMAC column are the same as described in 3.E.I ., except that 6 M urea is included in all buffers used and 10 column volumes of buffer C are used to wash the column after protein loading, instead of 50 column volumes.
  • the protein fractions eluted from the IMAC column with buffer D (buffer C containing 500 mM imidazole) are pooled.
  • Arginine is added to the solution to final concentration of 0.5 M and the mixture is dialyzed against PBS containing 0.5 M arginine and various concentrations of urea (4 M, 3 M, 2 M, 1 M, and 0.5 M) to progressively decrease the concentration of urea.
  • the final dialysate is filtered through a 0.22 ⁇ m membrane and stored at -45°C.
  • a first alternative involves the use of a mild denaturant, N-octyl glucoside (NOG). Briefly, a pellet obtained in 3.E. is homogenized in 5 mM imidazole, 500 mM sodium chloride, 20 mM Tris-HCl (pH 7.9) by microfluidization at a pressure of 15,000 psi and is clarified by centrifugation at 4,000-5,000 x g. The pellet is recovered, resuspended in 50 mM NaP0 4 (pH 7.5) containing 1-2% weight
  • NOG N-octyl glucoside
  • the NOG-soluble impurities are removed by centrifugation.
  • the pellet is extracted once more by repeating the preceding extraction step.
  • the pellet is dissolved in 8 M urea, 50 mM Tris (pH 8.0).
  • the urea-solubilized protein is diluted with an equal volume of 2 M arginine, 50 mM Tris (pH 8.0), and is dialyzed against 1 M arginine for 24-48 hours to remove the urea.
  • the final dialysate is filtered through a 0.22 ⁇ m membrane and stored at -45°C.
  • a second alternative involves the use of a strong denaturant, such as guanidine hydrochloride. Briefly, a pellet obtained in 3.E.
  • a protection test is described above that was canied out for testing the protective activity of the purified, native proteins. This test can also be used for testing the protective efficacy of recombinant proteins. Alternatively, the following test can be used.
  • mice Groups of 10 OF1 mice (IFF A Credo) are immunized rectally with 25 ⁇ g of the purified recombinant protein, admixed with 1 ⁇ g of cholera toxin (Berna) in physiological buffer. Mice are immunized on days 0, 7, 14, and 21. Fourteen days after the last immunization, the mice are challenged with H. pylori strain ORV2001 grown in liquid media (the cells are grown on agar plates, as described in LA., and, after harvest, the cells are resuspended in Brucella broth; the flasks are then incubated overnight at 37 °C). Fourteen days after challenge, the mice are sacrificed and their stomachs are removed. The amount of H. pylori is determined by measuring the urease activity in the stomach and by culture. 3.G. Production of monospecific polyclonal antibodies 3.G.I. Hyperimmune rabbit antiserum
  • New Zealand rabbits are injected both subcutaneously and intramuscularly with 100 ⁇ g of a purified fusion polypeptide, as obtained in 3.E.I . or 3.E.2., in the presence of Freund's complete adjuvant and in a total volume of approximately 2 ml. Twenty one and 42 days after the initial injection, booster doses, which are identical to priming doses, except that Freund's incomplete adjuvant is used, are administered in the same way. Fifteen days after the last injection, animal serum is recovered, decomplemented, and filtered through a 0.45 ⁇ m membrane. 3.G.2. Mouse hyperimmune ascites fluid
  • mice are injected subcutaneously with 10-50 ⁇ g of a purified fusion polypeptide, as obtained in 3.E.I . or 3.E.2., in the presence of Freund's complete adjuvant and in a volume of approximately 200 ⁇ l. Seven and 14 days after the initial injection, booster doses, which are identical to the priming doses, except that Freund's incomplete adjuvant is used, are administered in the same way. Twenty one and 28 days after the initial infection, mice receive 50 ⁇ g of the antigen alone intraperitoneally.
  • mice are also injected intraperitoneally with sarcoma 180/TG cells CM26684 (Lennette et al, Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections , 5th Ed., Washington DC, American Public Health Association, 1979). Ascites fluid is collected 10-13 days after the last injection.
  • EXAMPLE 4 Methods for producing transcriptional fusions lacking His- tags Methods for amplification and cloning of DNA encoding the polypeptides of the invention as transcriptional fusions lacking His-tags are described as follows. Two PCR primers for each clone are designed based upon the sequences of the polynucleotides that encode them (SEQ ID NOs: 1-21 (odd numbers), 65, and 67). These primers can be used to amplify DNA encoding the polypeptides of the invention from any Helicobacter pylori strain, including, for example, ORV2001 and the H. pylori strain deposited with the American Type Culture Collection (ATCC, Rockville, Maryland) as ATCC number 43579, as well as from other Helicobacter species.
  • ATCC American Type Culture Collection
  • the N-terminal primers are designed to include the ribosome binding site of the target gene, the ATG start site, the signal sequence (if any), and the cleavage site.
  • the N-terminal primers can include a 5' clamp and restriction endonuclease recognition site, such as that for BamHl (GGATCC), which facilitates subsequent cloning.
  • the C-terminal primers can include a restriction endonuclease recognition site, such as that fo ⁇ Xhol (CTCGAG), which can be used in subsequent cloning, and a TAA stop codon. Specific primers that can be used are listed above.
  • Amplification of a genes encoding the polypeptides of the invention can be canied out using Vent DNA polymerase (New England Biolabs) or Taq DNA polymerase (Appligene) under the conditions described above in Example 3.
  • Vent DNA polymerase New England Biolabs
  • Taq DNA polymerase Appligene
  • Thermalase DNA polymerase or Pwo DNA polymerase can be used, according to instructions provided by the manufacturers.
  • a single PCR product for each clone is amplified and can be cloned into BamHl-XhoI cleaved pET24, resulting in construction of transcriptional fusions that permit expression of the proteins without ⁇ is-tags.
  • the expressed products can be purified as denatured proteins that are refolded by dialysis into 1 M arginine.
  • Cloning into pET 24 allows transcription of genes from the T7 promoter, which is supplied by the vector, but relies upon binding of the RNA-specific DNA polymerase to the intrinsic ribosome binding site of the genes, and thereby expression of the complete ORF.
  • the amplification, digestion, and cloning protocols are as described above for constructing translational fusions.
  • An immune serum as prepared as is described in section 3.G., is applied to a protein A Sepharose Fast Flow column (Pharmacia) equilibrated in 100 mM Tris-HCl (pH 8.0). The resin is washed by applying 10 column volumes of 100 mM Tris-HCl and 10 volumes of 10 mM Tris-HCl (pH 8.0) to the column. IgG antibodies are eluted with 0.1 M glycine buffer (pH 3.0) and are collected in 5 ml fractions to each of which is added 0.25 ml 1 M Tris-HCl (pH 8.0).
  • the optical density of the eluate is measured at 280 nm and the fractions containing the IgG antibodies are pooled, dialyzed against 50 mM Tris-HCl (pH 8.0), and, if necessary, stored frozen at -70°C.
  • CNBr-activated Sepharose 4B gel (1 g of dried gel provides for approximately 3.5 ml of hydrated gel; gel capacity is from 5 to 10 mg coupled IgG/ml of gel) manufactured by Pharmacia (17-0430- 01) is suspended in 1 mM HC1 buffer and washed using a buchner by adding small quantities of 1 mM HC1 buffer. The total volume of buffer is 200 ml per gram of gel.
  • Purified IgG antibodies are dialyzed for 4 hours at 20+5 °C against 50 volumes of 500 mM sodium phosphate buffer (pH 7.5). The antibodies are then diluted in 500 mM phosphate buffer (pH 7.5) to a final concentration of 3 mg/ml.
  • IgG antibodies are mixed with the gel overnight at 5+3 °C.
  • the gel is packed into a chromatography column and is washed with 2 column volumes of 500 mM phosphate buffer (pH 7.5), and 1 column volume of 50 mM sodium phosphate buffer, containing 500 mM NaCl (pH 7.5).
  • the gel is then transfened to a tube, mixed with 100 mM ethanolamine (pH 7.5) for 4 hours at room temperature, and washed twice with 2 column volumes of PBS.
  • the gel is then stored in 1/10,000 PBS/merthiolate.
  • the amount of IgG antibodies coupled to the gel is determined by measuring the optical density (OD) at 280 nm of the IgG solution and the direct eluate, plus washings.
  • OD optical density
  • the adsorbed gel is washed with 2 to 6 volumes of 10 mM sodium phosphate buffer (pH 6.8) and the antigen is eluted with 100 mM glycine buffer (pH 2.5).
  • the eluate is recovered in 3 ml fractions, to each of which is added 150 ⁇ l of 1 M sodium phosphate buffer (pH 8.0). Abso ⁇ tion is measured at 280 nm for each fraction; those fractions containing the antigen are pooled and stored at -20°C.
  • EXAMPLE 6 The GHPO 1360 polypeptide is useful as a serodiagnostic tool for H. pylori infection
  • H. pylori proteins The reactivity of patient sera against H. pylori proteins was analyzed by immunoblot technique. Briefly, total lysate of H. pylori strain ORV2001 was subjected to SDS-PAGE electrophoresis (BioRad protean II system) on a 12.5% gel. Proteins were electrotransfened onto a nitrocellulose paper for immunoblot assay. After blocking, the nitrocellulose paper was incubated with patient sera (1 :500 diluted in blocking buffer) for one hour at room temperature, washed, and further incubated with peroxidase-conjugated goat anti-human IgG. The positive bands were revealed by incubation with the appropriate substrates. The results showed that the H. y/or.
  • MOLECULE TYPE Genomic DNA
  • FEATURE
  • MOLECULE TYPE Genomic DNA
  • FEATURE
  • AGT GTC AAC GGA ACC AAA ACC ACA ACC CAA ACC ATA GAC GGC AAA AGC 903 Ser Val Asn Gly Thr Lys Thr Thr Thr Gin Thr He Asp Gly Lys Ser 200 205 210 215
  • MOLECULE TYPE Genomic DNA
  • FEATURE
  • AAAC ATG AAA AAA CAC ATC CTT TCA TTA GCT TTA GGC TCG CTT TTA GTT 409
  • AGC ACG CAA ACA ACC GCG ACA ACC ACG CAA GAC GGC GTA ACG ATC ACC 1129 Ser Thr Gin Thr Thr Ala Thr Thr Thr Gin Asp Gly Val Thr He Thr 220 225 230 235
  • CTTTTTTA AACCCTCTTT TTTTAAGGGG TTTCTTTTTA AAGCTTTTTTTT GAAGTCTTTT 2859
  • MOLECULE TYPE Genomic DNA
  • FEATURE
  • ATC AAA AAC GCT GAA ATA GCC GTA GAG CAA TCC AAA ATC GTT ACC GCC 1193 He Lys Asn Ala Glu He Ala Val Glu Gin Ser Lys He Val Thr Ala 295 300 305
  • AAAAATACTC AAAGGCGTTT TTTGCAATCT AAATAAAAAA TTAGCGTTAT TCAAGCGATC 2527
  • MOLECULE TYPE Genomic DNA
  • FEATURE

Abstract

The invention provides 76 kDa, 32 kDa, and 50 kDa Helicobacter polypeptides, which can be used in vaccination methods for preventing or treating Helicobacter infection, and polynucleotides that encode these polypeptides. The invention also provides diagnostic methods employing these polypeptides.

Description

76 kDa. 32 kDa. and 50 kDa HELICOBACTER POLYPEPTIDES AND CORRESPONDING POLYNUCLEOTIDE MOLECULES The invention relates to Helicobacter polypeptides and corresponding polynucleotide molecules that can be used in methods to prevent or treat Helicobacter infection in mammals, such as humans.
Background of the Invention Helicobacter is a genus of spiral, gram-negative bacteria that colonize the gastrointestinal tracts of mammals. Several species colonize the stomach, most notably H. pylori, H. heilmanii, H. felis, and H. mustelae. Although H. pylori is the species most commonly associated with human infection, H. heilmanii and H. felis have also been isolated from humans, but at lower frequencies than H. pylori. Helicobacter infects over 50% of adult populations in developed countries and nearly 100% in developing countries and some Pacific rim countries, making it one of the most prevalent infections worldwide.
Helicobacter is routinely recovered from gastric biopsies of humans with histological evidence of gastritis and peptic ulceration. Indeed, H. pylori is now recognized as an important pathogen of humans, in that the chronic gastritis it causes is a risk factor for the development of peptic ulcer diseases and gastric carcinoma. It is thus highly desirable to develop safe and effective vaccines for preventing and treating Helicobacter infection.
A number of Helicobacter antigens have been characterized or isolated. These include urease, which is composed of two structural subunits of approximately 30 and 67 kDa (Ηu et al, Infect. Immun. 58:992, 1990; Dunn et al, J. Biol. Chem. 265:9464, 1990; Evans et al, Microbial Pathogenesis 10: 15, 1991 ; Labigne et al, J. Bact., 173: 1920, 1991); the 87 kDa vacuolar cytotoxin (VacA) (Cover et al, J. Biol. Chem. 267: 10570, 1992; Phadnis et al, Infect. Immun. 62: 1557, 1994; WO 93/18150); a 128 kDa immunodominant antigen associated with the cytotoxin (CagA, also called TagA; WO 93/18150; U.S. Patent No. 5,403,924); 13 and 58 kDa heat shock proteins HspA and HspB (Suerbaum et al, Mol. Microbiol. 14:959, 1994; WO 93/18150); a 54 kDa catalase (Hazell et al, J. Gen. Microbiol.137:57, 1991); a 15 kDa histidine-rich protein (Hpn) (Gilbert et al, Infect. Immun. 63:2682, 1995); a 20 kDa membrane-associated lipoprotein (Kostrcynska et al, J. Bact. 176:5938, 1994); a 30 kDa outer membrane protein (Bolin et al, J. Clin. Microbiol. 33:381, 1995); a lactoferrin receptor (FR 2,724,936); and several porins, designated HopA, HopB, HopC, HopD, and HopE, which have molecular weights of 48-67 kDa (Exner et al, Infect. Immun. 63:1567, 1995; Doig et al, J. Bact. 177:5447, 1995). Some of these proteins have been proposed as potential vaccine antigens. In particular, urease is believed to be a vaccine candidate (WO 94/9823; WO 95/22987; WO 95/3824; Michetti et al, Gastroenterology 107: 1002, 1994). Nevertheless, it is thought that several antigens may ultimately be necessary in a vaccine.
Summary of the Invention The invention provides polynucleotide molecules that encode a family of 76 kDa Helicobacter polypeptides, designated GHPO 386, GHPO 789, GHPO 1516, GHPO 1197, GHPO 1180, GHPO 896, GHPO 711, GHPO 190, GHPO 185, GHPO 1417, and GHPO 1414, a 32 kDa polypeptide, designated GHPO 1360, and a 50 kDa polypeptide, designated GHPO 750, which can be used, e.g., in methods to prevent, treat, or diagnose Helicobacter infection. The polypeptides include those having the amino acid sequences shown in SEQ ID NOs:2-22 (even numbers), 66, and 68. Those skilled in the art will understand that the invention also includes polynucleotide molecules that encode mutants and derivatives of these polypeptides, which can result from the addition, deletion, or substitution of non-essential amino acids, as is described further below.
In addition to the polynucleotide molecules described above, the invention includes the corresponding polypeptides (i.e., polypeptides encoded by the polynucleotide molecules of the invention, or fragments thereof), and monospecific antibodies that specifically bind to these polypeptides. The present invention has many applications and includes expression cassettes, vectors, and cells transformed or transfected with the polynucleotides of the invention. Accordingly, the present invention provides (i) methods for producing polypeptides of the invention in recombinant host systems and related expression cassettes, vectors, and transformed or transfected cells; (ii) live vaccine vectors, such as pox virus, Salmonella typhimurium, and Vibrio cholerae vectors, that contain polynucleotides of the invention (such vaccine vectors being useful in, e.g., methods for preventing or treating Helicobacter infection) in combination with a diluent or carrier, and related pharmaceutical compositions and associated therapeutic and/or prophylactic methods; (iii) therapeutic and/or prophylactic methods involving administration of polynucleotide molecules, either in a naked form or formulated with a delivery vehicle, polypeptides or mixtures of polypeptides, or monospecific antibodies of the invention, and related pharmaceutical compositions; (iv) methods for detecting the presence of Helicobacter in biological samples, which can involve the use of polynucleotide molecules, monospecific antibodies, or polypeptides of the invention; and (v) methods for purifying polypeptides of the invention by antibody-based affinity chromatography. Brief Description of the Drawings Figure 1 is an alignment of the predicted amino acid sequences of GHPO 386 (SEQ ID NO:2), GHPO 789 (SEQ ID NO:4), and GHPO 1516 (SEQ ID NO:6), as well as a consensus sequence for the 76 kDa protein family. Figure 2 is an alignment of the predicted amino acid sequences of
GHPO 1197 (SEQ ID NO: 8), GHPO 1180 (SEQ ID NO: 10), GHPO 896 (SEQ ID NO: 12), GHPO 711 (SEQ ID NO: 14), GHPO 190 (SEQ ID NO: 16), GHPO 185 (SEQ ID NO: 18), GHPO 1417 (SEQ ID NO:20), and GHPO 1414 (SEQ ID NO:22), as well as a consensus sequence for the 76 kDa protein family.
Detailed Description
Open reading frames (ORFs) encoding a family of new, full length, membrane-associated 76 kDa polypeptides, designated GHPO 386, GHPO 789, GHPO 1516, GHPO 1197, GHPO 1180, GHPO 896, GHPO 711, GHPO 190, GHPO 185, GHPO 1417, and GHPO 1414, a 32 kDa polypeptide, designated GHPO 1360, and a 50 kDa polypeptide, designated GHPO 750, have been identified in the H. pylori genome. The amino acid sequences of the 76 kDa polypeptides are aligned in Figures 1 and 2. The 76 kDa, 32 kDa, and 50 kDa polypeptides can be used, for example, in vaccination methods for preventing or treating Helicobacter infection. For example, GHPO 750, GHPO 1360, GHPO 190, and GHPO 1516 have been shown to be protective antigens. By "protective antigen" is meant an antigen that is capable of reducing the infection level after challenge, relative to a positive control. Absolute protection from infection, although included in the invention, is not required.
The polypeptides of the invention (except GHPO 750, see below) are secreted polypeptides that can be produced in their mature forms (i.e., as polypeptides that have been exported through class II or class III secretion pathways) or as precursors that include a signal peptide, which can be removed in the course of excretion/secretion by cleavage at the N-terminal end of the mature form. (The cleavage site is located at the C-terminal end of the signal peptide, adjacent to the mature form.) The cleavage site for the polypeptides of the invention and, thus, the first amino acid of the mature polypeptides, was putatively determined.
According to a first aspect of the invention, there are provided isolated polynucleotides that encode the precursor and mature forms of Helicobacter GHPO 386, GHPO 789, GHPO 1516, GHPO 1 197, GHPO 1180, GHPO 896, GHPO 711, GHPO 190, GHPO 185, GHPO 1417, GHPO 1414, GHPO 1360, and GHPO 750.
An isolated polynucleotide of the invention encodes: (i) a polypeptide having an amino acid sequence that is homologous to a Helicobacter amino acid sequence of a polypeptide associated with the Helicobacter membrane, the Helicobacter amino acid sequence being selected from the group consisting of the amino acid sequences shown:
-in SEQ ID NO:2, beginning with an amino acid in any one of positions -19 to 5, preferably in position -19 or position 1, and ending with an amino acid in position 689 (GHPO 386); -in SEQ ID NO: 4, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 713 (GHPO 789);
-in SEQ ID NO: 6, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 725 (GHPO 1516); -in SEQ ID NO: 8, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 691 (GHPO 1197);
-in SEQ ID NO: 10, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 652 (GHPO 1180);
-in SEQ ID NO: 12, beginning with an amino acid in any one of positions - 18 to 5, preferably in position - 18 or position 1 , and ending with an amino acid in position 673 (GHPO 896); -in SEQ ID NO: 14, beginning with an amino acid in any one of positions -21 to 5, preferably in position -21 or position 1, and ending with an amino acid in position 619 (GHPO 711);
-in SEQ ID NO: 16, beginning with an amino acid in any one of positions -17 to 5, preferably in position -17 or position 1, and ending with an amino acid in position 635 (GHPO 190);
-in SEQ ID NO: 18, beginning with an amino acid in any one of positions -19 to 5, preferably in position -19 or position 1, and ending with an amino acid in position 626 (GHPO 185);
-in SEQ ID NO:20, beginning with an amino acid in any one of positions -16 to 5, preferably in position -16 or position 1, and ending with an amino acid in position 467 (GHPO 1417);
-in SEQ ID NO: 22, beginning with an amino acid in any one of positions -18 to 5, preferably in position -18 or position 1 , and ending with an amino acid in position 673 (GHPO 1414); -in SEQ ID NO:66, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 279 (GHPO 1360); and -in SEQ ID NO:68, beginning with an amino acid in position 1 and ending with an amino acid in position 399 (GHPO 750); or (ii) a derivative of the polypeptide.
The term "isolated polynucleotide" is defined as a polynucleotide that is removed from the environment in which it naturally occurs. For example, a naturally- occurring DNA molecule present in the genome of a living bacteria or as part of a gene bank is not isolated, but the same molecule, separated from the remaining part of the bacterial genome, as a result of, e.g., a cloning event (amplification), is "isolated." Typically, an isolated DNA molecule is free from DNA regions (e.g., coding regions) with which it is immediately contiguous, at the 5' or 3' ends, in the naturally occurring genome. Such isolated polynucleotides can be part of a vector or a composition and still be isolated, as such a vector or composition is not part of its natural environment. A polynucleotide of the invention can consist of RNA or DNA (e.g., cDNA, genomic DNA, or synthetic DNA), or modifications or combinations of RNA or DNA. The polynucleotide can be double-stranded or single-stranded and, if single-stranded, can be the coding (sense) strand or the non-coding (antisense) strand. The sequences that encode polypeptides of the invention, as shown in SEQ ID NOs:2-22 (even numbers), 66, and 68, can be (a) the coding sequence as shown in SEQ ID NOs: 1-21 (odd numbers), 65, and 67; (b) a ribonucleotide sequence derived by transcription of (a); or (c) a different coding sequence that, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptides as the polynucleotide molecules having the sequences illustrated in any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67. The polypeptides of the invention can be ones that are naturally secreted or excreted by, e.g., H. felis, H. mustelae, H. heilmanii, or H. pylori. By "polypeptide" or "protein" is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Both terms are used interchangeably in the present application. By "homologous amino acid sequence" is meant an amino acid sequence that differs from an amino acid sequence shown in any of SEQ ID NOs:2-22 (even numbers), 66, and 68, or an amino acid sequence encoded by the nucleotide sequence of any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67, by one or more non-conservative amino acid substitutions, deletions, or additions located at positions at which they do not destroy the specific antigenicity of the polypeptide. Preferably, such a sequence is at least 75%, more preferably at least 80%, and most preferably at least 90% identical to an amino acid sequence shown in any of SEQ ID NOs:2-22 (even numbers), 66, and 68. Homologous amino acid sequences include sequences that are identical or substantially identical to an amino acid sequence as shown in any of SEQ ID NOs:2-22 (even numbers), 66, and 68. By "amino acid sequence that is substantially identical" is meant a sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, and most preferably at least 99% identical to an amino acid sequence of reference and that differs from the sequence of reference, if at all, by a majority of conservative amino acid substitutions.
Conservative amino acid substitutions typically include substitutions among amino acids of the same class. These classes include, for example, amino acids having uncharged polar side chains, such as asparagine, glutamine, serine, threonine, and tyrosine; amino acids having basic side chains, such as lysine, arginine, and histidine; amino acids having acidic side chains, snch as aspartic acid and glutamic acid; and amino acids having nonpolar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine.
Homology can be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Similar amino acid sequences are aligned to obtain the maximum degree of homology (i.e., identity). To this end, it may be necessary to artificially introduce gaps into the sequence. Once the optimal alignment has been set up, the degree of homology (i.e., identity) is established by recording all of the positions in which the amino acids of both sequences are identical, relative to the total number of positions.
Homologous polynucleotide sequences are defined in a similar way. Preferably, a homologous sequence is one that is at least 45%, more preferably at least 60%, and most preferably at least 85% identical to a coding sequence of any of SEQ ID NOs: l-21 (odd numbers), 65, and 67.
Polypeptides having a sequence homologous to one of the sequences shown in SEQ ID NOs:2-22 (even numbers), 66, and 68 include naturally- occurring allelic variants, as well as mutants or any other non-naturally occurring variants that are analogous in terms of antigenicity, to a polypeptide having a sequence as shown in SEQ ID NOs:2-22 (even numbers), 66, and 68.
As is known in the art, an allelic variant is an alternate form of a polypeptide that is characterized as having a substitution, deletion, or addition of one or more amino acids that does not alter the biological function of the polypeptide. By "biological function" is meant a function of the polypeptide in the cells in which it naturally occurs, even if the function is not necessary for the growth or survival of the cells. For example, the biological function of a porin is to allow the entry into cells of compounds present in the extracellular medium. The biological function is distinct from the antigenic function. A polypeptide can have more than one biological function.
Allelic variants are very common in nature. For example, a bacterial species, e.g., H. pylori, is usually represented by a variety of strains that differ from each other by minor allelic variations. Indeed, a polypeptide that fulfills the same biological function in different strains can have an amino acid sequence that is not identical in each of the strains. Such an allelic variation can be equally reflected at the polynucleotide level. Support for the use of allelic variants of polypeptide antigens comes from, e.g., studies of the Helicobacter urease antigen. The amino acid sequence of Helicobacter urease varies widely from species to species, yet cross-species protection occurs, indicating that the urease molecule, when used as an immunogen, is highly tolerant of amino acid variations. Even among different strains of the single species H. pylori, there are amino acid sequence variations.
For example, although the amino acid sequences of the UreA and UreB subunits of H. pylori and H. felis ureases differ from one another by 26.5% and 11.8%, respectively (Ferrero et al, Molecular Microbiology 9(2):323-333, 1993), it has been shown that H. pylori urease protects mice from H. felis infection (Michetti et al, Gastroenterology 107:1002, 1994). In addition, it has been shown that the individual structural subunits of urease, UreA and UreB, which contain distinct amino acid sequences, are both protective antigens against Helicobacter infection (Michetti et al, supra). Similarly, Cuenca et al. (Gastroenterology 110:1770, 1996) showed that therapeutic immunization of H. wMste/αe-infected ferrets with H. pylori urease was effective at eradicating H. mustelae infection. Further, several urease variants have been reported to be effective vaccine antigens, including, e.g., recombinant UreA + UreB apoenzyme expressed from pORV142 (UreA and UreB sequences derived from H. pylori strain CPM630; Lee et al, J. Infect. Dis.l72: 161, 1995); recombinant UreA + UreB apoenzyme expressed from pORV214 (UreA and UreB sequences differ from H. pylori strain CPM630 by one and two amino acid changes, respectively; Lee et al, supra, 1995); a UreA-glutathione-S-transferase fusion protein (UreA sequence from H. pylori strain ATCC 43504; Thomas et al, Acta Gastro-Enterologica Belgica 56:54, 1993); UreA + UreB holoenzyme purified from H. pylori strain NCTC11637 (Marchetti et al, Science 267: 1655, 1995); a UreA-MBP fusion protein (UreA from H. pylori strain 85P; Ferrero et al, Infection and Immunity 62:4981 , 1994); a UreB-MBP fusion protein (UreB from H. pylori strain 85P; Ferrero et al, supra); a UreA-MBP fusion protein (UreA from H. felis strain ATCC 49179; Ferrero et al, supra); a UreB-MBP fusion protein (UreB from H. felis strain ATCC 49179; Ferrero et al, supra); and a 37 kDa fragment of UreB containing amino acids 220-569 (Dore-Davin et al, "A 37 kD fragment of UreB is sufficient to confer protection against Helicobacter felis infection in mice"). Finally, Thomas et al. (supra) showed that oral immunization of mice with crude sonicates of H. pylori protected mice from subsequent challenge with H. felis.
Polynucleotides, e.g., DNA molecules, encoding allelic variants can easily be obtained by polymerase chain reaction (PCR) amplification of genomic bacterial DNA extracted by conventional methods. This involves the use of synthetic oligonucleotide primers matching sequences that are upstream and downstream of the 5' and 3' ends of the coding region. Suitable primers can be designed based on the nucleotide sequence information provided in SEQ ID NOs: 1-21 (odd numbers), 65, and 67. Typically, a primer consists of 10 to 40, preferably 15 to 25 nucleotides. It can also be advantageous to select primers containing C and G nucleotides in proportions sufficient to ensure efficient hybridization, e.g., an amount of C and G nucleotides of at least 40%, preferably 50%, of the total nucleotide amount. Those skilled in the art can readily design primers that can be used to isolate the polynucleotides of the invention from different Helicobacter strains.
As an example, primers useful for cloning a polynucleotide molecule encoding a polypeptide having the amino acid sequence of unprocessed GHPO 386 (SEQ ID NO:2), including a signal peptide, are shown in SEQ ID NO:23 (matching at the 5' end) and in SEQ ID NO:25 (matching at the 3' end).
Primers useful for cloning a DNA molecule encoding a polypeptide having the amino acid sequence of mature GHPO 386 (amino acids 1-689 of SEQ ID NO:2), lacking a signal peptide, are shown in SEQ ID NO:24 (matching at the 5' end) and in SEQ ID NO:25 (matching at the 3' end). Primers useful for cloning a DNA molecule encoding a polypeptide having the amino acid sequence of GHPO 1360 (SEQ ID NO:66), are shown in SEQ ID NO:78 (matching at the 5' end) and in SEQ ID NO: 79 (matching at the 3' end). Use of these primers enables amplification of the entire gene encoding GHPO 1360. Primers having sequences shown in SEQ ID NO: 82 (matching at the 5' end of the coding sequence corresponding to the mature protein) and SEQ ID NO:79 (matching at the 3' end) can be used to amplify the portion of the gene encoding mature GHPO 1360. Experimental conditions for carrying out PCR can readily be determined by one skilled in the art and illustrations of carrying out PCR are provided in Examples 3 and 4. Thus, the first aspect of the invention includes: (i) isolated polynucleotide molecules (e.g., DNA molecules) that can be amplified and/or cloned using the polymerase chain reaction from a Helicobacter, e.g., H. pylori, genome using either:
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:23, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
NO:25 (unprocessed GHPO 386);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:26, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:28 (unprocessed GHPO 789); - a 5' oligonucleotide primer having a sequence as shown in SEQ ID
NO:29, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:31 (unprocessed GHPO 1516);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:32, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:34 (unprocessed GHPO 1197);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:35, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:37 (unprocessed GHPO 1180);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:38, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
NO:40 (unprocessed GHPO 896);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:41, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:43 (unprocessed GHPO 711); - a 5' oligonucleotide primer having a sequence as shown in SEQ ID
NO: 44, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:46 (unprocessed GHPO 190); - a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:47, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:49 (unprocessed GHPO 185);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:50, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
NO: 52 (unprocessed GHPO 1417);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO: 53, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:55 (unprocessed GHPO 1414); - a 5' oligonucleotide primer having a sequence as shown in SEQ ID
NO: 78, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:79 (unprocessed GHPO 1360); or
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO: 80, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO: 81 (GHPO 750); and
(ii) isolated polynucleotide molecules (e.g., DNA molecules) that can be amplified and/or cloned by the polymerase chain reaction from a Helicobacter, e.g., H. pylori, genome using either:
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:24, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
NO:25 (mature GHPO 386);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:27, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:28 (mature GHPO 789); - a 5' oligonucleotide primer having a sequence as shown in SEQ ID
NO:30, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:31 (mature GHPO 1516); - a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:33, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:34 (mature GHPO 1197);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO: 36, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
NO:37 (mature GHPO 1180);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO: 39, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:40 (mature GHPO 896); - a 5' oligonucleotide primer having a sequence as shown in SEQ ID
NO:42, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:43 (mature GHPO 711);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO: 45, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:46 (mature GHPO 190);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:48, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:49 (mature GHPO 185);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:51, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
NO:52 (mature GHPO 1417);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO: 54, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:55 (mature GHPO 1414); or - a 5' oligonucleotide primer having a sequence as shown in SEQ ID
NO: 82, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:79 (mature GHPO 1360). The 5' ends of the primers described above can advantageously include a restriction endonuclease recognition site that contains, typically, 4 to 6 nucleotides. For example, the sequences 5'-GGATCC-3' (BamHT) or 5'- CTCGAG-3' (Xhol) can be used. Restriction sites can be selected by those skilled in the art so that the amplified DNA, when digested, if necessary, can be conveniently cloned into an appropriately digested vector, such as a plasmid vector. In addition, a 5' clamp (e.g., GCC) can be included in the primers 5' to the restriction endonuclease recognition site.
Useful homologs that do not occur naturally can be designed using known methods for identifying regions of an antigen that are likely to be tolerant of amino acid sequence changes and/or deletions. For example, sequences of the antigen from different species can be compared to identify conserved sequences.
Polypeptide derivatives that are encoded by polynucleotides of the invention include, e.g., fragments, polypeptides having large internal deletions derived from full-length polypeptides, and fusion proteins. Polypeptide fragments of the invention can be derived from a polypeptide having a sequence homologous to the sequences of any of SEQ ID NOs:2-22 (even numbers), 66, and 68, to the extent that the fragments retain the substantial antigenicity of the parent polypeptide (specific antigenicity). Polypeptide derivatives can also be constructed by large internal deletions that remove a substantial part of the parent polypeptide, while retaining specific antigenicity. Generally, polypeptide derivatives should be about at least 12 amino acids in length to maintain antigenicity. Advantageously, they can be at least 20 amino acids, preferably at least 50 amino acids, more preferably at least 75 amino acids, and most preferably at least 100 amino acids in length. Useflil polypeptide derivatives, e.g., polypeptide fragments, can be designed using computer-assisted analysis of amino acid sequences in order to identify sites in protein antigens having potential as surface-exposed, antigenic regions (Hughes et al, Infect. Immun. 60(9):3497, 1992). For example, the Laser Gene Program from DNA Star can be used to obtain hydrophilicity, antigenic index, and intensity index plots for the polypeptides of the invention. This program can also be used to obtain information about homologies of the polypeptides with known protein motifs. One skilled in the art can readily use the information provided in such plots to select peptide fragments for use as vaccine antigens. For example, fragments spanning regions of the plots in which the antigenic index is relatively high can be selected. One can also select fragments spanning regions in which both the antigenic index and the intensity plots are relatively high. Fragments containing conserved sequences, particularly hydrophilic conserved sequences, can also be selected. Polypeptide fragments and polypeptides having large internal deletions can be used for revealing epitopes that are otherwise masked in the parent polypeptide and that may be of importance for inducing a protective T cell-dependent immune response. Deletions can also remove immunodominant regions of high variability among strains. It is an accepted practice in the field of immunology to use fragments and variants of protein immunogens as vaccines, as all that is required to induce an immune response to a protein is a small (e.g., 8 to 10 amino acids) immunogenic region of the protein. This has been done for a number of vaccines against pathogens other than Helicobacter. For example, short synthetic peptides corresponding to surface-exposed antigens of pathogens such as murine mammary tumor virus (peptide containing 11 amino acids; Dion et al, Virology 179:474-477, 1990), Semliki Forest virus (peptide containing 16 amino acids; Snijders et al, J. Gen. Virol. 72:557-565, 1991), and canine parvovirus (2 overlapping peptides, each containing 15 amino acids; Langeveld et al, Vaccine 12(15): 1473-1480, 1994) have been shown to be effective vaccine antigens against their respective pathogens. Polynucleotides encoding polypeptide fragments and polypeptides having large internal deletions can be constructed using standard methods (see, e.g., Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994), for example, by PCR, including inverse PCR, by restriction enzyme treatment of the cloned DNA molecules, or by the method of Kunkel et al (Proc. Natl. Acad. Sci. USA 82:448, 1985; biological material available at Stratagene).
A polypeptide derivative can also be produced as a fusion polypeptide that contains a polypeptide or a polypeptide derivative of the invention fused, e.g., at the - or C-terminal end, to any other polypeptide (hereinafter referred to as a peptide tail). Such a product can be easily obtained by translation of a genetic fusion, i.e., a hybrid gene. Vectors for expressing fusion polypeptides are commercially available, and include the pMal-c2 or pMal-p2 systems of New England Biolabs, in which the peptide tail is a maltose binding protein, the glutathione-S-transferase system of Pharmacia, or the His-Tag system available from Novagen. These and other expression systems provide convenient means for further purification of polypeptides and derivatives of the invention.
Another particular example of fusion polypeptides included in invention includes a polypeptide or polypeptide derivative of the invention fused to a polypeptide having adjuvant activity, such as, e.g., subunit B of either cholera toxin or E. coli heat-labile toxin. Several possibilities can be used for producing such fusion proteins. First, the polypeptide of the invention can be fused to the
N-terminal end or, preferably, to the C-terminal end of the polypeptide having adjuvant activity. Second, a polypeptide fragment of the invention can be fused within the amino acid sequence of the polypeptide having adjuvant activity. Spacer sequences can also be included, if desired.
As stated above, the polynucleotides of the invention encode Helicobacter polypeptides in precursor or mature form. They can also encode hybrid precursors containing heterologous signal peptides, which can mature into polypeptides of the invention. By "heterologous signal peptide" is meant a signal peptide that is not found in the naturally-occurring precursor of a polypeptide of the invention.
A polynucleotide of the invention hybridizes, preferably under stringent conditions, to a polynucleotide having a sequence as shown in any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67. Hybridization procedures are, e.g., described by Ausubel et al. (supra); Silhavy et al. (Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1984); and Davis et al. (A Manual for Genetic Engineering: Advanced Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1980). Important parameters that can be considered for optimizing hybridization conditions are reflected in the following formula, which facilitates calculation of the melting temperature (Tm), which is the temperature above which two complementary DNA strands separate from one another (Casey et al, Nucl. Acid Res. 4: 1539, 1977): Tm = 81.5 + 0.5 -x (% G+C) + 1.6 log (positive ion concentration) - 0.6 x (% formamide). Under appropriate stringency conditions, hybridization temperature (Th) is approximately 20 to 40°C, 20 to 25 °C, or, preferably, 30 to 40°C below the calculated Tm. Those skilled in the art will understand that optimal temperature and salt conditions can be readily determined empirically in preliminary experiments using conventional procedures. For example, stringent conditions can be achieved, both for pre-hybridizing and hybridizing incubations, (i) within 4-16 hours at 42 °C, in 6 x SSC containing
50% formamide or (ii) within 4-16 hours at 65 °C in an aqueous 6 x SSC solution (1 M NaCl, 0.1 M sodium citrate (pH 7.0)). For polynucleotides containing 30 to 600 nucleotides, the above formula is used and then is coπected by subtracting (600/polynucleotide size in base pairs). Stringency conditions are defined by a Th that is 5 to 10 °C below Tm.
Hybridization conditions with oligonucleotides shorter than 20-30 bases do not precisely follow the rules set forth above. In such cases, the formula for calculating the Tm is as follows: Tm = 4 x (G+C) + 2 (A+T). For example, an 18 nucleotide fragment of 50% G+C would have an approximate Tm of 54°C.
A polynucleotide molecule of the invention, containing RNA, DNA, or modifications or combinations thereof, can have various applications. For example, a polynucleotide molecule can be used (i) in a process for producing the encoded polypeptide in a recombinant host system, (ii) in the construction of vaccine vectors such as poxviruses, which are further used in methods and compositions for preventing and/or treating Helicobacter infection, (iii) as a vaccine agent, in a naked form or formulated with a delivery vehicle, and (iv) in the construction of attenuated Helicobacter strains that can over-express a polynucleotide of the invention or express it in a non-toxic, mutated form. According to a second aspect of the invention, there is therefore provided (i) an expression cassette containing a polynucleotide molecule of the invention placed under the control of elements (e.g., a promoter) required for expression; (ii) an expression vector containing an expression cassette of the invention; (iii) a procaryotic or eucaryotic cell transformed or transfected with an expression cassette and or vector of the invention; as well as (iv) a process for producing a polypeptide or polypeptide derivative encoded by a polynucleotide of the invention, which involves culturing a procaryotic or eucaryotic cell transformed or transfected with an expression cassette and/or vector of the invention, under conditions that allow expression of the polynucleotide molecule of the invention and, recovering the encoded polypeptide or polypeptide derivative from the cell culture. A recombinant expression system can be selected from procaryotic and eucaryotic hosts. Eucaryotic hosts include, for example, yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris), mammalian cells (e.g., COS1, NIH3T3, or JEG3 cells), arthropods cells (e.g., Spodoptera frugiperda (SF9) cells), and plant cells. Preferably, a procaryotic host such as E. coli is used. Bacterial and eucaryotic cells are available from a number of different sources that are known to those skilled in the art, e.g., the American Type Culture Collection (ATCC; Rockville, Maryland).
The choice of the expression cassette will depend on the host system selected, as well as the features desired for the expressed polypeptide. For example, it may be useful to produce a polypeptide of the invention in a particular lipidated form or any other form. Typically, an expression cassette includes a constitutive or inducible promoter that is functional in the selected host system; a ribosome binding site; a start codon (ATG); if necessary, a region encoding a signal peptide, e.g., a lipidation signal peptide; a polynucleotide molecule of the invention; a stop codon; and, optionally, a 3' terminal region (translation and/or transcription terminator). The signal pepti de-encoding region is adjacent to the polynucleotide of the invention and is placed in the proper reading frame. The signal pepti de-encoding region can be homologous or heterologous to the polynucleotide molecule encoding the mature polypeptide and it can be specific to the secretion apparatus of the host used for expression. The open reading frame constituted by the polynucleotide molecule of the invention, alone or together with the signal peptide, is placed under the control of the promoter so that transcription and translation occur in the host system. Promoters and signal peptide-encoding regions are widely known and available to those skilled in the art and include, for example, the promoter of Salmonella typhimurium (and derivatives) that is inducible by arabinose (promoter araB) and is functional in Gram-negative bacteria such as E. coli (U.S. Patent No. 5,028,530; Cagnon et al, Protein Engineering 4(7): 843, 1991); the promoter of the bacteriophage T7 RNA polymerase gene, which is functional in a number of E. coli strains expressing T7 polymerase (U.S. Patent No. 4,952,496); the OspA lipidation signal peptide; and RlpB lipidation signal peptide (Takase et al, J. Bact. 169:5692, 1987).
The expression cassette is typically part of an expression vector, which is selected for its ability to replicate in the chosen expression system. Expression vectors (e.g., plasmids or viral vectors) can be chosen from, for example, those described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985, Supp. 1987) and can purchased from various commercial sources. Methods for transforming or transfecting host cells with expression vectors are well known in the art and will depend on the host system selected, as described in Ausubel et al (supra).
Upon expression, a recombinant polypeptide of the invention (or a polypeptide derivative) is produced and remains in the intracellular compartment, is secreted/excreted in the extracellular medium or in the periplasmic space, or is embedded in the cellular membrane. The polypeptide can then be recovered in a substantially purified form from the cell extract or from the supernatant after centrifugation of the cell culture. Typically, the recombinant polypeptide can be purified by antibody-based affinity purification or by any other method known to a person skilled in the art, such as by genetic fusion to a small affinity-binding domain. Antibody-based affinity purification methods are also available for purifying a polypeptide of the invention extracted from a Helicobacter strain. Antibodies useful for immunoaffinity purification of the polypeptides of the invention can be obtained using methods described below. Polynucleotides of the invention can also be used in DNA vaccination methods, using either a viral or bacterial host as gene delivery vehicle (live vaccine vector) or administering the gene in a free form, e.g., inserted into a plasmid. Therapeutic or prophylactic efficacy of a polynucleotide of the invention can be evaluated as is described below. Accordingly, in a third aspect of the invention, there is provided (i) a vaccine vector such as a poxvirus, containing a polynucleotide molecule of the invention placed under the control of elements required for expression; (ii) a composition of matter containing a vaccine vector of the invention, together with a diluent or carrier; (iii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a vaccine vector of the invention; (iv) a method for inducing an immune response against Helicobacter in a mammal (e.g., a human; alternatively, the method can be used in veterinary applications for treating or preventing Helicobacter infection of animals, e.g., cats or birds), which involves administering to the mammal an immunogenically effective amount of a vaccine vector of the invention to elicit an immune response, e.g., a protective or therapeutic immune response to Helicobacter; and (v) a method for preventing and/or treating a Helicobacter (e.g., H. pylori, H. felis, H. mustelae, or H. heilmanii) infection, which involves administering a prophylactic or therapeutic amount of a vaccine vector of the invention to an individual in need. Additionally, the third aspect of the invention encompasses the use of a vaccine vector of the invention in the preparation of a medicament for preventing and/or treating Helicobacter infection.
A vaccine vector of the invention can express one or several polypeptides or derivatives of the invention, as well as at least one additional Helicobacter antigen such as a urease apoenzyme or a subunit, fragment, homolog, mutant, or derivative thereof. In addition, it can express a cytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12), that enhances the immune response. Thus, a vaccine vector can include an additional polynucleotide molecules encoding, e.g., urease subunit A, B, or both, or a cytokine, placed under the control of elements required for expression in a mammalian cell.
Alternatively, a composition of the invention can include several vaccine vectors, each of which being capable of expressing a polypeptide or derivative of the invention. A composition can also contain a vaccine vector capable of expressing an additional Helicobacter antigen such as urease apoenzyme, a subunit, fragment, homolog, mutant, or derivative thereof, or a cytokine such as IL-2 or IL-12.
In vaccination methods for treating or preventing infection in a mammal, a vaccine vector of the invention can be administered by any conventional route in use in the vaccine field, for example, to a mucosal (e.g., ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract) surface or via a parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route. Prefened routes depend upon the choice of the vaccine vector. The administration can be achieved in a single dose or repeated at intervals. The appropriate dosage depends on various parameters that are understood by those skilled in the art, such as the nature of the vaccine vector itself, the route of administration, and the condition of the mammal to be vaccinated (e.g., the weight, age, and general health of the mammal).
Live vaccine vectors that can be used in the invention include viral vectors, such as adenoviruses and poxviruses, as well as bacterial vectors, e.g., Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bilie de Calmette- Guerin (BCG), and Streptococcus. An example of an adenovirus vector, as well as a method for constructing an adenovirus vector capable of expressing a polynucleotide molecule of the invention, is described in U.S. Patent No. 4,920,209. Poxvirus vectors that can be used in the invention include, e.g., vaccinia and canary pox viruses, which are described in U.S. Patent No. 4,722,848 and U.S. Patent No. 5,364,773, respectively (also see, e.g., Tartaglia et al, Virology 188:217, 1992, for a description of a vaccinia virus vector, and Taylor et al, Vaccine 13:539, 1995, for a description of a canary poxvirus vector). Poxvirus vectors capable of expressing a polynucleotide of the invention can be obtained by homologous recombination, as described in Kieny et al (Nature 312: 163, 1984) so that the polynucleotide of the invention is inserted in the viral genome under appropriate conditions for expression in mammalian cells. Generally, the dose of viral vector vaccine, for therapeutic or prophylactic use, can be from about lxl 04 to about 1x10", advantageously from about lxlO7 to about lxlO10, or, preferably, from about lxlO7 to about lxl 09 plaque-forming units per kilogram. Preferably, viral vectors are administered parenterally, for example, in 3 doses that are 4 weeks apart. Those skilled in the art will recognize that it is preferable to avoid adding a chemical adjuvant to a composition containing a viral vector of the invention and thereby minimizing the immune response to the viral vector itself.
Non-toxicogenic Vibrio cholerae mutant strains that can be used in live oral vaccines are described by Mekalanos et al. (Nature 306:551, 1983) and in U.S. Patent No. 4,882,278 (strain in which a substantial amount of the coding sequence of each of the two ctxA alleles has been deleted so that no functional cholerae toxin is produced); WO 92/11354 (strain in which the z'rgA locus is inactivated by mutation; this mutation can be combined in a single strain with ctxA mutations); and WO 94/1533 (deletion mutant lacking functional ctxA and attRSl DNA sequences). These strains can be genetically engineered to express heterologous antigens, as described in WO 94/19482. An effective vaccine dose of a V. cholerae strain capable of expressing a polypeptide or polypeptide derivative encoded by a polynucleotide molecule of the invention can contain, e.g., about lxlO5 to about lxlO9, preferably about lxlO6 to about lxl 08 viable bacteria in an appropriate volume for the selected route of administration. Preferred routes of administration include all mucosal routes, but, most preferably, these vectors are administered intranasally or orally.
Attenuated Salmonella typhimurium strains, genetically engineered for recombinant expression of heterologous antigens, and their use as oral vaccines, are described by Nakayama et al. (Bio/Technology 6:693, 1988) and in WO 92/11361. Preferred routes of administration for these vectors include all mucosal routes. Most preferably, the vectors are administered intranasally or orally. Others bacterial strains useful as vaccine vectors are described by
High et al. (EMBO 11 : 1991, 1992) and Sizemore et al. (Science 270:299, 1995; Shigellaβexneri); Medaglini et al. (Proc. Natl. Acad. Sci. USA 92:6868, 1995; (Streptococcus gordonii); Flynn (Cell. Mol. Biol. 40 (suppl. I):31, 1194), and in WO 88/6626, WO 90/0594, WO 91/13157, WO 92/1796, and WO 92/21376 (Bacille Calmette Guerin). In bacterial vectors, a polynucleotide of the invention can be inserted into the bacterial genome or it can remain in a free state, for example, carried on a plasmid.
An adjuvant can also be added to a composition containing a bacterial vector vaccine. A number of adjuvants that can be used are known to those skilled in the art. For example, preferred adjuvants can be selected from the list provided below. According to a fourth aspect of the invention, there is also provided
(i) a composition of matter containing a polynucleotide of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a polynucleotide of the invention; (iii) a method for inducing an immune response against Helicobacter, in a mammal, by administering to the mammal an immunogenically effective amount of a polynucleotide of the invention to elicit an immune response, e.g., a protective immune response to Helicobacter; and (iv) a method for preventing and or treating a Helicobacter (e.g., H. pylori, H. felis, H. mustelae, or H. heilmanii) infection, by administering a prophylactic or therapeutic amount of a polynucleotide of the invention to an individual in need of such treatment. Additionally, the fourth aspect of the invention encompasses the use of a polynucleotide of the invention in the preparation of a medicament for preventing and/or treating Helicobacter infection. The fourth aspect of the invention preferably includes the use of a polynucleotide molecule placed under conditions for expression in a mammalian cell, e.g., in a plasmid that is unable to replicate in mammalian cells and to substantially integrate into a mammalian genome. Polynucleotides (for example, DNA or RNA molecules) of the invention can also be administered as such to a mammal as a vaccine. When a DNA molecule of the invention is used, it can be in the form of a plasmid that is unable to replicate in a mammalian cell and unable to integrate into the mammalian genome. Typically, a DNA molecule is placed under the control of a promoter suitable for expression in a mammalian cell. The promoter can function ubiquitously or tissue-specifically. Examples of non-tissue specific promoters include the early Cytomegalovirus (CMV) promoter (U.S. Patent No. 4,168,062) and the Rous Sarcoma Virus promoter (Norton et al, Molec. Cell Biol. 5:281, 1985). The desmin promoter (Li et al, Gene 78:243, 1989; Li et al, J. Biol. Chem. 266:6562, 1991 ; Li et al, J. Biol. Chem. 268: 10403, 1993) is tissue-specific and drives expression in muscle cells. More generally, useful promoters and vectors are described, e.g., in WO 94/21797 and by Hartikka et al. (Human Gene Therapy 7: 1205, 1996). For DNA/RNA vaccination, the polynucleotide of the invention can encode a precursor or a mature form of a polypeptide of the invention. When it encodes a precursor form, the precursor sequence can be homologous or heterologous. In the latter case, a eucaryotic leader sequence can be used, such as the leader sequence of the tissue-type plasminogen factor (tPA). A composition of the invention can contain one or several polynucleotides of the invention. It can also contain at least one additional polynucleotide encoding another Helicobacter antigen, such as urease subunit A, B, or both, or a fragment, derivative, mutant, or analog thereof. A polynucleotide encoding a cytokine, such as interleukin-2 (IL-2) or interleukin- 12 (IL-12), can also be added to the composition so that the immune response is enhanced. These additional polynucleotides are placed under appropriate control for expression. Advantageously, DNA molecules of the invention and/or additional DNA molecules to be included in the same composition are earned in the same plasmid.
Standard methods can be used in the preparation of therapeutic polynucleotides of the invention. For example, a polynucleotide can be used in a naked form, free of any delivery vehicles, such as anionic liposomes, cationic lipids, microparticles, e.g., gold microparticles, precipitating agents, e.g., calcium phosphate, or any other transfection-facilitating agent. In this case, the polynucleotide can be simply diluted in a physiologically acceptable solution, such as sterile saline or sterile buffered saline, with or without a carrier. When present, the caπier preferably is isotonic, hypotonic, or weakly hypertonic, and has a relatively low ionic strength, such as provided by a sucrose solution, e.g., a solution containing 20% sucrose.
Alternatively, a polynucleotide can be associated with agents that assist in cellular uptake. It can be, e.g., (i) complemented with a chemical agent that modifies cellular permeability, such as bupivacaine (see, e.g.,
WO 94/16737), (ii) encapsulated into liposomes, or (iii) associated with cationic lipids or silica, gold, or tungsten microparticles.
Anionic and neutral liposomes are well-known in the art (see, e.g.,
Liposomes: A Practical Approach, RPC New Ed, IRL Press, 1990, for a detailed description of methods for making liposomes) and are useful for delivering a large range of products, including polynucleotides.
Cationic lipids can also be used for gene delivery. Such lipids include, for example, Lipofectin™, which is also known as DOTMA (N-[l-
(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (1,2- bis(oleyloxy)-3-(trimethylammonio)propane), DDAB
(dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycyl spermine), and cholesterol derivatives. A description of these cationic lipids can be found in EP 187,702, WO 90/11092, U.S. Patent No. 5,283,185, WO 91/15501, WO 95/26356, and U.S. Patent No. 5,527,928. Cationic lipids for gene delivery are preferably used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine; WO 90/11092). Other transfection- facilitating compounds can be added to a formulation containing cationic liposomes. A number of them are described in, e.g., WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/2397. They include, e.g., spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane (see, for example, WO 93/18759) and membrane-permeabilizing compounds such as GALA, Gramicidine S, and cationic bile salts (see, for example, WO 93/19768).
Gold or tungsten microparticles can also be used for gene delivery, as described in WO 91/359, WO 93/17706, and by Tang et al. (Nature 356: 152, 1992). In this case, the microparticle-coated polynucleotides can be injected via intradermal or intraepidermal routes using a needleless injection device
("gene gun"), such as those described in U.S. Patent No. 4,945,050, U.S. Patent No. 5,015,580, and WO 94/24263.
The amount of DNA to be used in a vaccine recipient depends, e.g., on the strength of the promoter used in the DNA construct, the immunogenicity of the expressed gene product, the condition of the mammal intended for administration (e.g., the weight, age, and general health of the mammal), the mode of administration, and the type of formulation. In general, a therapeutically or prophylactically effective dose from about 1 μg to about 1 mg, preferably, from about 10 μg to about 800 μg, and, more preferably, from about 25 μg to about 250 μg, can be administered to human adults. The administration can be achieved in a single dose or repeated at intervals. The route of administration can be any conventional route used in the vaccine field. As general guidance, a polynucleotide of the invention can be administered via a mucosal surface, e.g., an ocular, intranasal, pulmonary, oral, intestinal, rectal, vaginal, or urinary tract surface, or via a parenteral route, e.g., by an intravenous, subcutaneous, intraperitoneal, intradermal, intraepidermal, or intramuscular route. The choice of administration route will depend on, e.g., the formulation that is selected. A polynucleotide formulated in association with bupivacaine is advantageously administered into muscle. When a neutral or anionic liposome or a cationic lipid, such as DOTMA, is used, the formulation can be advantageously injected via intravenous, intranasal (for example, by aerosolization), intramuscular, intradermal, and subcutaneous routes. A polynucleotide in a naked form can advantageously be administered via the intramuscular, intradermal, or subcutaneous routes. Although not absolutely required, such a composition can also contain an adjuvant. A systemic adjuvant that does not require concomitant administration in order to exhibit an adjuvant effect is preferable.
The sequence information provided in the present application enables the design of specific nucleotide probes and primers that can be used in diagnostic methods. Accordingly, in a fifth aspect of the invention, there is provided a nucleotide probe or primer having a sequence found in, or derived by degeneracy of the genetic code from, a sequence shown in any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67, or a complementary sequence thereof. ' The term "probe" as used in the present application refers to DNA (preferably single stranded) or RNA molecules (or modifications or combinations thereof) that hybridize under the stringent conditions, as defined above, to polynucleotide molecules having sequences homologous to those shown in any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67, or to a complementary or anti-sense sequence of any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67. Generally, probes are significantly shorter than the full- length sequences shown in any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67. For example, they can contain from about 5 to about 100, preferably from about 10 to about 80 nucleotides. In particular, probes have sequences that are at least 75%, preferably at least 85%, more preferably 95% homologous to a portion of a sequence as shown in any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67, or a sequence complementary to such sequences.
Probes can contain modified bases, such as inosine, methyl-5- deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, or diamino-2, 6- purine. Sugar or phosphate residues can also be modified or substituted. For example, a deoxyribose residue can be replaced by a poly amide (Nielsen et al, Science 254: 1497, 1991) and phosphate residues can be replaced by ester groups such as diphosphate, alkyl, arylphosphonate, and phosphorothioate esters. In addition, the 2'-hydroxyl group on ribonucleotides can be modified by addition of, e.g., alkyl groups.
Probes of the invention can be used in diagnostic tests, or as capture or detection probes. Such capture probes can be immobilized on solid supports, directly or indirectly, by covalent means or by passive adsoφtion. A detection probe can be labeled by a detectable label, for example a label selected from radioactive isotopes; enzymes, such as peroxidase and alkaline phosphatase; enzymes that are able to hydrolyze a chromogenic, fluorogenic, or luminescent substrate; compounds that are chromogenic, fluorogenic, or luminescent; nucleotide base analogs; and biotin. Probes of the invention can be used in any conventional hybridization method, such as in dot blot methods (Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1982), Southern blot methods (Southern, J. Mol. Biol. 98:503, 1975), northern blot methods (identical to Southern blot to the exception that RNA is used as a target), or a sandwich method (Dunn et al, Cell 12:23, 1977). As is known in the art, the latter technique involves the use of a specific capture probe and a specific detection probe that have nucleotide sequences that are at least partially different from each other.
Primers used in the invention usually contain about 10 to 40 nucleotides and are used to initiate enzymatic polymerization of DNA in an amplification process (e.g., PCR), an elongation process, or a reverse transcription method. In a diagnostic method involving PCR, the primers can be labeled.
Thus, the invention also encompasses (i) a reagent containing a probe of the invention for detecting and/or identifying the presence of Helicobacter in a biological material; (ii) a method for detecting and/or identifying the presence of Helicobacter in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA or RNA is extracted from the material and denatured, and (c) the sample is exposed to a probe of the invention, for example, a capture probe, a detection probe, or both, under stringent hybridization conditions, so that hybridization is detected; and (iii) a method for detecting and/or identifying the presence of Helicobacter in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA is extracted therefrom, (c) the extracted DNA is contacted with at least one, or, preferably two, primers of the invention, and amplified by the polymerase chain reaction, and (d) an amplified DNA - molecule is produced.
As mentioned above, polypeptides that can be produced by expression of the polynucleotides of the invention can be used as vaccine antigens. Accordingly, a sixth aspect of the invention features a substantially purified polypeptide or polypeptide derivative having an amino acid sequence encoded by a polynucleotide of the invention.
A "substantially purified polypeptide" is defined as a polypeptide that is separated from the environment in which it naturally occurs and/or a polypeptide that is free of most of the other polypeptides that are present in the environment in which it was synthesized. The polypeptides of the invention can be purified from a natural source, such as a Helicobacter strain, or can be produced using recombinant methods. Homologous polypeptides or polypeptide derivatives encoded by polynucleotides of the invention can be screened for specific antigenicity by testing cross-reactivity with an antiserum raised against a polypeptide having an amino acid sequence as shown in any of SEQ ID NOs:2-22 (even numbers), 66, and 68. Briefly, a monospecific hyperimmune antiserum can be raised against a purified reference polypeptide as such or as a fusion polypeptide, for example, an expression product of MBP, GST, or His-tag systems, or a synthetic peptide predicted to be antigenic. The homologous polypeptide or derivative that is screened for specific antigenicity can be produced as such or as a fusion polypeptide. In the latter case, and if the antiserum is also raised against a fusion polypeptide, two different fusion systems are employed.
Specific antigenicity can be determined using a number of methods, including Western blot (Towbin et al, Proc. Natl. Acad. Sci. USA 76:4350, 1979), dot blot, and ELISA methods, as described below.
In a Western blot assay, the product to be screened, either as a purified preparation or a total E. coli extract, is fractionated by SDS-PAGE, as described, for example, by Laemmli (Nature 227:680, 1970). After being transferred to a filter, such as a nitrocellulose membrane, the material is incubated with the monospecific hyperimmune antiserum, which is diluted in a range of dilutions from about 1 :50 to about 1 :5000, preferably from about 1 : 100 to about 1 :500. Specific antigenicity is shown once a band coπesponding to the product exhibits reactivity at any of the dilutions in the range.
In an ELISA assay, the product to be screened can be used as the coating antigen. A purified preparation is preferred, but a whole cell extract can also be used. Briefly, about 100 μl of a preparation of about 10 μg protein/ml is distributed into wells of a 96-well ELISA plate. The plate is incubated for about 2 hours at 37°C, then overnight at 4°C. The plate is washed with phosphate buffered saline (PBS) containing 0.05% Tween 20 (PBS/Tween buffer) and the wells are saturated with 250 μl PBS containing 1% bovine serum albumin (BSA), to prevent non-specific antibody binding. After 1 hour of incubation at 37 °C, the plate is washed with PBS/Tween buffer. The antiserum is serially diluted in PBS/Tween buffer containing 0.5% BSA, and 100 μl dilutions are added to each well. The plate is incubated for 90 minutes at 37 °C, washed, and evaluated using standard methods. For example, a goat anti-rabbit peroxidase conjugate can be added to the wells when the specific antibodies used were raised in rabbits. Incubation is carried out for about 90 minutes at 37° C and the plate is washed. The reaction is developed with the appropriate substrate and the reaction is measured by colorimetry (absorbance measured spectrophotometrically). Under these experimental conditions, a positive reaction is shown once an O.D. value of 1.0 is detected with a dilution of at least about 1 :50, preferably of at least about 1 :500.
In a dot blot assay, a purified product is prefened, although a whole cell extract can be used. Briefly, a solution of the product at a concentration of about 100 μg/ml is serially diluted two-fold with 50 mM Tris-HCl (pH 7.5). One hundred μl of each dilution is applied to a filter, such as a 0.45 μm nitrocellulose membrane, set in a 96- well dot blot apparatus (Biorad). The buffer is removed by applying vacuum to the system. Wells are washed by addition of 50 mM Tris-HCl (pH 7.5) and the membrane is air-dried. The membrane is saturated in blocking buffer (50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 10 g/L skim milk) and incubated with an antiserum diluted from about 1 : 50 to about 1 : 5000, preferably about 1 :500. The reaction is detected using standard methods. For example, a goat anti -rabbit peroxidase conjugate can be added to the wells when rabbit antibodies are used. Incubation is carried out for about 90 minutes at 37 °C and the blot is washed. The reaction is developed with the appropriate substrate and stopped. The reaction is then measured visually by the appearance of a colored spot, e.g., by colorimetry. Under these experimental conditions, a positive reaction is associated with detection of a colored spot for reactions carried out with a dilution of at least about 1 :50, preferably, of at least about 1 :500. Therapeutic or prophylactic efficacy of a polypeptide or polypeptide derivative of the invention can be evaluated as is described below.
According to a seventh aspect of the invention, there is provided (i) a composition of matter containing a polypeptide of the invention together with a diluent or carrier; (ii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a polypeptide of the invention; (iii) a method for inducing an immune response against Helicobacter in a mammal by administering to the mammal an immunogenically effective amount of a polypeptide of the invention to elicit an immune response, e.g., a protective immune response to Helicobacter; and (iv) a method for preventing and/or treating a Helicobacter (e.g., H. pylori, H. felis, H. mustelae, orH. heilmanii) infection, by administering a prophylactic or therapeutic amount of a polypeptide of the invention to an individual in need of such treatment. Additionally, this aspect of the invention includes the use of a polypeptide of the invention in the preparation of a medicament for preventing and/or treating Helicobacter infection.
The immunogenic compositions of the invention can be administered by any conventional route in use in the vaccine field, for example, to a mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface or via a parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route. The choice of the administration route depends upon a number of parameters, such as the adjuvant used. For example, if a mucosal adjuvant is used, the intranasal or oral route will be prefened, and if a lipid formulation or an aluminum compound is used, a parenteral route will be preferred. In the latter case, the subcutaneous or intramuscular route is most preferred. The choice of administration route can also depend upon the nature of the vaccine agent. For example, a polypeptide of the invention fused to CTB or to LTB will be best administered to a mucosal surface.
A composition of the invention can contain one or several polypeptides or derivatives of the invention. It can also contain at least one additional Helicobacter antigen, such as the urease apoenzyme, or a subunit, fragment, homolog, mutant, or derivative thereof.
For use in a composition of the invention, a polypeptide or polypeptide derivative can be formulated into or with liposomes, such as neutral or anionic liposomes, microspheres, ISCOMS, or virus-like particles (VLPs), to facilitate delivery and/or enhance the immune response. These compounds are readily available to those skilled in the art; for example; see Liposomes: A Practical Approach (supra). Adjuvants other than liposomes can also be used in the invention and are well known in the art (see, for example, the list provided below).
Administration can be achieved in a single dose or repeated as necessary at intervals that can be determined by one skilled in the art. For example, a priming dose can be followed by three booster doses at weekly or monthly intervals. An appropriate dose depends on various parameters, including the nature of the recipient (e.g., whether the recipient is an adult or an infant), the particular vaccine antigen, the route and frequency of administration, the presence/absence or type of adjuvant, and the desired effect (e.g., protection and/or treatment), and can be readily determined by one skilled in the art. In general, a vaccine antigen of the invention can be administered mucosally in an amount ranging from about 10 μg to about 500 mg, preferably from about 1 mg to about 200 mg. For a parenteral route of administration, the dose usually should not exceed about 1 mg, and is, preferably, about 100 μg. When used as components of a vaccine, the polynucleotides and polypeptides of the invention can be used sequentially as part of a multi-step immunization process. For example, a mammal can be initially primed with a vaccine vector of the invention, such as a pox virus, e.g., via a parenteral route, and then boosted twice with a polypeptide encoded by the vaccine vector, e.g., via the mucosal route. In another example, liposomes associated with a polypeptide or polypeptide derivative of the invention can be used for priming, with boosting being earned out mucosally using a soluble polypeptide or polypeptide derivative of the invention, in combination with a mucosal - adjuvant (e.g., LT).
Polypeptides and polypeptide derivatives of the invention can also be used as diagnostic reagents for detecting the presence of anti-Helicobacter antibodies, e.g., in blood samples. Such polypeptides can be about 5 to about 80, preferably, about 10 to about 50 amino acids in length and can be labeled or unlabeled, depending upon the diagnostic method. Diagnostic methods involving such a reagent are described below. Upon expression of a polynucleotide molecule of the invention, a polypeptide or polypeptide derivative is produced and can be purified using known methods. For example, the polypeptide or polypeptide derivative can be produced as a fusion protein containing a fused tail that facilitates purification. The fusion product can be used to immunize a small mammal, e.g., a mouse or a rabbit, in order to raise monospecific antibodies against the polypeptide or polypeptide derivative. The eighth aspect of the invention thus provides a monospecific antibody that binds to a polypeptide or polypeptide derivative of the invention.
By "monospecific antibody" is meant an antibody that is capable of reacting with a unique, naturally-occurring Helicobacter polypeptide. An antibody of the invention can be polyclonal or monoclonal. Monospecific antibodies can be recombinant, e.g., chimeric (e.g., consisting of a variable region of murine origin and a human constant region), humanized (e.g., a human immunoglobulin constant region and a variable region of animal, e.g., murine, origin), and/or single chain. Both polyclonal and monospecific antibodies can also be in the form of immunoglobulin fragments, e.g., F(ab)'2 or Fab fragments. The antibodies of the invention can be of any isotype, e.g., IgG or IgA, and polyclonal antibodies can be of a single isotype or can contain a mixture of isotypes. The antibodies of the invention, which can be raised to a polypeptide or polypeptide derivative of the invention, can be produced and identified using standard immunological assays, e.g., Western blot assays, dot blot assays, or ELISA (see, e.g., Coligan et al, Current Protocols in Immunology, John Wiley & Sons, Inc., New York, NY, 1994). The antibodies can be used in diagnostic methods to detect the presence of Helicobacter antigens in a sample, such as a biological sample. The antibodies can also be used in affinity chromatography methods for purifying a polypeptide or polypeptide derivative of the invention. As is discussed further below, the antibodies can also be used in prophylactic and therapeutic passive immunization methods.
Accordingly, a ninth aspect of the invention provides (i) a reagent for detecting the presence of Helicobacter in a biological sample that contains an antibody, polypeptide, or polypeptide derivative of the invention; and (ii) a diagnostic method for detecting the presence of Helicobacter in a biological sample, by contacting the biological sample with an antibody, a polypeptide, or a polypeptide derivative of the invention, so that an immune complex is formed, and detecting the complex as an indication of the presence of Helicobacter in the sample or the organism from which the sample was derived. The immune complex is formed between a component of the sample and the antibody, polypeptide, or polypeptide derivative, and that any unbound material can be removed prior to detecting the complex. A polypeptide reagent can be used for detecting the presence of anti-Helicobacter antibodies in a sample, e.g., a blood sample, while an antibody of the invention can be used for screening a sample, such as a gastric extract or biopsy sample, for the presence of Helicobacter polypeptides.
For use in diagnostic methods, the reagent (e.g., the antibody, polypeptide, or polypeptide derivative of the invention) can be in a free ^tate or can be immobilized on a solid support, such as, for example, on the interior surface of a tube or on the surface, or within pores, of a bead. Immobilization can be achieved using direct or indirect means. Direct means include passive adsoφtion (i.e., non-covalent binding) or covalent binding between the support and the reagent. By "indirect means" is meant that an anti-reagent compound that interacts with the reagent is first attached to the solid support. For example, if a polypeptide reagent is used, an antibody that binds to it can serve as an anti-reagent, provided that it binds to an epitope that is not involved in recognition of antibodies in biological samples. Indirect means can also employ a ligand-receptor system, for example, a molecule, such as a vitamin, can be grafted onto the polypeptide reagent and the corresponding receptor can be immobilized on the solid phase. This concept is illustrated by the well known biotin-streptavidin system. Alternatively, indirect means can be used, e.g., by adding to the reagent a peptide tail, chemically or by genetic engineering, and immobilizing the grafted or fused product by passive adsorption or covalent linkage of the peptide tail.
According to a tenth aspect of the invention, there is provided a process for purifying, from a biological sample, a polypeptide or polypeptide derivative of the invention, which involves carrying out antibody-based affinity chromatography with the biological sample, wherein the antibody is a monospecific antibody of the invention.
For use in a purification process of the invention, the antibody can be polyclonal or monospecific, and preferably is of the IgG type. Purified IgGs can be prepared from an antiserum using standard methods (see, e.g., Coligan et al, supra). Conventional chromatography supports, as well as standard methods for grafting antibodies, are described, for example, by Harlow et al (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988).
Briefly, a biological sample, such as an H. pylori extract, preferably in a buffer solution, is applied to a chromatography material, which is, preferably, equilibrated with the buffer used to dilute the biological sample, so that the polypeptide or polypeptide derivative of the invention (i.e., the antigen) is allowed to adsorb onto the material. The chromatography material, such as a gel or a resin coupled to an antibody of the invention, can be in batch form or in a column. The unbound components are washed off and the antigen is eluted with an appropriate elution buffer, such as a glycine buffer, a buffer containing a chaotropic agent, e.g., guanidine HC1, or a buffer having high salt concentration (e.g., 3 M MgCl2). Eluted fractions are recovered and the presence of the antigen is detected, e.g., by measuring the absorbance at 280 nm.
An antibody of the invention can be screened for therapeutic efficacy as follows. According to an eleventh aspect of the invention, there is provided (i) a composition of matter containing a monospecific antibody of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a monospecific antibody of the invention; and (iii) a method for treating or preventing Helicobacter (e.g., H. pylori, H. felis, H. mustelae, or H. heilmanii) infection, by administering a therapeutic or prophylactic amount of a monospecific antibody of the invention to an individual in need of such treatment. In addition, the eleventh aspect of the invention includes the use of a monospecific antibody of the invention in the preparation of a medicament for treating or preventing Helicobacter infection.
The monospecific antibody can be polyclonal or monoclonal, and is, preferably, predominantly of the IgA isotype. In passive immunization methods, the antibody is administered to a mucosal surface of a mammal, e.g., the gastric mucosa, e.g., orally or intragastrically, optionally, in the presence of a bicarbonate buffer. Alternatively, systemic administration, not requiring a bicarbonate buffer, can be carried out. A monospecific antibody of the invention can be administered as a single active agent or as a mixture with at least one additional monospecific antibody specific for a different Helicobacter polypeptide. The amount of antibody and the particular regimen used can be readily determined by one skilled in the art. For example, daily administration of about 100 to 1,000 mg of antibody over one week, or three doses per day of about 100 to 1,000 mg of antibody over two or three days, can be effective regimens for most p poses.
Therapeutic or prophylactic efficacy can be evaluated using standard methods in the art, e.g., by measuring induction of a mucosal immune response or induction of protective and/or therapeutic immunity, using, e.g., the H. felis mouse model and the procedures described by Lee et al. (Eur. J. Gastroenterology & Hepatology 7:303, 1995) or Lee et al. (J. Infect. Dis. 172: 161, 1995). Those skilled in the art will recognize that the H. felis strain of the model can be replaced with another Helicobacter strain. For example, the efficacy of polynucleotide molecules and polypeptides from H. pylori is, preferably, evaluated in a mouse model using an H. pylori strain. Protection can be determined by comparing the degree of Helicobacter infection in the gastric tissue assessed by, for example, urease activity, bacterial counts, or gastritis, to that of a control group. Protection is shown when infection is reduced by comparison to the control group. Such an evaluation can be made for polynucleotides, vaccine vectors, polypeptides, and polypeptide derivatives, as well as for antibodies of the invention.
For example, various doses of an antibody of the invention can be administered to the gastric mucosa of mice previously challenged with an H. pylori strain, as described, e.g., by Lee et al. (supra). Then, after an appropriate period of time, the bacterial load of the mucosa can be estimated by assessing urease activity, as compared to a control. Reduced urease activity indicates that the antibody is therapeutically effective.
Adjuvants that can be used in any of the vaccine compositions described above are described as follows. Adjuvants for parenteral administration include, for example, aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate. The antigen can be precipitated with, or adsorbed onto, the aluminum compound using standard methods. Other adjuvants, such as RIBI (ImmunoChem, Hamilton, MT), can also be used in parenteral administration. Adjuvants that can be used for mucosal administration include, for example, bacterial toxins, e.g., the cholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridium difficile toxin A, the pertussis toxin (PT), and combinations, subunits, toxoids, or mutants thereof. For example, a purified preparation of native cholera toxin subunit B (CTB) can be used. Fragments, homologs, derivatives, and fusions to any of these toxins can also be used, provided that they retain adjuvant activity. Preferably, a mutant having reduced toxicity is used. Suitable mutants are described, e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant). Additional LT mutants that can be used in the methods and compositions of the invention include, e.g., Ser-63-Lys, Ala-69-Gly, Glu-110-Asp, and Glu-112-Asp mutants. Other adjuvants, such as the bacterial monophosphoryl lipid A (MPLA) of, e.g., E. coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri; saponins, and polylactide glycolide (PLGA) microspheres, can also be used in mucosal administration. Adjuvants useful for both mucosal and parenteral administrations, such as polyphosphazene (WO 95/2415), can also be used. Any pharmaceutical composition of the invention, containing a polynucleotide, polypeptide, polypeptide derivative, or antibody of the invention, can be manufactured using standard methods. It can be formulated with a pharmaceutically acceptable diluent or carrier, e.g., water or a saline solution, such as PBS, optionally, including a bicarbonate salt, such as sodium bicarbonate, e.g., 0.1 to 0.5 M. Bicarbonate can advantageously be added to compositions intended for oral or intragastric administration. In general, a diluent or caπier can be selected on the basis of the mode and route of administration, and standard pharmaceutical practice. Suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use in pharmaceutical formulations, are described in Remington 's Pharmaceutical Sciences, a standard reference text in this field and in the USP/NF.
The invention also includes methods in which gastroduodenal infections, such as Helicobacter infection, are treated by oral administration of a Helicobacter polypeptide of the invention and a mucosal adjuvant, in combination with an antibiotic, an antisecretory agent, a bismuth salt, an antacid, sucralfate, or a combination thereof. Examples of such compounds that can be administered with the vaccine antigen and an adjuvant are antibiotics, including, e.g., macrolides, tetracyclines, β-lactams, aminoglycosides, quinolones, penicillins, and derivatives thereof (specific examples of antibiotics that can be used in the invention include, e.g., amoxicillin, clarithromycin, tetracycline, metronidizole, erythromycin, cefuroxime, and erythromycin); antisecretory agents, including, e.g., H2- receptor antagonists (e.g., cimetidine, ranitidine, famotidine, nizatidine and roxatidine), proton pump inhibitors (e.g., omeprazole, lansoprazole, and pantoprazole), prostaglandin analogs (e.g., misoprostil and enprostil), and anticholinergic agents (e.g., pirenzepine, telenzepine, carbenoxolone, and proglumide); and bismuth salts, including colloidal bismuth subcitrate, tripotassium dicitrate bismuthate, bismuth subsalicylate, bicitropeptide, and pepto-bismol (see, e.g., Goodwin et al, Helicobacter pylori, Biology and Clinical Practice, CRC Press, Boca Raton, FL, pp 366-395, 1993; Physicians' Desk Reference, 49th edn., Medical Economics Data Production Company, Montvale, New Jersey, 1995). In addition, compounds containing more than one of the above-listed components coupled together, e.g., ranitidine coupled to bismuth subcitrate, can be used. The invention also includes compositions for carrying out these methods, i.e., compositions containing a Helicobacter antigen (or antigens) of the invention, an adjuvant, and one or more of the above-listed compounds, in a pharmaceutically acceptable carrier or diluent. Amounts of the above-listed compounds used in the methods and compositions of the invention can readily be determined by one skilled in the art. In addition, one skilled in the art can readily design treatment/immunization schedules. For example, the non- vaccine components can be administered on days 1-14, and the vaccine antigen + adjuvant can be administered on days 7, 14, 21, and 28.
Methods and pharmaceutical compositions of the invention can be used to treat or to prevent Helicobacter infections and, accordingly, gastroduodenal diseases associated with these infections, including acute, chronic, and atrophic gastritis, and peptic ulcer diseases, e.g., gastric and duodenal ulcers.
A 76 kDa protein band containing GHPO 386, GHPO 789, and GHPO 1516 (hereinafter the "purified 76 kDa proteins"), GHPO 1360,- and GHPO 750 were purified from Helicobacter pylori strain ATCC number 43579 (American Type Culture Collection, Rockville, Maryland) by immunoaffinity- based chromatography using the methods described below in Example 1, and were shown to be effective vaccine antigens as follows.
Groups of 10 mice each were orally immunized with 1, 5, or 25 μg of the purified 76 kDa proteins, purified GHPO 1360, or purified GHPO 750 in combination with 5 μg of the heat-labile enterotoxin (LT) of E. coli. Twenty five μg of recombinant urease, in combination with 5 μg LT, was used as a positive control, and 5 μg of LT in PBS was used as a negative control. The immunizations were carried out four times each, on days 0, 7, 14, and 21 of the experiment. On day 33, blood samples were collected from the mice and, on day 34, saliva samples were collected. On day 35, all of the mice were challenged by intragastric administration of 1 x 107 streptomycin-resistant, mouse-adapted H. pylori. On day 49, additional saliva samples were collected and, about two weeks after challenge, on days 52-53, the mice were sacrificed. Stomachs were removed from the mice and were analyzed for Helicobacter infection by measuring urease activity in the intact stomach tissue and by a quantitative culture study (Table 1).
Briefly, these studies showed that the gastric urease activities in samples from mice immunized with all three amounts of the purified 76 kDa proteins (i.e., 1, 5, and 25 μg), in combination with LT, were generally lower than the gastric urease activities of samples from mice immunized with LT alone or mice that were not treated prior to challenge. Levels of gastric urease activity generally decreased with increasing amounts of the protein administered, with the gastric urease activity levels for the 25 μg doses generally approaching those of mice immunized with 25 μg of recombinant urease and LT.
The quantitative culture analyses showed that the levels of Helicobacter detected in the stomachs of mice immunized with the purified 76 kDa proteins, purified GHPO 1360, or purified GHPO 750, which generally decreased with increasing dosages, were less than the levels detected in the stomachs of control mice that were immunized with LT alone or untreated before Helicobacter challenge (Tables 1 and 2). The percentages of mice protected by immunization with the purified 76 kDa proteins, purified GHPO 1360, or purified GHPO 750 met or approached the percentages of mice protected by treatment with urease (Tables 1 and 2). These results show that the purified 76 kDa proteins, GHPO 1360, and GHPO 750 are effective vaccine antigens for use in preventing Helicobacter infection.
Table 1
Figure imgf000051_0001
Table 2
Figure imgf000052_0001
The invention is further illustrated by the following examples.
Example 1 describes purification of GHPO 1516 (76 kDa), GHPO 1360 (32 kDa), and GHPO 750 (50 kDa) from Helicobacter cultures. Example 2 describes identification of genes, e.g., genes encoding 76 kDa proteins, such as GHPO 386, GHPO 789, GHPO 1516, GHPO 1197, GHPO 1180, GHPO 896, GHPO 711, GHPO 190, GHPO 185, GHPO 1417, and GHPO 1414, a 32 kDa protein (GHPO 1360), and a 50 kDa protein (GHPO 750) in the Helicobacter genome, as well as identification of signal sequences, and primer design for amplification of genes lacking signal sequences. Example 3 describes cloning of DNA encoding GHPO 386, GHPO 789, GHPO 1516, GHPO 896, GHPO 1360, and GHPO 750 into a vector that provides a histidine tag, and production and purification of the resulting his-tagged fusion proteins. Example 4 describes methods for cloning DNA encoding the polypeptides of the invention so that they can be produced without His-tags, Example 5 describes methods for purifying recombinant polypeptides of the invention, and Example 6 describes use of the GHPO 1360 polypeptide as a serodiagnostic tool for H. pylori infection
EXAMPLE 1: Purification and partial sequence analysis of GHPO 1516 (76 kDa), GHPO 1360 (32 kDa), and GHPO 750 (50 kDa) protein from Helicobacter pylori
l.A. Culture and initial purification steps
Frozen seeds from H. pylori strain ATCC 43579 are used to seed a 75 cm2 flask containing a biphasic medium (a solid phase made of Colombia gelose containing 6% fresh sheep blood and a liquid phase made of triptcase soja containing 20% fetal calf serum). After 24 hours of culturing under microaerophilic conditions, the liquid phase is used for seeding several 75 cm2 flasks containing biphasic medium lacking sheep blood. After 24 hours of culture, the liquid phase is used to seed a 2 L biofermentor in triptcase soja liquid phase containing 10 g/L beta-cyclodextrine. At OD 1.5-1.8, this culture is diluted in a 10 L biofermentor containing the liquid medium. After 24 hours, the bacteria are spun in a centrifuge at 4,000 x g for 30 minutes at 4°C. A 10 L culture contains about 20 to 30 g (wet weight) bacteria.
The pellet obtained using the method described above is washed with 500 ml PBS (7.650 g NaCl, 0.724 g disodium phosphate, and 0.210 g monopotassium phosphate for one liter (pH 7.2)) for a one liter culture. The bacteria are then spun in a centrifuge again under the same conditions.
The pellet (Cl) is suspended in 1% N-octyl-D-glucopyranoside (NOG; 30 ml/L; Sigma). The bacterial suspension is incubated for 1 hour at room temperature while stirring, spun in a centrifuge at 17,600 x g for 30 minutes at 4°C, and the pellet (C2) is recovered.
The supernatant (S2) is dialyzed against PBS overnight at 4°C while stirring. The precipitate is recovered by centrifugation at 2,600 x g for 30 minutes at 4°C. The supernatant (S2d) is discarded and the pellet (Cs2d) is recovered and stored at -20°C.
The pellet (C2) is resuspended in 20 mM Tris-HCl buffer (pH 7.5) and
100 μM Pefabloc (Buffer A), and is homogenized with an ultra-turrax (3821, Janke and Kungel). Lysozyme and EDTA are added at 0.1 mg/ml and 1 mM, respectively.
The homogenate is sonicated three times for 2 minutes each at 4°C, and then is spun in an ultracentrifuge at 210,000 x g for 30 minutes at 4°C. The supernatant (S3), which contains the cytoplasmic and periplasmic proteins, is eliminated, while the pellet is recovered, washed with buffer A, and spun in an ultracentrifuge at 210,000 x g for 30 minutes at 4°C. The supernatant (S4) is eliminated and the pellet (C4) is stored at -20°C. This pellet (C4) contains membrane proteins.
The pellet (C4) is washed in 50 mM NaC03 (pH 9.5) and 100 μM Pefabloc (buffer B). The suspension is spun in an ultracentrifuge at
210,000 x g for 30 minutes at 4°C. The supernatant (S5) is eliminated, and the pellet (C5) is then washed and spun in an ultracentrifuge as is described above. The supernatant (S6) is eliminated and the pellet (C6) is stored at -20°C. l.B. Purification of the proteins of membrane fraction C4 by preparative SDS-PAGE
SDS-PAGE is carried out according to the method of Laemmli (supra), using a biphasic gel consisting of a 5% polyacrylamide concentrating gel and a 10% polyacrylamide separating gel. The membrane fraction C4 is resuspended in buffer A, diluted in an equal volume of 2x sample buffer, and heated for 5 minutes at 95°C. About 19 mg of protein is applied to the gel (16 x 12 cm; 5 mm thick). Pre-migration is carried out for 2 hours at 50 V, and is followed by migration overnight at 65 V. After Coomassie blue staining, five major bands are revealed that have apparent molecular weights of 87, 76, 54, 50, and 32 kDa. Bands at 50 and 32 kDa appear to be slightly contaminated with bands at 47 and 35 kDa, respectively.
A band corresponding to the purified 76 kDa proteins, 32 kDa protein (GHPO 1360), or 50 kDa protein (GHPO 750) is cut out from the gel and is pounded with an ultra-turrax in 10-20 ml extraction buffer (25 mM Tris- HCl (pH 8.8), 8 M urea, 10% SDS, 100 μM phenyl methyl sulfonyl fluoride (PMSF), and 10 μM Pefabloc (buffer C)).
Each homogenate is filtered through a Millipore AP20 filter under 7 bars at room temperature, washed with 5-10 ml buffer C, and then filtered again. Each filtrate is precipitated with three volumes of a 50/50 mixture of 75% methanol and 75% isopropanol, and then is spun in a centrifuge at 240,000 x g for 16 hours at 10°C.
Each pellet is resuspended in 2 ml of 10 mM NaP04 (pH 7.0) containing 1 M NaCl, 0.1% Sarkosyl, 100 μM PMSF, and 6 M urea (buffer D). The solubilized sample is dialyzed, in order, against 100 ml buffer D containing 4 M urea, 100 ml buffer D containing 2 M urea and 0.5% Sarkosyl, and twice against 100 ml buffer D that does not contain urea or Sarkosyl. The dialyses are earned out for 1 hour each while stirring at room temperature. The last dialysate is incubated for 30 minutes in an ice bath, and then is spun in a centrifuge at low speed for 10 minutes at 4°C. The supernatant is recovered, filtered through a Millipore filter (0.45 μm), and stored at -20°C.
l.C. Purification of the 76 kDa, 32 kDa, or 50 kDa protein by immunoaffinity-based chromatography l.C.l. Antiserum preparation
Specific polyclonal serum against the purified 76 kDa proteins, the 32 kDa protein (GHPO 1360), or the 50 kDa protein (GHPO 750), which are purified by preparative SDS-PAGE, is prepared by hyperimmunizing rabbits as follows. On day 0, a preparation containing 50 μg of the protein mixed with complete Freund's adjuvant is administered subcutaneously to the rabbits at multiple sites. The rabbits are boosted at days 21 and 42 with 25 μg of the protein in incomplete Freund's adjuvant, and are sacrificed at day 60. Complement is removed from the serum by heating for 30 minutes at 56°C. The hyperimmune serum is then sterilized by filtration through a Millipore membrane (0.22 μm).
I.C.2. IgG purification
The hyperimmune serum prepared as described above is applied to a Protein A Sepharose Fast Flow column (Pharmacia) that is equilibrated with
100 mM Tris-HCl (pH 8.0). The column is washed with 10 column volumes of 100 mM Tris-HCl (pH 8.0), and then with 10 column volumes of 10 mM Tris- HCl (pH 8.0). IgGs are eluted in 0.1 M glycine buffer (pH 3.0), and are collected as 5 ml fractions, to each of which 0.25 ml of Tris-HCl (pH 8.0) is added. The optical density of each fraction is measured at 280 nm, the IgG- containing fractions are pooled together and, if necessary, frozen at -70ΘC. l.C.3. Preparation of the column
An appropriate amount of CNBr-activated Sepharose 4B gel (Pharmacia; reference: 17-0430-01) is suspended in 1 mM NaCl buffer (1 g dry gel provides for 3.5 ml hydrated gel; 5 to 10 mg IgGs can be retained per ml of hydrated gel). The gel is then washed using a buchner by adding small quantities of 1 mM HC1. The total volume of 1 mM HC1 that is used amounts to 200 ml/g of gel.
Purified IgGs are dialyzed for 4 hours at room temperature against 50 volumes of 500 mM sodium phosphate buffer (pH 7.5). The IgGs are then diluted to 3 mg/ml with the same buffer. IgGs are incubated with the gel overnight at 5+3 °C while stirring. The gel is packed in a chromatography column and is washed with 2 column volumes of 500 mM phosphate buffer (pH 7.5). The gel is then transferred to a tube and is incubated with 100 mM ethanolamine (pH 7.5), and then it is washed with 2 column volumes of PBS. The gel can be stored in PBS/merthiolate, 1/10,000. I.C.4. Adsorption and elution
The 76 kDa protein is adsorbed and eluted as follows. The membrane fraction Cs2d is suspended in 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, and then is filtered through a 0.45 μm membrane. The supernatant is applied to the column, which is equilibrated with 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, at a flow rate of about 10 ml/hour. The column is washed with 20 column volumes of 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, and then with 2 to 6 volumes 10 mM phosphate buffer (pH 6.8).
The antigen is eluted with 100 mM glycine buffer (pH 2.5). The eluate is collected in 3 ml fractions, to each of which is added 150 μl 1 M phosphate buffer (pH 8.0). The optical density of each fraction is measured at 280 nm, fractions containing the 76 kDa protein are pooled, and stored at -70°C.
Analysis by 10% SDS-PAGE reveals a single band at 76 kDa. N- terminal sequence was canied out on this purified 76 kDa preparation, and the sequence obtained is as follows: EDDGFYTSVGYQIGEAAQMV (SEQ ID NO:58).
The 32 kDa protein (GHPO 1360) or the 50 kDa protein (GHPO 750) is purified by immunoaffinity-based chromatography as follows. In order to separate the 32 or 50 kDa protein from the contaminating proteins (the 47 and 35 kDa proteins, respectively), membrane fraction C4 is solubilized in 50 mM NaC03 (pH 9.5) for 30 minutes at room temperature under stirring and the preparation is centrifuged for 30 minutes at 200,000 x g at 4°C. The 47 and 35 kDa proteins are insoluble in the NaC03 buffer and are eliminated in the pellet.
The supernatant is dialyzed against 50 mM Tris-HCL (pH 8.0), 2 mM EDTA, and then is filtered through a 0.45 μm membrane. The filtered supernatant is applied to the column, which is equilibrated with 50 mM Tris- HCL (pH 8.0), 2 mM EDTA, at a flow rate of about 10 ml/hour. The column is washed with 20 column volumes of 50 mM Tris-HCL (pH 8.0), 2 mM EDTA, and then with 2 to 6 volumes of 10 mM phosphate buffer (pH 6.8).
The antigen is eluted with 100 mM glycine buffer (pH 2.5). The eluate is collected in 3 ml fractions, to each of which is added 150 μl 1 M phosphate buffer (pH 8.0). The optical density of each fraction is measured at 280 nm, and fractions containing the 50 or 32 kDa protein are pooled and stored at -70°C.
Analysis of the purified protein by 10% SDS-PAGE reveals single bands at 50 and 32 kDa. N-terminal sequencing is carried out with the purified 50 kDa protein preparation. The sequence found is as follows: MKEKFNRTKPHVNIGTIGHVDH (SEQ ID NO:73). Similarly, N-terminal and internal sequencing is carried out with the purified 32 kDa preparation. The sequences found are as follows: AHNANNATHNTKK (SEQ ID NO: 74) and KPAHNA (SEQ ID NO:75) (N-terminal), and IDKQPKAKK (SEQ ID NO:76) and FWAKKQAE (SEQ ID NO:77) (internal).
l.D. Purification of the 76 kDa protein from membrane fraction Cs2d and purification of the 32 kDa and 50 kDa proteins from membrane fraction C4
The 76 kDa protein can also be purified as follows. A 40 ml Q- Sepharose column (diameter: 2.5 cm; height: 8 cm) is prepared according to the manufacturer's instructions (Pharmacia). The column is washed and equilibrated with buffer B, containing 50 mM NaCO, (pH 9.5), 100 μM Pefabloc, and 0.1% Zwittergent 3-14. The chromatography is monitored by measuring absorbance at 280 nm at the column exit.
One hundred and forty mg of protein from the membrane fraction Cs2d resuspended in buffer B are applied to the column. The column is washed with 0.1 M NaCl in buffer B, and then a 0.1-0.5 M NaCl gradient is applied to the column. The fraction eluted between 0.35 and 0.45 M NaCl is further purified on a 10 ml S-Sepharose column (diameter: 1.5 cm; height: 5 cm; up to 10 mg protein/ml of gel), which is prepared according to the manufacturer's instructions (Pharmacia). The fraction obtained is dialyzed against 50 mM acetate (pH 5.0) containing 100 μM Pefabloc and 0.1% Zwittergent 3-14, and then is applied to the column, which is equilibrated with the acetate buffer.
The column is washed with the acetate buffer until the absorbance at 280 nm is stabilized (about 3 column volumes are required). Proteins are eluted with a 0-0.5 M NaCl gradient in acetate buffer. The fraction eluted at
0.15 M NaCl is enriched with the 76 kDa protein.
The 32 kDa protein (GHPO 1360) can also be purified as follows.
Membrane fraction C4 is solubilized in 50 mM NaC03 buffer (pH 9.5) at room temperature for 30 minutes under stirring. The suspension is then centrifuged at 200,000 x g for 30 minutes at 4°C. This allows the 32 and 35 kDa proteins to be separated, since the 35 kDa protein is insoluble in the NaC03 buffer. The supernatant is dialyzed against 50 mM NaP04 buffer (pH 7.0), and then is applied to an SP-Sepharose column, which is equilibrated with the NaP04 buffer. The column is washed with the NaP04 buffer, and then an 0-0.5 M
NaCl gradient is applied to the column. The fraction eluted between 0.26 and
0.31 M contains the 32 kDa protein.
The 50 kDa protein can also be purified as follows. Membrane fraction
C4 is solubilized in 50 mM NaC03 buffer (pH 9.5) at room temperature for 30 minutes while stirring. The suspension is then centrifuged at 200,000 x g for 30 minutes at 4°C. This allows the 50 and 47 kDa proteins to be separated, since the 47 kDa protein is insoluble in the NaC03 buffer. The supernatant is dialyzed against 50 mM NaP04 buffer (pH 7.0).
A 40 ml Q-Sepharose column (diameter: 2.5 cm; height: 8 cm) is prepared according to the manufacturer's instructions (Pharmacia), washed, and equilibrated with buffer B (pH 9.5) (50 mM NaC03 , 100 μM Pefabloc, and
0.1% Zwittergent 3-14).
The chromatography is monitored by UV detection at 280 nm at the column exit. One hundred and forty mg of protein solubilized as is described above are applied to the column, which is then washed with buffer B until the absorbance at 280 nm is stabilized. The proteins are eluted with a 0.1-
0.5 M NaCl gradient in buffer B (10 fold Vτ), which is followed by washing in buffer B containing 0.5, and then 1, M NaCl (2 fold Vτ). The fractions are recovered, analyzed by SDS-PAGE, and pooled according to their electrophoretic profiles.
Fraction 9, which conesponds to the beginning of the washing at 1 M NaCl and contains acidic proteins, is further purified as follows. A 10 ml DEAE Sepharose column (diameter: 1.5 cm, height: 5 cm) is prepared according to the manufacturer's instructions (Pharmacia) (up to 10 mg protein/ml of gel). The column is washed and equilibrated with buffer B. Chromatography is monitored as is described above. Fraction 9 is dialyzed against buffer B and contains about 10 mg protein.
Fraction 9 is applied to the DEAE-Sepharose column. The column is washed with buffer B until the absorbance at 280 nm is stabilized. The proteins are eluted with a 0-0.5 M NaCl gradient in buffer B (10 fold Vτ), followed by washing in buffer B, containing 1 M NaCl (2 fold Vτ). Fractions are recovered and analyzed by SDS-PAGE. The 50 kDa protein is found in the fractions eluted at 0.3-0.4 M NaCl.
EXAMPLE 2: Identification of genes in the H. pylori genome, such as genes encoding the 76 kDa proteins, the 32 kDa protein (GHPO 1360), and the 50 kDa protein (GHPO 750) identification of signal sequences, and primer design for amplification of genes lacking signal sequences
2.A. Creating H. pylori genomic databases
The H. pylori genome was provided as a text file containing a single contiguous string of nucleotides that had been determined to be 1.76 Megabases in length. The complete genome was split into 17 separate files using the program SPLIT (Creativity in Action), giving rise to 16 contigs, each containing 100,000 nucleotides, and a 17th contig containing the remaining 76,000 nucleotides. A header was added to each of the 17 files using the format: >hpg0.txt (representing contig 1), .hpgl .txt (representing contig 2), etc. The resulting 17 files, named hpgO through hpglό, were then copied together to form one file that represented the plus strand of the complete H. pylori genome. The constructed database was given the designation "Η." A negative strand database of the H. pylori genome was created similarly by first creating a reverse complement of the positive strand using the program SeqPup (D.G. Gilbert, Indiana University Biology Department) and then performing the same procedure as described above for the plus strand. This database was given the designation "N."
The regions predicted to encode open reading frames (ORFs) were defined for the complete H. pylori genome using the program GENEMARK™ (Borodovsky et al, Comp. Chem. 17: 123, 1993). A database was created from a text file containing an annotated version of all ORFs predicted to be encoded by the H. pylori genome for both the plus and minus strands, and was given the designation "O." Each ORF was assigned a number indicating its location on the genome and its position relative to other genes. No manipulation of the text file was required.
2.B. Searching the H. pylori databases
The databases constructed as is described above were searched using the program FASTA (Pearson et al, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988). FASTA was used for searching either a DNA sequence against either of the gene databases ("Η" and/or "N"), or a peptide sequence against the ORF library ("O"). TFASTX was used to search a peptide sequence against all possible reading frames of a DNA database ("Η" and/or "N" libraries). - Potential frameshifts also being resolved, FASTX was used for searching the translated reading frames of a DNA sequence against either a DNA database, or a peptide sequence against the protein database.
2.C. Isolation of DNA sequences from the H. pylori genome The FASTA searches against the constructed DNA databases identified exact nucleotide coordinates on one or more of the isolated contigs, and therefore the location of the target DNA. Once the exact location of the target sequence was known, the contig identified to carry the gene was exported into the software package MapDraw (DNAStar, Inc.) and the gene was isolated. Gene sequences with flanking DNA was then excised and copied into the EditSeq. Software package (DNAStar, Inc.) for further analysis.
2.D. Identification of signal sequences
The deduced protein encoded by a target gene sequence is analyzed using the PROTEAN software package (DNAStar, Inc.). This analysis predicts those areas of the protein that are hydrophobic by using the Kyte-Doolittle algorithm, and identifies any potential polar residues preceding the hydrophobic core region, which is typical for many signal sequences. For confirmation, the target protein is then searched against a PROSITE database (DNAStar, Inc.) consisting of motifs and signatures. Characteristic of many signal sequences and hydrophobic regions in general, is the identification of predicted prokaryotic lipid attachment sites. Where confirmation between the two approaches is apparent at the N-terminus of any protein, putative cleavage sites are sought. Specifically, this includes the presence of either an Alanine (A), Serine (S), or Glycine (G) residue immediately after the core hydrophobic region. In the case of lipoproteins, a Cysteine (C) residue would be identified as the +1 residue, post-cleavage.
2.E. Rational design of PCR primers based on the identification of signal sequences In order to clone gene sequences as N-terminus translational fusions for the generation of recombinant proteins with N-terminal Histidine tags, the gene sequence that specifies the signal sequence is omitted. The 5'-end of the gene- specific portion of the N-terminal primer is designed to start at the first codon beyond the cleavage site. In the case of lipoproteins, the 5'-end of the N- terminal primer begins at the second codon, immediately after the modifiable residue at position +1 post-cleavage. The omission of the signal sequence from the recombinant allows for one-step purification, and potential problems associated with insertion of signal sequences in the membrane of the host strain carrying the hybrid construct are avoided.
EXAMPLE 3 : Preparation of isolated DNA encoding GHPO 386, GHPO 789, GHPO 1516, GHPO 896, GHPO 1360, and GHPO 750, and production of these proteins as a histidine-tagged fusion proteins
3.A. Preparation of genomic DNA from Helicobacter pylori
Helicobacter pylori strain ORV2001, stored in LB medium containing 50%) glycerol at -70°C, is grown on Colombia agar containing 7% sheep blood for 48 hours under microaerophilic conditions (8-10% C02, 5-7% 02, and 85- 87% N2). Cells are harvested, washed with PBS (pH 7.2), and DNA is then extracted from the cells using the Rapid Prep Genomic DNA Isolation kit (Pharmacia Biotech). 3.B. PCR amplification
DNA encoding GHPO 386, GHPO 789, GHPO 1516, GHPO 896, GHPO 1360, and GHPO odd numbers), 65, and 67 is amplified from genomic DNA, as can be prepared as is described above, by the Polymerase Chain Reaction (PCR) using the following primers: GHPO 386:
N-terminal primer: 5'-CTGAATTCGATTTCAAGGAGAAAACATGAAA-3' (SEQ ID NO:59); and C-terminal primer:
5'-CCGCTCGAGTTAGTAAGCGAACACATAATT-3* (SEQ ID NO:60). GHPO 789:
N-terminal primer: 5'-CGCGGATCCGAATCCAATTTAATCCAAAAAGG-3' (SEQ ID NO:61); and
C-terminal primer: 5'-CCGCTCGAGTTAGTAAGCGAACACATAGTTCAA-3' (SEQ ID NO:62). GHPO 1516:
N-terminal primer: 5'-CGCGGATCCGAATCCAATTTAATCCAAAAAGG-3' (SEQ ID NO:56); and
C-terminal primer: 5'-CCGCTCGAGTTAAGTAAGCGAACACATATTCAA-3' (SEQ ID NO:57). GHPO 896: N-terminal primer:
5'-CGCGGATCCGAAGTTTCTTTGTATCAAAG-3' (SEQ ID NO:63); and
C-terminal primer: 5'-CCGCTCGAGTTAGTAAGCAAACACATAATTGTG-3' (SEQ ID NO: 64). GHPO 1360:
N-terminal primer: 5'-CGCGGATCCGAATGAAAAAAAATATCTTAAAT-3' (SEQ ID NO:69); and
C-terminal primer: 5*-CCGCTCGAGTTACTTGTTGATAACAATTTT-3' (SEQ ID NO:70). GHPO 750:
N-terminal primer: 5*-CGCGGATCCGAATGGCAAAAGAAAAGTTTAAC-3' (SEQ ID NO:71); and
C-terminal primer: 5'-CCGCTCGAGTTATTCAATAATATTGCTCAC-3* (SEQ ID NO:72). GHPO 711 : N-terminal primer:
5'-GGGAATTCAAAAAAACGAAAAAAACG-3' (SEQ ID NO:83); and
C-terminal primer: 5'-CCCCTCGAGTTAATAGGCAAACAC-3' (SEQ ID NO:84).
The N-terminal and C-terminal primers for each clone both include a 5' clamp and a restriction enzyme recognition sequence for cloning puφoses (BamHl (GGATCC) andXhol (CTCGAG) recognition sequences).
Amplification of gene-specific DNA is carried out using a heat-stable DNA Polymerase (e.g., Thermalase DNA Polymerase (Amresco)) according to the manufacturer's instructions. The reaction mixture, which is brought to a final volume of 100 μl with distilled water, is as follows: dNTPs mix 200 μM
1 Ox ThermoPol buffer 10 μl primers 300 nM each
DNA template 50 ng
DNA polymerase 2 units
Appropriate amplification reaction conditions can readily be determined by one skilled in the art. In the present case, the following conditions were used. For GHPO 386 and GHPO 789, in a reaction containing Taq DNA polymerase (Appligene), a denaturing step was canied out at 95 °C for 30 seconds, followed by an annealing step at 50 °C for one minute, and an extension step at 72 °C for 2 minutes and 30 seconds. Twenty five cycles were canied out. For GHPO 896, in a reaction containing Taq DNA polymerase, a denaturing step was carried out at 97 °C for 30 seconds, followed by an annealing step at 50°C for one minute, and an extension step at 72°C for 2 minutes and 30 seconds. Twenty five cycles were canied out. The same reaction conditions were used for GHPO 1516 as GHPO 896, except that Vent DNA polymerase was used for clone GHPO 1516, instead of Taq DNA polymerase, and the annealing temperature was 55 °C. For GHPO 1360 and GHPO 750, Thermalase DNA polymerase was used. A denaturing step was canied out at 95 °C for 30 seconds, followed by an annealing step at 55 °C for one minute, and an extension step at 72 °C for 2 minutes. Thirty cycles were carried out. For GHPO 711, Vent DNA polymerase was used. A denaturing step was canied out at 94 °C for 30 seconds, followed by an annealing step at 50°C for 30 seconds, and an extension step at 72°C for 1 minute. Twenty five cycles were canied out. 3.C. Transformation and selection of transformants
A single PCR product is thus amplified and is then digested at 37 °C for 2 hours with BamHl and_A7røI concunently in a 20 μl reaction volume. The digested product is ligated to similarly cleaved pET28a (Novagen) that is dephosphorylated prior to the ligation by treatment with Calf Intestinal Alkaline Phosphatase (CIP). The gene fusion constructed in this manner allows one-step affinity purification of the resulting fusion protein because of the presence of histidine residues at the N-terminus of the fusion protein, which are encoded by the vector.
The ligation reaction (20 μl) is canied out at 14 °C overnight and then is used to transform 100 μl fresh E. coli XL 1 -blue competent cells (Novagen). The cells are incubated on ice for 2 hours, then heat-shocked at 42 °C for 30 seconds, and returned to ice for 90 seconds. The samples are then added to 1 ml LB broth in the absence of selection and grown at 37 °C for 2 hours. The cells are then plated out on LB agar containing kanamycin (50 μg/ml) at a lOx and neat dilution and incubated overnight at 37 °C. The following day, 50 colonies are picked onto secondary plates and incubated at 37 °C overnight. Five colonies are picked into 3 ml LB broth supplemented with kanamycin (100 μg/ml) and are grown overnight at 37°C. Plasmid DNA is extracted using the Quiagen mini -prep, method and is quantitated by agarose gel electrophoresis.
PCR is performed with the gene-specific primers under the conditions stated above and transformant DNA is confirmed to contain the desired insert. If PCR-positive, one of the five plasmid DNA samples (500 ng) extracted from the E. coli XL 1 -blue cells is used to transform competent BL21 (λDE3) E. coli competent cells (Novagen; as described previously). Transformants (10) are picked onto selective kanamycin (50 μg/ml) containing LB agar plates and stored as a research stock in LB containing 50% glycerol.
3.D. Purification of recombinant proteins
One ml of frozen glycerol stock prepared as described in 3. C. is used to inoculate 50 ml of LB medium containing 25 μg/ml of kanamycin in a 250 ml Erlenmeyer flask. The flask is incubated at 37°C for 2 hours or until the absorbance at 600 nm (OD600) reaches 0.4-1.0. The culture is stopped from growing by placing the flask at 4°C overnight. The following day, 10 ml of the overnight culture are used to inoculate 240 ml LB medium containing kanamycin (25 μg/ml), with the initial OD600 about 0.02-0.04. Four flasks are inoculated for each ORF.
The cells are grown to an OD600 of 1.0 (about 2 hours at 37°C), a 1 ml sample is harvested by centrifugation, and the sample is analyzed by SDS- PAGE to detect any leaky expression. The remaining culture is induced with 1 mM IPTG and the induced cultures are grown for an additional 2 hours at 37°C.
The final OD600 is taken and the cells are harvested by centrifugation at 5,000 x g for 15 minutes at 4°C. The supernatant is discarded and the pellets are resuspended in 50 mM Tris-HCl (pH 8.0), 2 mM EDTA. Two hundred and fifty ml of buffer are used for a 1 L culture and the cells are recovered by centrifugation at 12,000 x g for 20 minutes. The supernatant is discarded and the pellets are stored at -45°C.
3. E. Protein purification
Pellets obtained from 3.D. are thawed and resuspended in 95 ml of 50 mM Tris-HCl (pH 8.0). Pefabloc and lysozyme are added to final concentrations of 100 μM and 100 μg/ml, respectively. The mixture is homogenized with magnetic stining at 5°C for 30 minutes. Benzonase (Merck) is added at a 1 U/ml final concentration, in the presence of 10 mM MgCl2, to ensure total digestion of the DNA. The suspension is sonicated (Branson Sonifier 450) for 3 cycles of 2 minutes each at maximum output. The homogenate is spun in a centrifuge at 19,000 x g for 15 minutes and both the supernatant and the pellet are analyzed by SDS-PAGE to detect the cellular location of the target protein in the soluble or insoluble fractions, as is described further below. 3.E.I. Soluble fraction
If the target protein is produced in a soluble form (i.e., in the supernatant obtained in 3.E.) NaCl and imidazole are added to the supernatant to final concentrations of 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, and 10 mM imidazole (buffer A). The mixture is filtered through a 0.45 μm membrane and loaded onto an IMAC column (Pharmacia HiTrap chelating Sepharose; 1 ml) that has been charged with nickel ions according to the manufacturer's recommendations. After loading, the column is washed with 50 column volumes of buffer A and the recombinant target protein is eluted with 5 ml of buffer B (50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 500 mM imidazole). The elution profile is monitored by measuring the absorbance of the fractions at 280 nm. Fractions conesponding to the protein peak are pooled, dialyzed against PBS containing 0.5 M arginine, filtered through a 0.22 μm membrane, and stored at -45°C. 3.E.2. Insoluble fraction If the target protein is expressed in the insoluble fraction (pellets obtained from 3.E.), purification is conducted under denaturing conditions. NaCl, imidazole, and urea are added to the resuspended pellet to final concentrations of 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 10 mM imidazole, and 6 M urea (buffer C). After complete solubilization, the mixture is filtered through a 0.45 μm membrane and loaded onto an IMAC column.
The purification procedures on the IMAC column are the same as described in 3.E.I ., except that 6 M urea is included in all buffers used and 10 column volumes of buffer C are used to wash the column after protein loading, instead of 50 column volumes.
The protein fractions eluted from the IMAC column with buffer D (buffer C containing 500 mM imidazole) are pooled. Arginine is added to the solution to final concentration of 0.5 M and the mixture is dialyzed against PBS containing 0.5 M arginine and various concentrations of urea (4 M, 3 M, 2 M, 1 M, and 0.5 M) to progressively decrease the concentration of urea. The final dialysate is filtered through a 0.22 μm membrane and stored at -45°C.
Alternatively, when the above purification process is not as efficient as it should be, two other processes may be used as follows. A first alternative involves the use of a mild denaturant, N-octyl glucoside (NOG). Briefly, a pellet obtained in 3.E. is homogenized in 5 mM imidazole, 500 mM sodium chloride, 20 mM Tris-HCl (pH 7.9) by microfluidization at a pressure of 15,000 psi and is clarified by centrifugation at 4,000-5,000 x g. The pellet is recovered, resuspended in 50 mM NaP04 (pH 7.5) containing 1-2% weight
/volume NOG, and homogenized. The NOG-soluble impurities are removed by centrifugation. The pellet is extracted once more by repeating the preceding extraction step. The pellet is dissolved in 8 M urea, 50 mM Tris (pH 8.0). The urea-solubilized protein is diluted with an equal volume of 2 M arginine, 50 mM Tris (pH 8.0), and is dialyzed against 1 M arginine for 24-48 hours to remove the urea. The final dialysate is filtered through a 0.22 μm membrane and stored at -45°C. A second alternative involves the use of a strong denaturant, such as guanidine hydrochloride. Briefly, a pellet obtained in 3.E. is homogenized in 5 mM imidazole, 500 mM sodium chloride, 20 mM Tris-HCl (pH 7.9) by microfluidization at a pressure of 15,000 psi and clarified by centrifugation at 4,000-5,000 x g. The pellet is recovered, resuspended in 6 M guanidine hydrochloride, and passed through an IMAC column charged with Ni"1-1". The bound antigen is eluted with 8 M urea (pH 8.5). Beta-mercaptoethanol is added to the eluted protein to a final concentration of 1 mM, then the eluted protein is passed through a Sephadex G-25 column equilibrated in 0.1 M acetic acid. Protein eluted from the column is slowly added to 4 volumes of 50 mM phosphate buffer (pH 7.0). The protein remains in solution.
3.F. Evaluation of the protective activity of the purified protein
A protection test is described above that was canied out for testing the protective activity of the purified, native proteins. This test can also be used for testing the protective efficacy of recombinant proteins. Alternatively, the following test can be used.
Groups of 10 OF1 mice (IFF A Credo) are immunized rectally with 25 μg of the purified recombinant protein, admixed with 1 μg of cholera toxin (Berna) in physiological buffer. Mice are immunized on days 0, 7, 14, and 21. Fourteen days after the last immunization, the mice are challenged with H. pylori strain ORV2001 grown in liquid media (the cells are grown on agar plates, as described in LA., and, after harvest, the cells are resuspended in Brucella broth; the flasks are then incubated overnight at 37 °C). Fourteen days after challenge, the mice are sacrificed and their stomachs are removed. The amount of H. pylori is determined by measuring the urease activity in the stomach and by culture. 3.G. Production of monospecific polyclonal antibodies 3.G.I. Hyperimmune rabbit antiserum
New Zealand rabbits are injected both subcutaneously and intramuscularly with 100 μg of a purified fusion polypeptide, as obtained in 3.E.I . or 3.E.2., in the presence of Freund's complete adjuvant and in a total volume of approximately 2 ml. Twenty one and 42 days after the initial injection, booster doses, which are identical to priming doses, except that Freund's incomplete adjuvant is used, are administered in the same way. Fifteen days after the last injection, animal serum is recovered, decomplemented, and filtered through a 0.45 μm membrane. 3.G.2. Mouse hyperimmune ascites fluid
Ten mice are injected subcutaneously with 10-50 μg of a purified fusion polypeptide, as obtained in 3.E.I . or 3.E.2., in the presence of Freund's complete adjuvant and in a volume of approximately 200 μl. Seven and 14 days after the initial injection, booster doses, which are identical to the priming doses, except that Freund's incomplete adjuvant is used, are administered in the same way. Twenty one and 28 days after the initial infection, mice receive 50 μg of the antigen alone intraperitoneally. On day 21, mice are also injected intraperitoneally with sarcoma 180/TG cells CM26684 (Lennette et al, Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections , 5th Ed., Washington DC, American Public Health Association, 1979). Ascites fluid is collected 10-13 days after the last injection.
EXAMPLE 4: Methods for producing transcriptional fusions lacking His- tags Methods for amplification and cloning of DNA encoding the polypeptides of the invention as transcriptional fusions lacking His-tags are described as follows. Two PCR primers for each clone are designed based upon the sequences of the polynucleotides that encode them (SEQ ID NOs: 1-21 (odd numbers), 65, and 67). These primers can be used to amplify DNA encoding the polypeptides of the invention from any Helicobacter pylori strain, including, for example, ORV2001 and the H. pylori strain deposited with the American Type Culture Collection (ATCC, Rockville, Maryland) as ATCC number 43579, as well as from other Helicobacter species.
The N-terminal primers are designed to include the ribosome binding site of the target gene, the ATG start site, the signal sequence (if any), and the cleavage site. The N-terminal primers can include a 5' clamp and restriction endonuclease recognition site, such as that for BamHl (GGATCC), which facilitates subsequent cloning. Similarly, the C-terminal primers can include a restriction endonuclease recognition site, such as that foτXhol (CTCGAG), which can be used in subsequent cloning, and a TAA stop codon. Specific primers that can be used are listed above.
Amplification of a genes encoding the polypeptides of the invention can be canied out using Vent DNA polymerase (New England Biolabs) or Taq DNA polymerase (Appligene) under the conditions described above in Example 3. Alternatively, Thermalase DNA polymerase or Pwo DNA polymerase (Boehringer Mannheim) can be used, according to instructions provided by the manufacturers.
A single PCR product for each clone is amplified and can be cloned into BamHl-XhoI cleaved pET24, resulting in construction of transcriptional fusions that permit expression of the proteins without Ηis-tags. The expressed products can be purified as denatured proteins that are refolded by dialysis into 1 M arginine. Cloning into pET 24 allows transcription of genes from the T7 promoter, which is supplied by the vector, but relies upon binding of the RNA-specific DNA polymerase to the intrinsic ribosome binding site of the genes, and thereby expression of the complete ORF. The amplification, digestion, and cloning protocols are as described above for constructing translational fusions.
EXAMPLE 5: Purification of the polypeptides of the invention by immunoaffinity
5.A. Purification of specific IgGs
An immune serum, as prepared as is described in section 3.G., is applied to a protein A Sepharose Fast Flow column (Pharmacia) equilibrated in 100 mM Tris-HCl (pH 8.0). The resin is washed by applying 10 column volumes of 100 mM Tris-HCl and 10 volumes of 10 mM Tris-HCl (pH 8.0) to the column. IgG antibodies are eluted with 0.1 M glycine buffer (pH 3.0) and are collected in 5 ml fractions to each of which is added 0.25 ml 1 M Tris-HCl (pH 8.0). The optical density of the eluate is measured at 280 nm and the fractions containing the IgG antibodies are pooled, dialyzed against 50 mM Tris-HCl (pH 8.0), and, if necessary, stored frozen at -70°C.
5.B. Preparation of the column
An appropriate amount of CNBr-activated Sepharose 4B gel (1 g of dried gel provides for approximately 3.5 ml of hydrated gel; gel capacity is from 5 to 10 mg coupled IgG/ml of gel) manufactured by Pharmacia (17-0430- 01) is suspended in 1 mM HC1 buffer and washed using a buchner by adding small quantities of 1 mM HC1 buffer. The total volume of buffer is 200 ml per gram of gel. Purified IgG antibodies are dialyzed for 4 hours at 20+5 °C against 50 volumes of 500 mM sodium phosphate buffer (pH 7.5). The antibodies are then diluted in 500 mM phosphate buffer (pH 7.5) to a final concentration of 3 mg/ml. IgG antibodies are mixed with the gel overnight at 5+3 °C. The gel is packed into a chromatography column and is washed with 2 column volumes of 500 mM phosphate buffer (pH 7.5), and 1 column volume of 50 mM sodium phosphate buffer, containing 500 mM NaCl (pH 7.5). The gel is then transfened to a tube, mixed with 100 mM ethanolamine (pH 7.5) for 4 hours at room temperature, and washed twice with 2 column volumes of PBS. The gel is then stored in 1/10,000 PBS/merthiolate. The amount of IgG antibodies coupled to the gel is determined by measuring the optical density (OD) at 280 nm of the IgG solution and the direct eluate, plus washings.
5.C. Adsorption and elution of the antigen An antigen solution in 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, for example, the supernatant obtained in 3.E.I . or the solubilized pellet obtained in 3.E.2., after centrifugation and filtration through a 0.45 μm membrane, is applied to a column equilibrated with 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, at a flow rate of about 10 ml/hour. The column is then washed with 20 volumes of 50 mM Tris-HCl (pH 8.0), 2 mM EDTA. Alternatively, adsoφtion can be achieved by mixing overnight at 5+3 °C.
The adsorbed gel is washed with 2 to 6 volumes of 10 mM sodium phosphate buffer (pH 6.8) and the antigen is eluted with 100 mM glycine buffer (pH 2.5). The eluate is recovered in 3 ml fractions, to each of which is added 150 μl of 1 M sodium phosphate buffer (pH 8.0). Absoφtion is measured at 280 nm for each fraction; those fractions containing the antigen are pooled and stored at -20°C.
EXAMPLE 6: The GHPO 1360 polypeptide is useful as a serodiagnostic tool for H. pylori infection
The reactivity of patient sera against H. pylori proteins was analyzed by immunoblot technique. Briefly, total lysate of H. pylori strain ORV2001 was subjected to SDS-PAGE electrophoresis (BioRad protean II system) on a 12.5% gel. Proteins were electrotransfened onto a nitrocellulose paper for immunoblot assay. After blocking, the nitrocellulose paper was incubated with patient sera (1 :500 diluted in blocking buffer) for one hour at room temperature, washed, and further incubated with peroxidase-conjugated goat anti-human IgG. The positive bands were revealed by incubation with the appropriate substrates. The results showed that the H. y/or. -positive ulcer patient sera react specifically with proteins having molecular weights between 50 and 60 kDa and about 30 to 35 kDa. To identify the nature of these proteins, the reactivities of the patient sera were analyzed by immunoblot assay against purified proteins with similar molecular weights: urease (67 kDa and 30 kDa), catalase (54 kDa), heat-shock protein B (60 kDa), and the GHPO 1360 polypeptide (32 kDa) expressed and purified as described in Example 5. All patient sera showed strong reactivity against the GHPO 1360 polypeptide, but the reactivities against other purified proteins were quite variable. These results show that the GHPO 1360 polypeptide is a useful antigen for use in diagnosis of H. pylori infection. Other embodiments are within the following claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: MERIEUX ORAVAX SOCIETE EN NOM COLLECTIF PASTEUR MERIEUX SERUMS ET VACCINS S.A., ET AL.
[ii) TITLE OF THE INVENTION: 76 kDa, 30 kDa, and 50 kDa
Helicobacter Polypeptides and Corresponding Polynucleotide Molecules
[iii) NUMBER OF SEQUENCES: 84
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Clark & Elbing LLP
(B) STREET: 176 Federal Street
(C) CITY: Boston
(D) STATE: MA
(E) COUNTRY: USA
(F) ZIP: 02110
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US98/
(B) FILING DATE: 31-MAR-98
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/834,666
(B) FILING DATE: 01-APR-1997
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/831,310
(B) FILING DATE: 01-APR-1997
(vii: ) ATTORNEY/AGENT INFORMATION: ( NAME: Clark, Paul T. (B REGISTRATION NUMBER: 30,162 (C REFERENCE/DOCKET NUMBER: 06132/037WO1
(ix) TELECOMMUNICATION INFORMATION: (A TELEPHONE: 617-428-0200 (B TELEFAX: 617-428-7045 (C TELEX :
(2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2798 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 328...2451 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 328...385 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
TGGTCCTGGC ATTCCGAGGT TCGAATCCTT GCACCCCAGC CATTTTTCCT TATTTTTTGG 60
CGCGGAGTAG AGCAGTCCGG TAGCTCGTTG GGCTCATAAC CCAAAGGTCA GTGGTTCAAA 120
TCCATTCTCC GCAACCAATC CTTTAAACCA CACCACCACC AAACGAACCA AACGAAACAA 180
AAAGCATCAA AATCAAAAAA ATGACAAAAT TTTTAAGAAA ATGACAAAAA AAAAAAAAAC 240
GATTTTATGC TATATTAACG AAATCTTGTG ATAAGATCTT ATTCTTTTAA AAGACTTATC 300
TAACCATTTT AATTTCAAGG AGAAAAC ATG AAA AAA ACC CTT TTA CTC TCT CTC 354
Met Lys Lys Thr Leu Leu Leu Ser Leu -15
TCT CTC TCT CTC TCG TTT TTG CTC CAC GCT GAA GAC GAC GGC TTT TAC 402 Ser Leu Ser Leu Ser Phe Leu Leu His Ala Glu Asp Asp Gly Phe Tyr -10 -5 1 5
ACA AGC GTG GGC TAT CAA ATC GGT GAA GCC GCT CAA ATG GTG AAA AAC 450 Thr Ser Val Gly Tyr Gin lie Gly Glu Ala Ala Gin Met Val Lys Asn 10 15 20
ACC AAA GGC ATT CAA GAG CTT TCA GAC AAT TAT GAA AAG CTG AAC AAT 498 Thr Lys Gly lie Gin Glu Leu Ser Asp Asn Tyr Glu Lys Leu Asn Asn 25 30 35
CTT TTG AAT AAT TAC AGC ACC CTA AAC ACC CTT ATC AAA TTG TCC GCT 546 Leu Leu Asn Asn Tyr Ser Thr Leu Asn Thr Leu lie Lys Leu Ser Ala 40 45 50
GAT CCG AGC GCG ATT AAC GAC GCA AGG GAT AAT CTA GGC TCA AGC TCT 594 Asp Pro Ser Ala lie Asn Asp Ala Arg Asp Asn Leu Gly Ser Ser Ser 55 60 65 70
AGG AAT TTG CTT GAT GTC AAA ACC AAT TCC CCC GCG TAT CAA GCC GTG 642 Arg Asn Leu Leu Asp Val Lys Thr Asn Ser Pro Ala Tyr Gin Ala Val 75 80 85
CTT TTA GCA CTC AAT GCT GCA GTG GGG TTG TGG CAA GTT ACA AGC TAC 690 Leu Leu Ala Leu Asn Ala Ala Val Gly Leu Trp Gin Val Thr Ser Tyr 90 95 100
GCT TTT ACT GCT TGT GGT CCT GGC AGT AAC GAG AAT GCG AAT GGA GGG 738 Ala Phe Thr Ala Cys Gly Pro Gly Ser Asn Glu Asn Ala Asn Gly Gly 105 110 115
ATC CAA ACT TTT AAT AAT GTG CCA GGA CAA GAT ACG ACG ACC ATC ACT 786 lie Gin Thr Phe Asn Asn Val Pro Gly Gin Asp Thr Thr Thr lie Thr 120 125 130
TGC AAT TCG TAT TAT GAG CCA GGA CAT GGT GGG CCT ATA TCC ACT GCA 834 Cys Asn Ser Tyr Tyr Glu Pro Gly His Gly Gly Pro lie Ser Thr Ala 135 140 145 150
AAT TAT GCG AAA ATC AAT CAA GCC TAT CAA ATC ATC CAA AAG GCT TTG 882 Asn Tyr Ala Lys lie Asn Gin Ala Tyr Gin lie lie Gin Lys Ala Leu 155 160 165
ACA GCC AAT GGA GCT AAT GGA GAT GGG GTC CCC GTT TTA AGC AAC ACC 930 Thr Ala Asn Gly Ala Asn Gly Asp Gly Val Pro Val Leu Ser Asn Thr 170 175 180
ACT ACA AAA CTT GAT TTC ACT ATC AAT GGA GAC AAA AGA ACG GGG GGC 978 Thr Thr Lys Leu Asp Phe Thr lie Asn Gly Asp Lys Arg Thr Gly Gly 185 190 195
AAA CCA AAT ACA CCT GAA AAG TTC CCA TGG AGT GAT GGG AAA TAT ATT 1026 Lys Pro Asn Thr Pro Glu Lys Phe Pro Trp Ser Asp Gly Lys Tyr lie 200 205 210
CAC ACC CAA TGG ATT AAC ACA ATA GTA ACA CCA ACA GAA ACA AAT ATC 1074 His Thr Gin Trp lie Asn Thr lie Val Thr Pro Thr Glu Thr Asn lie 215 220 225 230
AAC ACA GAA AAT AAC GCT CAA GAG CTT TTA AAA CAA GCG AGC ATC ATT 1122 Asn Thr Glu Asn Asn Ala Gin Glu Leu Leu Lys Gin Ala Ser lie lie 235 240 245
ATC ACT ACC CTA AAT GAG GCA TGC CCA AAC TTC CAA AAT GGT GGT AGA 1170 lie Thr Thr Leu Asn Glu Ala Cys Pro Asn Phe Gin Asn Gly Gly Arg 250 255 260
AGT TAT TGG CAA GGG ATA AGC GGC AAT GGG ACA ATG TGC GGG ATG TTT 1218 Ser Tyr Trp Gin Gly lie Ser Gly Asn Gly Thr Met Cys Gly Met Phe 265 270 275
AAG AAT GAA ATC AGC GCG ATC CAA GGC ATG ATC GCT AAC GCT CAA GAA 1266 Lys Asn Glu lie Ser Ala lie Gin Gly Met lie Ala Asn Ala Gin Glu _ 280 285 290
GCT GTC GCG CAA AGC AAA ATC GTT AGT GAA AAC GCG CAA AAT CAA AAC 1314 Ala Val Ala Gin Ser Lys lie Val Ser Glu Asn Ala Gin Asn Gin Asn 295 300 305 310
AAC TTG GAT ACT GGA AAA CCA TTC AAC CCT TAC ACG GAC GCC AGC TTT 1362 Asn Leu Asp Thr Gly Lys Pro Phe Asn Pro Tyr Thr Asp Ala Ser Phe 315 320 325
GCG CAA AGC ATG CTC AAA AAC GCT CAA GCG CAA GCA GAG ATT TTA AAC 1410 Ala Gin Ser Met Leu Lys Asn Ala Gin Ala Gin Ala Glu lie Leu Asn 330 335 340
CAA GCC GAA CAA GTA GTA AAA AAC TTT GAA AAA ATC CCT ACA GCC TTT 1458 Gin Ala Glu Gin Val Val Lys Asn Phe Glu Lys lie Pro Thr Ala Phe 345 350 355
GTA TCA GAC TCT TTA GGG GTG TGT TAT GAA GTG CAA GGG GGT GAG CGT 1506 Val Ser Asp Ser Leu Gly Val Cys Tyr Glu Val Gin Gly Gly Glu Arg 360 365 370
AGG GGC ACC AAT CCA GGT CAG GTA ACT TCT AAC ACT TGG GGA GCC GGT 1554 Arg Gly Thr Asn Pro Gly Gin Val Thr Ser Asn Thr Trp Gly Ala Gly 375 380 385 390
TGC GCG TAT GTG AAA CAA ACC ATA ACG AAT TTA GAC AAC AGC ATC GCT 1602 Cys Ala Tyr Val Lys Gin Thr lie Thr Asn Leu Asp Asn Ser lie Ala 395 400 405
CAC TTT GGC ACT CAA GAG CAG CAG ATA CAG CAA GCC GAA AAC ATC GCT 1650 His Phe Gly Thr Gin Glu Gin Gin lie Gin Gin Ala Glu Asn He Ala 410 415 420
GAC ACT CTA GTG AAT TTC AAA TCT AGA TAC AGC GAA TTA GGC AAC ACC 1698 Asp Thr Leu Val Asn Phe Lys Ser Arg Tyr Ser Glu Leu Gly Asn Thr 425 430 435
TAT AAC AGC ATC ACC ACC GCG CTC TCC AAA GTC CCT AAC GCG CAA AGC 1746 Tyr Asn Ser He Thr Thr Ala Leu Ser Lys Val Pro Asn Ala Gin Ser 440 445 450
TTG CAA AAC GTG GTG AGC AAA AAG AAT AAC CCC TAT AGC CCT CAA GGC 1794 Leu Gin Asn Val Val Ser Lys Lys Asn Asn Pro Tyr Ser Pro Gin Gly 455 460 465 470
ATA GAG ACC AAT TAC TAC CTC AAT CAA AAT TCT TAC AAC CAA ATC CAA 1842 He Glu Thr Asn Tyr Tyr Leu Asn Gin Asn Ser Tyr Asn Gin He Gin 475 480 485
ACC ATC AAC CAA GAA CTA GGG CGT AAC CCC TTT AGG AAA GTG GGC ATC 1890 Thr He Asn Gin Glu Leu Gly Arg Asn Pro Phe Arg Lys Val Gly He 490 495 500
GTC AAT TCT CAA ACC AAC AAT GGT GCC ATG AAT GGG ATC GGT ATT CAG 1938 Val Asn Ser Gin Thr Asn Asn Gly Ala Met Asn Gly He Gly He Gin 505 510 515
GTG GGC TAT AAG CAA TTC TTT GGC CAA AAA AGA AAA TGG GGC GCT AGG 1986 Val Gly Tyr Lys Gin Phe Phe Gly Gin Lys Arg Lys Trp Gly Ala Arg 520 525 530
TAT TAC GGC TTT TTT GAC TAC AAC CAT GCG TTC ATT AAA TCC AGC TTC 2034 Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Phe He Lys Ser Ser Phe 535 540 545 550 TTC AAC TCG GCT TCT GAT GTG TGG ACT TAT GGT TTT GGA GCG GAC GCT 2082
Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly Ala Asp Ala 555 560 565
CTT TAT AAC TTC ATC AAC GAT AAA GCC ACC AAT TTC TTA GGC AAA AAC 2130 Leu Tyr Asn Phe He Asn Asp Lys Ala Thr Asn Phe Leu Gly Lys Asn 570 575 580
AAC AAG CTT TCC GTG GGG CTT TTT GGA GGG ATT GCG TTA GCG GGC ACT 2178 Asn Lys Leu Ser Val Gly Leu Phe Gly Gly He Ala Leu Ala Gly Thr 585 590 595
TCA TGG CTT AAT TCT GAG TAT GTG AAT TTA GCC ACC GTG AAT AAC GTC 2226 Ser Trp Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val Asn Asn Val 600 605 610
TAT AAC GCT AAA ATG AAT GTG GCG AAT TTC CAA TTC TTA TTC AAT ATG 2274 Tyr Asn Ala Lys Met Asn Val Ala Asn Phe Gin Phe Leu Phe Asn Met 615 620 625 630
GGA GTG AGG ATG AAT TTA GCC AGA TCC AAG AAA AAA GGC AGC GAT CAT 2322 Gly Val Arg Met Asn Leu Ala Arg Ser Lys Lys Lys Gly Ser Asp His 635 640 645
GCG GCT CAG CAT GGG ATT GAA CTA GGG CTT AAA ATC CCC ACC ATC AAC 2370 Ala Ala Gin His Gly He Glu Leu Gly Leu Lys He Pro Thr He Asn 650 655 660
ACG AAC TAT TAT TCT TTC ATG GGG GCT GAA CTC AAA TAC AGA AGG CTT 2418 Thr Asn Tyr Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg Arg Leu 665 670 675
TAT AGC GTG TAT TTG AAT TAT GTG TTC GCT TAC TAAGCTTTTT GTGAAACTCC 2471 Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr 680 685
CTTTTTAAGG GGTTTTTTTT TGAACTCTCT TTTTAAATTC TCTTTTTAAA GAGATTTCTT 2531
TTTTTTAAGC TTTTTTTTGA ATTCTTTTTT TTGAATTCTT TGTTTTTAAG CTTTTTTTAA 2591
ACCCTTTCGT TTTTAAACTC CCTTTTTTAA GGGATTTCTT TTTTTAAACT CTTTTTTTTT 2651
AAACTCTTTT TTTTAAACCC TCTTTTTTTA AGGGATTTCT TTTTAAAGCT TTTTTGAAGT 2711
CTTTTTTTAA ATTCTTTTTT TGGOGGTTTG ATCTTTCTTT TTGCCAATCC CCACTACTTT 2771
CGCTTTTTAA TCTTTAGGTT TTATTTT 2798
(2) INFORMATION FOR SEQ ID NO : 2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 708 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...19 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :
Met Lys Lys Thr Leu Leu Leu Ser Leu Ser Leu Ser Leu Ser Phe Leu
-15 -10 -5
Leu His Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gin He
1 5 10
Gly Glu Ala Ala Gin Met Val Lys Asn Thr Lys Gly He Gin Glu Leu
15 20 25
Ser Asp Asn Tyr Glu Lys Leu Asn Asn Leu Leu Asn Asn Tyr Ser Thr 30 35 40 45
Leu Asn Thr Leu He Lys Leu Ser Ala Asp Pro Ser Ala He Asn Asp
50 55 60
Ala Arg Asp Asn Leu Gly Ser Ser Ser Arg Asn Leu Leu Asp Val Lys
65 70 75
Thr Asn Ser Pro Ala Tyr Gin Ala Val Leu Leu Ala Leu Asn Ala Ala
80 85 90
Val Gly Leu Trp Gin Val Thr Ser Tyr Ala Phe Thr Ala Cys Gly Pro
95 100 105
Gly Ser Asn Glu Asn Ala Asn Gly Gly He Gin Thr Phe Asn Asn Val 110 115 120 125
Pro Gly Gin Asp Thr Thr Thr He Thr Cys Asn Ser Tyr Tyr Glu Pro
130 135 140
Gly His Gly Gly Pro He Ser Thr Ala Asn Tyr Ala Lys He Asn Gin
145 150 155
Ala Tyr Gin He He Gin Lys Ala Leu Thr Ala Asn Gly Ala Asn Gly
160 165 170
Asp Gly Val Pro Val Leu Ser Asn Thr Thr Thr Lys Leu Asp Phe Thr
175 180 185
He Asn Gly Asp Lys Arg Thr Gly Gly Lys Pro Asn Thr Pro Glu Lys 190 195 200 205
Phe Pro Trp Ser Asp Gly Lys Tyr He His Thr Gin Trp He Asn Thr
210 215 220
He Val Thr Pro Thr Glu Thr Asn He Asn Thr Glu Asn Asn Ala Gin
225 230 235
Glu Leu Leu Lys Gin Ala Ser He He He Thr Thr Leu Asn Glu Ala
240 245 250
Cys Pro Asn Phe Gin Asn Gly Gly Arg Ser Tyr Trp Gin Gly He Ser
255 260 265
Gly Asn Gly Thr Met Cys Gly Met Phe Lys Asn Glu He Ser Ala He 270 275 280 285
Gin Gly Met He Ala Asn Ala Gin Glu Ala Val Ala Gin Ser Lys He
290 295 300
Val Ser Glu Asn Ala Gin Asn Gin Asn Asn Leu Asp Thr Gly Lys Pro
305 310 315
Phe Asn Pro Tyr Thr Asp Ala Ser Phe Ala Gin Ser Met Leu Lys Asn
320 325 330
Ala Gin Ala Gin Ala Glu He Leu Asn Gin Ala Glu Gin Val Val Lys
335 340 345
Asn Phe Glu Lys He Pro Thr Ala Phe Val Ser Asp Ser Leu Gly Val 350 355 360 365
Cys Tyr Glu Val Gin Gly Gly Glu Arg Arg Gly Thr Asn Pro Gly Gin
370 375 380
Val Thr Ser Asn Thr Trp Gly Ala Gly Cys Ala Tyr Val Lys Gin Thr 385 390 395
He Thr Asn Leu Asp Asn Ser He Ala His Phe Gly Thr Gin Glu Gin
400 405 410
Gin He Gin Gin Ala Glu Asn He Ala Asp Thr Leu Val Asn Phe Lys
415 420 425
Ser Arg Tyr Ser Glu Leu Gly Asn Thr Tyr Asn Ser He Thr Thr Ala 430 435 440 445
Leu Ser Lys Val Pro Asn Ala Gin Ser Leu Gin Asn Val Val Ser Lys
450 455 460
Lys Asn Asn Pro Tyr Ser Pro Gin Gly He Glu Thr Asn Tyr Tyr Leu
465 470 475
Asn Gin Asn Ser Tyr Asn Gin He Gin Thr He Asn Gin Glu Leu Gly
480 485 490
Arg Asn Pro Phe Arg Lys Val Gly He Val Asn Ser Gin Thr Asn Asn
495 500 505
Gly Ala Met Asn Gly He Gly He Gin Val Gly Tyr Lys Gin Phe Phe 510 515 520 525
Gly Gin Lys Arg Lys Trp Gly Ala Arg Tyr Tyr Gly Phe Phe Asp Tyr
530 535 540
Asn His Ala Phe He Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp Val
545 550 555
Trp Thr Tyr Gly Phe Gly Ala Asp Ala Leu Tyr Asn Phe He Asn Asp
560 565 570
Lys Ala Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Val Gly Leu
575 580 585
Phe Gly Gly He Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Glu Tyr 590 595 600 605
Val Asn Leu Ala Thr Val Asn Asn Val Tyr Asn Ala Lys Met Asn Val
610 615 620
Ala Asn Phe Gin Phe Leu Phe Asn Met Gly Val Arg Met Asn Leu Ala
625 630 635
Arg Ser Lys Lys Lys Gly Ser Asp His Ala Ala Gin His Gly He Glu
640 645 650
Leu Gly Leu Lys He Pro Thr He Asn Thr Asn Tyr Tyr Ser Phe Met
655 660 665
Gly Ala Glu Leu Lys Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr 670 675 680 685
Val Phe Ala Tyr
(2) INFORMATION FOR SEQ ID NO : 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2699 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 199...2397 (D) OTHER INFORMATION: (A) NAME/KEY: Signal Sequence
(B) LOCATION: 199...259 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
TAAAATCCAA TTAAAAGCGT TCAAAGGTAA CGCAAAAAAA CAAAAAATGA CGCAATTTTT 60
TCAAAATGAC AAAAAAAAAC GCTTTATGCT ATAATACCCC AAATACATTC TAATAGCAAA 120
TGCGTTCTAA TGCAAATGCA TTCCAATGTA TGAAATCCCT AATACTAAAT CCAATTTAAT 180
CCAAAAAGGA GAAAAAAC ATG AAA AAA CAC ATC CTT TCA TTA GCT TTA GGC 231
Met Lys Lys His He Leu Ser Leu Ala Leu Gly -20 -15 -10
TCG CTT TTA GTT TCC ACT TTG AGC GCT GAA GAC GAC GGC TTT TAC ACA 279 Ser Leu Leu Val Ser Thr Leu Ser Ala Glu Asp Asp Gly Phe Tyr Thr
-5 1 5
AGC GTA GGC TAT CAG ATC GGT GAA GCC GCT CAA ATG GTA ACA AAC ACC 327 Ser Val Gly Tyr Gin He Gly Glu Ala Ala Gin Met Val Thr Asn Thr 10 15 20
AAA GGC ATC CAA CAG CTT TCA GAC AAT TAT GAA AAT TTG AAC AAC CTT 375 Lys Gly He Gin Gin Leu Ser Asp Asn Tyr Glu Asn Leu Asn Asn Leu 25 30 35
TTA ACG AGA TAC AGC ACC CTA AAC ACC CTT ATC AAA TTG TCC GCT GAT 423 Leu Thr Arg Tyr Ser Thr Leu Asn Thr Leu He Lys Leu Ser Ala Asp 40 45 50 55
CCG AGC GCA ATT AAT GCG GTG CGG GAA AAT CTG GGC GCG AGC GCG AAG 471 Pro Ser Ala He Asn Ala Val Arg Glu Asn Leu Gly Ala Ser Ala Lys 60 65 70
AAT TTG ATC GGC GAT AAA GCC AAC TCC CCC GCC TAT CAA GCC GTG CTT 519 Asn Leu He Gly Asp Lys Ala Asn Ser Pro Ala Tyr Gin Ala Val Leu 75 80 85
TTA GCG ATC AAC GCG GCG GTA GGG TTT TGG AAT GTC GTG GGC TAT GTG 567 Leu Ala He Asn Ala Ala Val Gly Phe Trp Asn Val Val Gly Tyr Val 90 95 100
ACG CAA TGT GGG GGT AAC GCC AAT GGT CAA GAA AGC ACC TCT TCA ACC 615 Thr Gin Cys Gly Gly Asn Ala Asn Gly Gin Glu Ser Thr Ser Ser Thr 105 110 115
ACC ATC TTC AAC AAC GAG CCA GGG TAT CGA TCC ACT TCC ATC ACT TGT 663 Thr He Phe Asn Asn Glu Pro Gly Tyr Arg Ser Thr Ser He Thr Cys 120 125 130 135
TCT TTG AAC GGG CAT AAG CCT GGA TAC TAT GGC CCT ATG AGC ATT GAG 711 Ser Leu Asn Gly His Lys Pro Gly Tyr Tyr Gly Pro Met Ser He Glu 140 145 150
AAT TTT AAA AAG CTT AAC GAA GCC TAT CAG ATC CTC CAA ACG GCT TTA 759 Asn Phe Lys Lys Leu Asn Glu Ala Tyr Gin He Leu Gin Thr Ala Leu 155 160 165
AAA AAC GGC TTA CCC GCG CTC AAA GAA AAC AAC GGG AAG GTC AGT GTA 807 Lys Asn Gly Leu Pro Ala Leu Lys Glu Asn Asn Gly Lys Val Ser Val 170 175 180
ACC TAT ACC TAC ACA TGC TCA GGG CAA GGG AAT AAT AAC TGC TCG CCA 855 Thr Tyr Thr Tyr Thr Cys Ser Gly Gin Gly Asn Asn Asn Cys Ser Pro 185 190 195
AGT GTC AAC GGA ACC AAA ACC ACA ACC CAA ACC ATA GAC GGC AAA AGC 903 Ser Val Asn Gly Thr Lys Thr Thr Thr Gin Thr He Asp Gly Lys Ser 200 205 210 215
GTA ACC ACC ACG ATC AGT TCA AAA GTG GTT GGT AGC ATC GCT AGT GGC 951 Val Thr Thr Thr He Ser Ser Lys Val Val Gly Ser He Ala Ser Gly 220 225 230
AAC ACA TCA CAT GTC ATC ACC AAC AAA TTA GAC GGT GTG CCT GAT AGC 999 Asn Thr Ser His Val He Thr Asn Lys Leu Asp Gly Val Pro Asp Ser 235 240 245
GCT CAA GCG CTC TTA GCG CAA GCG AGC ACG CTC ATC AAC ACC ATC AAC 1047 Ala Gin Ala Leu Leu Ala Gin Ala Ser Thr Leu He Asn Thr He Asn 250 255 260
GAA GCA TGC CCG TAT TTC CAT GCT ACT AAT AGT AGT GAG GCT AAC GCC 1095 Glu Ala Cys Pro Tyr Phe His Ala Thr Asn Ser Ser Glu Ala Asn Ala 265 270 275
CCA AAA TTC TCT ACT ACT ACT GGG AAA ATA TGC GGC GCT TTT TCA GAA 1143 Pro Lys Phe Ser Thr Thr Thr Gly Lys He Cys Gly Ala Phe Ser Glu 280 285 290 295
GAA ATC AGC GCG ATC CAA AAG ATG ATC ACG GAC GCG CAA GAG CTA GTT 1191 Glu He Ser Ala He Gin Lys Met He Thr Asp Ala Gin Glu Leu Val 300 305 310
AAT CAA ACG AGC GTC ATT AAC AGC AAC GAA CAA TCA ACT CCG GTA GGC 1239 Asn Gin Thr Ser Val He Asn Ser Asn Glu Gin Ser Thr Pro Val Gly 315 320 325
AAT AAT AAT GGC AAG CCT TTC AAC CCT TTC ACG GAC GCA AGT TTT GCG 1287 Asn Asn Asn Gly Lys Pro Phe Asn Pro Phe Thr Asp Ala Ser Phe Ala 330 335 340
CAA GGC ATG CTC GCT AAC GCT AGC GCG CAA GCT AAA ATG CTC AAT TTA 1335 Gin Gly Met Leu Ala Asn Ala Ser Ala Gin Ala Lys Met Leu Asn Leu 345 350 355
GCC CAT CAG GTG GGG CAA GCC ATT AAC CCA GAG AAT CTT AGC GAG AAT 138"3 Ala His Gin Val Gly Gin Ala He Asn Pro Glu Asn Leu Ser Glu Asn 360 365 370 375
TTT AAA AAT TTT GTT ACA GGC TTT TTA GCC ACA TGC AAT AAC AAA TCA. 1431 Phe Lys Asn Phe Val Thr Gly Phe Leu Ala Thr Cys Asn Asn Lys Ser 380 385 390 ACA GCT GGC ACT GGT GGC ACA CAA GGT TCA GCT CCA GGC ACA GTG ACC 1479 Thr Ala Gly Thr Gly Gly Thr Gin Gly Ser Ala Pro Gly Thr Val Thr 395 400 405
ACT CAA ACT TTC GCT TCT GGT TGC GCG TAT GTG GAG CAA ACC CTA ACG 1527 Thr Gin Thr Phe Ala Ser Gly Cys Ala Tyr Val Glu Gin Thr Leu Thr 410 415 420
AAC TTA GGC AAC AGC ATC GCT CAC TTT GGC ACT CAA GAG CAG CAG ATA 1575 Asn Leu Gly Asn Ser He Ala His Phe Gly Thr Gin Glu Gin Gin He 425 430 435
CAG CAA GCC GAA AAC ATC GCT GAC ACT CTA GTG AAT TTC AAA TCT AGA 1623 Gin Gin Ala Glu Asn He Ala Asp Thr Leu Val Asn Phe Lys Ser Arg 440 445 450 455
TAC AGC GAA TTA GGC AAC ACC TAT AAC AGC ATC ACC ACC GCG CTC TCC 1671 Tyr Ser Glu Leu Gly Asn Thr Tyr Asn Ser He Thr Thr Ala Leu Ser 460 465 470
AAA GTC CCT AAC GCG CAA AGC TTG CAA AAC GTG GTG AGC AAA AAG AAT 1719 Lys Val Pro Asn Ala Gin Ser Leu Gin Asn Val Val Ser Lys Lys Asn 475 480 485
AAC CCC TAT AGC CCT CAA GGC ATA GAG ACC AAT TAC TAC CTC AAT CAA 1767 Asn Pro Tyr Ser Pro Gin Gly He Glu Thr Asn Tyr Tyr Leu Asn Gin 490 495 500
AAT TCT TAC AAC CAA ATC CAA ACC ATC AAC CAA GAA CTA GGG CGT AAC 1815 Asn Ser Tyr Asn Gin He Gin Thr He Asn Gin Glu Leu Gly Arg Asn 505 510 515
CCC TTT AGG AAA GTG GGC ATC GTC AAT TCT CAA ACC AAC AAT GGT GCC 1863 Pro Phe Arg Lys Val Gly He Val Asn Ser Gin Thr Asn Asn Gly Ala 520 525 530 535
ATG AAT GGG ATC GGT ATT CAG GTG GGC TAT AAG CAA TTC TTT GGC CAA 1911 Met Asn Gly He Gly He Gin Val Gly Tyr Lys Gin Phe Phe Gly Gin 540 545 550
AAA AGA AAA TGG GGC GCT AGG TAT TAC GGC TTT TTT GAT TAC AAC CAT 1959 Lys Arg Lys Trp Gly Ala Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His 555 560 565
GCG TTC ATC AAA TCC AGC TTT TTC AAC TCG GCT TCT GAC GTG TGG ACT 2007 Ala Phe He Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp Val Trp Thr 570 575 580
TAT GGT TTT GGA GCG GAC GCG CTT TAT AAC TTC ATC AAC GAT AAA GCC 2055 Tyr Gly Phe Gly Ala Asp Ala Leu Tyr Asn Phe He Asn Asp Lys Ala 585 590 595
ACC AAT TTC TTA GGC AAA AAC AAC AAG CTT TCT TTG GGG CTT TTT GGC 2103 Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Leu Gly Leu Phe Gly 600 605 610 615
GGG ATT GCG TTA GCG GGC ACT TCA TGG CTC AAT TCT GAG TAC GTG AAT 2151 Gly He Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Glu Tyr Val Asn 620 625 630
TTA GCC ACC GTG AAT AAC GTC TAT AAC GCT AAA ATG AAT GTG GCG AAT 2199
Leu Ala Thr Val Asn Asn Val Tyr Asn Ala Lys Met Asn Val Ala Asn 635 640 645
TTC CAA TTC TTA TTC AAT ATG GGA GTG AGG ATG AAT TTA GCC AGA TCC 2247
Phe Gin Phe Leu Phe Asn Met Gly Val Arg Met Asn Leu Ala Arg Ser
650 655 660
AAG AAA AAA GGC AGC GAT CAT GCA GCT CAG CAT GGG ATT GAG TTA GGG 2295
Lys Lys Lys Gly Ser Asp His Ala Ala Gin His Gly He Glu Leu Gly 665 670 675
CTT AAA ATC CCC ACC ATC AAC ACG AAC TAT TAT TCC TTT ATG GGG GCT 2343
Leu Lys He Pro Thr He Asn Thr Asn Tyr Tyr Ser Phe Met Gly Ala
680 685 690 695
GAA CTC AAA TAC AGA AGG CTC TAT AGC GTG TAT TTG AAC TAT GTG TTC 2391
Glu Leu Lys Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe 700 705 710
GCT TAC TAATGTTTGG CTCTTTGTGA AACTCCCTTT TTAAGGGGTT TTTTTTTGAA CT 2449 Ala Tyr
CTCTTTTTAA ATTCTCTTTT TAAAGAGATT TCTTTTTTTT AAGCTTTTTT TTGAATTCTT 2509
TTTTTTTGAA TTCTTTGTTT TTAAGCTTTT TTTAAACCCT TTCGTTTTTA AACTCCCTTT 2569
TTTAAGGGAT TTCTTTTTTT GAACTCCCTT TTTTGAACCC TTTTTTTTAA ACCCTCTTTT 2629
TTTAAGGGGT TTCTTTTTAA AGCTTTTTTG AAGTCTTTTT TTAAATTCTT TTTTTGGGGG 2689
TTTGATCTTT 2699
(2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 733 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...20 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
Met Lys Lys His He Leu Ser Leu Ala Leu Gly Ser Leu Leu Val Ser
-20 -15 -10 -5.
Thr Leu Ser Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gin
1 5 10 Ile Gly Glu Ala Ala Gin Met Val Thr Asn Thr Lys Gly He Gin Gin
15 20 25
Leu Ser Asp Asn Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr Ser
30 35 40
Thr Leu Asn Thr Leu He Lys Leu Ser Ala Asp Pro Ser Ala He Asn 45 50 55 60
Ala Val Arg Glu Asn Leu Gly Ala Ser Ala Lys Asn Leu He Gly Asp
65 70 75
Lys Ala Asn Ser Pro Ala Tyr Gin Ala Val Leu Leu Ala He Asn Ala
80 85 90
Ala Val Gly Phe Trp Asn Val Val Gly Tyr Val Thr Gin Cys Gly Gly
95 100 105
Asn Ala Asn Gly Gin Glu Ser Thr Ser Ser Thr Thr He Phe Asn Asn
110 115 120
Glu Pro Gly Tyr Arg Ser Thr Ser He Thr Cys Ser Leu Asn Gly His 125 130 135 140
Lys Pro Gly Tyr Tyr Gly Pro Met Ser He Glu Asn Phe Lys Lys Leu
145 150 155
Asn Glu Ala Tyr Gin He Leu Gin Thr Ala Leu Lys Asn Gly Leu Pro
160 165 170
Ala Leu Lys Glu Asn Asn Gly Lys Val Ser Val Thr Tyr Thr Tyr Thr
175 180 185
Cys Ser Gly Gin Gly Asn Asn Asn Cys Ser Pro Ser Val Asn Gly Thr
190 195 200
Lys Thr Thr Thr Gin Thr He Asp Gly Lys Ser Val Thr Thr Thr He 205 210 215 220
Ser Ser Lys Val Val Gly Ser He Ala Ser Gly Asn Thr Ser His Val
225 230 235
He Thr Asn Lys Leu Asp Gly Val Pro Asp Ser Ala Gin Ala Leu Leu
240 245 250
Ala Gin Ala Ser Thr Leu He Asn Thr He Asn Glu Ala Cys Pro Tyr
255 260 265
Phe His Ala Thr Asn Ser Ser Glu Ala Asn Ala Pro Lys Phe Ser Thr
270 275 280
Thr Thr Gly Lys He Cys Gly Ala Phe Ser Glu Glu He Ser Ala He 285 290 295 300
Gin Lys Met He Thr Asp Ala Gin Glu Leu Val Asn Gin Thr Ser Val
305 310 315
He Asn Ser Asn Glu Gin Ser Thr Pro Val Gly Asn Asn Asn Gly Lys
320 325 330
Pro Phe Asn Pro Phe Thr Asp Ala Ser Phe Ala Gin Gly Met Leu Ala
335 340 345
Asn Ala Ser Ala Gin Ala Lys Met Leu Asn Leu Ala His Gin Val Gly
350 355 360
Gin Ala He Asn Pro Glu Asn Leu Ser Glu Asn Phe Lys Asn Phe Val 365 370 375 380
Thr Gly Phe Leu Ala Thr Cys Asn Asn Lys Ser Thr Ala Gly Thr Gly
385 390 395
Gly Thr Gin Gly Ser Ala Pro Gly Thr Val Thr Thr Gin Thr Phe Ala
400 405 410
Ser Gly Cys Ala Tyr Val Glu Gin Thr Leu Thr Asn Leu Gly Asn Ser
415 420 425
He Ala His Phe Gly Thr Gin Glu Gin Gin He Gin Gin Ala Glu Asn
430 435 440
He Ala Asp Thr Leu Val Asn Phe Lys Ser Arg Tyr Ser Glu Leu Gly 445 450 455 460
Asn Thr Tyr Asn Ser He Thr Thr Ala Leu Ser Lys Val Pro Asn Ala 465 470 475
Gin Ser Leu Gin Asn Val Val Ser Lys Lys Asn Asn Pro Tyr Ser Pro
480 485 490
Gin Gly He Glu Thr Asn Tyr Tyr Leu Asn Gin Asn Ser Tyr Asn Gin
495 500 505
He Gin Thr He Asn Gin Glu Leu Gly Arg Asn Pro Phe Arg Lys Val
510 515 520
Gly He Val Asn Ser Gin Thr Asn Asn Gly Ala Met Asn Gly He Gly 525 530 535 540
He Gin Val Gly Tyr Lys Gin Phe Phe Gly Gin Lys Arg Lys Trp Gly
545 550 555
Ala Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Phe He Lys Ser
560 565 570
Ser Phe Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly Ala
575 580 585
Asp Ala Leu Tyr Asn Phe He Asn Asp Lys Ala Thr Asn Phe Leu Gly
590 595 600
Lys Asn Asn Lys Leu Ser Leu Gly Leu Phe Gly Gly He Ala Leu Ala 605 610 615 620
Gly Thr Ser Trp Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val Asn
625 630 635
Asn Val Tyr Asn Ala Lys Met Asn Val Ala Asn Phe Gin Phe Leu Phe
640 645 650
Asn Met Gly Val Arg Met Asn Leu Ala Arg Ser Lys Lys Lys Gly Ser
655 660 665
Asp His Ala Ala Gin His Gly He Glu Leu Gly Leu Lys He Pro Thr
670 675 680
He Asn Thr Asn Tyr Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg 685 690 695 700
Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr 705 710
(2) INFORMATION FOR SEQ ID NO : 5 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2915 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 365...2597 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 365...425 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 TTTTAGGCGA CAAAATCGCT TATGTTGGGG ATAAAGGCAA CCCGCACAAT TTCGCTCACA 60
AGAAATAAAC CGCTCATAAG GGGCAAACGC CCCAAAAAAG CGATTTTTAA AGAGGTTACG 120
GCAAAATCAA GCTCTTTAGT ATTTAATCTT AAAAAATGCT AAAAGCCTTT TTATGGGCTA 180
ACACCACACA AAAAGCATCA AAATCAAAAA AATGACAAAA TTTTTAAGAA AATGACAAAA 240
AAAAACGCTT TATGCTATAA TACCCCAAAT ACATTCTAAT AGCAAATGCG TTCTAATGCA 300
AATGCATTCC AATGTATGAA ATCCCTAATA CTAAATCCAA TTTAATCCAA AAAGGAGAAA 360
AAAC ATG AAA AAA CAC ATC CTT TCA TTA GCT TTA GGC TCG CTT TTA GTT 409
Met Lys Lys His He Leu Ser Leu Ala Leu Gly Ser Leu Leu Val
-20 -15 -10
TCC ACT TTG AGC GCT GAA GAC GAC GGC TTT TAC ACA AGC GTA GGC TAT 457 Ser Thr Leu Ser Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr -5 1 5 10
CAG ATC GGT GAA GCC GCT CAA ATG GTA ACA AAC ACC AAA GGC ATC CAA 505 Gin He Gly Glu Ala Ala Gin Met Val Thr Asn Thr Lys Gly He Gin 15 20 25
CAG CTT TCA GAC AAT TAT GAA AAT TTG AAC AAC CTT TTA ACG AGA TAC 553 Gin Leu Ser Asp Asn Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr 30 35 40
AGC ACC CTA AAC ACC CTT ATC AAA TTG TCC GCT GAT CCG AGC GCA ATT 601 Ser Thr Leu Asn Thr Leu He Lys Leu Ser Ala Asp Pro Ser Ala He 45 50 55
AAT GCG GTG CGG GAA AAT CTG GGC GCG AGC ACG AAG AAT TTG ATC GGC 649 Asn Ala Val Arg Glu Asn Leu Gly Ala Ser Thr Lys Asn Leu He Gly 60 65 70 75
GAT AAA GCC AAC TCC CCG GCG TAT CAA GCC GTG TTT TTA GCG ATC AAC 697 Asp Lys Ala Asn Ser Pro Ala Tyr Gin Ala Val Phe Leu Ala He Asn 80 85 90
GCG GCG GTA GGG TTG TGG AAT ACC ATC GGC TAT GCG GTC ATG TGC GGG 745 Ala Ala Val Gly Leu Trp Asn Thr He Gly Tyr Ala Val Met Cys Gly 95 100 105
AAC GGG AAC GGC ACA GAG AGT GGG CCT GGC AGC GTG ATC TTT AAT GAC 793 Asn Gly Asn Gly Thr Glu Ser Gly Pro Gly Ser Val He Phe Asn Asp 110 115 120
CAA CCA GGA CAG GAT TCC ACG CAA ATT ACT TGC AAC CGC TTT GAA TCA 841 Gin Pro Gly Gin Asp Ser Thr Gin He Thr Cys Asn Arg Phe Glu Ser 125 130 135
ACT GGG CCT GGT AAA AGC ATG TCT ATT GAT GAA TTC AAA AAA CTC AAT 889 Thr Gly Pro Gly Lys Ser Met Ser He Asp Glu Phe Lys Lys Leu Asn 140 145 150 155
GAA GCC TAT CAA ATC ATC CAG CAA GCT TTA AAA AAT CAA AGT GGG TTT 93-7 Glu Ala Tyr Gin He He Gin Gin Ala Leu Lys Asn Gin Ser Gly Phe 160 165 170
CCT GAA TTA GGC GGG AAC GGC ACA AAA GTG AGT GTT AAT TAC AAT TAC 985 Pro Glu Leu Gly Gly Asn Gly Thr Lys Val Ser Val Asn Tyr Asn Tyr" 175 180 185 GAA TGC AGA CAA ACT GCT GAT ATC AAC GGC GGT GTG TAT CAG TTC TGC 1033 Glu Cys Arg Gin Thr Ala Asp He Asn Gly Gly Val Tyr Gin Phe Cys 190 195 200
AAG GCT AAA AAT GGT AGT AGT AGC AGT AGT AAT GGC GGT AAT GGC AGT 1081 Lys Ala Lys Asn Gly Ser Ser Ser Ser Ser Asn Gly Gly Asn Gly Ser 205 210 215
AGC ACG CAA ACA ACC GCG ACA ACC ACG CAA GAC GGC GTA ACG ATC ACC 1129 Ser Thr Gin Thr Thr Ala Thr Thr Thr Gin Asp Gly Val Thr He Thr 220 225 230 235
ACT ACC TAT AAT AAT AAC AAA GCC ACC GTC AAA TTT GAC ATC ACC AAT 1177 Thr Thr Tyr Asn Asn Asn Lys Ala Thr Val Lys Phe Asp He Thr Asn 240 245 250
AAC GCT GAA CAG CTG TTA AAT CAA GCG GCA AAC ATC ATG CAA GTC CTT 1225 Asn Ala Glu Gin Leu Leu Asn Gin Ala Ala Asn He Met Gin Val Leu 255 260 265
AAT ACG CAA TGC CCT TTA GTG CGT TCC ACG AAT AAC GAA AAC ACT CCA 1273 Asn Thr Gin Cys Pro Leu Val Arg Ser Thr Asn Asn Glu Asn Thr Pro 270 275 280
GGG GGT GGT CAA CCA TGG GGT TTA AGC ACA TCC GGG AAT GCG TGC AGC 1321 Gly Gly Gly Gin Pro Trp Gly Leu Ser Thr Ser Gly Asn Ala Cys Ser 285 290 295
ATC TTC CAA CAA GAA TTT AGC CAG GTT ACT AGC ATG ATC AAA AAC GCC 1369 He Phe Gin Gin Glu Phe Ser Gin Val Thr Ser Met He Lys Asn Ala 300 305 310 315
CAA GAA ATA ATC GCG CAA AGC AAA ATC GTT AGT GAA AAC GCG CAA AAT 1417 Gin Glu He He Ala Gin Ser Lys He Val Ser Glu Asn Ala Gin Asn 320 325 330
CAA AAC AAC TTG GAT ACT GGA AAA CCA TTC AAC CCT TAC ACG GAC GCC 1465 Gin Asn Asn Leu Asp Thr Gly Lys Pro Phe Asn Pro Tyr Thr Asp Ala 335 340 345
AGC TTT GCG CAA AGC ATG CTC AAA AAC GCT CAA GCG CAA GCA GAG ATG 1513 Ser Phe Ala Gin Ser Met Leu Lys Asn Ala Gin Ala Gin Ala Glu Met 350 355 360
TTC AAT TTG AGC GAA CAA GTG AAA AAG AAC TTG GAA GTC ATG AAA AAC 1561 Phe Asn Leu Ser Glu Gin Val Lys Lys Asn Leu Glu Val Met Lys Asn 365 370 375
AAC AAT AAT GTT AAC GAG AAA TTA GCA GGA TTT GGG AAA GAA GAA GTA 1609 Asn Asn Asn Val Asn Glu Lys Leu Ala Gly Phe Gly Lys Glu Glu Val" - 380 385 390 395
ATG ACC AAT TTT GTT AGC GCC TTT TTG GCA AGC TGC AAA GAT GGT GGC 1657 Met Thr Asn Phe Val Ser Ala Phe Leu Ala Ser Cys Lys Asp Gly Gly 400 405 410
ACA TTG CCT AAT GCA GGG GTT ACT TCT AAC ACT TGG GGG GCG GGT TGC 1705 Thr Leu Pro Asn Ala Gly Val Thr Ser Asn Thr Trp Gly Ala Gly Cys 415 420 425
GCG TAT GTG GGA GAG ACG ATA AGC GCC CTA ACC AAC AGC ATC GCT CAC 1753 Ala Tyr Val Gly Glu Thr He Ser Ala Leu Thr Asn Ser He Ala His 430 435 440
TTT GGC ACT CAA GAG CAG CAG ATA CAG CAA GCC GAA AAC ATC GCT GAC 1801 Phe Gly Thr Gin Glu Gin Gin He Gin Gin Ala Glu Asn He Ala Asp 445 450 455
ACT CTA GTG AAT TTC AAA TCT AGA TAC AGC GAA TTA GGC AAC ACC TAT 1849 Thr Leu Val Asn Phe Lys Ser Arg Tyr Ser Glu Leu Gly Asn Thr Tyr 460 465 470 475
AAC AGC ATC ACC ACC GCG CTC TCC AAA GTC CCT AAC GCG CAA AGC TTG 1897 Asn Ser He Thr Thr Ala Leu Ser Lys Val Pro Asn Ala Gin Ser Leu 480 485 490
CAA AAC GTG GTG AGC AAA AAG AAT AAC CCC TAT AGC CCT CAA GGC ATA 1945 Gin Asn Val Val Ser Lys Lys Asn Asn Pro Tyr Ser Pro Gin Gly He 495 500 505
GAG ACC AAT TAC TAC CTC AAT CAA AAT TCT TAC AAC CAA ATC CAA ACC 1993 Glu Thr Asn Tyr Tyr Leu Asn Gin Asn Ser Tyr Asn Gin He Gin Thr 510 515 520
ATC AAC CAA GAA CTA GGG CGT AAC CCC TTT AGG AAA GTG GGC ATC GTC 2041 He Asn Gin Glu Leu Gly Arg Asn Pro Phe Arg Lys Val Gly He Val 525 530 535
AAT TCT CAA ACC AAC AAT GGT GCC ATG AAT GGG ATC GGC ATT CAG GTG 2089 Asn Ser Gin Thr Asn Asn Gly Ala Met Asn Gly He Gly He Gin Val 540 545 550 555
GGC TAT AAG CAA TTC TTT GGC CAA AAA AGA AAA TGG GGC GCT AGG TAT 2137 Gly Tyr Lys Gin Phe Phe Gly Gin Lys Arg Lys Trp Gly Ala Arg Tyr 560 565 570
TAC GGC TTT TTT GAT TAC AAC CAT GCG TTC ATC AAA TCC AGC TTT TTC 2185 Tyr Gly Phe Phe Asp Tyr Asn His Ala Phe He Lys Ser Ser Phe Phe 575 580 585
AAC TCG GCT TCT GAC GTG TGG ACT TAT GGT TTT GGA GCG GAC GCG CTT 2233 Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly Ala Asp Ala Leu 590 595 600
TAT AAC TTC ATC AAC GAT AAA GCC ACC AAT TTC TTA GGC AAA AAC AAC 2281 Tyr Asn Phe He Asn Asp Lys Ala Thr Asn Phe Leu Gly Lys Asn Asn
605 610 615 - -
AAG CTT TCT TTG GGG CTT TTT GGC GGG ATT GCG TTA GCG GGC ACT TCA 2329 Lys Leu Ser Leu Gly Leu Phe Gly Gly He Ala Leu Ala Gly Thr Ser 620 625 630 635
TGG CTC AAT TCT GAG TAC GTG AAT TTA GCC ACC GTG AAT AAC GTC TAT 2377 Trp Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val Asn Asn Val Tyr 640 645 650
AAC GCT AAA ATG AAT GTG GCG AAT TTC CAA TTC TTA TTC AAT ATG GGA 2425 Asn Ala Lys Met Asn Val Ala Asn Phe Gin Phe Leu Phe Asn Met Gly 655 660 665
GTG AGG ATG AAT TTA GCC AGA TCC AAG AAA AAA GGC AGC GAT CAT GCA 2473 Val Arg Met Asn Leu Ala Arg Ser Lys Lys Lys Gly Ser Asp His Ala 670 675 680
GCT CAG CAT GGG ATT GAG TTA GGG CTT AAA ATC CCC ACC ATC AAC ACG 2521 Ala Gin His Gly He Glu Leu Gly Leu Lys He Pro Thr He Asn Thr 685 690 695
AAC TAT TAT TCC TTT ATG GGG GCT GAA CTC AAA TAC AGA AGG CTC TAT 2569 Asn Tyr Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg Arg Leu Tyr 700 705 710 715
AGC GTG TAT TTG AAT NAT GTG TTC GCT TAC TAAGCTTTTT GTGAAACTCC 2619 Ser Val Tyr Leu Asn Xaa Val Phe Ala Tyr 720 725
CTTTTTAAGG GGTTTTTTTT TGAACTCTCT TTTAAATTCT CTTTTTAAAG AGATTTCTTT 2679
TTTTAAGCTT TTTTTTGAAC TTTTTTTTGA ATTCTTTGTT TTTAAGCTTT TTTTAAACCC 2739
TTTCGTTTTT AAACTCCCTT TTTTAAGGGA TTTCTTTTTT TGAACTCCCT TTTTTGAACC 2799
CTTTTTTTTA AACCCTCTTT TTTTAAGGGG TTTCTTTTTA AAGCTTTTTT GAAGTCTTTT 2859
TTTAAATTCT TTTTTTGGGG GTTTGATCTT TCTTTTTGCC AATCCCCACT ACTTTC 2915
(2) INFORMATION FOR SEQ ID NO : 6
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 745 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...20 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :
Met Lys Lys His He Leu Ser Leu Ala Leu Gly Ser Leu Leu Val Ser -20 -15 -10 -5
Thr Leu Ser Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gin
1 5 10
He Gly Glu Ala Ala Gin Met Val Thr Asn Thr Lys Gly He Gin Gin
15 20 25
Leu Ser Asp Asn Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr Ser 30 35 40
Thr Leu Asn Thr Leu He Lys Leu Ser Ala Asp Pro Ser Ala He Asn 45 50 55 60
Ala Val Arg Glu Asn Leu Gly Ala Ser Thr Lys Asn Leu He Gly Asp
65 70 75
Lys Ala Asn Ser Pro Ala Tyr Gin Ala Val Phe Leu Ala He Asn Ala
80 85 90
Ala Val Gly Leu Trp Asn Thr He Gly Tyr Ala Val Met Cys Gly Asn
95 100 105
Gly Asn Gly Thr Glu Ser Gly Pro Gly Ser Val He Phe Asn Asp Gin
110 115 120
Pro Gly Gin Asp Ser Thr Gin He Thr Cys Asn Arg Phe Glu Ser Thr 125 130 135 140
Gly Pro Gly Lys Ser Met Ser He Asp Glu Phe Lys Lys Leu Asn Glu
145 150 155
Ala Tyr Gin He He Gin Gin Ala Leu Lys Asn Gin Ser Gly Phe Pro
160 165 170
Glu Leu Gly Gly Asn Gly Thr Lys Val Ser Val Asn Tyr Asn Tyr Glu
175 180 185
Cys Arg Gin Thr Ala Asp He Asn Gly Gly Val Tyr Gin Phe Cys Lys
190 195 200
Ala Lys Asn Gly Ser Ser Ser Ser Ser Asn Gly Gly Asn Gly Ser Ser 205 210 215 220
Thr Gin Thr Thr Ala Thr Thr Thr Gin Asp Gly Val Thr He Thr Thr
225 230 235
Thr Tyr Asn Asn Asn Lys Ala Thr Val Lys Phe Asp He Thr Asn Asn
240 245 250
Ala Glu Gin Leu Leu Asn Gin Ala Ala Asn He Met Gin Val Leu Asn
255 260 265
Thr Gin Cys Pro Leu Val Arg Ser Thr Asn Asn Glu Asn Thr Pro Gly
270 275 280
Gly Gly Gin Pro Trp Gly Leu Ser Thr Ser Gly Asn Ala Cys Ser He 285 290 295 300
Phe Gin Gin Glu Phe Ser Gin Val Thr Ser Met He Lys Asn Ala Gin
305 310 315
Glu He He Ala Gin Ser Lys He Val Ser Glu Asn Ala Gin Asn Gin
320 325 330
Asn Asn Leu Asp Thr Gly Lys Pro Phe Asn Pro Tyr Thr Asp Ala Ser
335 340 345
Phe Ala Gin Ser Met Leu Lys Asn Ala Gin Ala Gin Ala Glu Met Phe
350 355 360
Asn Leu Ser Glu Gin Val Lys Lys Asn Leu Glu Val Met Lys Asn Asn 365 370 375 380
Asn Asn Val Asn Glu Lys Leu Ala Gly Phe Gly Lys Glu Glu Val Met
385 390 395
Thr Asn Phe Val Ser Ala Phe Leu Ala Ser Cys Lys Asp Gly Gly Thr
400 405 410
Leu Pro Asn Ala Gly Val Thr Ser Asn Thr Trp Gly Ala Gly Cys Ala
415 420 425
Tyr Val Gly Glu Thr He Ser Ala Leu Thr Asn Ser He Ala His Phe -
430 435 440
Gly Thr Gin Glu Gin Gin He Gin Gin Ala Glu Asn He Ala Asp Thr 445 450 455 460
Leu Val Asn Phe Lys Ser Arg Tyr Ser Glu Leu Gly Asn Thr Tyr Asn
465 470 475
Ser He Thr Thr Ala Leu Ser Lys Val Pro Asn Ala Gin Ser Leu Gin 480 485 490 Asn Val Val Ser Lys Lys Asn Asn Pro Tyr Ser Pro Gin Gly He Glu
495 500 505
Thr Asn Tyr Tyr Leu Asn Gin Asn Ser Tyr Asn Gin He Gin Thr He
510 515 520
Asn Gin Glu Leu Gly Arg Asn Pro Phe Arg Lys Val Gly He Val Asn 525 530 535 540
Ser Gin Thr Asn Asn Gly Ala Met Asn Gly He Gly He Gin Val Gly
545 550 555
Tyr Lys Gin Phe Phe Gly Gin Lys Arg Lys Trp Gly Ala Arg Tyr Tyr
560 565 570
Gly Phe Phe Asp Tyr Asn His Ala Phe He Lys Ser Ser Phe Phe Asn
575 580 585
Ser Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly Ala Asp Ala Leu Tyr
590 595 600
Asn Phe He Asn Asp Lys Ala Thr Asn Phe Leu Gly Lys Asn Asn Lys 605 610 615 620
Leu Ser Leu Gly Leu Phe Gly Gly He Ala Leu Ala Gly Thr Ser Trp
625 630 635
Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val Asn Asn Val Tyr Asn
640 645 650
Ala Lys Met Asn Val Ala Asn Phe Gin Phe Leu Phe Asn Met Gly Val
655 660 665
Arg Met Asn Leu Ala Arg Ser Lys Lys Lys Gly Ser Asp His Ala Ala
670 675 680
Gin His Gly He Glu Leu Gly Leu Lys He Pro Thr He Asn Thr Asn 685 690 695 700
Tyr Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg Arg Leu Tyr Ser
705 710 715
Val Tyr Leu Asn Xaa Val Phe Ala Tyr 720 725
(2) INFORMATION FOR SEQ ID NO : 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2603 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 210...2342 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 210...270 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 : ATGACCTTTA TTGGTTTAAT ATTTGTTTAG AAATAACACA AAAACCTTTT TTTTTTTTTT 60 TGAAAGGGCA AAAACGCCTA ATTAATATCA AAATCCCATG AATTTATACT ATATTAACGA 120
AAGCTTGCGG TATGGTTTCA CCTAAAGACA CACTTCCGCA AGATTTACTA ACAATTTCAA 180
TCTTATTTCA AGTAATAAAA GGAGAAAAC ATG AAG AAA AAA TTT CTG TCA TTA 233
Met Lys Lys Lys Phe Leu Ser Leu -20 -15
ACC TTA GGT TCG CTT TTA GTT TCC GCT TTA AGC GCT GAA GAC AAC GGC 281 Thr Leu Gly Ser Leu Leu Val Ser Ala Leu Ser Ala Glu Asp Asn Gly -10 -5 1
TTT TTT GTG AGT GCG GGC TAT CAA ATC GGT GAA TCC GCT CAA ATG GTG 329 Phe Phe Val Ser Ala Gly Tyr Gin He Gly Glu Ser Ala Gin Met Val 5 10 15 20
AAA AAC ACT AAA GGC ATT CAA GAT CTT TCA GAT AGC TAT GAA AGA CTG 377 Lys Asn Thr Lys Gly He Gin Asp Leu Ser Asp Ser Tyr Glu Arg Leu 25 30 35
AAC AAT CTT TTA ACG AGT TAT AGT GCC CTA AAC ACT CTT ATT AGG CAG 425 Asn Asn Leu Leu Thr Ser Tyr Ser Ala Leu Asn Thr Leu He Arg Gin 40 45 50
TCC GCC GAC CCC AAC GCT ATC AAT AAC GCA AGG GGC AAT TTG AAC GCT 473 Ser Ala Asp Pro Asn Ala He Asn Asn Ala Arg Gly Asn Leu Asn Ala 55 60 65
AGT GCG AAG AAT TTG ATC AAT GAT AAA AAG AAT TCC CCG GCG TAT CAA 521 Ser Ala Lys Asn Leu He Asn Asp Lys Lys Asn Ser Pro Ala Tyr Gin 70 75 80
GCG GTG CTT TTA GCC TTG AAT GCG GCA GCG GGG TTG TGG CAA GTC ATG 569 Ala Val Leu Leu Ala Leu Asn Ala Ala Ala Gly Leu Trp Gin Val Met 85 90 95 100
AGC TAT TCG ATC AGC GTT TGT GGC CCT GGC TCT GAC AAA AAT AAA AAT 617 Ser Tyr Ser He Ser Val Cys Gly Pro Gly Ser Asp Lys Asn Lys Asn 105 110 115
GGG GGC GTC CAA ACC TTT GAA AAT GTG CCG TCA AAT GGG GGG ACT ACC 665 Gly Gly Val Gin Thr Phe Glu Asn Val Pro Ser Asn Gly Gly Thr Thr 120 125 130
ATT GCT TGC GAT TCA TTT TAT GAA CCA GGA AAG TGG AGC GGT ATA TCC 713 He Ala Cys Asp Ser Phe Tyr Glu Pro Gly Lys Trp Ser Gly He Ser 135 140 145
ACT GAA AAT TAC GCA AAA ATC AAT AAA GCC TAT CAA ATC ATC CAA AAG 761 Thr Glu Asn Tyr Ala Lys He Asn Lys Ala Tyr Gin He He Gin Lys 150 155 160
GCT TTT GGA GCA AGC GGG CAA GAT ATT CCT GCC TTA AGC GAC ACC AAA 809 Ala Phe Gly Ala Ser Gly Gin Asp He Pro Ala Leu Ser Asp Thr Lys 165 170 175 180
GAA CTT AAT TTT GAA ATT AAA GGG AAA AAA AAT GAT AGC GTC CAG CCA 857 Glu Leu Asn Phe Glu He Lys Gly Lys Lys Asn Asp Ser Val Gin Pro 185 190 195 GGA GAA AGA TGG AAA TTC CCA TGG ACT AAT GGA AAA TTT GTT TCA GTC 905 Gly Glu Arg Trp Lys Phe Pro Trp Thr Asn Gly Lys Phe Val Ser Val 200 205 210
AAG TGG GTG AAT GGG AAG TAT GAA GAA ATT AAA GAA GAC ATC AAA GTG 953 Lys Trp Val Asn Gly Lys Tyr Glu Glu He Lys Glu Asp He Lys Val 215 220 225
TCA AAT AAC GCT CAA GAG CTT TTA AAA CAG GCT AGC ACT ATT TTA ACC 1001 Ser Asn Asn Ala Gin Glu Leu Leu Lys Gin Ala Ser Thr He Leu Thr 230 235 240
ACT CTT AAT GAA GCA TGC CCA TGG TTG AGT AAT GGT GGT GCA GGC AAT 1049 Thr Leu Asn Glu Ala Cys Pro Trp Leu Ser Asn Gly Gly Ala Gly Asn 245 250 255 260
GTG GCC GGT GGC AAT AGT TTA TGG GCC GGA ATA GAT AAA GGC GAC GGG 1097 Val Ala Gly Gly Asn Ser Leu Trp Ala Gly He Asp Lys Gly Asp Gly 265 270 275
AGC GCA TGC GGG ATT TTT AAA AAT GAA ATC AGC GCG ATT CAA GAC ATG 1145 Ser Ala Cys Gly He Phe Lys Asn Glu He Ser Ala He Gin Asp Met 280 285 290
ATC AAA AAC GCT GAA ATA GCC GTA GAG CAA TCC AAA ATC GTT ACC GCC 1193 He Lys Asn Ala Glu He Ala Val Glu Gin Ser Lys He Val Thr Ala 295 300 305
AAC GCG CAA AAC CAG CAC AAC CTA GAC ACT GGG AAA GCA TTC AAC CCC 1241 Asn Ala Gin Asn Gin His Asn Leu Asp Thr Gly Lys Ala Phe Asn Pro 310 315 320
TAT AAA GAC GCC AAC TTC GCC CAA AGC ATG TTC GCT AAC GCT AGA GCG 1289 Tyr Lys Asp Ala Asn Phe Ala Gin Ser Met Phe Ala Asn Ala Arg Ala 325 330 335 340
CAA GCG GAG ATT TTA AAC CGC GCT CAA GCA GTG GTG AAG GAC TTT GAA 1337 Gin Ala Glu He Leu Asn Arg Ala Gin Ala Val Val Lys Asp Phe Glu 345 350 355
AGA ATC CCT GCA GCG TTC GTG AAA GAC TCT TTA GGA GTA TGC CAT GAA 1385 Arg He Pro Ala Ala Phe Val Lys Asp Ser Leu Gly Val Cys His Glu 360 365 370
AAG GGT AGC GAC GGC AAT CTC CGT GGC ACG CCA TCT GGC ACG GTT ACT 1433 Lys Gly Ser Asp Gly Asn Leu Arg Gly Thr Pro Ser Gly Thr Val Thr 375 380 385
TCT AAC ACT TGG GGA GCC GGC TGC GCG TAT GTG GGA GAA ACC GTA ACG 1481 Ser Asn Thr Trp Gly Ala Gly Cys Ala Tyr Val Gly Glu Thr Val Thr" 390 395 400
AAT CTA AAA AAC AGC ATC GCT CAT TTT GGC GAC CAA GCG GAG CGA ATC 1529 Asn Leu Lys Asn Ser He Ala His Phe Gly Asp Gin Ala Glu Arg He 405 410 415 420
CAT AAT GCG CGA AAT CTC GCC TAC ACT TTA GCG AAT TTC AGC GGC CAG 1577 His Asn Ala Arg Asn Leu Ala Tyr Thr Leu Ala Asn Phe Ser Gly Gin 425 430 435
TAC AAA AAG CTA GGC GAA CAC TAT GAC AGC ATC ACA GCG GCG CTC TCT 1625
Tyr Lys Lys Leu Gly Glu His Tyr Asp Ser He Thr Ala Ala Leu Ser 440 445 450
AGC TTG CCT GAT GCG CAA TCT TTA CAA AAT GTG GTG AGC AAA AAG ACT 1673
Ser Leu Pro Asp Ala Gin Ser Leu Gin Asn Val Val Ser Lys Lys Thr 455 460 465
AAC CCT AAC AGC CCG CAA GGC ATA CAG GAT AAT TAC TAC ATT GAC TCC 1721
Asn Pro Asn Ser Pro Gin Gly He Gin Asp Asn Tyr Tyr He Asp Ser 470 475 480
AAC ATC CAT TCT CAA GTG CAA TCT AGG AGT CAA GAA CTC GGC AGT AAC 1769
Asn He His Ser Gin Val Gin Ser Arg Ser Gin Glu Leu Gly Ser Asn
485 490 495 500
CCT TTC AGA CGC GCC GGG CTA ATC GCC GCT TCT ACC ACC AAT AAC GGC 1817
Pro Phe Arg Arg Ala Gly Leu He Ala Ala Ser Thr Thr Asn Asn Gly 505 510 515
GCG ATG AAT GGG ATT GGC TTT CAA GTG GGC TAT AAG CAA TTC TTT GGG 1865
Ala Met Asn Gly He Gly Phe Gin Val Gly Tyr Lys Gin Phe Phe Gly 520 525 530
AAA AAC AAA CGA TGG GGC GCG AGA TAC TAC GGC TTT GTG GAT TAC AAC 1913
Lys Asn Lys Arg Trp Gly Ala Arg Tyr Tyr Gly Phe Val Asp Tyr Asn 535 540 545
CAC ACC TAT AAC AAG TCC CAA TTT TTC AAC TCC GAT TCT GAT GTT TGG 1961
His Thr Tyr Asn Lys Ser Gin Phe Phe Asn Ser Asp Ser Asp Val Trp 550 555 560
ACT TAT GGC GTG GGG AGC GAT TTG TTA GTG AAT TTC ATC AAC GAT AAA 2009
Thr Tyr Gly Val Gly Ser Asp Leu Leu Val Asn Phe He Asn Asp Lys
565 570 575 580
GCC ACT AAA CAC AAT AAA ATT TCT TTT GGC GCG TTT GGC GGT ATC CAA 2057
Ala Thr Lys His Asn Lys He Ser Phe Gly Ala Phe Gly Gly He Gin 585 590 595
CTA GCC GGG ACT TCA TGG CTT AAT TCT CAG TAT GTG AAT TTA GCG AAT 2105
Leu Ala Gly Thr Ser Trp Leu Asn Ser Gin Tyr Val Asn Leu Ala Asn 600 605 610
GTG AAC AAT TAT TAT AAA GCT AAA ATC AAC ACC TCT AAC TTC CAA TTC 2153
Val Asn Asn Tyr Tyr Lys Ala Lys He Asn Thr Ser Asn Phe Gin Phe 615 620 625
TTA TTC AAT CTG GGC TTA AGG ACC AAT CTC GCC AGA AAT AAA AGA ATA 2201
Leu Phe Asn Leu Gly Leu Arg Thr Asn Leu Ala Arg Asn Lys Arg He 630 635 640
GGC GCT GAT CAT AGC GCG CAA CAT GGC ATG GAA TTA GGC GTG AAG ATC 2249
Gly Ala Asp His Ser Ala Gin His Gly Met Glu Leu Gly Val Lys He 645 650 655 660
CCC ACG ATC AAC ACA AAT TAC TAT TCT TTG CTA GGC ACT ACC TTG CAA 2297 Pro Thr He Asn Thr Asn Tyr Tyr Ser Leu Leu Gly Thr Thr Leu Gin 665 670 675
TAC AGA AGG CTT TAT AGC GTG TAT CTC AAC TAT GTG TTT GCT TAC TAAAA 2347 Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr 680 685 690
GCTTAAACTC CTTTTTAAAC TCCCTTTTTA GGGGGTTTAA TCTTTTTAAC TGACTTTTCT 2407
TTTAGCTTTT TTTAATTTTT TCCACCAAAC AAAGTTTTTT GACTTCAAGC GTTAATCACA 2467
AAAAATACTC AAAGGCGTTT TTTGCAATCT AAATAAAAAA TTAGCGTTAT TCAAGCGATC 2527
ATTTTAAACC ACCCAAGCAA GAAACCCCAA ACATCTTTAG CGTTCGCGCG CTCCACTAAC 2587
CAAAAAACGC CCCAAA 2603
(2) INFORMATION FOR SEQ ID NO : 8 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 711 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...20 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
Met Lys Lys Lys Phe Leu Ser Leu Thr Leu Gly Ser Leu Leu Val Ser -20 -15 -10 -5
Ala Leu Ser Ala Glu Asp Asn Gly Phe Phe Val Ser Ala Gly Tyr Gin
1 5 10
He Gly Glu Ser Ala Gin Met Val Lys Asn Thr Lys Gly He Gin Asp
15 20 25
Leu Ser Asp Ser Tyr Glu Arg Leu Asn Asn Leu Leu Thr Ser Tyr Ser
30 35 40
Ala Leu Asn Thr Leu He Arg Gin Ser Ala Asp Pro Asn Ala He Asn 45 50 55 60
Asn Ala Arg Gly Asn Leu Asn Ala Ser Ala Lys Asn Leu He Asn Asp
65 70 75
Lys Lys Asn Ser Pro Ala Tyr Gin Ala Val Leu Leu Ala Leu Asn Ala
80 85 90
Ala Ala Gly Leu Trp Gin Val Met Ser Tyr Ser He Ser Val Cys Gly
95 100 105
Pro Gly Ser Asp Lys Asn Lys Asn Gly Gly Val Gin Thr Phe Glu Asn
110 115 120
Val Pro Ser Asn Gly Gly Thr Thr He Ala Cys Asp Ser Phe Tyr Glu 125 130 135 140
Pro Gly Lys Trp Ser Gly He Ser Thr Glu Asn Tyr Ala Lys He Asn 145 150 155
Lys Ala Tyr Gin He He Gin Lys Ala Phe Gly Ala Ser Gly Gin Asp
160 165 170
He Pro Ala Leu Ser Asp Thr Lys Glu Leu Asn Phe Glu He Lys Gly
175 180 185
Lys Lys Asn Asp Ser Val Gin Pro Gly Glu Arg Trp Lys Phe Pro Trp
190 195 200
Thr Asn Gly Lys Phe Val Ser Val Lys Trp Val Asn Gly Lys Tyr Glu 205 210 215 220
Glu He Lys Glu Asp He Lys Val Ser Asn Asn Ala Gin Glu Leu Leu
225 230 235
Lys Gin Ala Ser Thr He Leu Thr Thr Leu Asn Glu Ala Cys Pro Trp
240 245 250
Leu Ser Asn Gly Gly Ala Gly Asn Val Ala Gly Gly Asn Ser Leu Trp
255 260 265
Ala Gly He Asp Lys Gly Asp Gly Ser Ala Cys Gly He Phe Lys Asn
270 275 280
Glu He Ser Ala He Gin Asp Met He Lys Asn Ala Glu He Ala Val 285 290 295 300
Glu Gin Ser Lys He Val Thr Ala Asn Ala Gin Asn Gin His Asn Leu
305 310 315
Asp Thr Gly Lys Ala Phe Asn Pro Tyr Lys Asp Ala Asn Phe Ala Gin
320 325 330
Ser Met Phe Ala Asn Ala Arg Ala Gin Ala Glu He Leu Asn Arg Ala
335 340 345
Gin Ala Val Val Lys Asp Phe Glu Arg He Pro Ala Ala Phe Val Lys
350 355 360
Asp Ser Leu Gly Val Cys His Glu Lys Gly Ser Asp Gly Asn Leu Arg 365 370 375 380
Gly Thr Pro Ser Gly Thr Val Thr Ser Asn Thr Trp Gly Ala Gly Cys
385 390 395
Ala Tyr Val Gly Glu Thr Val Thr Asn Leu Lys Asn Ser He Ala His
400 405 410
Phe Gly Asp Gin Ala Glu Arg He His Asn Ala Arg Asn Leu Ala Tyr
415 420 425
Thr Leu Ala Asn Phe Ser Gly Gin Tyr Lys Lys Leu Gly Glu His Tyr
430 435 440
Asp Ser He Thr Ala Ala Leu Ser Ser Leu Pro Asp Ala Gin Ser Leu 445 450 455 460
Gin Asn Val Val Ser Lys Lys Thr Asn Pro Asn Ser Pro Gin Gly He
465 470 475
Gin Asp Asn Tyr Tyr He Asp Ser Asn He His Ser Gin Val Gin Ser
480 485 490
Arg Ser Gin Glu Leu Gly Ser Asn Pro Phe Arg Arg Ala Gly Leu He
495 500 505
Ala Ala Ser Thr Thr Asn Asn Gly Ala Met Asn Gly He Gly Phe Gin
510 515 520
Val Gly Tyr Lys Gin Phe Phe Gly Lys Asn Lys Arg Trp Gly Ala Arg 525 530 535 540
Tyr Tyr Gly Phe Val Asp Tyr Asn His Thr Tyr Asn Lys Ser Gin Phe
545 550 555
Phe Asn Ser Asp Ser Asp Val Trp Thr Tyr Gly Val Gly Ser Asp Leu
560 565 570
Leu Val Asn Phe He Asn Asp Lys Ala Thr Lys His Asn Lys He Ser
575 580 585
Phe Gly Ala Phe Gly Gly He Gin Leu Ala Gly Thr Ser Trp Leu Asn 590 595 600 Ser Gin Tyr Val Asn Leu Ala Asn Val Asn Asn Tyr Tyr Lys Ala Lys 605 610 615 620
He Asn Thr Ser Asn Phe Gin Phe Leu Phe Asn Leu Gly Leu Arg Thr
625 630 635
Asn Leu Ala Arg Asn Lys Arg He Gly Ala Asp His Ser Ala Gin His
640 645 650
Gly Met Glu Leu Gly Val Lys He Pro Thr He Asn Thr Asn Tyr Tyr
655 660 665
Ser Leu Leu Gly Thr Thr Leu Gin Tyr Arg Arg Leu Tyr Ser Val Tyr
670 675 680
Leu Asn Tyr Val Phe Ala Tyr 685 690
(2) INFORMATION FOR SEQ ID NO : 9 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2427 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 232...2247 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 232...292 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :
AAAACGCGCA GCAAAAAATC TCTGTTAAGC TTTTATCATT AGCGTTCCAT TGAAACAAAA 60
TCTAAAAACC CTTTCCAATA CCACCCAAAC AAACGCGCAA AAAATGCAAA AATTCTAAAT 120
TTTCTCCAAA TGACAAAAAA AAAAAAAACG ATTTTATGCT ACAATGCTTT TAATACATTC 180
TTACTTAATG TATAAAATCT CAATCACTCA ATTTAATTTC AAAGGATATT T ATG AAA 237
Met Lys -20
AAA ACC CTT TTA CTC TCT CTC TCT CTC TCT CTC TCG TCA TCG CTT TTA 285 Lys Thr Leu Leu Leu Ser Leu Ser Leu Ser Leu Ser Ser Ser Leu Leu -15 -10 -5
AAC GCT GAA GAC AAC GGC TTT TTT ATC AGC GCG GGC TAT CAA ATC GGT " 333 Asn Ala Glu Asp Asn Gly Phe Phe He Ser Ala Gly Tyr Gin He Gly 1 5 10
GAA GCC GCT CAA ATG GTG AAA AAC ACC GGC GAA TTG AAA AAA CTT TCA 381 Glu Ala Ala Gin Met Val Lys Asn Thr Gly Glu Leu Lys Lys Leu Ser 15 20 25 30 GAC ACT TAT GAG AAT TTG AGC AAC CTT TTA ACC AAT TTT AAC AAC CTC 429 Asp Thr Tyr Glu Asn Leu Ser Asn Leu Leu Thr Asn Phe Asn Asn Leu 35 40 45
AAT CAA GCG GTA ACG AAC GCG AGC AGC CCT TCA GAA ATC AAT GCC ACG 477 Asn Gin Ala Val Thr Asn Ala Ser Ser Pro Ser Glu He Asn Ala Thr 50 55 60
ATC GAT AAT TTA AAA GCA AAC ACG CAA GGG CTG ATT GGC GAA AAA ACC 525 He Asp Asn Leu Lys Ala Asn Thr Gin Gly Leu He Gly Glu Lys Thr 65 70 75
AAT TCC CCG GCG TAT CAA GCG GTG TAT TTG GCG CTC AAT GCG GCG GTG 573 Asn Ser Pro Ala Tyr Gin Ala Val Tyr Leu Ala Leu Asn Ala Ala Val 80 85 90
GGG CTG TGG AAT GTG ATA GCC TAT AAT GTC CAA TGC GGT CCT GGT AAG 621 Gly Leu Trp Asn Val He Ala Tyr Asn Val Gin Cys Gly Pro Gly Lys 95 100 105 110
AGT GGG GAT CAA AGC GTA ATT TTT GAT GGC CAA CCA GGA CAT GAT TCA 669 Ser Gly Asp Gin Ser Val He Phe Asp Gly Gin Pro Gly His Asp Ser 115 120 125
AGA TCC ATT AAT TGC AAT TTA ACC GGT TAT AAC AAC GGG GTT AGC GGC 717 Arg Ser He Asn Cys Asn Leu Thr Gly Tyr Asn Asn Gly Val Ser Gly 130 135 140
CCT TTA TCC ATT GAC AAT TTT AAA ACG CTT AAT CAA GCT TAT CAA ACT 765 Pro Leu Ser He Asp Asn Phe Lys Thr Leu Asn Gin Ala Tyr Gin Thr 145 150 155
ATC CAA CAA GCT TTA AAA CAA GAT AGC GGA TTT CCT GTT TTG GAT AGT 813 He Gin Gin Ala Leu Lys Gin Asp Ser Gly Phe Pro Val Leu Asp Ser 160 165 170
AAA GGA AAA CAA GTA ACT ATA AAA ATA ACA ACA CAA ACT AAT GGA GCT 861 Lys Gly Lys Gin Val Thr He Lys He Thr Thr Gin Thr Asn Gly Ala 175 180 185 190
AAT AAA AGT GAA ACT ACT ACT ACT ACT ACT ACT ACT AAT GAC GCT CAA 909 Asn Lys Ser Glu Thr Thr Thr Thr Thr Thr Thr Thr Asn Asp Ala Gin 195 200 205
ACC CTT TTG CAA GAA GCC AGT AAA ATG ATA AGC GTC CTC ACT ACA AAC 957 Thr Leu Leu Gin Glu Ala Ser Lys Met He Ser Val Leu Thr Thr Asn 210 215 220
TGC CCA TGG GTA AAT ACC GCT CAT AAC TCA AAC GGG GGT GCA CCG TGG 1005 Cys Pro Trp Val Asn Thr Ala His Asn Ser Asn Gly Gly Ala Pro Trp 225 230 235
AAT TTA AAT ACG ACA GGG AAT GTG TGT CAG GTT TTT GCC ACG GAG TTT 1053 Asn Leu Asn Thr Thr Gly Asn Val Cys Gin Val Phe Ala Thr Glu Phe 240 245 250
AGC GCC GTT ACT AGC ATG ATC AAA AAC GCG CAA GAA ATC GTA ACG CAA 1101 Ser Ala Val Thr Ser Met He Lys Asn Ala Gin Glu He Val Thr Gin 255 260 265 270
GCT CAA AGC CTT AAC AAC CCG CAA AGC AAT CAA AAC GCG CCG AAA GAT 1149 Ala Gin Ser Leu Asn Asn Pro Gin Ser Asn Gin Asn Ala Pro Lys Asp 275 280 285
TTC AAT CCT TAC ACC TCT GCT GAT AGG GCT TTC GCT CAA AAC ATG CTC 1197 Phe Asn Pro Tyr Thr Ser Ala Asp Arg Ala Phe Ala Gin Asn Met Leu 290 295 300
AAT CAC GCG CAA GCG CAA GCC AAG ATG CTT GAA CTA GCC GAT CAA ATG 1245 Asn His Ala Gin Ala Gin Ala Lys Met Leu Glu Leu Ala Asp Gin Met 305 310 315
AAA AAA GAC CTT AAC ACT ATC CCA AAA CAA TTT ATC ACA AAC TAC TTG 1293 Lys Lys Asp Leu Asn Thr He Pro Lys Gin Phe He Thr Asn Tyr Leu 320 325 330
GCA GCT TGC CGC AAT GGG GGT GGG ACA TTA CCT GAT GCA GGG GTT ACT 1341 Ala Ala Cys Arg Asn Gly Gly Gly Thr Leu Pro Asp Ala Gly Val Thr 335 340 345 350
TCT AAC ACT TGG GGG GCC GGT TGC GCC TAT GTG GAA GAG ACG ATA ACC 1389 Ser Asn Thr Trp Gly Ala Gly Cys Ala Tyr Val Glu Glu Thr He Thr 355 360 365
GCC CTA AAT AAC AGC CTT GCG CAT TTT GGC ACT CAA GCC GAT CAA ATC 1437 Ala Leu Asn Asn Ser Leu Ala His Phe Gly Thr Gin Ala Asp Gin He 370 375 380
AAG CAA TCT GAG TTG TTG GCG CGC ACG ATA CTT GAT TTT AGA GGC AGC 1485 Lys Gin Ser Glu Leu Leu Ala Arg Thr He Leu Asp Phe Arg Gly Ser 385 390 395
CTT AAG GAT TTA AAC AAC ACT TAT AAC AGC ATC ACC ACG ACC GCT TCA 1533 Leu Lys Asp Leu Asn Asn Thr Tyr Asn Ser He Thr Thr Thr Ala Ser 400 405 410
AAC ACG CCC AAT TCC CCA TTC CTT AAA AAT TTG ATA AGC CAA TCC ACT 1581 Asn Thr Pro Asn Ser Pro Phe Leu Lys Asn Leu He Ser Gin Ser Thr 415 420 425 430
AAC CCT AAT AAC CCC GGG GGC TTA CAG GCC GTT TAT CAA GTC AAC CAA 1629 Asn Pro Asn Asn Pro Gly Gly Leu Gin Ala Val Tyr Gin Val Asn Gin 435 440 445
AGC GCT TAT TCG CAA TTA TTA AGC GCC ACG CAA GAA TTA GGG CAT AAC 1677 Ser Ala Tyr Ser Gin Leu Leu Ser Ala Thr Gin Glu Leu Gly His Asn 450 455 460
CCT TTC AGA CGC GTT GGC TTA ATC AGC TCT CAA ACC AAC AAC GGT GCG 1725 Pro Phe Arg Arg Val Gly Leu He Ser Ser Gin Thr Asn Asn Gly Ala 465 470 475
ATG AAT GGG ATC GGC GTG CAA ATA GGG TAT AAA CAA TTT TTT GGT GAA 1773 Met Asn Gly He Gly Val Gin He Gly Tyr Lys Gin Phe Phe Gly Glu 4 80 4 85 4 90
AAA AGA AGA TGG GGG TTA AGG TAT TAT GGT TTT TTT GAT TAC AAC CAT 1821 Lys Arg Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His 495 500 505 510
GCT TAT ATC AAA TCC AGC TTT TTC AAC TCC GCC TCT GAT GTG TTC ACT 1869 Ala Tyr He Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp Val Phe Thr 515 520 525
TAT GGG GTA GGA ACA GAT GTC CTC TAT AAC TTT ATC AAC GAT AAA GCC 1917 Tyr Gly Val Gly Thr Asp Val Leu Tyr Asn Phe He Asn Asp Lys Ala 530 535 540
ACC AAA AAC AAT AAG ATT TCT TTT GGG GTG TTT GGG GGG ATT GCG TTA 1965 Thr Lys Asn Asn Lys He Ser Phe Gly Val Phe Gly Gly He Ala Leu 545 550 555
GCT GGC ACT TCG TGG CTT AAT TCT CAA TAC GTG AAT TTA GCG ACA TTC 2013 Ala Gly Thr Ser Trp Leu Asn Ser Gin Tyr Val Asn Leu Ala Thr Phe 560 565 570
AAT AAT TTT TAC AGC GCT AAA ATG AAT GTG GCG AAT TTC CAA TTC TTA 2061 Asn Asn Phe Tyr Ser Ala Lys Met Asn Val Ala Asn Phe Gin Phe Leu 575 580 585 590
TTC AAC TTG GGC TTG AGA ATG AAT CTC GCT AAA AAC AAA AAG AAA GCG 2109 Phe Asn Leu Gly Leu Arg Met Asn Leu Ala Lys Asn Lys Lys Lys Ala 595 600 605
AGC GAT CAT GTA GCT CAG CAT GGC GTG GAA CTA GGC GTG AAG ATC CCT 2157 Ser Asp His Val Ala Gin His Gly Val Glu Leu Gly Val Lys He Pro 610 615 620
ACG ATC AAC ACG AAT TAC TAT TCT TTG CTA GGC ACT CAA CTC CAA TAC 2205 Thr He Asn Thr Asn Tyr Tyr Ser Leu Leu Gly Thr Gin Leu Gin Tyr 625 630 635
CGC AGG CTT TAT AGC GTG TAT TTG AAT TAT GTG TTT GCT TAC TAATATCTG 2256 Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr 640 645 650
TCTTTTTGTG AAACTCCCTT TTTAAGGGAT TTTTTTTGAA GCCTTTCTTT TTTTAAACCC 2316 TCTTTTTTGG GGGTCAAGCG TAAAATTCAC CCCTATCCCT TTAAGAAAAT AAAATAAAAG 2376 AAAATGCGTT TTATAACAAA ATAAGATCTA AAACAATAAA ACAAAAACCC A 2427
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 672 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE: (A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...20 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Met Lys Lys Thr Leu Leu Leu Ser Leu Ser Leu Ser Leu Ser Ser Ser -20 -15 -10 -5
Leu Leu Asn Ala Glu Asp Asn Gly Phe Phe He Ser Ala Gly Tyr Gin
1 5 10
He Gly Glu Ala Ala Gin Met Val Lys Asn Thr Gly Glu Leu Lys Lys
15 20 25
Leu Ser Asp Thr Tyr Glu Asn Leu Ser Asn Leu Leu Thr Asn Phe Asn
30 35 40
Asn Leu Asn Gin Ala Val Thr Asn Ala Ser Ser Pro Ser Glu He Asn 45 50 55 60
Ala Thr He Asp Asn Leu Lys Ala Asn Thr Gin Gly Leu He Gly Glu
65 70 75
Lys Thr Asn Ser Pro Ala Tyr Gin Ala Val Tyr Leu Ala Leu Asn Ala
80 85 90
Ala Val Gly Leu Trp Asn Val He Ala Tyr Asn Val Gin Cys Gly Pro
95 100 105
Gly Lys Ser Gly Asp Gin Ser Val He Phe Asp Gly Gin Pro Gly His
110 115 120
Asp Ser Arg Ser He Asn Cys Asn Leu Thr Gly Tyr Asn Asn Gly Val 125 130 135 140
Ser Gly Pro Leu Ser He Asp Asn Phe Lys Thr Leu Asn Gin Ala Tyr
145 150 155
Gin Thr He Gin Gin Ala Leu Lys Gin Asp Ser Gly Phe Pro Val Leu
160 165 170
Asp Ser Lys Gly Lys Gin Val Thr He Lys He Thr Thr Gin Thr Asn
175 180 185
Gly Ala Asn Lys Ser Glu Thr Thr Thr Thr Thr Thr Thr Thr Asn Asp
190 195 200
Ala Gin Thr Leu Leu Gin Glu Ala Ser Lys Met He Ser Val Leu Thr 205 210 215 220
Thr Asn Cys Pro Trp Val Asn Thr Ala His Asn Ser Asn Gly Gly Ala
225 230 235
Pro Trp Asn Leu Asn Thr Thr Gly Asn Val Cys Gin Val Phe Ala Thr
240 245 250
Glu Phe Ser Ala Val Thr Ser Met He Lys Asn Ala Gin Glu He Val
255 260 265
Thr Gin Ala Gin Ser Leu Asn Asn Pro Gin Ser Asn Gin Asn Ala Pro
270 275 280
Lys Asp Phe Asn Pro Tyr Thr Ser Ala Asp Arg Ala Phe Ala Gin Asn 285 290 295 300
Met Leu Asn His Ala Gin Ala Gin Ala Lys Met Leu Glu Leu Ala Asp
305 310 315
Gin Met Lys Lys Asp Leu Asn Thr He Pro Lys Gin Phe He Thr Asn
320 325 330
Tyr Leu Ala Ala Cys Arg Asn Gly Gly Gly Thr Leu Pro Asp Ala Gly
335 340 345
Val Thr Ser Asn Thr Trp Gly Ala Gly Cys Ala Tyr Val Glu Glu Thr
350 355 360
He Thr Ala Leu Asn Asn Ser Leu Ala His Phe Gly Thr Gin Ala Asp 365 370 375 380
Gin He Lys Gin Ser Glu Leu Leu Ala Arg Thr He Leu Asp Phe Arg
385 390 395
Gly Ser Leu Lys Asp Leu Asn Asn Thr Tyr Asn Ser He Thr Thr Thr
400 405 410
Ala Ser Asn Thr Pro Asn Ser Pro Phe Leu Lys Asn Leu He Ser Gin
415 420 425
Ser Thr Asn Pro Asn Asn Pro Gly Gly Leu Gin Ala Val Tyr Gin Val
430 435 440
Asn Gin Ser Ala Tyr Ser Gin Leu Leu Ser Ala Thr Gin Glu Leu Gly 445 450 455 460
His Asn Pro Phe Arg Arg Val Gly Leu He Ser Ser Gin Thr Asn Asn
465 470 475
Gly Ala Met Asn Gly He Gly Val Gin He Gly Tyr Lys Gin Phe Phe
480 485 490
Gly Glu Lys Arg Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr
495 500 505
Asn His Ala Tyr He Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp Val
510 515 520
Phe Thr Tyr Gly Val Gly Thr Asp Val Leu Tyr Asn Phe He Asn Asp 525 530 535 540
Lys Ala Thr Lys Asn Asn Lys He Ser Phe Gly Val Phe Gly Gly He
545 550 555
Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gin Tyr Val Asn Leu Ala
560 565 570
Thr Phe Asn Asn Phe Tyr Ser Ala Lys Met Asn Val Ala Asn Phe Gin
575 580 585
Phe Leu Phe Asn Leu Gly Leu Arg Met Asn Leu Ala Lys Asn Lys Lys
590 595 600
Lys Ala Ser Asp His Val Ala Gin His Gly Val Glu Leu Gly Val Lys 605 610 615 620
He Pro Thr He Asn Thr Asn Tyr Tyr Ser Leu Leu Gly Thr Gin Leu
625 630 635
Gin Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr 640 645 650
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2429 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 205...2277 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 205...259 (D) OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
TGAAAGAAGA CTGATTAGTC TTTCTTTTAG GGGCGATTCA AGCCTTAAAA GCCGGGTCAA 60
AATCCCCATT TTTCCCAATT TTTACAAAAA AAAAAAAAAC AAAATCTCTA AAATTTAGAG 120
CTAAAATTAG CCATAAAATT CCATTTATTG CTTATAATAT GAAGTTTCTT TGTATCAAAG 180
AAAAATCTAT TAAAAGGAGA AAAC ATG AAA AAA TCC CTC TTA CTC TCT CTT 231
Met Lys Lys Ser Leu Leu Leu Ser Leu -15 -10
TCT CTC ATC GCT TCC TTA TCA AGA GCT GAA GAT GAC GGA TTT TAT ACG 279 Ser Leu He Ala Ser Leu Ser Arg Ala Glu Asp Asp Gly Phe Tyr Thr -5 1 5
AGT GTG GGC TAT CAG ATC GGT GAA GCG GTC CAA CAA GTG AAA AAC ACA 327 Ser Val Gly Tyr Gin He Gly Glu Ala Val Gin Gin Val Lys Asn Thr 10 15 20
GGA GCA TTG CAA AAT CTT GCA GAC AGA TAC GAT AAC TTA AAC AAC CTT 375 Gly Ala Leu Gin Asn Leu Ala Asp Arg Tyr Asp Asn Leu Asn Asn Leu 25 30 35
TTA AAC CAA TAC AAT TAT TTA AAT TCC TTA GTC AAT TTA GCC AGC ACG 423 Leu Asn Gin Tyr Asn Tyr Leu Asn Ser Leu Val Asn Leu Ala Ser Thr 40 45 50 55
CCG AGC GCG ATC ACC GGT GCG ATT GAT AAT TTA AGC TCA AGC GCG ATT 471 Pro Ser Ala He Thr Gly Ala He Asp Asn Leu Ser Ser Ser Ala He 60 65 70
AAC CTC ACT AGC GCC ACC ACC ACT TCC CCC GCC TAT CAA GCT GTG GCT 519 Asn Leu Thr Ser Ala Thr Thr Thr Ser Pro Ala Tyr Gin Ala Val Ala 75 80 85
TTA GCG CTC AAT GCC GCT GTG GGC ATG TGG CAA GTC ATA GCC CTT TTT 567 Leu Ala Leu Asn Ala Ala Val Gly Met Trp Gin Val He Ala Leu Phe 90 95 100
ATT GGC TGT GGC CCT GGC CCT ACC AAT AAT CAA AGC TAT CAA TCG TTT 615 He Gly Cys Gly Pro Gly Pro Thr Asn Asn Gin Ser Tyr Gin Ser Phe 105 110 115
GGT AAC ACA CCA GCC CTT AAT GGG ACC ACC ACC ACT TGC AAT CAA GCA 663 Gly Asn Thr Pro Ala Leu Asn Gly Thr Thr Thr Thr Cys Asn Gin Ala 120 125 130 135
TAT GGG ACA GGC CCT AAT GGC ATC CTA TCT ATT GAT GAA TAC CAA AAA 711 Tyr Gly Thr Gly Pro Asn Gly He Leu Ser He Asp Glu Tyr Gin Lys 140 145 150
CTC AAC CAA GCT TAT CAG ATC ATC CAA ACC GCT TTA AAC CAA AAT CAA 759 Leu Asn Gin Ala Tyr Gin He He Gin Thr Ala Leu Asn Gin Asn Gin 155 160 165
GGG GGT GGG ATG CCT GCC TTG AAT GAC ACC ACC AAA ACA GGG GTA GTC 807 Gly Gly Gly Met Pro Ala Leu Asn Asp Thr Thr Lys Thr Gly Val Val 170 175 180
AAC ATA CAA CAA ACC AAT TAT AGG ACC ACC ACA CAA AAC AAT ATC ATA 855
Asn He Gin Gin Thr Asn Tyr Arg Thr Thr Thr Gin Asn Asn He He 185 190 195
GAG CAT TAT TAT ACA GAG AAT GGG AAA GAG ATC CCA GTC TCT TAT TCA 903
Glu His Tyr Tyr Thr Glu Asn Gly Lys Glu He Pro Val Ser Tyr Ser
200 205 210 215
GGC GGA TCA TCA TTC TCG CCT ACA ATA CAA TTG ACA TAC CAT AAT AAC 951
Gly Gly Ser Ser Phe Ser Pro Thr He Gin Leu Thr Tyr His Asn Asn
220 225 230
GCT GAA AAC CTT TTG CAA CAA GCC GCC ACT ATC ATG CAA GTC CTT ATT 999
Ala Glu Asn Leu Leu Gin Gin Ala Ala Thr He Met Gin Val Leu He 235 240 245
ACT CAA AAG CCG CAT GTG CAA ACG AGC AAT GGC GGT AAA GCG TGG GGG 1047
Thr Gin Lys Pro His Val Gin Thr Ser Asn Gly Gly Lys Ala Trp Gly 250 255 260
TTG AGT TCT ACG CCT GGG AAT GTG ATG GAT ATT TTT GGT CCT TCT TTT 1095
Leu Ser Ser Thr Pro Gly Asn Val Met Asp He Phe Gly Pro Ser Phe 265 270 275
AAC GCT ATT AAT GAG ATG ATT AAA AAC GCT CAA ACA GCC CTA GCA AAA 1143
Asn Ala He Asn Glu Met He Lys Asn Ala Gin Thr Ala Leu Ala Lys
280 285 290 295
ACC CAA CAG CTT AAC GCT AAT GAA AAC GCC CAA ATC ACG CAA CCC AAC 1191
Thr Gin Gin Leu Asn Ala Asn Glu Asn Ala Gin He Thr Gin Pro Asn
300 305 310
AAT TTC AAC CCC TAC ACC TCT AAA GAC AAA GGG TTC GCT CAA GAA ATG 1239
Asn Phe Asn Pro Tyr Thr Ser Lys Asp Lys Gly Phe Ala Gin Glu Met 315 320 325
CTC AAT AGA GCT GAA GCT CAA GCA GAG ATT TTA AAT TTA GCT AAG CAA 1287
Leu Asn Arg Ala Glu Ala Gin Ala Glu He Leu Asn Leu Ala Lys Gin 330 335 340
GTA GCG AAC AAT TTC CAC AGC ATT CAA GGG CCT ATT CAA GGG GAT TTA 1335
Val Ala Asn Asn Phe His Ser He Gin Gly Pro He Gin Gly Asp Leu 345 350 355
GAA GAA TGT AAA GCA GGA TCG GCT GGC GTG ATC ACT AAT AAC ACT TGG 1383
Glu Glu Cys Lys Ala Gly Ser Ala Gly Val He Thr Asn Asn Thr Trp
360 365 370 375
GGT TCA GGT TGC GCG TTT GTG AAA GAA ACT TTA AAC TCT TTA GAG CAA 14-31
Gly Ser Gly Cys Ala Phe Val Lys Glu Thr Leu Asn Ser Leu Glu Gin
380 385 390
CAC ACC GCT TAT TAC GGC AAC CAG GTC AAT CAG GAT AGG GCT TTG GCT 1479
His Thr Ala Tyr Tyr Gly Asn Gin Val Asn Gin Asp Arg Ala Leu Ala 395 400 405 CAA ACC ATT TTG AAT TTT AAA GAA GCC CTT AAC ACC CTG AAT AAA GAC 1527 Gin Thr He Leu Asn Phe Lys Glu Ala Leu Asn Thr Leu Asn Lys Asp 410 415 420
TCA AAA GCG ATC AAT AGC GGT ATC TCC AAC TTG CCT AAC GCT AAA TCT 1575 Ser Lys Ala He Asn Ser Gly He Ser Asn Leu Pro Asn Ala Lys Ser 425 430 435
CTT CAA AAC ATG ACG CAT GCC ACT CAA AAC CCT AAT TCC CCA GAA GGT 1623 Leu Gin Asn Met Thr His Ala Thr Gin Asn Pro Asn Ser Pro Glu Gly 440 445 450 455
CTG CTC ACT TAT TCT TTG GAT TCA AGC AAA TAC AAC CAG CTC CAA ACC 1671 Leu Leu Thr Tyr Ser Leu Asp Ser Ser Lys Tyr Asn Gin Leu Gin Thr 460 465 470
ATC GCG CAA GAA TTG GGC AAA AAC CCT TTC AGG CGC TTT GGC GTG ATT 1719 He Ala Gin Glu Leu Gly Lys Asn Pro Phe Arg Arg Phe Gly Val He 475 480 485
GAC TTT CAA AAC AAC AAC GGC GCA ATG AAC GGG ATC GGC GTG CAA GTG 1767 Asp Phe Gin Asn Asn Asn Gly Ala Met Asn Gly He Gly Val Gin Val 490 495 500
GGT TAT AAA CAA TTC TTT GGT AAA AAA AGG AAT TGG GGG TTA AGG TAT 1815 Gly Tyr Lys Gin Phe Phe Gly Lys Lys Arg Asn Trp Gly Leu Arg Tyr 505 510 515
TAT GGT TTC TTT GAT TAT AAC CAT GCT TAT ATC AAA TCT AAT TTT TTC 1863 Tyr Gly Phe Phe Asp Tyr Asn His Ala Tyr He Lys Ser Asn Phe Phe 520 525 530 535
AAC TCC GCT TCT GAT GTG TGG ACT TAT GGG GTG GGT ATG GAC GCT CTC 1911 Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Val Gly Met Asp Ala Leu 540 545 550
TAT AAC TTC ATC AAC GAT AAA AAC ACC AAC TTT TTA GGC AAG AAC AAC 1959 Tyr Asn Phe He Asn Asp Lys Asn Thr Asn Phe Leu Gly Lys Asn Asn 555 560 565
AAG CTT TCA GTA GGG CTT TTT GGA GGC TTT GCG TTA GCC GGG ACT TCG 2007 Lys Leu Ser Val Gly Leu Phe Gly Gly Phe Ala Leu Ala Gly Thr Ser 570 575 580
TGG CTT AAT TCC CAA CAA GTG AAT TTG ACC ATG ATG AAT GGC ATT TAT 2055 Trp Leu Asn Ser Gin Gin Val Asn Leu Thr Met Met Asn Gly He Tyr 585 590 595
AAC GCT AAT GTC AGC ACT TCT AAC TTC CAA TTT TTG TTT GAT TTA GGC 2103 Asn Ala Asn Val Ser Thr Ser Asn Phe Gin Phe Leu Phe Asp Leu Gly 600 605 610 615
TTG AGA ATG AAC CTC GCT AGG CCT AAG AAA AAA GAC AGC GAT CAT GCC 2151 Leu Arg Met Asn Leu Ala Arg Pro Lys Lys Lys Asp Ser Asp His Ala 620 625 630
GCT CAG CAT GGC ATT GAA CTA GGT TTT AAG ATC CCC ACG ATC AAC ACC 2199 Ala Gin His Gly He Glu Leu Gly Phe Lys He Pro Thr He Asn Thr 635 640 645
AAC TAT TAT TCT TTC ATG GGC GCT AAA CTA GAA TAC AGA AGG ATG TAT 2247 Asn Tyr Tyr Ser Phe Met Gly Ala Lys Leu Glu Tyr Arg Arg Met Tyr 650 655 660
AGC CTT TTT CTC AAT TAT GTG TTT GCT TAC TAAAAATTCT TTTTGAACCC CTC 2300 Ser Leu Phe Leu Asn Tyr Val Phe Ala Tyr 665 670
TTTTTTTGGG GGAGTGTTGC AAAAATGCCC CCCTATTTGC TTGTGAGTTT TGGTTAAAAT 2360 TTTAGTTACC CACGCTTAAA AAGCGCCAAG CCTTTTACAC ACAACTCCTT TAATTTTGTT 2420 TTTAAGAAA 2429
(2) INFORMATION FOR SEQ ID NO : 12 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 691 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...18 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 12 :
Met Lys Lys Ser Leu Leu Leu Ser Leu Ser Leu He Ala Ser Leu Ser
-15 -10 -5
Arg Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gin He Gly
1 5 10
Glu Ala Val Gin Gin Val Lys Asn Thr Gly Ala Leu Gin Asn Leu Ala 15 20 25 30
Asp Arg Tyr Asp Asn Leu Asn Asn Leu Leu Asn Gin Tyr Asn Tyr Leu
35 40 45
Asn Ser Leu Val Asn Leu Ala Ser Thr Pro Ser Ala He Thr Gly Ala
50 55 60
He Asp Asn Leu Ser Ser Ser Ala He Asn Leu Thr Ser Ala Thr Thr
65 70 75
Thr Ser Pro Ala Tyr Gin Ala Val Ala Leu Ala Leu Asn Ala Ala Val
80 85 90
Gly Met Trp Gin Val He Ala Leu Phe He Gly Cys Gly Pro Gly Pro 95 100 105 110
Thr Asn Asn Gin Ser Tyr Gin Ser Phe Gly Asn Thr Pro Ala Leu Asn
115 120 125
Gly Thr Thr Thr Thr Cys Asn Gin Ala Tyr Gly Thr Gly Pro Asn Gly
130 135 140
He Leu Ser He Asp Glu Tyr Gin Lys Leu Asn Gin Ala Tyr Gin He 145 150 155 He Gin Thr Ala Leu Asn Gin Asn Gin Gly Gly Gly Met Pro Ala Leu
160 165 170
Asn Asp Thr Thr Lys Thr Gly Val Val Asn He Gin Gin Thr Asn Tyr 175 180 185 190
Arg Thr Thr Thr Gin Asn Asn He He Glu His Tyr Tyr Thr Glu Asn
195 200 205
Gly Lys Glu He Pro Val Ser Tyr Ser Gly Gly Ser Ser Phe Ser Pro
210 215 220
Thr He Gin Leu Thr Tyr His Asn Asn Ala Glu Asn Leu Leu Gin Gin
225 230 235
Ala Ala Thr He Met Gin Val Leu He Thr Gin Lys Pro His Val Gin
240 245 250
Thr Ser Asn Gly Gly Lys Ala Trp Gly Leu Ser Ser Thr Pro Gly Asn 255 260 265 270
Val Met Asp He Phe Gly Pro Ser Phe Asn Ala He Asn Glu Met He
275 280 285
Lys Asn Ala Gin Thr Ala Leu Ala Lys Thr Gin Gin Leu Asn Ala Asn
290 295 300
Glu Asn Ala Gin He Thr Gin Pro Asn Asn Phe Asn Pro Tyr Thr Ser
305 310 315
Lys Asp Lys Gly Phe Ala Gin Glu Met Leu Asn Arg Ala Glu Ala Gin
320 325 330
Ala Glu He Leu Asn Leu Ala Lys Gin Val Ala Asn Asn Phe His Ser 335 340 345 350
He Gin Gly Pro He Gin Gly Asp Leu Glu Glu Cys Lys Ala Gly Ser
355 360 365
Ala Gly Val He Thr Asn Asn Thr Trp Gly Ser Gly Cys Ala Phe Val
370 375 380
Lys Glu Thr Leu Asn Ser Leu Glu Gin His Thr Ala Tyr Tyr Gly Asn
385 390 395
Gin Val Asn Gin Asp Arg Ala Leu Ala Gin Thr He Leu Asn Phe Lys
400 405 410
Glu Ala Leu Asn Thr Leu Asn Lys Asp Ser Lys Ala He Asn Ser Gly 415 420 425 430
He Ser Asn Leu Pro Asn Ala Lys Ser Leu Gin Asn Met Thr His Ala
435 440 445
Thr Gin Asn Pro Asn Ser Pro Glu Gly Leu Leu Thr Tyr Ser Leu Asp
450 455 460
Ser Ser Lys Tyr Asn Gin Leu Gin Thr He Ala Gin Glu Leu Gly Lys
465 470 475
Asn Pro Phe Arg Arg Phe Gly Val He Asp Phe Gin Asn Asn Asn Gly
480 485 490
Ala Met Asn Gly He Gly Val Gin Val Gly Tyr Lys Gin Phe Phe Gly 495 500 505 510
Lys Lys Arg Asn Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn
515 520 525
His Ala Tyr He Lys Ser Asn Phe Phe Asn Ser Ala Ser Asp Val Trp
530 535 540
Thr Tyr Gly Val Gly Met Asp Ala Leu Tyr Asn Phe He Asn Asp Lys
545 550 555
Asn Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Val Gly Leu Phe
560 565 570
Gly Gly Phe Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gin Gin Val 575 580 585 590
Asn Leu Thr Met Met Asn Gly He Tyr Asn Ala Asn Val Ser Thr Ser.
595 600 605
Asn Phe Gin Phe Leu Phe Asp Leu Gly Leu Arg Met Asn Leu Ala Arg 610 615 620
Pro Lys Lys Lys Asp Ser Asp His Ala Ala Gin His Gly He Glu Leu
625 630 635
Gly Phe Lys He Pro Thr He Asn Thr Asn Tyr Tyr Ser Phe Met Gly
640 645 650
Ala Lys Leu Glu Tyr Arg Arg Met Tyr Ser Leu Phe Leu Asn Tyr Val 655 660 665 670
Phe Ala Tyr
(2) INFORMATION FOR SEQ ID NO : 13
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2270 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 130...2049 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 130...193 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
ATTGAGCGCA TCAAAACACC CTAAAACTTT TTTGAAATCC AATAAATTTA TGTTATAATT 60
AAACGCATTG TAAATAAATT CTCATTTTGA TACATTTTTA CAATAAAACA TTACTTTAAG 120
GAACATCTT ATG AAA AAA ACG AAA AAA ACG ATT CTG CTT TCT CTA ACT CTC 171
Met Lys Lys Thr Lys Lys Thr He Leu Leu Ser Leu Thr Leu
-20 -15 -10
GCG GCG TCA TTG CTC CAT GCT GAA GAC AAC GGC GTT TTT TTA AGC GTG 219 Ala Ala Ser Leu Leu His Ala Glu Asp Asn Gly Val Phe Leu Ser Val
-5 1 5
GGT TAT CAA ATC GGT GAA GCG GTT CAA AAA GTG AAA AAC GCC GAC AAG 267 Gly Tyr Gin He Gly Glu Ala Val Gin Lys Val Lys Asn Ala Asp Lys 10 15 20 25
GTG CAA AAA CTT TCA GAC ACT TAT GAA CAA TTA AGC CGG CTT TTA ACC " 315 Val Gin Lys Leu Ser Asp Thr Tyr Glu Gin Leu Ser Arg Leu Leu Thr 30 35 40
AAC GAT AAT GGC ACA AAC TCA AAG ACA AGC GCG CAA ATC AAC CAA GCG 363 Asn Asp Asn Gly Thr Asn Ser Lys Thr Ser Ala Gin He Asn Gin Ala 45 50 55 GTT AAT AAT TTG AAC GAA CGC GCA AAA ACT TTA GCC GGT GGG ACA ACC 411 Val Asn Asn Leu Asn Glu Arg Ala Lys Thr Leu Ala Gly Gly Thr Thr 60 65 70
AAT TCC CCT GCC TAT CAA GCC ACG CTT TTA GCG TTG AGA TCG GTG TTA 459 Asn Ser Pro Ala Tyr Gin Ala Thr Leu Leu Ala Leu Arg Ser Val Leu 75 80 85
GGG CTA TGG AAT AGC ATG GGT TAT GCG GTC ATA TGC GGA GGT TAT ACC 507 Gly Leu Trp Asn Ser Met Gly Tyr Ala Val He Cys Gly Gly Tyr Thr 90 95 100 105
AAA AGT CCA GGC GAA AAC AAT CAA AAA GAT TTC CAC TAC ACC GAT GAG 555 Lys Ser Pro Gly Glu Asn Asn Gin Lys Asp Phe His Tyr Thr Asp Glu 110 115 120
AAT GGC AAT GGC ACT ACA ATC AAT TGC GGT GGG AGC ACA AAT AGT AAT 603 Asn Gly Asn Gly Thr Thr He Asn Cys Gly Gly Ser Thr Asn Ser Asn 125 130 135
GGC ACT CAT AGT TCT AGT GGC ACA AAT ACA TTA AAA GCA GAC AAA AAT 651 Gly Thr His Ser Ser Ser Gly Thr Asn Thr Leu Lys Ala Asp Lys Asn 140 145 150
GTT TCT CTA TCT ATT GAG CAA TAT GAA AAA ATC CAT GAA GCT TAT CAG 699 Val Ser Leu Ser He Glu Gin Tyr Glu Lys He His Glu Ala Tyr Gin 155 160 165
ATT CTT TCA AAA GCT TTA AAA CAA GCC GGG CTT GCT CCT TTA AAT AGC 747 He Leu Ser Lys Ala Leu Lys Gin Ala Gly Leu Ala Pro Leu Asn Ser 170 175 180 185
AAA GGG GAA AAG TTA GAA GCG CAT GTA ACC ACA TCA AAA CCA GAA AAT 795 Lys Gly Glu Lys Leu Glu Ala His Val Thr Thr Ser Lys Pro Glu Asn 190 195 200
AAT AGT CAA ACT AAA ACG ACA ACT TCT GTT ATT GAT ACG ACT AAT GAT 843 Asn Ser Gin Thr Lys Thr Thr Thr Ser Val He Asp Thr Thr Asn Asp 205 210 215
GCG CAA AAT CTT TTG ACT CAA GCG CAA ACG ATT GTC AAT ACC CTT AAA 891 Ala Gin Asn Leu Leu Thr Gin Ala Gin Thr He Val Asn Thr Leu Lys 220 225 230
GAT TAT TGC CCC ATG TTG ATA GCG AAA TCT AGT AGT GAA AGT AGT GGC 939 Asp Tyr Cys Pro Met Leu He Ala Lys Ser Ser Ser Glu Ser Ser Gly 235 240 245
GCA GCT ACT ACA AAC GCC CCT TCA TGG CAA ACA GCC GGT GGC GGC AAA 987 Ala Ala Thr Thr Asn Ala Pro Ser Trp Gin Thr Ala Gly Gly Gly Lys 250 255 260 265
AAT TCA TGT GCG ACT TTT GGT GCG GAG TTT AGT GCC GCT TCA GAC ATG 1035 Asn Ser Cys Ala Thr Phe Gly Ala Glu Phe Ser Ala Ala Ser Asp Met 270 275 280
ATT AAT AAT GCG CAA AAA ATC GTT CAA GAA ACC CAA CAA CTC AGC GCC 1083 He Asn Asn Ala Gin Lys He Val Gin Glu Thr Gin Gin Leu Ser Ala 285 290 295
AAC CAA CCA AAA AAT ATC ACA CAA CCC CAT AAT CTC AAC CTT AAC ACC 1131 Asn Gin Pro Lys Asn He Thr Gin Pro His Asn Leu Asn Leu Asn Thr 300 305 310
CCT AGC AGT CTT ACG GCT TTA GCT CAA AAA ATG CTC AAA AAT GCG CAA 1179 Pro Ser Ser Leu Thr Ala Leu Ala Gin Lys Met Leu Lys Asn Ala Gin 315 320 325
TCT CAA GCA GAA ATT TTA AAA CTA GCC AAT CAA GTG GAG AGC GAT TTT 1227 Ser Gin Ala Glu He Leu Lys Leu Ala Asn Gin Val Glu Ser Asp Phe 330 335 340 345
AAC AAA CTT TCT TCA GGC CAT CTT AAA GAC TAC ATA GGG AAA TGC GAT 1275 Asn Lys Leu Ser Ser Gly His Leu Lys Asp Tyr He Gly Lys Cys Asp 350 355 360
GCG AGC GCT ATA AGC AGT GCG AAT ATG ACA ATG CAA AAT CAA AAG AAC 1323 Ala Ser Ala He Ser Ser Ala Asn Met Thr Met Gin Asn Gin Lys Asn 365 370 375
AAT TGG GGG AAC GGG TGT GCT GGC GTG GAA GAA ACT CTG TCT TCA TTA 1371 Asn Trp Gly Asn Gly Cys Ala Gly Val Glu Glu Thr Leu Ser Ser Leu 380 385 390
AAA ACA AGT GCC GCT GAT TTT AAC AAC CAA ACG CCA CAA ATC AAT CAA 1419 Lys Thr Ser Ala Ala Asp Phe Asn Asn Gin Thr Pro Gin He Asn Gin 395 400 405
GCG CAA AAC CTA GCC AAC ACC CTT ATT CAA GAA CTT GGC AAC AAC CCT 1467 Ala Gin Asn Leu Ala Asn Thr Leu He Gin Glu Leu Gly Asn Asn Pro 410 415 420 425
TTT AGG AAT ATG GGC ATG ATC GCT TCT TCA ACC ACG AAT AAC GGC GCC 1515 Phe Arg Asn Met Gly Met He Ala Ser Ser Thr Thr Asn Asn Gly Ala 430 435 440
TTG AAT GGC CTT GGG GTG CAA GTG GGT TAT AAG CAA TTT TTT GGG GAA 1563 Leu Asn Gly Leu Gly Val Gin Val Gly Tyr Lys Gin Phe Phe Gly Glu 445 450 455
AAG AAA AGA TGG GGG TTA AGG TAT TAT GGT TTC TTT GAT TAC AAC CAC 1611 Lys Lys Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His 460 465 470
GCC TAT ATC AAA TCC AAT TTC TTT AAC TCG GCT TCT GAT GTG TGG ACT 1659 Ala Tyr He Lys Ser Asn Phe Phe Asn Ser Ala Ser Asp Val Trp Thr 475 480 485
TAT GGG GTG GGC AGC GAT TTA TTG TTT AAT TTC ATC AAT GAT AAA AAC 1707 Tyr Gly Val Gly Ser Asp Leu Leu Phe Asn Phe He Asn Asp Lys Asn 490 495 500 505
ACC AAC TTT TTA GGC AAG AAT AAC AAG ATT TCA GTG GGA TTT TTT GGA 1755 Thr Asn Phe Leu Gly Lys Asn Asn Lys He Ser Val Gly Phe Phe Gly 510 515 520
GGT ATC GCC TTA GCA GGG ACT TCA TGG CTT AAT TCT CAA TTC GTG AAT 1803
Gly He Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gin Phe Val Asn 525 530 535
TTA AAA ACC ATC AGC AAT GTT TAT AGC GCT AAA GTG AAT ACG GCT AAC 1851
Leu Lys Thr He Ser Asn Val Tyr Ser Ala Lys Val Asn Thr Ala Asn
540 545 550
TTC CAA TTT TTA TTC AAT TTG GGC TTG AGA ACC AAT CTC GCT AGA CCT 1899
Phe Gin Phe Leu Phe Asn Leu Gly Leu Arg Thr Asn Leu Ala Arg Pro 555 560 565
AAG AAA AAA GAT AGT CAT CAT GCG GCT CAA CAT GGC ATG GAA TTG GGC 1947
Lys Lys Lys Asp Ser His His Ala Ala Gin His Gly Met Glu Leu Gly 570 575 580 585
GTG AAA ATC CCT ACC ATT AAC ACG AAT TAT TAT TCT TTT CTA GAC ACT 1995
Val Lys He Pro Thr He Asn Thr Asn Tyr Tyr Ser Phe Leu Asp Thr 590 595 600
AAA CTA GAA TAT CGA AGG CTT TAT AGC GTG TAT CTC AAT TAT GTG TTT 2043
Lys Leu Glu Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe 605 610 615
GCC TAT TAAAAACCCT CTTTTTAAAA AAGGGGGGGC TTTAAAAAAC CTCTAAAGAT AA 2101 Ala Tyr
AAATTTTCAA AAAACAATCA TTAAACCCTA AAAAAGAAAT TTTAAGGTAT AATGCTTTCG 2161 CCATTTTTAA TTTTCCATGG CAAACTCCTT TTTAGAATTT ATCCCCATAA TCGCTCTTAT 2221 GGGGCGTTTG TTTTGCAACA ATCTTTTCGA AACTATCCAA CAAGCTTTA 2270
(2) INFORMATION FOR SEQ ID NO : 14 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 640 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...21 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 14 :
Met Lys Lys Thr Lys Lys Thr He Leu Leu Ser Leu Thr Leu Ala Ala
-20 -15 -10
Ser Leu Leu His Ala Glu Asp Asn Gly Val Phe Leu Ser Val Gly Tyr -5 1 5 10
Gin He Gly Glu Ala Val Gin Lys Val Lys Asn Ala Asp Lys Val Gin
15 20 25
Lys Leu Ser Asp Thr Tyr Glu Gin Leu Ser Arg Leu Leu Thr Asn Asp
30 35 40
Asn Gly Thr Asn Ser Lys Thr Ser Ala Gin He Asn Gin Ala Val Asn
45 50 55
Asn Leu Asn Glu Arg Ala Lys Thr Leu Ala Gly Gly Thr Thr Asn Ser 60 65 70 75
Pro Ala Tyr Gin Ala Thr Leu Leu Ala Leu Arg Ser Val Leu Gly Leu
80 85 90
Trp Asn Ser Met Gly Tyr Ala Val He Cys Gly Gly Tyr Thr Lys Ser
95 100 105
Pro Gly Glu Asn Asn Gin Lys Asp Phe His Tyr Thr Asp Glu Asn Gly
110 115 120
Asn Gly Thr Thr He Asn Cys Gly Gly Ser Thr Asn Ser Asn Gly Thr
125 130 135
His Ser Ser Ser Gly Thr Asn Thr Leu Lys Ala Asp Lys Asn Val Ser 140 145 150 155
Leu Ser He Glu Gin Tyr Glu Lys He His Glu Ala Tyr Gin He Leu
160 165 170
Ser Lys Ala Leu Lys Gin Ala Gly Leu Ala Pro Leu Asn Ser Lys Gly
175 180 185
Glu Lys Leu Glu Ala His Val Thr Thr Ser Lys Pro Glu Asn Asn Ser
190 195 200
Gin Thr Lys Thr Thr Thr Ser Val He Asp Thr Thr Asn Asp Ala Gin
205 210 215
Asn Leu Leu Thr Gin Ala Gin Thr He Val Asn Thr Leu Lys Asp Tyr 220 225 230 235
Cys Pro Met Leu He Ala Lys Ser Ser Ser Glu Ser Ser Gly Ala Ala
240 245 250
Thr Thr Asn Ala Pro Ser Trp Gin Thr Ala Gly Gly Gly Lys Asn Ser
255 260 265
Cys Ala Thr Phe Gly Ala Glu Phe Ser Ala Ala Ser Asp Met He Asn
270 275 280
Asn Ala Gin Lys He Val Gin Glu Thr Gin Gin Leu Ser Ala Asn Gin
285 290 295
Pro Lys Asn He Thr Gin Pro His Asn Leu Asn Leu Asn Thr Pro Ser 300 305 310 315
Ser Leu Thr Ala Leu Ala Gin Lys Met Leu Lys Asn Ala Gin Ser Gin
320 325 330
Ala Glu He Leu Lys Leu Ala Asn Gin Val Glu Ser Asp Phe Asn Lys
335 340 345
Leu Ser Ser Gly His Leu Lys Asp Tyr He Gly Lys Cys Asp Ala Ser
350 355 360
Ala He Ser Ser Ala Asn Met Thr Met Gin Asn Gin Lys Asn Asn Trp
365 370 375
Gly Asn Gly Cys Ala Gly Val Glu Glu Thr Leu Ser Ser Leu Lys Thr 380 385 390 395
Ser Ala Ala Asp Phe Asn Asn Gin Thr Pro Gin He Asn Gin Ala Gin
400 405 410
Asn Leu Ala Asn Thr Leu He Gin Glu Leu Gly Asn Asn Pro Phe Arg
415 420 425
Asn Met Gly Met He Ala Ser Ser Thr Thr Asn Asn Gly Ala Leu Asn
430 435 440
Gly Leu Gly Val Gin Val Gly Tyr Lys Gin Phe Phe Gly Glu Lys Lys 445 450 455 Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Tyr 460 465 470 475
He Lys Ser Asn Phe Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly
480 485 490
Val Gly Ser Asp Leu Leu Phe Asn Phe He Asn Asp Lys Asn Thr Asn
495 500 505
Phe Leu Gly Lys Asn Asn Lys He Ser Val Gly Phe Phe Gly Gly He
510 515 520
Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gin Phe Val Asn Leu Lys
525 530 535
Thr He Ser Asn Val Tyr Ser Ala Lys Val Asn Thr Ala Asn Phe Gin 540 545 550 555
Phe Leu Phe Asn Leu Gly Leu Arg Thr Asn Leu Ala Arg Pro Lys Lys
560 565 570
Lys Asp Ser His His Ala Ala Gin His Gly Met Glu Leu Gly Val Lys
575 580 585
He Pro Thr He Asn Thr Asn Tyr Tyr Ser Phe Leu Asp Thr Lys Leu
590 595 600
Glu Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr 605 610 615
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2248 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 173...2128 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 173...224 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
TGGTTTTATC GTTACAAAAT TCAACATTTC AAAGATAAAT AAGTTAAAAT ACCCCAAAAT 60
CTTTTTTTTT TTTTTGAAAT CCAATCAATT TATAGTAAAA TTAGGTTCAT TGTAAATATA 120
TTATCACTTC ATGATATTCT TACAACAAAA ACATTACTTT AAGGAACATT TT ATG AAA 178
Met Lys
AAG ACA ATT CTG CTC TCT CTC TCT GCT TCA TCG CTC TTG CAC GCT GAA 226
Lys Thr He Leu Leu Ser Leu Ser Ala Ser Ser Leu Leu His Ala Glu
-15 -10 -5 1 .
GAC AAC GGC TTT TTT GTG AGC GCC GGC TAT CAA ATC GGC GAA GCG GTG 274 Asp Asn Gly Phe Phe Val Ser Ala Gly Tyr Gin He Gly Glu Ala Val
5 10 15
CAA ATG GTC AAA AAC ACC GGT GAA TTG AAA AAC TTG AAC GAA AAA TAC 322
Gin Met Val Lys Asn Thr Gly Glu Leu Lys Asn Leu Asn Glu Lys Tyr 20 25 30
GAG CAA TTA AGC CAG TAT TTA AAT CAA GTG GCT TCG TTG AAG CAA AGC 370
Glu Gin Leu Ser Gin Tyr Leu Asn Gin Val Ala Ser Leu Lys Gin Ser 35 40 45
ATT CAA AAC GCC AAC AAC ATT GAG CTG GTC AAT AGC TCT TTA AAC TAT 418
He Gin Asn Ala Asn Asn He Glu Leu Val Asn Ser Ser Leu Asn Tyr
50 55 60 65
TTA AAA AGC TTT ACC AAC AAC AAC TAT AAC AGC ACC ACC CAA TCG CCC 466
Leu Lys Ser Phe Thr Asn Asn Asn Tyr Asn Ser Thr Thr Gin Ser Pro 70 75 80
ATC TTT AAT GCC GTG CAA GCC GTT ATC ACT TCG GTA TTG GGT TTT TGG 514
He Phe Asn Ala Val Gin Ala Val He Thr Ser Val Leu Gly Phe Trp 85 90 95
AGT CTT TAT GCG GGG AAT TAC TTC ACT TTT TTT GTG GGT AAA AAG GTG 562
Ser Leu Tyr Ala Gly Asn Tyr Phe Thr Phe Phe Val Gly Lys Lys Val 100 105 110
GGT GAT AGT GGG CAA CCC GCT AGT GTC CAG GGT AAC CCT CCT TTT AAA 610
Gly Asp Ser Gly Gin Pro Ala Ser Val Gin Gly Asn Pro Pro Phe Lys 115 120 125
ACG ATT ATA GAG AAC TGC TCA GGA ATT GAA AAC TGC GCT ATG GAT CAA 658
Thr He He Glu Asn Cys Ser Gly He Glu Asn Cys Ala Met Asp Gin
130 135 140 145
ACC ACT TAT GAT AAG ATG AAA AAA CTC GCT GAA GAC CTC CAA GCG GCT 706
Thr Thr Tyr Asp Lys Met Lys Lys Leu Ala Glu Asp Leu Gin Ala Ala 150 155 160
CAA ACA AAC TCT GCC ACT AAA GGC AAC AAT CTT TGC GCT TTA TCC GGG 754
Gin Thr Asn Ser Ala Thr Lys Gly Asn Asn Leu Cys Ala Leu Ser Gly 165 170 175
TGT GCT GCA ACA GAC TCA ACA TCA AAC CCA CCA AAC TCA ACC GTG AGC 802
Cys Ala Ala Thr Asp Ser Thr Ser Asn Pro Pro Asn Ser Thr Val Ser 180 185 190
AAC GCT CTT AAT TTG GCG CAA CAG CTT ATG GAT TTA ATC GCA AAC ACT 850
Asn Ala Leu Asn Leu Ala Gin Gin Leu Met Asp Leu He Ala Asn Thr 195 200 205
AAA ACG GCT ATG ATG TGG AAA AAT ATC GTC ATC AGT GGC GTT TCA AAC 898
Lys Thr Ala Met Met Trp Lys Asn He Val He Ser Gly Val Ser Asn
210 215 220 225
ACA TCC GGT GCT ATC ACA TCC ACT AAT TAC CCA ACG CAA TAC GCG GTG 946
Thr Ser Gly Ala He Thr Ser Thr Asn Tyr Pro Thr Gin Tyr Ala Val 23 0 235 24 0
TTT AAC AAC ATT AAG GCG ATG ATA CCC ATT TTG CAA CAA GCG GTT ACG 994 Phe Asn Asn He Lys Ala Met He Pro He Leu Gin Gin Ala Val Thr 245 250 255
CTT TCT CAA AGC AAC CAC ACC CTA TCT GCT AGC TTG CAA GCT CAA GCC 1042 Leu Ser Gin Ser Asn His Thr Leu Ser Ala Ser Leu Gin Ala Gin Ala 260 265 270
ACA GGA TCT CAA ACA AAC CCT AAA TTC GCT AAA GAC ATC TAC ACT TTC 1090 Thr Gly Ser Gin Thr Asn Pro Lys Phe Ala Lys Asp He Tyr Thr Phe 275 280 285
GCT CAA AAC CAA AAG CAA GTC ATC TCT TAC GCT CAA GAC ATT TTC AAC 1138 Ala Gin Asn Gin Lys Gin Val He Ser Tyr Ala Gin Asp He Phe Asn 290 295 300 305
CTC TTT AAT TCT ATC CCT GCA GAG CAG TAT AAG TAT CTA GAG AAA GCT 1186 Leu Phe Asn Ser He Pro Ala Glu Gin Tyr Lys Tyr Leu Glu Lys Ala 310 315 320
TAC TTG AAA ATA CCC AAT GCG GGT TCA ACG CCT ACT AAC CCT TAC AGA 1234 Tyr Leu Lys He Pro Asn Ala Gly Ser Thr Pro Thr Asn Pro Tyr Arg 325 330 335
CAA GTG GTG AAT TTA AAC CAA GAA GTT CAG ACG ATT AAA AAC AAT GTG 1282 Gin Val Val Asn Leu Asn Gin Glu Val Gin Thr He Lys Asn Asn Val 340 345 350
AGT TAT TAT GGT AAC CGG GTG GAT GCG GCT TTA AGC GTG GCT AGA GAT 1330 Ser Tyr Tyr Gly Asn Arg Val Asp Ala Ala Leu Ser Val Ala Arg Asp 355 360 365
GTT TAT AAC CTA AAA TCC AAT CAA GCA GAA ATC GTA ACC GCC TAT AAC 1378 Val Tyr Asn Leu Lys Ser Asn Gin Ala Glu He Val Thr Ala Tyr Asn 370 375 380 385
GAC GCT AAG ACT TTG AGC GAA GAG ATT TCT AAA CTC CCG CAC AAT CAA 1426 Asp Ala Lys Thr Leu Ser Glu Glu He Ser Lys Leu Pro His Asn Gin 390 395 400
GTC AAT ACA AAA GAC ATT GTT ACA CTA CCT TAC GAT AAA AAC GCC CCA 1474 Val Asn Thr Lys Asp He Val Thr Leu Pro Tyr Asp Lys Asn Ala Pro 405 410 415
GCA GCA GGC CAA TCC AAC TAC CAA ATC AAC CCA GAG CAG CAA TCC AAT 1522 Ala Ala Gly Gin Ser Asn Tyr Gin He Asn Pro Glu Gin Gin Ser Asn 420 425 430
CTT AAC CAA GCT TTA GCA GCG ATG AGC AAT AAC CCC TTT AAA AAA GTG 1570 Leu Asn Gin Ala Leu Ala Ala Met Ser Asn Asn Pro Phe Lys Lys Val 435 440 445
GGC ATG ATC AGC TCT CAA AAC AAT AAC GGC GCT TTG AAC GGG CTT GGC 1618 Gly Met He Ser Ser Gin Asn Asn Asn Gly Ala Leu Asn Gly Leu Gly 450 455 460 465 GTG CAA GTG GGT TAT AAG CAA TTC TTT GGC GAA AGC AAA AGA TGG GGG 1666 Val Gin Val Gly Tyr Lys Gin Phe Phe Gly Glu Ser Lys Arg Trp Gly 470 475 480
TTA AGG TAT TAC GGA TTC TTT GAT TAC AAC CAC GGC TAC ATC AAA TCC 1714 Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Gly Tyr He Lys Ser 485 490 495
AGC TTC TTT AAC TCT TCT TCT GAT ATA TGG ACT TAT GGC GGT GGG AGC 1762 Ser Phe Phe Asn Ser Ser Ser Asp He Trp Thr Tyr Gly Gly Gly Ser 500 505 510
GAT TTG TTA GTG AAT ATT ATC AAC GAT AGC ATC ACA AGA AAG AAC AAC 1810 Asp Leu Leu Val Asn He He Asn Asp Ser He Thr Arg Lys Asn Asn 515 520 525
AAG CTC TCC GTG GGT CTT TTT GGA GGC ATC CAA CTA GCA GGG ACT ACA 1858 Lys Leu Ser Val Gly Leu Phe Gly Gly He Gin Leu Ala Gly Thr Thr 530 535 540 545
TGG CTT AAT TCT CAA TAC GTG AAT TTA ACC GCG TTC AAT AAC CCT TAC 1906 Trp Leu Asn Ser Gin Tyr Val Asn Leu Thr Ala Phe Asn Asn Pro Tyr 550 555 560
AGC GCG AAA GTC AAT GCT ACC AAT TTC CAA TTC TTG TTC AAT CTC GGC 1954 Ser Ala Lys Val Asn Ala Thr Asn Phe Gin Phe Leu Phe Asn Leu Gly 565 570 575
TTG AGG ACG AAT CTC GCT ACA GCT AGG AAA AAA GAC AGC GAA CAT TCC 2002 Leu Arg Thr Asn Leu Ala Thr Ala Arg Lys Lys Asp Ser Glu His Ser 580 585 590
GCG CAA CAT GGC ATT GAA TTG GGT ATT AAA ATC CCC ACC ATT ACC ACG 2050 Ala Gin His Gly He Glu Leu Gly He Lys He Pro Thr He Thr Thr 595 600 605
AAT TAC TAT TCT TTT CTA GGC ACT CAA TTG CAA TAC AGA AGG CTC TAT 2098 Asn Tyr Tyr Ser Phe Leu Gly Thr Gin Leu Gin Tyr Arg Arg Leu Tyr 610 615 620 625
AGC GTG TAT CTC AAT TAT GTG TTC GCT TAC TGAGTGATTC AAGCTCTCTT CTT 2151 Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr 630 635
TAAGGGGGTT TAGAAAAATC GCAACGCCAA GCTTTTTATC GTTGGTGATA AAATCTACAA 2211 AACTAACGGC GCGACAACAA ACCCTAACGC TACGCTC 2248
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 652 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal ( ix ) FEATURE :
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...17 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Met Lys Lys Thr He Leu Leu Ser Leu Ser Ala Ser Ser Leu Leu His
-15 -10 -5
Ala Glu Asp Asn Gly Phe Phe Val Ser Ala Gly Tyr Gin He Gly Glu
1 5 10 15
Ala Val Gin Met Val Lys Asn Thr Gly Glu Leu Lys Asn Leu Asn Glu
20 25 30
Lys Tyr Glu Gin Leu Ser Gin Tyr Leu Asn Gin Val Ala Ser Leu Lys
35 40 45
Gin Ser He Gin Asn Ala Asn Asn He Glu Leu Val Asn Ser Ser Leu
50 55 60
Asn Tyr Leu Lys Ser Phe Thr Asn Asn Asn Tyr Asn Ser Thr Thr Gin
65 70 75
Ser Pro He Phe Asn Ala Val Gin Ala Val He Thr Ser Val Leu Gly 80 85 90 95
Phe Trp Ser Leu Tyr Ala Gly Asn Tyr Phe Thr Phe Phe Val Gly Lys
100 105 110
Lys Val Gly Asp Ser Gly Gin Pro Ala Ser Val Gin Gly Asn Pro Pro
115 120 125
Phe Lys Thr He He Glu Asn Cys Ser Gly He Glu Asn Cys Ala Met
130 135 140
Asp Gin Thr Thr Tyr Asp Lys Met Lys Lys Leu Ala Glu Asp Leu Gin
145 150 155
Ala Ala Gin Thr Asn Ser Ala Thr Lys Gly Asn Asn Leu Cys Ala Leu 160 165 170 175
Ser Gly Cys Ala Ala Thr Asp Ser Thr Ser Asn Pro Pro Asn Ser Thr
180 185 190
Val Ser Asn Ala Leu Asn Leu Ala Gin Gin Leu Met Asp Leu He Ala
195 200 205
Asn Thr Lys Thr Ala Met Met Trp Lys Asn He Val He Ser Gly Val
210 215 220
Ser Asn Thr Ser Gly Ala He Thr Ser Thr Asn Tyr Pro Thr Gin Tyr
225 230 235
Ala Val Phe Asn Asn He Lys Ala Met He Pro He Leu Gin Gin Ala 240 245 250 255
Val Thr Leu Ser Gin Ser Asn His Thr Leu Ser Ala Ser Leu Gin Ala
260 265 270
Gin Ala Thr Gly Ser Gin Thr Asn Pro Lys Phe Ala Lys Asp He Tyr
275 280 285
Thr Phe Ala Gin Asn Gin Lys Gin Val He Ser Tyr Ala Gin Asp He
290 295 300
Phe Asn Leu Phe Asn Ser He Pro Ala Glu Gin Tyr Lys Tyr Leu Glu
305 310 315
Lys Ala Tyr Leu Lys He Pro Asn Ala Gly Ser Thr Pro Thr Asn Pro 320 325 330 335
Tyr Arg Gin Val Val Asn Leu Asn Gin Glu Val Gin Thr He Lys Asn
340 345 350
Asn Val Ser Tyr Tyr Gly Asn Arg Val Asp Ala Ala Leu Ser Val Ala 355 360 365
Arg Asp Val Tyr Asn Leu Lys Ser Asn Gin Ala Glu He Val Thr Ala
370 375 380
Tyr Asn Asp Ala Lys Thr Leu Ser Glu Glu He Ser Lys Leu Pro His
385 390 395
Asn Gin Val Asn Thr Lys Asp He Val Thr Leu Pro Tyr Asp Lys Asn 400 405 410 415
Ala Pro Ala Ala Gly Gin Ser Asn Tyr Gin He Asn Pro Glu Gin Gin
420 425 430
Ser Asn Leu Asn Gin Ala Leu Ala Ala Met Ser Asn Asn Pro Phe Lys
435 440 445
Lys Val Gly Met He Ser Ser Gin Asn Asn Asn Gly Ala Leu Asn Gly
450 455 460
Leu Gly Val Gin Val Gly Tyr Lys Gin Phe Phe Gly Glu Ser Lys Arg
465 470 475
Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Gly Tyr He 480 485 490 495
Lys Ser Ser Phe Phe Asn Ser Ser Ser Asp He Trp Thr Tyr Gly Gly
500 505 510
Gly Ser Asp Leu Leu Val Asn He He Asn Asp Ser He Thr Arg Lys
515 520 525
Asn Asn Lys Leu Ser Val Gly Leu Phe Gly Gly He Gin Leu Ala Gly
530 535 540
Thr Thr Trp Leu Asn Ser Gin Tyr Val Asn Leu Thr Ala Phe Asn Asn
545 550 555
Pro Tyr Ser Ala Lys Val Asn Ala Thr Asn Phe Gin Phe Leu Phe Asn 560 565 570 575
Leu Gly Leu Arg Thr Asn Leu Ala Thr Ala Arg Lys Lys Asp Ser Glu
580 585 590
His Ser Ala Gin His Gly He Glu Leu Gly He Lys He Pro Thr He
595 600 605
Thr Thr Asn Tyr Tyr Ser Phe Leu Gly Thr Gin Leu Gin Tyr Arg Arg
610 615 620
Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr 625 630 635
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2161 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 122...2056 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 122...179 (D) OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
CAAAAATCTT TTTTTTTTTT TTTTGAAATC CAATAAATTT ATGGTAAAGT TAAACATATT 60
GTAAATAAAT TTTAATTTCT ATTCATGTTT ACAATAAAAA AATTACTTTA AGGAACATTT 120
T ATG AAA AAG ACA ATT CTA CTC TCT CTC TCT CTC TCG CTT TCA TCG CTC 169 Met Lys Lys Thr He Leu Leu Ser Leu Ser Leu Ser Leu Ser Ser Leu -15 -10 -5
TTG CAC GCT GAA GAC AAC GGC TTT TTT GTG AGC GCC GGC TAT CAA ATC 217 Leu His Ala Glu Asp Asn Gly Phe Phe Val Ser Ala Gly Tyr Gin He 1 5 10
GGC GAA CGG GTG CAA ATG GTC AAA AAC ACC GGC GAA TTG AAA AAC TTG 265 Gly Glu Arg Val Gin Met Val Lys Asn Thr Gly Glu Leu Lys Asn Leu 15 20 25
AAC GAA AAA TAC GAG CAA TTA AGC CAA TCT TTA GCC CAA CTG GCT TCG 313 Asn Glu Lys Tyr Glu Gin Leu Ser Gin Ser Leu Ala Gin Leu Ala Ser 30 35 40 45
TTA AAA AAA AGC ATT CAA ACG GCG AAC AAC ATT CAG GCT GTC AAC AAT 361 Leu Lys Lys Ser He Gin Thr Ala Asn Asn He Gin Ala Val Asn Asn 50 55 60
GCT TTA AGC GAT TTA AAA AGC TTT GCG AGT AAC AAC CAC ACA AAC AAA 409 Ala Leu Ser Asp Leu Lys Ser Phe Ala Ser Asn Asn His Thr Asn Lys 65 70 75
GAA ACA TCG CCC ATC TAC AAC ACC GCG CAA GCT GTT ATC ACT TCA GTA 457 Glu Thr Ser Pro He Tyr Asn Thr Ala Gin Ala Val He Thr Ser Val 80 85 90
TTG GCT TTT TGG AGT CTT TAT GCA GGG AAC GCT ACC AGT TTT CAT GTG 505 Leu Ala Phe Trp Ser Leu Tyr Ala Gly Asn Ala Thr Ser Phe His Val 95 100 105
ACC GGT TTG AAT GAT GGA TCT AAT GCT CCT CTT GGA AGA ATC CAT CAA 553 Thr Gly Leu Asn Asp Gly Ser Asn Ala Pro Leu Gly Arg He His Gin 110 115 120 125
GAT GGG AAC TGC ACA GGA TTA CAA CAA TGT TTT ATG AAT AAA GAA ACT 601 Asp Gly Asn Cys Thr Gly Leu Gin Gin Cys Phe Met Asn Lys Glu Thr 130 135 140
TAT GAT AAA ATG AAA GCG CTT GCC GAA AAT CTC CAA AAA GCT CAA GGC 649 Tyr Asp Lys Met Lys Ala Leu Ala Glu Asn Leu Gin Lys Ala Gin Gly 145 150 155
AAT CTC TGT GCC TTA TCA GAA TGC CCT AGC GAT CAA TTA AAT GGA AAC 697 Asn Leu Cys Ala Leu Ser Glu Cys Pro Ser Asp Gin Leu Asn Gly Asn 160 165 170
AAT GGA AAC AAA ACT TCC ATG ACT AAA GCT CTT GAA ACC GCG CAA CAG. 745 Asn Gly Asn Lys Thr Ser Met Thr Lys Ala Leu Glu Thr Ala Gin Gin 175 180 185 CTT ATG GAT TTA ATC GCA AAC ACT AAA ACG GCT ATG ATG TGG AAA AAT 793 Leu Met Asp Leu He Ala Asn Thr Lys Thr Ala Met Met Trp Lys Asn 190 195 200 205
ATC GTC ATC GCA GGT GTT ACA AAC AGA CCC GGT GGT GCT GGC GCT ATC 841 He Val He Ala Gly Val Thr Asn Arg Pro Gly Gly Ala Gly Ala He 210 215 220
ACA TCC ACT GGT CCT GTA ACC GAC TAT GCG GTG TTT AAC AAC ATT AAG 889 Thr Ser Thr Gly Pro Val Thr Asp Tyr Ala Val Phe Asn Asn He Lys 225 230 235
GCG ATG ATA CCC ATT TTG CAA CAA GCG GTT ACG CTT TCT CAA AGC AAC 937 Ala Met He Pro He Leu Gin Gin Ala Val Thr Leu Ser Gin Ser Asn 240 245 250
CAC ACC CTA TCT GCT AGC TTG CAA GCT CAA GCC ACA GGA TCT CAA ACA 985 His Thr Leu Ser Ala Ser Leu Gin Ala Gin Ala Thr Gly Ser Gin Thr 255 260 265
AAC CCT AAA TTC GCT AAA GAC ATC TAC ACT TTC GCT CAA AAC CAA AAG 1033 Asn Pro Lys Phe Ala Lys Asp He Tyr Thr Phe Ala Gin Asn Gin Lys 270 275 280 285
CAA GTC ATC TCT TAC GCT CAA GAC ATT TTC AAC CTC TTT AAT TCT ATC 1081 Gin Val He Ser Tyr Ala Gin Asp He Phe Asn Leu Phe Asn Ser He 290 295 300
CCT GCA GAG CAG TAT AAG TAT CTA GAG AAA GCT TAC TTG AAA ATA CCC 1129 Pro Ala Glu Gin Tyr Lys Tyr Leu Glu Lys Ala Tyr Leu Lys He Pro 305 310 315
AAT GCG GGT TCA ACG CCT ACT AAC CCT TAC AGA CAA GTG GTG AAT TTA 1177 Asn Ala Gly Ser Thr Pro Thr Asn Pro Tyr Arg Gin Val Val Asn Leu 320 325 330
AAC CAA GAA GTT CAG ACG ATT AAA AAC AAT GTG AGT TAT TAT GGT AAC 1225 Asn Gin Glu Val Gin Thr He Lys Asn Asn Val Ser Tyr Tyr Gly Asn 335 340 345
CGG GTG GAT GCG GCT TTA AGC GTG GCT AGA GAT GTT TAT AAC CTA AAA 1273 Arg Val Asp Ala Ala Leu Ser Val Ala Arg Asp Val Tyr Asn Leu Lys 350 355 360 365
TCC AAT CAA GCA GAA ATC GTA ACC GCC TAT AAC GAC GCT AAG ACT TTG 1321 Ser Asn Gin Ala Glu He Val Thr Ala Tyr Asn Asp Ala Lys Thr Leu 370 375 380
AGC GAA GAG ATT TCT AAA CTC CCG CAC AAT CAA GTC AAT ACA AAA GAC 1369 Ser Glu Glu He Ser Lys Leu Pro His Asn Gin Val Asn Thr Lys Asp 385 390 395
ATT GTT ACA CTA CCT TAC GAT AAA AAC GCC CCA GCA GCA GGC CAA TCC 1417 He Val Thr Leu Pro Tyr Asp Lys Asn Ala Pro Ala Ala Gly Gin Ser 400 405 410
AAC TAC CAA ATC AAC CCA GAG CAG CAA TCC AAT CTT AAC CAA GCT TTA 1465 Asn Tyr Gin He Asn Pro Glu Gin Gin Ser Asn Leu Asn Gin Ala Leu 415 420 425
GCA GCG ATG AGC AAT AAC CCC TTT AAA AAA GTG GGC ATG ATC AGC TCT 1513 Ala Ala Met Ser Asn Asn Pro Phe Lys Lys Val Gly Met He Ser Ser 430 435 440 445
CAA AAC AAT AAC GGC GCT TTG AAC GGG CTT GGC GTG CAA GTG GGT TAT 1561 Gin Asn Asn Asn Gly Ala Leu Asn Gly Leu Gly Val Gin Val Gly Tyr 450 455 460
AAG CAA TTC TTT GGC GAA AGC AAA AGA TGG GGG TTA AGG TAT TAC GGA 1609 Lys Gin Phe Phe Gly Glu Ser Lys Arg Trp Gly Leu Arg Tyr Tyr Gly 465 470 475
TTC TTT GAT TAC AAC CAC GGC TAC ATC AAA TCC AGC TTC TTT AAC TCT 1657 Phe Phe Asp Tyr Asn His Gly Tyr He Lys Ser Ser Phe Phe Asn Ser 480 485 490
TCT TCT GAT ATA TGG ACT TAT GGC GGT GGG AGC GAT TTG TTA GTG AAT 1705 Ser Ser Asp He Trp Thr Tyr Gly Gly Gly Ser Asp Leu Leu Val Asn 495 500 505
ATT ATC AAC GAT AGC ATC ACA AGA AAG AAC AAC AAG CTC TCC GTG GGT 1753 He He Asn Asp Ser He Thr Arg Lys Asn Asn Lys Leu Ser Val Gly 510 515 520 525
CTT TTT GGA GGC ATC CAA CTA GCA GGG ACT ACA TGG CTT AAT TCT CAA 1801 Leu Phe Gly Gly He Gin Leu Ala Gly Thr Thr Trp Leu Asn Ser Gin 530 535 540
TAC GTG AAT TTA ACC GCG TTC AAT AAC CCT TAC AGC GCG AAA GTC AAT 1849 Tyr Val Asn Leu Thr Ala Phe Asn Asn Pro Tyr Ser Ala Lys Val Asn 545 550 555
GCT ACC AAT TTC CAA TTC TTG TTC AAT CTC GGC TTG AGG ACG AAT CTC 1897 Ala Thr Asn Phe Gin Phe Leu Phe Asn Leu Gly Leu Arg Thr Asn Leu 560 565 570
GCT ACA GCT AGG AAA AAA GAC AGC GAA CAT TCC GCG CAA CAT GGC ATT 1945 Ala Thr Ala Arg Lys Lys Asp Ser Glu His Ser Ala Gin His Gly He 575 580 585
GAA TTG GGT ATT AAA ATC CCC ACC ATT ACC ACG AAT TAC TAT TCT TTT 1993 Glu Leu Gly He Lys He Pro Thr He Thr Thr Asn Tyr Tyr Ser Phe 590 595 600 605
CTA GGC ACT CAA TTG CAA TAC AGA AGG CTC TAT AGC GTG TAT CTC AAT 2041 Leu Gly Thr Gin Leu Gin Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn 610 615 620
TAT GTG TTC GCT TAT TAAAAAATCT TCTTTTTAAA ATAGGGGGAG CTTCATCAAA T 2097 Tyr Val Phe Ala Tyr 625
CTATTTTGAT AGTTATCAAT ATTTGATGAA AATAAAGTCA AAAACAAAAT AAACCAAATC 2157 ACCC 2161 (2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 645 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...19 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Met Lys Lys Thr He Leu Leu Ser Leu Ser Leu Ser Leu Ser Ser Leu
-15 -10 -5
Leu His Ala Glu Asp Asn Gly Phe Phe Val Ser Ala Gly Tyr Gin He
1 5 10
Gly Glu Arg Val Gin Met Val Lys Asn Thr Gly Glu Leu Lys Asn Leu
15 20 25
Asn Glu Lys Tyr Glu Gin Leu Ser Gin Ser Leu Ala Gin Leu Ala Ser 30 35 40 45
Leu Lys Lys Ser He Gin Thr Ala Asn Asn He Gin Ala Val Asn Asn
50 55 60
Ala Leu Ser Asp Leu Lys Ser Phe Ala Ser Asn Asn His Thr Asn Lys
65 70 75
Glu Thr Ser Pro He Tyr Asn Thr Ala Gin Ala Val He Thr Ser Val
80 85 90
Leu Ala Phe Trp Ser Leu Tyr Ala Gly Asn Ala Thr Ser Phe His Val
95 100 105
Thr Gly Leu Asn Asp Gly Ser Asn Ala Pro Leu Gly Arg He His Gin 110 115 120 125
Asp Gly Asn Cys Thr Gly Leu Gin Gin Cys Phe Met Asn Lys Glu Thr
130 135 140
Tyr Asp Lys Met Lys Ala Leu Ala Glu Asn Leu Gin Lys Ala Gin Gly
145 150 155
Asn Leu Cys Ala Leu Ser Glu Cys Pro Ser Asp Gin Leu Asn Gly Asn
160 165 170
Asn Gly Asn Lys Thr Ser Met Thr Lys Ala Leu Glu Thr Ala Gin Gin
175 180 185
Leu Met Asp Leu He Ala Asn Thr Lys Thr Ala Met Met Trp Lys Asn 190 195 200 205
He Val He Ala Gly Val Thr Asn Arg Pro Gly Gly Ala Gly Ala He
210 215 220
Thr Ser Thr Gly Pro Val Thr Asp Tyr Ala Val Phe Asn Asn He Lys
225 230 235
Ala Met He Pro He Leu Gin Gin Ala Val Thr Leu Ser Gin Ser Asn
240 245 250
His Thr Leu Ser Ala Ser Leu Gin Ala Gin Ala Thr Gly Ser Gin Thr
255 260 265
Asn Pro Lys Phe Ala Lys Asp He Tyr Thr Phe Ala Gin Asn Gin Lys 270 275 280 285
Gin Val He Ser Tyr Ala Gin Asp He Phe Asn Leu Phe Asn Ser He
290 295 300
Pro Ala Glu Gin Tyr Lys Tyr Leu Glu Lys Ala Tyr Leu Lys He Pro
305 310 315
Asn Ala Gly Ser Thr Pro Thr Asn Pro Tyr Arg Gin Val Val Asn Leu
320 325 330
Asn Gin Glu Val Gin Thr He Lys Asn Asn Val Ser Tyr Tyr Gly Asn
335 340 345
Arg Val Asp Ala Ala Leu Ser Val Ala Arg Asp Val Tyr Asn Leu Lys 350 355 360 365
Ser Asn Gin Ala Glu He Val Thr Ala Tyr Asn Asp Ala Lys Thr Leu
370 375 380
Ser Glu Glu He Ser Lys Leu Pro His Asn Gin Val Asn Thr Lys Asp
385 390 395
He Val Thr Leu Pro Tyr Asp Lys Asn Ala Pro Ala Ala Gly Gin Ser
400 405 410
Asn Tyr Gin He Asn Pro Glu Gin Gin Ser Asn Leu Asn Gin Ala Leu
415 420 425
Ala Ala Met Ser Asn Asn Pro Phe Lys Lys Val Gly Met He Ser Ser 430 435 440 445
Gin Asn Asn Asn Gly Ala Leu Asn Gly Leu Gly Val Gin Val Gly Tyr
450 455 460
Lys Gin Phe Phe Gly Glu Ser Lys Arg Trp Gly Leu Arg Tyr Tyr Gly
465 470 475
Phe Phe Asp Tyr Asn His Gly Tyr He Lys Ser Ser Phe Phe Asn Ser
480 485 490
Ser Ser Asp He Trp Thr Tyr Gly Gly Gly Ser Asp Leu Leu Val Asn
495 500 505
He He Asn Asp Ser He Thr Arg Lys Asn Asn Lys Leu Ser Val Gly 510 515 520 525
Leu Phe Gly Gly He Gin Leu Ala Gly Thr Thr Trp Leu Asn Ser Gin
530 535 540
Tyr Val Asn Leu Thr Ala Phe Asn Asn Pro Tyr Ser Ala Lys Val Asn
545 550 555
Ala Thr Asn Phe Gin Phe Leu Phe Asn Leu Gly Leu Arg Thr Asn Leu
560 565 570
Ala Thr Ala Arg Lys Lys Asp Ser Glu His Ser Ala Gin His Gly He
575 580 585
Glu Leu Gly He Lys He Pro Thr He Thr Thr Asn Tyr Tyr Ser Phe 590 595 600 605
Leu Gly Thr Gin Leu Gin Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn
610 615 620
Tyr Val Phe Ala Tyr 625
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1799 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: (A) NAME/KEY: Coding Sequence
(B) LOCATION: 185...1633 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 185...233 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 19 :
TACTCAAAAC ATTTTTCACT ATCAAAAACC TTTTTTTTAA ATCCAAAAAA AAAGCAAAAT 60
TTCTTAATTT TTGCTCAATT TTATTAAAAA TTCAATAAAT TTATGGCACA ATTTAAACTT 120
ATTGTAAATA AAGTTTCAAT TTGATACGAT TTTACAAACA AAACATTACT TTAAGGAACA 180
TTTT ATG AAA AAA ACG ATT TTA CTT TCT CTT ATG GTT TCA TCG CTC CTC 229 Met Lys Lys Thr He Leu Leu Ser Leu Met Val Ser Ser Leu Leu -15 -10 -5
GCT GAA AAT GAC GGC GTT TTT ATG AGC GTG GGC TAT CAA ATC GGC GAA 277 Ala Glu Asn Asp Gly Val Phe Met Ser Val Gly Tyr Gin He Gly Glu 1 5 10 15
GCG GTT CAA CAA GTG AAA AAC ACC GGC GAA ATC CAA AAA GTC TCC AAC 325 Ala Val Gin Gin Val Lys Asn Thr Gly Glu He Gin Lys Val Ser Asn 20 25 30
GCT TAC GAA AAT TTG AAC AAT CTT TTA ACC CGC TAT AAC GAA CTC AAA 373 Ala Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr Asn Glu Leu Lys 35 40 45
CAA ACG GCC TCT AAC ACC AAT TCA AGT ACC GCT CAA GCG ATT GAT AAT 421 Gin Thr Ala Ser Asn Thr Asn Ser Ser Thr Ala Gin Ala He Asp Asn 50 55 60
CTA AAA GAG AGC GCT AGC CGA TTG AAA ACG ACC CCC AAT AGC GCT AAT 469 Leu Lys Glu Ser Ala Ser Arg Leu Lys Thr Thr Pro Asn Ser Ala Asn 65 70 75
CAA GCC GTG TCT TCA GCG CTC AGC TCT GCG GTA GCC ATG TGG CAA GTA 517 Gin Ala Val Ser Ser Ala Leu Ser Ser Ala Val Ala Met Trp Gin Val 80 85 90 95
ATA GTC TCT AAT TTA GCC AAT AAC TCG CTA CCC ACT AGT GAA TAC AAC 565 He Val Ser Asn Leu Ala Asn Asn Ser Leu Pro Thr Ser Glu Tyr Asn 100 105 110
AAA ATC AAT GCG ATT TCT CAA TCG CTC CAA AAC ACC CTA GAA AAT AAA~ " 613 Lys He Asn Ala He Ser Gin Ser Leu Gin Asn Thr Leu Glu Asn Lys 115 120 125
AAC AAT GAT CTT AAA ATT GAA AAT GAC TAC GAC CAT CTT TTA ACT CAA 661 Asn Asn Asp Leu Lys He Glu Asn Asp Tyr Asp His Leu Leu Thr Gln_ 130 135 140 GCT AGC ACC ATT ATT AAT ACC CTT CAA AGC CAA TGC CCA GGC ATA GAC 709
Ala Ser Thr He He Asn Thr Leu Gin Ser Gin Cys Pro Gly He Asp 145 150 155
GGA GGC AAT GGC AAA CCA TGG GGC ATT AAT GCA AGC GGG AAC GCA TGC 757
Gly Gly Asn Gly Lys Pro Trp Gly He Asn Ala Ser Gly Asn Ala Cys
160 165 170 175
AAT ATT TTT GGC AAC ACC TTT AAC GCC ATC ACT AGC ATG ATA GAT AGC 805
Asn He Phe Gly Asn Thr Phe Asn Ala He Thr Ser Met He Asp Ser 180 185 190
GCT AAA AAA GCC GCC GCA GAT GCC CGA AGA ACT GCC CCA GAA AGT CCA 853
Ala Lys Lys Ala Ala Ala Asp Ala Arg Arg Thr Ala Pro Glu Ser Pro 195 200 205
AAC CAA CCA AGT GCG TTT AAC AAC GCT GAT TTC AAT AAA AAC CTT AAT 901
Asn Gin Pro Ser Ala Phe Asn Asn Ala Asp Phe Asn Lys Asn Leu Asn 210 215 220
CAA GTC TCA AGC GTT ATT AAT GAC ACG ATC TCT TAC CTC AAA GGG GAC 949
Gin Val Ser Ser Val He Asn Asp Thr He Ser Tyr Leu Lys Gly Asp 225 230 235
AAT TTA GCA ACC ATC TAC AAC ACC CTT CAA AAA ACG CCC GAT TCT AAA 997
Asn Leu Ala Thr He Tyr Asn Thr Leu Gin Lys Thr Pro Asp Ser Lys
240 245 250 255
GGG TTT CAA AGT TTG GTG AGC CGA TCT AGC TAT AGT TAT TCC CTC AAC 1045
Gly Phe Gin Ser Leu Val Ser Arg Ser Ser Tyr Ser Tyr Ser Leu Asn 260 265 270
GAA ACC CAA TAT TCT GAA TTC CAA ACT ACC ACC AAA GAG TTT GGC CAT 1093
Glu Thr Gin Tyr Ser Glu Phe Gin Thr Thr Thr Lys Glu Phe Gly His 275 280 285
AAC CCT TTT AGA AGC GTG GGT TTA ATC AAC TCT CAA AGC AAT AAC GGA 1141
Asn Pro Phe Arg Ser Val Gly Leu He Asn Ser Gin Ser Asn Asn Gly 290 295 300
GCG ATG AAT GGC GTG GGC GTG CAA TTA GGC TAT AAG CAA TTC TTT GGG 1189
Ala Met Asn Gly Val Gly Val Gin Leu Gly Tyr Lys Gin Phe Phe Gly 305 310 315
AAA AAT AAA TTT TTT GGG ATC CGT TAT TAT GCC TTT TTT GAT TAC AAC 1237
Lys Asn Lys Phe Phe Gly He Arg Tyr Tyr Ala Phe Phe Asp Tyr Asn
320 325 330 335
CAT GCC TAT ATC AAA TCC AAC TTT TTC AAC TCC GCT TCC AAT GTT TTC 1285
His Ala Tyr He Lys Ser Asn Phe Phe Asn Ser Ala Ser Asn Val Phe 340 345 350
ACT TAT GGC GCA GGC AGT GAT CTT TTA TTG AAT TTC ATC AAT GGC GGA 1333
Thr Tyr Gly Ala Gly Ser Asp Leu Leu Leu Asn Phe He Asn Gly Gly 355 360 365
TCC GAT AAA AAC CGC AAA GTC TCT TTT GGC ATT TTT GGA GGC ATC GCT 1381 Ser Asp Lys Asn Arg Lys Val Ser Phe Gly He Phe Gly Gly He Ala 370 375 380
CTA GCA GGC ACG ACA TGG CTT AAT TCC CAA TTT ATG AAT TTA AAA ACC 1429 Leu Ala Gly Thr Thr Trp Leu Asn Ser Gin Phe Met Asn Leu Lys Thr 385 390 395
ACC AAT AGC GCC TAC AGC GCT AAG ATC AAC AAC ACC AAT TTC CAA TTC 1477 Thr Asn Ser Ala Tyr Ser Ala Lys He Asn Asn Thr Asn Phe Gin Phe 400 405 410 415
TTA TTC AAT ACT GGT TTA AGG CTT CAA GGG ATT CAC CAT GGC GTT GAA 1525 Leu Phe Asn Thr Gly Leu Arg Leu Gin Gly He His His Gly Val Glu 420 425 430
TTA GGC GTG AAA ATC CCC ACC ATC AAC ACG AAT TAC TAT TCT TTC ATG 1573 Leu Gly Val Lys He Pro Thr He Asn Thr Asn Tyr Tyr Ser Phe Met 435 440 445
GGC GCT AAA TTA GCA TAC CGA AGA CTT TAT AGC GTG TAT TTC AAT TAT 1621 Gly Ala Lys Leu Ala Tyr Arg Arg Leu Tyr Ser Val Tyr Phe Asn Tyr 450 455 460
GTT TTG GCC TAT TGATATTGAA TCGGTTCTCA TTACTAATGA GGACAAAGCC AAACT 1678 Val Leu Ala Tyr 465
TTTTGGCTCT CAATGAATAA CGGCATCATT TTACTTGACT TTTTACAAAA AACACACTAA 1738 AATTTCTTTT TCTTTTTTGA GCGAAATTCC AGATTAGCTC AGCGGTAGAG TAGGCGGCTG 1798 T 1799
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 483 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...16 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Met Lys Lys Thr He Leu Leu Ser Leu Met Val Ser Ser Leu Leu Ala
-15 -10 -5
Glu Asn Asp Gly Val Phe Met Ser Val Gly Tyr Gin He Gly Glu Ala 1 5 10 15 -
Val Gin Gin Val Lys Asn Thr Gly Glu He Gin Lys Val Ser Asn Ala 20 25 30 Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr Asn Glu Leu Lys Gin
35 40 45
Thr Ala Ser Asn Thr Asn Ser Ser Thr Ala Gin Ala He Asp Asn Leu
50 55 60
Lys Glu Ser Ala Ser Arg Leu Lys Thr Thr Pro Asn Ser Ala Asn Gin 65 70 75 80
Ala Val Ser Ser Ala Leu Ser Ser Ala Val Ala Met Trp Gin Val He
85 90 95
Val Ser Asn Leu Ala Asn Asn Ser Leu Pro Thr Ser Glu Tyr Asn Lys
100 105 110
He Asn Ala He Ser Gin Ser Leu Gin Asn Thr Leu Glu Asn Lys Asn
115 120 125
Asn Asp Leu Lys He Glu Asn Asp Tyr Asp His Leu Leu Thr Gin Ala
130 135 140
Ser Thr He He Asn Thr Leu Gin Ser Gin Cys Pro Gly He Asp Gly 145 150 155 160
Gly Asn Gly Lys Pro Trp Gly He Asn Ala Ser Gly Asn Ala Cys Asn
165 170 175
He Phe Gly Asn Thr Phe Asn Ala He Thr Ser Met He Asp Ser Ala
180 185 190
Lys Lys Ala Ala Ala Asp Ala Arg Arg Thr Ala Pro Glu Ser Pro Asn
195 200 205
Gin Pro Ser Ala Phe Asn Asn Ala Asp Phe Asn Lys Asn Leu Asn Gin
210 215 220
Val Ser Ser Val He Asn Asp Thr He Ser Tyr Leu Lys Gly Asp Asn 225 230 235 240
Leu Ala Thr He Tyr Asn Thr Leu Gin Lys Thr Pro Asp Ser Lys Gly
245 250 255
Phe Gin Ser Leu Val Ser Arg Ser Ser Tyr Ser Tyr Ser Leu Asn Glu
260 265 270
Thr Gin Tyr Ser Glu Phe Gin Thr Thr Thr Lys Glu Phe Gly His Asn
275 280 285
Pro Phe Arg Ser Val Gly Leu He Asn Ser Gin Ser Asn Asn Gly Ala
290 295 300
Met Asn Gly Val Gly Val Gin Leu Gly Tyr Lys Gin Phe Phe Gly Lys 305 310 315 320
Asn Lys Phe Phe Gly He Arg Tyr Tyr Ala Phe Phe Asp Tyr Asn His
325 330 335
Ala Tyr He Lys Ser Asn Phe Phe Asn Ser Ala Ser Asn Val Phe Thr
340 345 350
Tyr Gly Ala Gly Ser Asp Leu Leu Leu Asn Phe He Asn Gly Gly Ser
355 360 365
Asp Lys Asn Arg Lys Val Ser Phe Gly He Phe Gly Gly He Ala Leu
370 375 380
Ala Gly Thr Thr Trp Leu Asn Ser Gin Phe Met Asn Leu Lys Thr Thr 385 390 395 400
Asn Ser Ala Tyr Ser Ala Lys He Asn Asn Thr Asn Phe Gin Phe Leu
405 410 415
Phe Asn Thr Gly Leu Arg Leu Gin Gly He His His Gly Val Glu Leu
420 425 430
Gly Val Lys He Pro Thr He Asn Thr Asn Tyr Tyr Ser Phe Met Gly
435 440 445
Ala Lys Leu Ala Tyr Arg Arg Leu Tyr Ser Val Tyr Phe Asn Tyr Val
450 455 460
Leu Ala Tyr 465 (2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2338 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 146...2218 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 146...200 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
ACTTAAAATT GTTTTTTTTT TTTTTCAAAA TATAAATTTT AAGCCAAAAA TAAGCATTTT 60 ATGGTAAAAT GGCGAACTTT CATAAACATG ACTATTATGG GAATGTCATG GGAATGTGAA 120 GAAAAATCTA TTAAAA GGA GAA AAC ATG AAA AAA TCC CTC TTA CTC TCT CTT 172
Met Lys Lys Ser Leu Leu Leu Ser Leu -18 -15 -10
TCT CTC ATC GCT TCC TTA TCA AGA GCT GAA GAT GAC GGA TTT TAT ACG 220 Ser Leu He Ala Ser Leu Ser Arg Ala Glu Asp Asp Gly Phe Tyr Thr
-5 1 5
AGT GTG GGC TAT CAG ATC GGT GAA GCG GTC CAA CAA GTG AAA AAC ACA 268 Ser Val Gly Tyr Gin He Gly Glu Ala Val Gin Gin Val Lys Asn Thr 10 15 20
GGA GCA TTG CAA AAT CTT GCA GAC AGA TAC GAT AAC TTA AAC AAC CTT 316 Gly Ala Leu Gin Asn Leu Ala Asp Arg Tyr Asp Asn Leu Asn Asn Leu 25 30 35
TTA AAC CAA TAC AAT TAT TTA AAT TCC TTA GTC AAT TTA GCC AGC ACG 364 Leu Asn Gin Tyr Asn Tyr Leu Asn Ser Leu Val Asn Leu Ala Ser Thr 40 45 50 55
CCG AGC GCG ATC ACC GGT GCG ATT GAT AAT TTA AGC TCA AGC GCG ATT 412 Pro Ser Ala He Thr Gly Ala He Asp Asn Leu Ser Ser Ser Ala He 60 65 70
AAC CTC ACT AGC GCC ACC ACC ACT TCC CCC GCC TAT CAA GCT GTG GCT 460 Asn Leu Thr Ser Ala Thr Thr Thr Ser Pro Ala Tyr Gin Ala Val Ala 75 80 85
TTA GCG CTC AAT GCC GCT GTG GGC ATG TGG CAA GTC ATA GCC CTT TTT 508 Leu Ala Leu Asn Ala Ala Val Gly Met Trp Gin Val He Ala Leu Phe 90 95 100
ATT GGC TGT GGC CCT GGC CCT ACC AAT AAT CAA AGC TAT CAA TCG TTT 556 He Gly Cys Gly Pro Gly Pro Thr Asn Asn Gin Ser Tyr Gin Ser Phe 105 110 115
GGT AAC ACA CCA GCC CTT AAT GGG ACC ACC ACC ACT TGC AAT CAA GCA 604 Gly Asn Thr Pro Ala Leu Asn Gly Thr Thr Thr Thr Cys Asn Gin Ala 120 125 130 135
TAT GGG ACA GGC CCT AAT GGC ATC CTA TCT ATT GAT GAA TAC CAA AAA 652 Tyr Gly Thr Gly Pro Asn Gly He Leu Ser He Asp Glu Tyr Gin Lys 140 145 150
CTC AAC CAA GCT TAT CAG ATC ATC CAA ACC GCT TTA AAC CAA AAT CAA 700 Leu Asn Gin Ala Tyr Gin He He Gin Thr Ala Leu Asn Gin Asn Gin 155 160 165
GGG GGT GGG ATG CCT GCC TTG AAT GAC ACC ACC AAA ACA GGG GTA GTC 748 Gly Gly Gly Met Pro Ala Leu Asn Asp Thr Thr Lys Thr Gly Val Val 170 175 180
AAC ATA CAA CAA ACC AAT TAT AGG ACC ACC ACA CAA AAC AAT ATC ATA 796 Asn He Gin Gin Thr Asn Tyr Arg Thr Thr Thr Gin Asn Asn He He 185 190 195
GAG CAT TAT TAT ACA GAG AAT GGG AAA GAG ATC CCA GTC TCT TAT TCA 844 Glu His Tyr Tyr Thr Glu Asn Gly Lys Glu He Pro Val Ser Tyr Ser 200 205 210 215
GGC GGA TCA TCA TTC TCG CCT ACA ATA CAA TTG ACA TAC CAT AAT AAC 892 Gly Gly Ser Ser Phe Ser Pro Thr He Gin Leu Thr Tyr His Asn Asn 220 225 230
GCT GAA AAC CTT TTG CAA CAA GCC GCC ACT ATC ATG CAA GTC CTT ATT 940 Ala Glu Asn Leu Leu Gin Gin Ala Ala Thr He Met Gin Val Leu He 235 240 245
ACT CAA AAG CCG CAT GTG CAA ACG AGC AAT GGC GGT AAA GCG TGG GGG 988 Thr Gin Lys Pro His Val Gin Thr Ser Asn Gly Gly Lys Ala Trp Gly 250 255 260
TTG AGT TCT ACG CCT GGG AAT GTG ATG GAT ATT TTT GGT CCT TCT TTT 1036 Leu Ser Ser Thr Pro Gly Asn Val Met Asp He Phe Gly Pro Ser Phe 265 270 275
AAC GCT ATT AAT GAG ATG ATT AAA AAC GCT CAA ACA GCC CTA GCA AAA 1084 Asn Ala He Asn Glu Met He Lys Asn Ala Gin Thr Ala Leu Ala Lys 280 285 290 295
ACC CAA CAG CTT AAC GCT AAT GAA AAC GCC CAA ATC ACG CAA CCC AAC 1132 Thr Gin Gin Leu Asn Ala Asn Glu Asn Ala Gin He Thr Gin Pro Asn 300 305 310
AAT TTC AAC CCC TAC ACC TCT AAA GAC AAA GGG TTC GCT CAA GAA ATG. 1180 Asn Phe Asn Pro Tyr Thr Ser Lys Asp Lys Gly Phe Ala Gin Glu Met 315 320 325 CTC AAT AGA GCT GAA GCT CAA GCA GAG ATT TTA AAT TTA GCT AAG CAA 1228
Leu Asn Arg Ala Glu Ala Gin Ala Glu He Leu Asn Leu Ala Lys Gin 330 335 340
GTA GCG AAC AAT TTC CAC AGC ATT CAA GGG CCT ATT CAA GGG GAT TTA 1276
Val Ala Asn Asn Phe His Ser He Gin Gly Pro He Gin Gly Asp Leu
345 350 355
GAA GAA TGT AAA GCA GGA TCG GCT GGC GTG ATC ACT AAT AAC ACT TGG 1324
Glu Glu Cys Lys Ala Gly Ser Ala Gly Val He Thr Asn Asn Thr Trp
360 365 370 375
GGT TCA GGT TGC GCG TTT GTG AAA GAA ACT TTA AAC TCT TTA GAG CAA 1372
Gly Ser Gly Cys Ala Phe Val Lys Glu Thr Leu Asn Ser Leu Glu Gin 380 385 390
CAC ACC GCT TAT TAC GGC AAC CAG GTC AAT CAG GAT AGG GCT TTG GCT 1420
His Thr Ala Tyr Tyr Gly Asn Gin Val Asn Gin Asp Arg Ala Leu Ala 395 400 405
CAA ACC ATT TTG AAT TTT AAA GAA GCC CTT AAC ACC CTG AAT AAA GAC 1468
Gin Thr He Leu Asn Phe Lys Glu Ala Leu Asn Thr Leu Asn Lys Asp 410 415 420
TCA AAA GCG ATC AAT AGC GGT ATC TCC AAC TTG CCT AAC GCT AAA TCT 1516
Ser Lys Ala He Asn Ser Gly He Ser Asn Leu Pro Asn Ala Lys Ser
425 430 435
CTT CAA AAC ATG ACG CAT GCC ACT CAA AAC CCT AAT TCC CCA GAA GGT 1564
Leu Gin Asn Met Thr His Ala Thr Gin Asn Pro Asn Ser Pro Glu Gly
440 445 450 455
CTG CTC ACT TAT TCT TTG GAT TCA AGC AAA TAC AAC CAG CTC CAA ACC 1612
Leu Leu Thr Tyr Ser Leu Asp Ser Ser Lys Tyr Asn Gin Leu Gin Thr 460 465 470
ATC GCG CAA GAA TTG GGC AAA AAC CCT TTC AGG CGC TTT GGC GTG ATT 1660
He Ala Gin Glu Leu Gly Lys Asn Pro Phe Arg Arg Phe Gly Val He 475 480 485
GAC TTT CAA AAC AAC AAC GGC GCA ATG AAC GGG ATC GGC GTG CAA GTG 1708
Asp Phe Gin Asn Asn Asn Gly Ala Met Asn Gly He Gly Val Gin Val 490 495 500
GGT TAT AAA CAA TTC TTT GGT AAA AAA AGG AAT TGG GGG TTA AGG TAT 1756
Gly Tyr Lys Gin Phe Phe Gly Lys Lys Arg Asn Trp Gly Leu Arg Tyr
505 510 515
TAT GGT TTC TTT GAT TAT AAC CAT GCT TAT ATC AAA TCT AAT TTT TTC 1804
Tyr Gly Phe Phe Asp Tyr Asn His Ala Tyr He Lys Ser Asn Phe Phe
520 525 530 535
AAC TCC GCT TCT GAT GTG TGG ACT TAT GGG GTG GGT ATG GAC GCT CTC 1852
Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Val Gly Met Asp Ala Leu 540 545 550
TAT AAC TTC ATC AAC GAT AAA AAC ACC AAC TTT TTA GGC AAG AAC AAC 1900 Tyr Asn Phe He Asn Asp Lys Asn Thr Asn Phe Leu Gly Lys Asn Asn 555 560 565
AAG CTT TCA GTA GGG CTT TTT GGA GGC TTT GCG TTA GCC GGG ACT TCG 1948 Lys Leu Ser Val Gly Leu Phe Gly Gly Phe Ala Leu Ala Gly Thr Ser 570 575 580
TGG CTT AAT TCC CAA CAA GTG AAT TTG ACC ATG ATG AAT GGC ATT TAT 1996 Trp Leu Asn Ser Gin Gin Val Asn Leu Thr Met Met Asn Gly He Tyr 585 590 595
AAC GCT AAT GTC AGC ACT TCT AAC TTC CAA TTT TTG TTT GAT TTA GGC 2044 Asn Ala Asn Val Ser Thr Ser Asn Phe Gin Phe Leu Phe Asp Leu Gly 600 605 610 615
TTG AGA ATG AAC CTC GCT AGG CCT AAG AAA AAA GAC AGC GAT CAT GCC 2092 Leu Arg Met Asn Leu Ala Arg Pro Lys Lys Lys Asp Ser Asp His Ala 620 625 630
GCT CAG CAT GGC ATT GAA CTA GGT TTT AAG ATC CCC ACG ATC AAC ACC 2140 Ala Gin His Gly He Glu Leu Gly Phe Lys He Pro Thr He Asn Thr 635 640 645
AAC TAT TAT TCT TTC ATG GGC GCT AAA CTA GAA TAC AGA AGG ATG TAT 2188 Asn Tyr Tyr Ser Phe Met Gly Ala Lys Leu Glu Tyr Arg Arg Met Tyr 650 655 660
AGC CTT TTT CTC AAT TAT GTG TTT GCT TAC TAAAAACTCT CTTTAAAAAA GGG 2241 Ser Leu Phe Leu Asn Tyr Val Phe Ala Tyr 665 670
GTTTGTTTAA AAACGCTTAA AAGCATTTTT AAAATTAAGC AGTAAAGAGC CTAGATAATC 2301 TCTTGCAACC GCTCTCAAGC GATAAAATTA AAGTGAT 2338
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 691 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...18 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Met Lys Lys Ser Leu Leu Leu Ser Leu Ser Leu He Ala Ser Leu Ser
-18 -15 -10 -5
Arg Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gin He Gly 1 5 10
Glu Ala Val Gin Gin Val Lys Asn Thr Gly Ala Leu Gin Asn Leu Ala 15 20 25 30
Asp Arg Tyr Asp Asn Leu Asn Asn Leu Leu Asn Gin Tyr Asn Tyr Leu
35 40 45
Asn Ser Leu Val Asn Leu Ala Ser Thr Pro Ser Ala He Thr Gly Ala
50 55 60
He Asp Asn Leu Ser Ser Ser Ala He Asn Leu Thr Ser Ala Thr Thr
65 70 75
Thr Ser Pro Ala Tyr Gin Ala Val Ala Leu Ala Leu Asn Ala Ala Val
80 85 90
Gly Met Trp Gin Val He Ala Leu Phe He Gly Cys Gly Pro Gly Pro 95 100 105 110
Thr Asn Asn Gin Ser Tyr Gin Ser Phe Gly Asn Thr Pro Ala Leu Asn
115 120 125
Gly Thr Thr Thr Thr Cys Asn Gin Ala Tyr Gly Thr Gly Pro Asn Gly
130 135 140
He Leu Ser He Asp Glu Tyr Gin Lys Leu Asn Gin Ala Tyr Gin He
145 150 155
He Gin Thr Ala Leu Asn Gin Asn Gin Gly Gly Gly Met Pro Ala Leu
160 165 170
Asn Asp Thr Thr Lys Thr Gly Val Val Asn He Gin Gin Thr Asn Tyr 175 180 185 190
Arg Thr Thr Thr Gin Asn Asn He He Glu His Tyr Tyr Thr Glu Asn
195 200 205
Gly Lys Glu He Pro Val Ser Tyr Ser Gly Gly Ser Ser Phe Ser Pro
210 215 220
Thr He Gin Leu Thr Tyr His Asn Asn Ala Glu Asn Leu Leu Gin Gin
225 230 235
Ala Ala Thr He Met Gin Val Leu He Thr Gin Lys Pro His Val Gin
240 245 250
Thr Ser Asn Gly Gly Lys Ala Trp Gly Leu Ser Ser Thr Pro Gly Asn 255 260 265 270
Val Met Asp He Phe Gly Pro Ser Phe Asn Ala He Asn Glu Met He
275 280 285
Lys Asn Ala Gin Thr Ala Leu Ala Lys Thr Gin Gin Leu Asn Ala Asn
290 295 300
Glu Asn Ala Gin He Thr Gin Pro Asn Asn Phe Asn Pro Tyr Thr Ser
305 310 315
Lys Asp Lys Gly Phe Ala Gin Glu Met Leu Asn Arg Ala Glu Ala Gin
320 325 330
Ala Glu He Leu Asn Leu Ala Lys Gin Val Ala Asn Asn Phe His Ser 335 340 345 350
He Gin Gly Pro He Gin Gly Asp Leu Glu Glu Cys Lys Ala Gly Ser
355 360 365
Ala Gly Val He Thr Asn Asn Thr Trp Gly Ser Gly Cys Ala Phe Val
370 375 380
Lys Glu Thr Leu Asn Ser Leu Glu Gin His Thr Ala Tyr Tyr Gly Asn
385 390 395
Gin Val Asn Gin Asp Arg Ala Leu Ala Gin Thr He Leu Asn Phe Lys
400 405 410
Glu Ala Leu Asn Thr Leu Asn Lys Asp Ser Lys Ala He Asn Ser Gly 415 420 425 430
He Ser Asn Leu Pro Asn Ala Lys Ser Leu Gin Asn Met Thr His Ala
435 440 445
Thr Gin Asn Pro Asn Ser Pro Glu Gly Leu Leu Thr Tyr Ser Leu Asp 450 455 460
Ser Ser Lys Tyr Asn Gin Leu Gin Thr He Ala Gin Glu Leu Gly Lys
465 470 475
Asn Pro Phe Arg Arg Phe Gly Val He Asp Phe Gin Asn Asn Asn Gly
480 485 490
Ala Met Asn Gly He Gly Val Gin Val Gly Tyr Lys Gin Phe Phe Gly 495 500 505 510
Lys Lys Arg Asn Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn
515 520 525
His Ala Tyr He Lys Ser Asn Phe Phe Asn Ser Ala Ser Asp Val Trp
530 535 540
Thr Tyr Gly Val Gly Met Asp Ala Leu Tyr Asn Phe He Asn Asp Lys
545 550 555
Asn Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Val Gly Leu Phe
560 565 570
Gly Gly Phe Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gin Gin Val 575 580 585 590
Asn Leu Thr Met Met Asn Gly He Tyr Asn Ala Asn Val Ser Thr Ser
595 600 605
Asn Phe Gin Phe Leu Phe Asp Leu Gly Leu Arg Met Asn Leu Ala Arg
610 615 620
Pro Lys Lys Lys Asp Ser Asp His Ala Ala Gin His Gly He Glu Leu
625 630 635
Gly Phe Lys He Pro Thr He Asn Thr Asn Tyr Tyr Ser Phe Met Gly
640 645 650
Ala Lys Leu Glu Tyr Arg Arg Met Tyr Ser Leu Phe Leu Asn Tyr Val 655 660 665 670
Phe Ala Tyr
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: TCAAGGAGAA AACATGAAAA AAACCC 26
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: GAAGACGACG GCTTTTACAC AAGCGT 26 (2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: AAAGCTTAGT AAGCGAACAC ATAA 24
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: AAGGAGAAAA AACATGAAAA AACACATCC 29
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GAAGACGACG GCTTTTACAC AAGCG 25
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: AACATTAGTA AGCGAACACA TAGTTC . 26
(2) INFORMATION FOR SEQ ID NO: 29: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: AAGGAGAAAA AACATGAAAA AACACATCC 29
(2) INFORMATION FOR SEQ ID NO: 30:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY, linear
(ii) MOLECULE TYPE: Genomic DNA (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 30: GAAGACGACG GCTTTTACAC AAGCGT 26
(2) INFORMATION FOR SEQ ID NO: 31.
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY, linear
(n) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 31 : AAAAGCTTAG TAAGCGAACA CAT 23
(2) INFORMATION FOR SEQ ID NO-.32:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: AAGGAGAAAA CATGAAGAAA AAATTT 26
(2) INFORMATION FOR SEQ ID NO: 33: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: GAAGACAACG GCTTTTTTGT GAGTG 25
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: AGCTTTTAGT AAGCAAACAC ATAGT 25
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: AAGGATATTT ATGAAAAAAA CCCTT 25
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
GAAGACAACG GCTTTTTTAT CAGCG 25
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(11) MOLECULE TYPE: Genomic DNA ( l) SEQUENCE DESCRIPTION: SEQ ID NO: 37: GATATTAGTA AGCAAACACA TAATTC 26
(2) INFORMATION FOR SEQ ID NO: 38:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE. Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 38 : AAGGAGAAAA CATGAAAAAA TCCCTCT 27
(2) INFORMATION FOR SEQ ID NO: 39:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS. single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 39 : GAAGATGACG GATTTTATAC GAGTGT 26
(2) INFORMATION FOR SEQ ID NO: 40:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS. single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: TTTTAGTAAG CAAACACATA ATTGAG 26
(2) INFORMATION FOR SEQ ID NO: 41:
(l) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: AAGGAACATC TTATGAAAAA AACG 24
(2) INFORMATION FOR SEQ ID NO: 42:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE- Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: GAAGACAACG GCGTTTTTTT AAGCG 25
(2) INFORMATION FOR SEQ ID NO: 43:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE. Genomic DNA (Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 43: GGTTTTTAAT AGGCAAACAC ATAAT 25
(2) INFORMATION FOR SEQ ID NO: 44:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: AAGGAACATT TTATGAAAAA GACAAT 26
(2) INFORMATION FOR SEQ ID NO: 45:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45: GAAGACAACG GCTTTTTTGT GAGCG 25
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: TCACTCAGTA AGCGAACACA TAA 23
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: AAGGAACATT TTATGAAAAA GACAA 25
(2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48: GAAGACAACG GCTTTTTTGT GAGCG 25
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: TTTTAATAAG CGAACACATA AAAGAG 26
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50: AAGGAACATT TTATGAAAAA AACGAT 26
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: GAAAATGACG GCGTTTTTAT GAGCG 25
(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52: ATATCAATAG GCCAAAACAT AATTGA 26
(2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: AAGGAGAAAA CATGAAAAAA TCCCTC 26
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: GAAGATGACG GATTTTATAC GAGTGT 26
(2) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55: TTTTAGTAAG CAAACACATA ATTGAG 26
(2) INFORMATION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56: CGCGGATCCG AATCCAATTT AATCCAAAAA GG 32
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57: CCGCTCGAGT TAAGTAAGCG AACACATATT CAA 33
(2) INFORMATION FOR SEQ ID NO: 58
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58
Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gin He Gly Glu Ala
1 5 10 15
Ala Gin Met Val 20
(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: CTGAATTCGA TTTCAAGGAG AAAACATGAA A 31
(2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60: CCGCTCGAGT TAGTAAGCGA ACACATAATT 30
(2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (11) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: CGCGGATCCG AATCCAATTT AATCCAAAAA GG 32
(2) INFORMATION FOR SEQ ID NO: 62:
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY, linear
(n) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62: CCGCTCGAGT TAGTAAGCGA ACACATAGTT CAA 33
(2) INFORMATION FOR SEQ ID NO:63:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(il) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: CGCGGATCCG AAGTTTCTTT GTATCAAAG 29
(2) INFORMATION FOR SEQ ID NO : 64 :
(l) SEQUENCE CHARACTERISTICS-
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: CCGCTCGAGT TAGTAAGCAA ACACATAATT GTG 33
(2) INFORMATION FOR SEQ ID NO: 65:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1149 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS. single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 106...1002 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 106...166 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
TTACTCTTTA ATGTGAGTTT TCTGTGTCAT GATAGCTGAT TTTGTTTTAA ATTTGCTATA 60 ATGTGAATTT AATGATGAAA ATTAGTTTAG AGTGGAGAAC ACACA ATG AAA AAA AAT 117
Met Lys Lys Asn -20 ATC TTA AAT TTA GCG TTA GTG GGT GCG TTG AGC ACG TCG TTT TTG ATG 165 He Leu Asn Leu Ala Leu Val Gly Ala Leu Ser Thr Ser Phe Leu Met
-15 -10 -5
GCT AAG CCG GCT CAT AAC GCA AAT AAC GCT ACG CAT AAC ACG AAA AAA 213 Ala Lys Pro Ala His Asn Ala Asn Asn Ala Thr His Asn Thr Lys Lys
1 5 10 15
ACG ACT GAT TCT TCA GCA GGC GTG TTA GCG ACA GTG GAT GGC AGA CCT 261 Thr Thr Asp Ser Ser Ala Gly Val Leu Ala Thr Val Asp Gly Arg Pro
20 25 30
ATC ACT AAA AGC GAT TTT GAC ATG ATT AAG CAA CGA AAT CCT AAT TTT 309 He Thr Lys Ser Asp Phe Asp Met He Lys Gin Arg Asn Pro Asn Phe
35 40 45
GAT TTT GAC AAG CTT AAA GAG AAA GAA AAA GAA GCC TTG ATT GAT CAA 357 Asp Phe Asp Lys Leu Lys Glu Lys Glu Lys Glu Ala Leu He Asp Gin
50 55 60
GCT ATT CGC ACC GCC CTT GTA GAA AAT GAA GCT AAA ACC GAG AAA TTG 405 Ala He Arg Thr Ala Leu Val Glu Asn Glu Ala Lys Thr Glu Lys Leu 65 70 75 80
GAC AGC ACT CCA GAA TTT AAA GCG ATG ATG GAA GCG GTT AAA AAA CAG 453 Asp Ser Thr Pro Glu Phe Lys Ala Met Met Glu Ala Val Lys Lys Gin
85 90 95
GCT TTA GTG GAA TTT TGG GCT AAA AAA CAG GCT GAA GAA GTG AAA AAA 501 Ala Leu Val Glu Phe Trp Ala Lys Lys Gin Ala Glu Glu Val Lys Lys
100 105 110
GTC CAA ATC CCA GAA AAA GAA ATG CAA GAT TTT TAC AAC GCT AAC AAA 549 Val Gin He Pro Glu Lys Glu Met Gin Asp Phe Tyr Asn Ala Asn Lys
115 120 125
GAT CAG CTT TTT GTC AAG CAA GAA GCC CAT GCT AGG CAT ATT TTA GTG 597 Asp Gin Leu Phe Val Lys Gin Glu Ala His Ala Arg His He Leu Val
130 135 140
AAA ACC GAA GAT GAG GCT AAA CGG ATT ATT TCT GAG ATT GAC AAA CAG 645 Lys Thr Glu Asp Glu Ala Lys Arg He He Ser Glu He Asp Lys Gin 145 150 155 160
CCA AAG GCT AAA AAA GAA GCT AAA TTC ATT GAG TTA GCC AAT CGG GAT 693 Pro Lys Ala Lys Lys Glu Ala Lys Phe He Glu Leu Ala Asn Arg Asp 165 170 175 ACG ATT GAT CCT AAC AGC AAG AAC GCG CAA AAT GGC GGT GAT TTG GGG 741
Thr He Asp Pro Asn Ser Lys Asn Ala Gin Asn Gly Gly Asp Leu Gly
180 185 190
AAA TTC CAA AAG AAC CAA ATG GCT CCG GAT TTT TCT AAA GCC GCT TTC 789
Lys Phe Gin Lys Asn Gin Met Ala Pro Asp Phe Ser Lys Ala Ala Phe
195 200 205
GCT TTA ACT CCT GGG GAT TAC ACT AAA ACC CCT GTT AAA ACA GAG TTT 837
Ala Leu Thr Pro Gly Asp Tyr Thr Lys Thr Pro Val Lys Thr Glu Phe
210 215 220
GGT TAT CAT ATT ATC TAT TTG ATT TCT AAA GAT AGC CCT GTA ACT TAT 885
Gly Tyr His He He Tyr Leu He Ser Lys Asp Ser Pro Val Thr Tyr
225 230 235 240
ACT TAT GAA CAG GCT AAA CCT ACC ATT AAG GGG ATG TTA CAA GAA AAG 933
Thr Tyr Glu Gin Ala Lys Pro Thr He Lys Gly Met Leu Gin Glu Lys
245 250 255
CTT TTC CAA GAA CGC ATG AAT CAA CGC ATT GAG GAA CTA AGA AAG CAC 981
Leu Phe Gin Glu Arg Met Asn Gin Arg He Glu Glu Leu Arg Lys His
260 265 270
GCT AAA ATT GTT ATC AAC AAG TAATTGATGA GGTGTTATCA TGTTAGTTAA AGGC 1036
Ala Lys He Val He Asn Lys
275 AATGAAATTT TATTGAAAGC CCATAAAGAA GGTTATGGGG TGGGGGCGTT TAATTTCGTG 1096 AATTTTGAAA TGCTAAACGC TATTTTTGAA GCAGGAAATG AGGAAAATTC CCC 1149
(2) INFORMATION FOR SEQ ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 299 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Signal Sequence
(B) LOCATION: 1...20 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
Met Lys Lys Asn He Leu Asn Leu Ala Leu Val Gly Ala Leu Ser Thr
-20 -15 -10 -5
Ser Phe Leu Met Ala Lys Pro Ala His Asn Ala Asn Asn Ala Thr His
1 5 10
Asn Thr Lys Lys Thr Thr Asp Ser Ser Ala Gly Val Leu Ala Thr Val
15 20 25
Asp Gly Arg Pro He Thr Lys Ser Asp Phe Asp Met He Lys Gin Arg
30 35 40
Asn Pro Asn Phe Asp Phe Asp Lys Leu Lys Glu Lys Glu Lys Glu Ala
45 50 55 60
Leu He Asp Gin Ala He Arg Thr Ala Leu Val Glu Asn Glu Ala Lys
65 70 75
Thr Glu Lys Leu Asp Ser Thr Pro Glu Phe Lys Ala Met Met Glu Ala 80 85 90
Val Lys Lys Gin Ala Leu Val Glu Phe Trp Ala Lys Lys Gin Ala Glu
95 100 105
Glu Val Lys Lys Val Gin He Pro Glu Lys Glu Met Gin Asp Phe Tyr
110 115 120
Asn Ala Asn Lys Asp Gin Leu Phe Val Lys Gin Glu Ala His Ala Arg 125 130 135 140
His He Leu Val Lys Thr Glu Asp Glu Ala Lys Arg He He Ser Glu
145 150 155
He Asp Lys Gin Pro Lys Ala Lys Lys Glu Ala Lys Phe He Glu Leu
160 165 170
Ala Asn Arg Asp Thr He Asp Pro Asn Ser Lys Asn Ala Gin Asn Gly
175 180 185
Gly Asp Leu Gly Lys Phe Gin Lys Asn Gin Met Ala Pro Asp Phe Ser
190 195 200
Lys Ala Ala Phe Ala Leu Thr Pro Gly Asp Tyr Thr Lys Thr Pro Val 205 210 215 220
Lys Thr Glu Phe Gly Tyr His He He Tyr Leu He Ser Lys Asp Ser
225 230 235
Pro Val Thr Tyr Thr Tyr Glu Gin Ala Lys Pro Thr He Lys Gly Met
240 245 250
Leu Gin Glu Lys Leu Phe Gin Glu Arg Met Asn Gin Arg He Glu Glu
255 260 265
Leu Arg Lys His Ala Lys He Val He Asn Lys 270 275
(2) INFORMATION FOR SEQ ID NO: 67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1448 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 118...1314 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:
CTCTTGAATG GCGATAAGAC AAAAATGTCT TAAATTTTGT GGTAGCATTT AGGAATACTT 60 AGGATTTTGT TTAGTATAAT TCTAAAATCC ATTTCAAAAA ATTAAGGAGA AATACAA ATG 120
Met _1 GCA AAA GAA AAG TTT AAC AGA ACT AAG CCG CAT GTT AAT ATT GGA ACC 168 Ala Lys Glu Lys Phe Asn Arg Thr Lys Pro His Val Asn He Gly Thr
5 10 15
ATT GGG CAT GTA GAC CAT GGT AAA ACG ACT TTG AGT GCA GCG ATT TCA 216 He Gly His Val Asp His Gly Lys Thr Thr Leu Ser Ala Ala He Ser
20 25 30
GCG GTG CTT TCT TTG AAA GGT CTT GCA GAA ATG AAA GAC TAT GAT AAT 264 Ala Val Leu Ser Leu Lys Gly Leu Ala Glu Met Lys Asp Tyr Asp Asn 35 40 45
ATT GAT AAC GCC CCT GAA GAA AAA GAA AGA GGG ATC ACT ATC GCT ACT 312
He Asp Asn Ala Pro Glu Glu Lys Glu Arg Gly He Thr He Ala Thr
50 55 60 65
TCT CAC ATT GAA TAT GAG ACT GAA AAC AGA CAC TAT GCG CAT GTG GAT 360
Ser His He Glu Tyr Glu Thr Glu Asn Arg His Tyr Ala His Val Asp
70 75 80
TGC CCA GGA CAC GCT GAC TAT GTA AAA AAC ATG ATC ACC GGT GCG GCG 408
Cys Pro Gly His Ala Asp Tyr Val Lys Asn Met He Thr Gly Ala Ala
85 90 95
CAA ATG GAC GGA GCG ATT TTG GTT GTT TCT GCA GCT GAT GGC CCT ATG 456
Gin Met Asp Gly Ala He Leu Val Val Ser Ala Ala Asp Gly Pro Met
100 105 110
CCT CAA ACT AGG GAG CAT ATC TTA TTG TCT CGT CAA GTA GGC GTG CCT 504
Pro Gin Thr Arg Glu His He Leu Leu Ser Arg Gin Val Gly Val Pro
115 120 125
CAC ATC GTT GTT TTC TTA AAC AAA CAA GAC ATG GTA GAT GAC CAA GAA 552
His He Val Val Phe Leu Asn Lys Gin Asp Met Val Asp Asp Gin Glu
130 135 140 145
TTG TTA GAA CTT GTA GAA ATG GAA GTG CGC GAA TTG TTG AGC GCG TAT 600
Leu Leu Glu Leu Val Glu Met Glu Val Arg Glu Leu Leu Ser Ala Tyr
150 155 160
GAA TTT CCT GGC GAT GAC ACT CCT ATC GTA GCG GGT TCA GCT TTA AGA 648
Glu Phe Pro Gly Asp Asp Thr Pro He Val Ala Gly Ser Ala Leu Arg
165 170 175
GCT TTA GAA GAA GCA AAG GCT GGT AAT GTG GGT GAA TGG GGT GAA AAA 696
Ala Leu Glu Glu Ala Lys Ala Gly Asn Val Gly Glu Trp Gly Glu Lys
180 185 190
GTG CTT AAA CTT ATG GCT GAA GTG GAT GCC TAT ATC CCT ACT CCA GAA 744
Val Leu Lys Leu Met Ala Glu Val Asp Ala Tyr He Pro Thr Pro Glu
195 200 205
AGA GAC ACT GAA AAA ACT TTC TTG ATG CCG GTT GAA GAT GTG TTC TCT 792
Arg Asp Thr Glu Lys Thr Phe Leu Met Pro Val Glu Asp Val Phe Ser
210 215 220 225
ATT GCG GGT AGA GGG ACT GTG GTT ACA GGT AGG ATT GAA AGA GGC GTG 840
He Ala Gly Arg Gly Thr Val Val Thr Gly Arg He Glu Arg Gly Val
230 235 240
GTG AAA GTA GGC GAT GAA GTG GAA ATC GTT GGT ATC AGA CCT ACA CAA 888
Val Lys Val Gly Asp Glu Val Glu He Val Gly He Arg Pro Thr Gin
245 250 255
AAA ACG ACT GTA ACC GGT GTA GAA ATG TTT AGG AAA GAG TTG GAA AAA 936
Lys Thr Thr Val Thr Gly Val Glu Met Phe Arg Lys Glu Leu Glu Lys
260 265 270
GGT GAA GCC GGC GAT AAT GTG GGC GTG CTT TTG AGA GGA ACT AAA AAA 984
Gly Glu Ala Gly Asp Asn Val Gly Val Leu Leu Arg Gly Thr Lys Lys
275 280 285
GAA GAA GTG GAA CGC GGT ATG GTT CTA TGC AAA CCA GGT TCT ATC ACT 1032
Glu Glu Val Glu Arg Gly Met Val Leu Cys Lys Pro Gly Ser He Thr
290 295 300 305
CCG CAC AAG AAA TTT GAG GGA GAA ATT TAT GTC CTT TCT AAA GAA GAA 1080
Pro His Lys Lys Phe Glu Gly Glu He Tyr Val Leu Ser Lys Glu Glu
310 315 320
GGC GGG AGA CAC ACT CCA TTC TTC ACC AAT TAC CGC CCG CAA TTC TAT 1128
Gly Gly Arg His Thr Pro Phe Phe Thr Asn Tyr Arg Pro Gin Phe Tyr
325 330 335
GTG CGC ACA ACT GAT GTG ACT GGC TCT ATC ACC CTT CCT GAA GGC GTA 1176
Val Arg Thr Thr Asp Val Thr Gly Ser He Thr Leu Pro Glu Gly Val 340 345 350
GAA ATG GTT ATG CCT GGC GAT AAT GTG AAA ATC ACT GTA GAG TTG ATT 1224 Glu Met Val Met Pro Gly Asp Asn Val Lys He Thr Val Glu Leu He
355 360 365
AGC CCT GTT GCG TTA GAG TTG GGA ACT AAA TTT GCG ATT CGT GAA GGC 1272 Ser Pro Val Ala Leu Glu Leu Gly Thr Lys Phe Ala He Arg Glu Gly 370 375 380 385
GGT AGG ACC GTT GGT GCT GGT GTT GTG AGC AAT ATT ATT GAA TAATATTAG 1323 Gly Arg Thr Val Gly Ala Gly Val Val Ser Asn He He Glu
390 395
CAAAAAGAGA GTTACCATAA AGGGTCATTA TGAAAGTTAA AATAGGGTTG AAGTGTTCTG 1383 ATTGTGAAGA TATCAATTAC AGCACAACCA AGAACGCTAA AACTAACACT GAAAAACTGG 1443 AGCTT 1448
(2) INFORMATION FOR SEQ ID NO: 68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 399 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:
Met Ala Lys Glu Lys Phe Asn Arg Thr Lys Pro His Val Asn He Gly
1 5 10 15
Thr He Gly His Val Asp His Gly Lys Thr Thr Leu Ser Ala Ala He
20 25 30
Ser Ala Val Leu Ser Leu Lys Gly Leu Ala Glu Met Lys Asp Tyr Asp
35 40 45
Asn He Asp Asn Ala Pro Glu Glu Lys Glu Arg Gly He Thr He Ala
50 55 60
Thr Ser His He Glu Tyr Glu Thr Glu Asn Arg His Tyr Ala His Val 65 70 75 80
Asp Cys Pro Gly His Ala Asp Tyr Val Lys Asn Met He Thr Gly Ala
85 90 95
Ala Gin Met Asp Gly Ala He Leu Val Val Ser Ala Ala Asp Gly Pro
100 105 110
Met Pro Gin Thr Arg Glu His He Leu Leu Ser Arg Gin Val Gly Val
115 120 125
Pro His He Val Val Phe Leu Asn Lys Gin Asp Met Val Asp Asp Gin
130 135 140
Glu Leu Leu Glu Leu Val Glu Met Glu Val Arg Glu Leu Leu Ser Ala 145 150 155 160
Tyr Glu Phe Pro Gly Asp Asp Thr Pro He Val Ala Gly Ser Ala Leu
165 170 175
Arg Ala Leu Glu Glu Ala Lys Ala Gly Asn Val Gly Glu Trp Gly Glu
180 185 190
Lys Val Leu Lys Leu Met Ala Glu Val Asp Ala Tyr He Pro Thr Pro
195 200 205
Glu Arg Asp Thr Glu Lys Thr Phe Leu Met Pro Val Glu Asp Val Phe
210 215 220
Ser He Ala Gly Arg Gly Thr Val Val Thr Gly Arg He Glu Arg Gly 225 230 235 240 Val Val Lys Val Gly Asp Glu Val Glu He Val Gly He Arg Pro Thr
245 250 255
Gin Lys Thr Thr Val Thr Gly Val Glu Met Phe Arg Lys Glu Leu Glu
260 265 270
Lys Gly Glu Ala Gly Asp Asn Val Gly Val Leu Leu Arg Gly Thr Lys
275 280 285
Lys Glu Glu Val Glu Arg Gly Met Val Leu Cys Lys Pro Gly Ser He
290 295 300
Thr Pro His Lys Lys Phe Glu Gly Glu He Tyr Val Leu Ser Lys Glu 305 310 315 320
Glu Gly Gly Arg His Thr Pro Phe Phe Thr Asn Tyr Arg Pro Gin Phe
325 330 335
Tyr Val Arg Thr Thr Asp Val Thr Gly Ser He Thr Leu Pro Glu Gly
340 345 350
Val Glu Met Val Met Pro Gly Asp Asn Val Lys He Thr Val Glu Leu
355 360 365
He Ser Pro Val Ala Leu Glu Leu Gly Thr Lys Phe Ala He Arg Glu
370 375 380
Gly Gly Arg Thr Val Gly Ala Gly Val Val Ser Asn He He Glu 385 390 395
(2) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69: CGCGGATCCG AATGAAAAAA AATATCTTAA AT 32
(2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70: CCGCTCGAGT TACTTGTTGA TAACAATTTT 30
(2) INFORMATION FOR SEQ ID NO: 71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: CGCGGATCCG AATGGCAAAA GAAAAGTTTA AC 32
(2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72: CCGCTCGAGT TATTCAATAA TATTGCTCAC 30
(2) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
Met Lys Glu Lys Phe Asn Arg Thr Lys Pro His Val Asn He Gly Thr
1 5 10 15
He Gly His Val Asp His 20
(2) INFORMATION FOR SEQ ID NO: 74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
Ala His Asn Ala Asn Asn Ala Thr His Asn Thr Lys Lys 1 5 10
(2) INFORMATION FOR SEQ ID NO: 75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
Lys Pro Ala His Asn Ala 1 5
(2) INFORMATION FOR SEQ ID NO: 76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
He Asp Lys Gin Pro Lys Ala Lys Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:
Phe Trp Ala Lys Lys Gin Ala Glu 1 5
(2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78: GTGGAGAACA CACAATGAAA AAAAATATC 29
(2) INFORMATION FOR SEQ ID NO: 79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79: GCTAATATTA TTCAATAATA TTGCTCACAA C 31
(2) INFORMATION FOR SEQ ID NO: 80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80: GGAGAAATAC AAATGGCAAA AGAAAAG 27
(2) INFORMATION FOR SEQ ID NO: 81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81: GCTAATATTA TTCAATAATA TTGCTCACAA C 31
(2) INFORMATION FOR SEQ ID NO: 82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82: CATAACGCAA ATAACGCTAC GCAT 24
(2) INFORMATION FOR SEQ ID NO: 83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: GGGAATTCAA AAAAACGAAA AAAACG 26
(2) INFORMATION FOR SEQ ID NO: 84: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 84 : CCCCTCGAGT TAATAGGCAA ACAC 24

Claims

What is claimed is:
1. An isolated polynucleotide that encodes:
(i) a polypeptide comprising an amino acid sequence that is homologous to the amino acid sequence of a Helicobacter membrane-associated polypeptide, wherein said amino acid sequence of said Helicobacter membrane-associated polypeptide is selected from the group consisting of the amino acid sequences as shown:
-in SEQ ID NO:2, beginning with an amino acid in any one of positions -19 to 5, preferably in position -19 or position 1, and ending with an amino acid in position 689 (GHPO 386);
-in SEQ ID NO:4, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 713 (GHPO 789);
-in SEQ ID NO:6, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 725 (GHPO 1516);
-in SEQ ID NO: 8, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 691 (GHPO 1197); -in SEQ ID NO: 10, beginning with an amino acid in any one of positions
-20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 652 (GHPO 1180);
-in SEQ ID NO: 12, beginning with an amino acid in any one of positions -18 to 5, preferably in position -18 or position 1, and ending with an amino acid in position 673 (GHPO 896);
-in SEQ ID NO: 14, beginning with an amino acid in any one of positions -21 to 5, preferably in position -21 or position 1, and ending with an amino acid in position 619 (GHPO 711);
-in SEQ ID NO: 16, beginning with an amino acid in any one of positions -17 to 5, preferably in position -17 or position 1, and ending with an amino acid in position 635 (GHPO 190);
-in SEQ ID NO: 18, beginning with an amino acid in any one of positions -19 to 5, preferably in position -19 or position 1, and ending with an amino acid in position 626 (GHPO 185);
-in SEQ ID NO:20, beginning with an amino acid in any one of positions - 16 to 5, preferably in position - 16 or position 1 , and ending with an amino acid in position 467 (GHPO 1417);
-in SEQ ID NO:22, beginning with an amino acid in any one of positions -18 to 5, preferably in position -18 or position 1, and ending with an amino acid in position 673 (GHPO 1414); - in SEQ ID NO: 66, beginning with an amino acid in any one of the positions from -20 to 5, preferably in position -20 or position 1 , and ending with an amino acid in position 279 (GHPO 1360); and
- in SEQ ID NO:68, beginning with an amino acid in position 1 and ending with an amino acid in position 399 (GHPO 750); or (ii) a derivative of the polypeptide.
2. An isolated polynucleotide that encodes:
(i) a polypeptide comprising an amino acid sequence that is homologous to an amino acid sequence selected from the group consisting of the amino acid sequences as shown: -in SEQ ID NO:2, beginning with amino acid in position -19 and ending with an amino acid in position 689 (GHPO 386); -in SEQ ID NO:4, beginning with an amino acid in position -20 and ending with an amino acid in position 713 (GHPO 789);
-in SEQ ID NO: 6, beginning with an amino acid in position -20 and ending with an amino acid in position 725 (GHPO 1516); -in SEQ ID NO: 8, beginning with an amino acid in position -20 and ending with an amino acid in position 691 (GHPO 1197);
-in SEQ ID NO: 10, beginning with an amino acid in position -20 and ending with an amino acid in position 652 (GHPO 1180);
-in SEQ ID NO: 12, beginning with an amino acid in position -18 and ending with an amino acid in position 673 (GHPO 896);
-in SEQ ID NO: 14, beginning with an amino acid in position -21 and ending with an amino acid in position 619 (GHPO 711);
-in SEQ ID NO: 16, beginning with an amino acid in position -17 and ending with an amino acid in position 635 (GHPO 190); -in SEQ ID NO: 18, beginning with an amino acid in position -19 and ending with an amino acid in position 626 (GHPO 185);
-in SEQ ID NO:20, beginning with an amino acid in position -16 and ending with an amino acid in position 467 (GHPO 1417);
-in SEQ ID NO:22, beginning with an amino acid in position -18 and ending with an amino acid in position 673 (GHPO 1414);
- in SEQ ID NO:66, beginning with an amino acid in position -20 and ending with an amino acid in position 279 (GHPO 1360); and
- in SEQ ID NO:68, beginning with an amino acid in position 1 and ending with an amino acid in position 399 (GHPO 750); or (ii) a derivative of the polypeptide.
3. The isolated polynucleotide of claim 1, which encodes the mature form of:
(i) a polypeptide comprising an amino acid sequence that is homologous to an amino acid sequence selected from the group consisting of the amino acid sequences as shown:
-in SEQ ID NO:2, beginning with an amino acid in any one of positions -19 to 5, preferably in position -19 or position 1, and ending with an amino acid in position 689 (GHPO 386);
-in SEQ ID NO:4, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 713 (GHPO 789);
-in SEQ ID NO:6, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 725 (GHPO 1516); -in SEQ ID NO: 8, beginning with an amino acid in any one of positions
-20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 691 (GHPO 1197);
-in SEQ ID NO: 10, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 652 (GHPO 1180);
-in SEQ ID NO: 12, beginning with an amino acid in any one of positions -18 to 5, preferably in position -18 or position 1, and ending with an amino acid in position 673 (GHPO 896);
-in SEQ ID NO: 14, beginning with an amino acid in any one of positions -21 to 5, preferably in position -21 or position 1, and ending with an amino acid in position 619 (GHPO 711);
-in SEQ ID NO: 16, beginning with an amino acid in any one of positions -17 to 5, preferably in position -17 or position 1, and ending with an amino acid in position 635 (GHPO 190);
-in SEQ ID NO: 18, beginning with an amino acid in any one of positions -19 to 5, preferably in position -19 or position 1, and ending with an amino acid in position 626 (GHPO 185);
-in SEQ ID NO:20, beginning with an amino acid in any one of positions -16 to 5, preferably in position -16 or position 1, and ending with an amino acid in position 467 (GHPO 1417);
-in SEQ ID NO:22, beginning with an amino acid in any one of positions -18 to 5, preferably in position -18 or position 1, and ending with an amino acid in position 673 (GHPO 1414);
- in SEQ ID NO: 66, beginning with an amino acid in any one of positions
-20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 279 (GHPO 1360); and
- in SEQ ID NO:68, beginning with an amino acid in position 1 and ending with an amino acid in position 399 (GHPO 750); or
(ii) a derivative of the polypeptide.
4. The isolated polynucleotide of claim 1, 2, or 3, wherein the polynucleotide is a DNA molecule.
5. The isolated polynucleotide of claim 1, which is a DNA molecule that can be amplified and/or cloned by polymerase chain reaction from an Helicobacter genome, using either: - a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:23, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:25 (unprocessed GHPO 386);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:26, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:28 (unprocessed GHPO 789);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:29, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:31 (unprocessed GHPO 1516); - a 5' oligonucleotide primer having a sequence as shown in SEQ ID
NO:32, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:34 (unprocessed GHPO 1197);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:35, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:37 (unprocessed GHPO 1180);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:38, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:40 (unprocessed GHPO 896);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:41 , and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:43 (unprocessed GHPO 711);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:44, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:46 (unprocessed GHPO 190); - a 5' oligonucleotide primer having a sequence as shown in SEQ ID
NO:47, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:49 (unprocessed GHPO 185); - a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:50, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:52 (unprocessed GHPO 1417);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:53, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:55 (unprocessed GHPO 1414);
- a 5' oligonucleotide primer comprising a sequence as shown in SEQ ID NO:78 and a 3' oligonucleotide primer comprising a sequence as shown in SEQ ID NO:79 (unprocessed GHPO 1360); - a 5' oligonucleotide primer having a sequence as shown in SEQ ID
NO:24, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:25 (mature GHPO 386);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:27, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:28 (mature GHPO 789);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO: 30, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:31 (mature GHPO 1516);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:33, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:34 (mature GHPO 1197);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:36, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:37 (mature GHPO 1180); - a 5' oligonucleotide primer having a sequence as shown in SEQ ID
NO: 39, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:40 (mature GHPO 896); - a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:42, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:43 (mature GHPO 711);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:45, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:46 (mature GHPO 190);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:48, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:49 (mature GHPO 185); - a 5' oligonucleotide primer having a sequence as shown in SEQ ID
NO:51 , and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:52 (mature GHPO 1417);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO: 54, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:55 (mature GHPO 1414);
- a 5' oligonucleotide primer comprising a sequence as shown in SEQ ID NO: 80 and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO: 81 (GHPO 750); or
- a 5' oligonucleotide primer comprising a sequence as shown in SEQ ID NO: 82 and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
NO:79 (mature GHPO 1360).
6. The isolated DNA molecule of claim 5, which can be amplified and/or cloned by the polymerase chain reaction from a Helicobacter pylori genome.
7. The isolated polynucleotide of claim 1 , which is a DNA molecule that encodes the mature form or a derivative of a polypeptide encoded by the DNA molecule of claim 5.
8. The isolated polynucleotide of claim 1, which is a DNA molecule that encodes the mature form or a derivative of a polypeptide encoded by the
DNA molecule of claim 6.
9. A compound, in a substantially purified form, that is the mature form or a derivative of a polypeptide comprising an amino acid sequence that is homologous to an amino acid sequence of a polypeptide associated with the Helicobacter membrane, which is selected from the group consisting of the amino acid sequences as shown:
-in SEQ ID NO:2, beginning with amino acid in position -19 and ending with an amino acid in position 689 (GHPO 386);
-in SEQ ID NO:4, beginning with an amino acid in position -20 and ending with an amino acid in position 713 (GHPO 789);
-in SEQ ID NO: 6, beginning with an amino acid in position -20 and ending with an amino acid in position 725 (GHPO 1516);
-in SEQ ID NO: 8, beginning with an amino acid in position -20 and ending with an amino acid in position 691 (GHPO 1197); -in SEQ ID NO: 10, beginning with an amino acid in position -20 and ending with an amino acid in position 652 (GHPO 1180);
-in SEQ ID NO: 12, beginning with an amino acid in position -1 and ending with an amino acid in position 673 (GHPO 896);
-in SEQ ID NO: 14, beginning with an amino acid in position -21 and ending with an amino acid in position 619 (GHPO 711); -in SEQ ID NO: 16, beginning with an amino acid in position -17 and ending with an amino acid in position 635 (GHPO 190);
-in SEQ ID NO: 18, beginning with an amino acid in position -19 and ending with an amino acid in position 626 (GHPO 185); -in SEQ ID NO:20, beginning with an amino acid in position -16 and ending with an amino acid in position 467 (GHPO 1417);
-in SEQ ID NO:22, beginning with an amino acid in position -18 and ending with an amino acid in position 673 (GHPO 1414);
- in SEQ ID NO: 66, beginning with an amino acid in position -20 and ending with an amino acid in position 279 (GHPO 1360); and
- in SEQ ID NO:68, beginning with an amino acid in position 1 and ending with an amino acid in position 399 (GHPO 750); or
(ii) a derivative of said polypeptide.
10. The compound of claim 9, which is the mature form or a derivative of a polypeptide encoded by a DNA molecule of claim 5.
11. The compound of claim 9, which is the mature form or a derivative of a polypeptide encoded by a DNA molecule of claim 6.
12. A pharmaceutical composition for preventing or treating Helicobacter infection in a mammal, said composition comprising a prophylactically or therapeutically effective amount of a compound of claim 9, 10, or 11 and a pharmaceutically acceptable diluent or carrier.
13. The composition of claim 12, further comprising an antibiotic, an antisecretory agent, a bismuth salt, or a combination thereof.
14. The composition of claim 13, wherein said antibiotic is selected from the group consisting of amoxicillin, clarithromycin, tetracycline, metronidizole, and erythromycin.
15. The composition of claim 13, wherein said bismuth salt is selected from the group consisting of bismuth subcitrate and bismuth subsalicylate.
16. The composition of claim 13, wherein said antisecretory agent is a proton pump inhibitor.
17. The composition of claim 16, wherein said proton pump inhibitor is selected from the group consisting of omeprazole, lansoprazole, and pantoprazole.
18. The composition of claim 13, wherein said antisecretory agent is an H2-receptor antagonist.
19. The composition of claim 18, wherein said H2-receptor antagonist is selected from the group consisting of ranitidine, cimetidine, famotidine, nizatidine, and roxatidine.
20. The composition of claim 13, wherein said antisecretory agent is a prostaglandin analog.
21. The composition of claim 20, wherein said prostaglandin analog is misoprostil or enprostil.
22. The composition of claim 12, which further comprises a prophylactically or therapeutically effective amount of a second Helicobacter polypeptide or a derivative thereof.
23. The composition of claim 22, wherein the second Helicobacter polypeptide is a Helicobacter urease, a subunit, or a derivative thereof.
24. The composition of claim 12, further comprising an adjuvant.
25. A pharmaceutical composition for preventing or treating Helicobacter infection in a mammal, said composition comprising a prophylactically or therapeutically effective amount of a polynucleotide of claim 1, 2, or 3 and a pharmaceutically acceptable canier or diluent.
26. A pharmaceutical composition for preventing or treating Helicobacter infection in a mammal, said composition comprising a prophylactically or therapeutically effective amount of a polynucleotide of claim 5, 6, or 7 and a pharmaceutically acceptable carrier or diluent.
27. A pharmaceutical composition for preventing or treating
Helicobacter infection in a mammal, said composition comprising a prophylactically or therapeutically effective amount of a polynucleotide of claim 8 and a pharmaceutically acceptable carrier or diluent.
28. A composition comprising a viral vector, in the genome of which is inserted a DNA molecule of claim 4, said DNA molecule being placed under conditions for expression in a mammalian cell and said viral vector being admixed with a physiologically acceptable diluent or carrier.
29. The composition of claim 28, wherein said viral vector is a poxvirus.
30. A composition that comprises a bacterial vector comprising a DNA molecule of claim 4, said DNA molecule being placed under conditions for expression and said bacterial vector being admixed with a physiologically acceptable diluent or canier.
31. The composition of claim 30, wherein said vector is selected from the group consisting of Shigella, Salmonella, Vibrio cholerae, Lactobacillus,
Bacille bilie de Calmette-Guerin, and Streptococcus.
32. The composition of claim 25, wherein said polynucleotide is a DNA molecule that is inserted in a plasmid that is unable to replicate and to substantially integrate in a mammalian genome and is placed under conditions for expression in a mammalian cell.
33. An expression cassette comprising a DNA molecule of claim 4, said DNA molecule being placed under conditions for expression in a procaryotic or eucaryotic cell.
34. A process for producing a compound of claim 9, which comprises culturing a procaryotic or eucaryotic cell transformed or transfected with an expression cassette of claim 33, and recovering said compound from the cell culture.
35. A pharmaceutical composition for preventing or treating Helicobacter infection in a mammal, said composition comprising a prophylactically or therapeutically effective amount of an antibody that binds to the compound of claim 9, 10, or 11 and a pharmaceutically acceptable canier or diluent.
PCT/US1998/006421 1997-04-01 1998-03-31 76 kDa, 32 kDa, AND 50 kDa HELICOBACTER POLYPEPTIDES AND CORRESPONDING POLYNUCLEOTIDE MOLECULES WO1998043479A1 (en)

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JP54196298A JP2001523954A (en) 1997-04-01 1998-03-31 76 kDa, 32 kDa, and 50 kDa Helicobacter polypeptides and corresponding polynucleotide molecules
EP98914395A EP0980204A4 (en) 1997-04-01 1998-03-31 76 kDa, 32 kDa, AND 50 kDa HELICOBACTER POLYPEPTIDES AND CORRESPONDING POLYNUCLEOTIDE MOLECULES

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US08/831,310 US20020026035A1 (en) 1997-04-01 1997-04-01 Helicobacter ghpo 1360 and ghpo 750 polypeptides and corresponding polynucleotide molecules
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WO2000000614A3 (en) * 1998-06-26 2000-05-04 American Cyanamid Co NOVEL ANTIGENS OF $i(HELICOBACTER PYLORI)
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WO2003059380A2 (en) * 2002-01-15 2003-07-24 Bio Life Science Forschungs Und Entwicklungsges M.B.H. Oral vaccination
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