JP2001517091A - Antigenic compositions and methods for detecting HELICOBACTER PYLORI - Google Patents

Abstract

  (57) [Summary] The present invention relates to a newly discovered H.264. For characterization and isolation of polypeptide antigens of pylori. H. encoding an antigen that is highly immunogenic. Disclosed herein is a cluster family of DNA replicas of parts of the genome of pylori. Also disclosed is a new antigenic protein recovered from H. pylori. The present invention also provides a method using the above-mentioned antigen.

Description

DETAILED DESCRIPTION OF THE INVENTION         HELICOBACTER PYLORI ANTIGENIC COMPOSITIONS AND DETECTION METHODSField of the invention   The present invention relates to one or more H.264. pylori antigens, polynucleotides encoding the antigens Peptide sequences, and diagnostic and diagnostic methods using such antigens and polynucleotides. And treatment methods. Background of the Invention   H. pylori can cause chronic superficial gastritis, peptic ulcer disease, and gastric adenocarcinoma A pathogen of the human stomach associated with atrophic gastritis, which has disappeared for most of the century. Ulcerative diseases are described in stress related rather than caused by pylori infection It was considered a ream. According to research, Human subjects taking pylori orally develop gastritis Developed and recovered after the infection was removed by antibiotic treatment At the indicated times, H. pneumoniae in peptic ulcer disease and gastric inflammation was observed. The causative role of pylori Some were approved (Warren and Marshall, 1983).   H. pylori is microaerobic, gram-negative, hypoproliferative, spiral or S-shaped An organism that infects the inner wall of the stomach and has flagella. H. pylori was originally 1 Cultured from a gastric biopsy in 982 and based on overall morphology, Campylobacter sp. Was placed in In 1989, a new genus Helicobacteracea was proposed and approved , H. pylori was the only member (Blaser, 1996). Currently Helicobacte Thirteen members of the r genus are recognized.   An unusual feature shared by all members of this genus is the 540kD cell surface enzyme Is the presence of the urease operon. Urease is It is one of the most abundant surface proteins produced. This enzyme converts urea to dioxide Multi-subunit urease with the function of hydrolyzing to carbon and ammonia (Cover, 1995). The resulting ammonia molecules surround the bacteria, thereby directing the bacteria Neutralize the acid in the vicinity of. Thus, urease is H. oleic at pH pH. pylori Important for survival and good colonization in the gastric environment.   H. pylori is one of the most common chronic bacterial infections in humans. H. pylo ri infection is found in more than 90% of patients with active gastritis and affects the gastric mucosa. H. The presence of pylori is associated with mucosa-associated lymphoid tissue lymphoma (Cover et al., 1996). In developed countries, about half of the population is H. pylori colonies Colonization is common in developing countries, even among children It is. In addition, 1-2 of 10 infected individuals develop peptic ulcer disease during their lives. Develops.   H. Current approaches to assessing pylori infection typically involve invasive techniques (eg, , Gastric biopsy specimen collection, culture, histology and detection of pre-form bacterial enzymes) . These approaches have a number of disadvantages: low sensitivity, inconvenience, and high cost. is there. Non-invasive approaches include urea breath test, UBT (Atherton et al., 1994) And anti-H. Various H. pylori antibodies to detect p. Serum test using pylori antigen Experiments. Urea breath test converts isotope urea to isotope carbon dioxide (analyte) And H. to ammonia. Depends on the presence of urease enzyme from pylori .¹³C (or¹⁴C) The urea breath test is fairly accurate, but the patient breath sample Mass spectrometer (an instrument that does not exist in most clinical settings) ). In addition, existing serum assays are routine Is still not sufficiently reliable for use in other situations, and Used in epidemiological studies to evaluate retrospectively.   H. as a human pathogen Importance of H. pylori and H. in the World Population pylori infection In view of the persistence of H. Reliable infection by pylori strains There is a need for effective methods and compositions for detection and treatment. Ideally Reagents for such assays are described in pylori is highly selective Should be easy and reproducible in addition to being highly specific and highly specific It is. In addition, the method is simple, convenient, and cost-effective, It should be reliable and show high sensitivity.Summary of the Invention   The present invention provides Discovery and discovery of a novel highly immunogenic polypeptide antigen of pylori And characterization. Furthermore, what forms part of the present invention has been previously recognized. There are 69 immunogenic cluster families that have not been identified. H. within the pylori genome The sequence and position of these cluster families of genes encode highly immunogenic antigens. H. was discovered to be Over 250 disclosed DNs of part of the pylori genome Determined based on A's replica. Also disclosed are protein mimics H. using material methodology. It is a natural antigenic protein recovered from pylori. Book The invention further relates to the use of one, several, many or each of the above antigens. Provide the law. What forms part of the present invention is described in Detect pylori infection One, several, or several of the antigenic proteins described herein for Diagnostic kits and methods using many or each, where assays are active It is effective for detecting H. pylori in various infectious states.   In one aspect, the invention relates to one or more of the polypeptides described herein. H. encoding a peptide antigen Includes polynucleotides of the pylori genome. Polinu With respect to nucleotides, some aspects of the present invention are described in US Pat. RNA and DN from pylori A polynucleotide, recombinant H. pylori polynucleotide, the above polynucleotide Recombinant vectors, including any of Includes transformed host cells.   These polynucleotides are encodes a pylori-specific polypeptide antigen You. The corresponding coding sequence can be used, for example, as a reagent in diagnostic tests and / or Enables the production of polypeptides useful as components of tin.   Preferred polynucleotides are SEQ ID NO: 43 (A22), SEQ ID NO: 38 (C1), Nos. 94 and 95 (Y124A), SEQ ID Nos. 169 and 172 (Y261A), SEQ ID No. 253 (c5), Sequence No. 20 (C7), SEQ ID Nos. 51 and 54 (B2), SEQ ID No. 60 (Y104B), and SEQ ID No. 9 8 (Y128D) Substantially pure, capable of selectively hybridizing under hybridization conditions Of the form, H. It is a DNA fragment encoding the pylori antigen. These antigens Encoding polynucleotides are sensitive and highly specific depending on the antigen obtained It is particularly preferred due to its properties.   Another polynucleotide contemplated by the present invention corresponds to SEQ ID NOs: 469-547 DNA fragments spanning one of the following DNA fragment clusters (representative , At least about 18 nucleotides in length) A substantially pure form of anti-hybrid that can selectively hybridize under Here is the DNA fragment that encodes the original:   (1). DNA fragments having a sequence spanning SEQ ID NOs: 222 and 223 (cluster -1, corresponding to SEQ ID NOs: 469, 470);   (2). DNA fragment having a sequence spanning SEQ ID NOs: 8, 135, 161, 162, 218 (Cluster 2, SEQ ID NO: 471);   (3). DNA fragment having a sequence spanning SEQ ID NOs: 11, 60 and 73 (cluster 3, SEQ ID NO: 472);   (Four). A DNA fragment having a sequence spanning SEQ ID NOs: 75, 127, 20, and 169 (class Star 4, SEQ ID NO: 473);   (Five). DNA fragments having a sequence spanning SEQ ID NOs: 175 and 176 (cluster -5, SEQ ID NOs: 474, 475);   (6). DNA fragments having sequences spanning SEQ ID NOs: 115, 3, and 227 Raster 6, SEQ ID NO: 476);   (7). A DNA fragment having a sequence spanning SEQ ID NO: 206 (cluster 7, sequence No. 477);   (8). A DNA fragment having a sequence spanning clones Y291 / T7 and Y291 / T3 ( Cluster 8, SEQ ID NO: 478);   (9). A DNA fragment having a sequence spanning SEQ ID NO: 35 (cluster 9, sequence Number 479);   (Ten). DNA having a sequence spanning SEQ ID NOs: 160, 100, 198, 159, 134, 197, 99 Fragment (cluster 10, SEQ ID NO: 480);   (11). DNA fragments having sequences spanning SEQ ID NOs: 201, 155, 202, and 156 (Cluster 11, SEQ ID NO: 481);   (12). DNA sequence having a sequence spanning SEQ ID NOs: 1, 114, 192, 130, 113, 191, 129 Fragment (cluster 12, SEQ ID NO: 482);   (13). DN having a sequence spanning SEQ ID NOs: 117, 116, 214, 203, 213, and 110 A fragment (cluster 13, SEQ ID NO: 483);   (14). A DNA fragment having a sequence spanning SEQ ID NOs: 158, 95, 94, and 157 Raster 14, SEQ ID NO: 484);   (15). DNA fragments having a sequence spanning SEQ ID NOs: 153 and 154 (class 15, SEQ ID NOs: 485,486);   (16). DNA fragments having the sequence spanning SEQ ID NOs: 140 and 141 (class 16, SEQ ID NO: 487);   (17). DNA fragment having a sequence spanning SEQ ID NOs: 181 and 182 (cluster 1 7, SEQ ID NO: 488);   (18). DNA fragment having a sequence spanning SEQ ID NOs: 138, 144, 15, and 137 G (cluster 18, SEQ ID NO: 489);   (19). DNA fragments having a sequence spanning SEQ ID NOs: 51 and 80 (cluster -19, SEQ ID NO: 490);   (20). DNA fragments having a sequence spanning SEQ ID NOs: 98 and 125 (cluster -20, SEQ ID NO: 491);   (twenty one). DNA fragments having a sequence spanning SEQ ID NOs: 43 and 28 (cluster 21 , SEQ ID NO: 492);   (twenty two). A DNA fragment having a sequence spanning SEQ ID NO: 6 (cluster 22, sequence No. 493);   (twenty three). A DNA fragment having a sequence spanning SEQ ID NOs: 78, 212, 142, and 79 (C Raster 23, SEQ ID NO: 494);   (twenty four). DNA fragment corresponding to SEQ ID NO: 495   (twenty five). A DNA fragment having a sequence spanning SEQ ID NO: 259 (cluster 25, Column number 496);   (26). A DNA fragment having a sequence spanning SEQ ID NO: 164 (cluster 26, Column number 497);   (27). A DNA fragment having the sequence spanning SEQ ID NO: 174 (cluster 27, sequence Column number 498);   (28). DNA fragments having sequences spanning SEQ ID NOs: 119, 83, 85, 136, 84, 120 and 24. Fragment (cluster 28, SEQ ID NO: 499);   (29). DNA fragments having sequences spanning SEQ ID NOs: 194, 108, 163, and 193 (Cluster 29, SEQ ID NO: 500);   (30). A DNA fragment having a sequence spanning SEQ ID NO: 224 (cluster 30, Column number 501);   (31). DN having a sequence spanning SEQ ID NOs: 150, 173, 199, 183, 230, and 253 A fragment (cluster 31, SEQ ID NO: 502);   (32). DNA fragment having a sequence spanning SEQ ID NOs: 122, 77, and 187 (class 32, SEQ ID NO: 503);   (33). DNA fragments having a sequence spanning SEQ ID NOs: 88, 184, and 89 (cluster -33, SEQ ID NO: 504);   (34). A DNA fragment having a sequence spanning SEQ ID NO: 91 (cluster 34, sequence Column number 505);   (35). DNA fragment having a sequence spanning SEQ ID NOs: 101, 92, 102, and 93 G (cluster 35, SEQ ID NOs: 506, 507, 508);   (36). DNA fragments having a sequence spanning SEQ ID NOs: 170 and 118 (class 36, SEQ ID NO: 509);   (37). SEQ ID NOs: 205, 104, 87, 204, 103, 126, 86, 264, 271, 151, and 15 A DNA fragment having two sequences (cluster 37, SEQ ID NO: 510);   (38). DNA fragment having a sequence spanning SEQ ID NOs: 151, 152, 271 and 264 (Cluster 38, SEQ ID NO: 511);   (39). DNA fragments having a sequence spanning SEQ ID NOs: 143 and 225 (class 39, SEQ ID NO: 512);   (40). DNA fragments having a sequence spanning SEQ ID NOs: 165 and 166 (class 40, SEQ ID NOs: 513, 514);   (41). A DNA fragment having a sequence spanning SEQ ID NO: 171 (cluster 41, Column number 515);   (42). DNA flag having a sequence spanning SEQ ID NOs: 209, 216, 217, 208, 215, 38 Ment (cluster 42, SEQ ID NO: 516);   (43). DNA fragment having a sequence spanning SEQ ID NOs: 177, 178, 179, 180 ( Cluster 43, SEQ ID NOs: 517, 518);   (44). A DNA fragment having a sequence spanning cluster 44 (SEQ ID NO: 519);   (45). A DNA fragment having a sequence spanning SEQ ID NOs: 186, 195, 185, 196 ( Cluster 45, SEQ ID NO: 520);   (46). A DNA fragment having a sequence spanning SEQ ID NO: 251 (cluster 46, sequence Column number 521);   (47). A DNA fragment having a sequence spanning cluster 47 (SEQ ID NOs: 522, 52) 3);   (48). DNA fragment having a sequence spanning SEQ ID NOs: 190 and 189 (cluster 4 8, SEQ ID NO: 524);   (49). A DNA fragment having a sequence spanning SEQ ID NO: 200 (cluster 49, Column number 525);   (50). A DNA fragment having a sequence spanning SEQ ID NOS: 211 and 210 (cluster 5 0, SEQ ID NO: 526);   (51). Has a sequence spanning SEQ ID NOs: 168, 81, 132, 82, 121, 133, 167, 228 DNA fragment (cluster 51, SEQ ID NO: 527);   (52). A DNA fragment having a sequence spanning SEQ ID NO: 146 (cluster 52, Column number 528);   (53). A DNA fragment having a sequence spanning cluster 53 (SEQ ID NO: 529);   (54). DNA fragments having a sequence spanning SEQ ID NOs: 221 and 220 (class 54, SEQ ID NO: 530);   (55). A DNA fragment having a sequence spanning SEQ ID NO: 74 (cluster 55, Column number 531);   (56). A DNA fragment having a sequence spanning SEQ ID NO: 72 (cluster 56, Column number 532);   (57). DNA fragments having a sequence spanning SEQ ID NOs: 70 and 71 (cluster -57, SEQ ID NO: 533);   (58). DNA fragments having a sequence spanning SEQ ID NOs: 96 and 97 (cluster 58 , SEQ ID NOs: 534,535);   (59). DNA fragments having a sequence spanning SEQ ID NOS: 105 and 106 (class 59, SEQ ID NOs: 536, 537);   (60). A DNA fragment having a sequence spanning SEQ ID NO: 107 (cluster 60, sequence Column number 538);   (61). A DNA fragment having a sequence spanning SEQ ID NO: 109 (cluster 61, Column number 539);   (62). A DNA fragment having a sequence spanning SEQ ID NOS: 111 and 112 (class Star 62, SEQ ID NO: 540);   (63). A DNA fragment having a sequence spanning cluster 63 (SEQ ID NO: 541);   (64). A DNA fragment having a sequence spanning SEQ ID NO: 58 (cluster 64, Column number 542);   (65). A DNA fragment having a sequence spanning cluster 65 (SEQ ID NO: 543);   (66). A DNA fragment having a sequence spanning SEQ ID NO: 90 (cluster 66, Column number 544);   (67). A DNA fragment having a sequence spanning SEQ ID NO: 13 (cluster 67, Column number 545);   (68). A DNA fragment having a sequence spanning SEQ ID NO: 47 (cluster 68, Column number 546);   (69). A DNA fragment having a sequence spanning SEQ ID NO: 16 (cluster 69, Column number 547).   According to another embodiment, H. pylori polynucleotide is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16 , SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 43, SEQ ID NO: 47 , SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 70-230, Sequence No. 248, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259 SEQ ID NO: 264, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 322, SEQ ID NO: 549 Stringent hybridization to DNA sequences selected from A polynucleotide capable of selectively hybridizing under hybridization conditions. here, These DNA sequences correspond to the H. cerevisiae encoding highly immunogenic proteins. pylori genome Corresponds to the cloned and sequenced region.   In a preferred embodiment, Polynucleotide encoding pylori antigen Are SEQ ID NO: 43 (A22), SEQ ID NO: 38 (C1), SEQ ID NOs: 94 and 95 (Y124A), SEQ ID NO: Nos. 169 and 172 (Y261A), SEQ ID NO: 253 (c5), SEQ ID NO: 20 (C7), SEQ ID NOs: 51 and 54 (B2), SEQ ID NO: 60 (Y104B), and SEQ ID NO: 98 (Y128D) The DNA sequence to be selected under stringent hybridization conditions. It is a polynucleotide that can hybridize alternatively.   Alternatively, H. The polynucleotide encoding the antigen of pylori is SEQ ID NO: 469- At least 18 contiguous regions across a cluster region selected from the group consisting of 547 Nucleotides.   In yet another embodiment, the present invention provides a method comprising the steps of: Poly encoding pylori antigen The nucleotides are SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NOs: 70 to 230, SEQ ID NO: 248, SEQ ID NO: 251, SEQ ID NO: 253, Sequence No. 255, SEQ ID No. 257, SEQ ID No. 259, SEQ ID No. 264, SEQ ID No. 270, SEQ ID No. 271 , SEQ ID NO: 322, SEQ ID NO: 549. Contains at least 18 contiguous nucleotides.   In yet another embodiment, Polynucleotide encoding pylori antigen Are SEQ ID NO: 43 (A22), SEQ ID NO: 38 (C1), SEQ ID NOs: 94 and 95 (Y124A), SEQ ID NO: Nos. 169 and 172 (Y261A), SEQ ID NO: 253 (c5), SEQ ID NO: 20 (C7), SEQ ID NOs: 51 and 54 (B2), SEQ ID NO: 60 (Y104B), and SEQ ID NO: 98 (Y128D) At least 18 contiguous nucleotides contained within the sequence of interest.   In another aspect, the present invention provides For immunoreactivity with pylori positive antiserum H. in substantially pure form, characterized by pylori polypeptide antigen No. The antigen of the present invention, in one embodiment, has the above characteristics, and typically Encoded by a polynucleotide that is at least 18 nucleotides in length. Sa More particularly, the antigen is a cluster selected from the group consisting of SEQ ID NOs: 469-547 Region and DNA sequences under stringent hybridization conditions. Polynucleotides of at least 18 nucleotides in length capable of selectively hybridizing Encoded by all or part of the code array.   Further antigens according to the invention are described in H.S. by the sequence encoding the pylori antigen Coded.   In a preferred embodiment, the antigen is SEQ ID NO: 43 (A22), SEQ ID NO: 38 (C1) , SEQ ID NOs: 94 and 95 (Y124A), SEQ ID NOs: 169 and 172 (Y261A), SEQ ID NO: 253 (c5), SEQ ID NO: 20 (C7), SEQ ID NOS: 51, 54 (B2), SEQ ID NO: 60 (Y104B), and sequences No. 98 (Y128D) and a DNA sequence selected from the group consisting of At least 18 nuclei capable of selectively hybridizing under hybridization conditions Encoded by a polynucleotide of length leotide.   Alternatively, the invention provides a cluster selected from the group consisting of SEQ ID NOs: 340-468. H. containing at least six consecutive amino acids contained within an antigenic sequence pylori anti Including the original. Here, the antigen is in a substantially purified form and pyl ori Characterized by immunoreactivity with positive antisera.   In certain embodiments, H. pylori antigen has SEQ ID NOs: 2, 4, 5, 7, 9, 10, 12 , 14, 17, 21, 25-28, 36, 37, 39, 44, 48, 55, 59, 61, 69, 249, 250, 252 254, 256, 258, 260-263, 265-269, 323, 324, and 550-554 At least six consecutive amino acids contained within the polypeptide sequence selected from Contains acids.   In yet another embodiment, pylori antigen consists of SEQ ID NOs: 555-602 At least six consecutive amino acids contained within a polypeptide sequence selected from the group Nonoic acid.   Alternatively, H. The pylori antigen is a polio selected from the group consisting of SEQ ID NOS: 555-602. Includes a repeptide sequence.   In one particular preferred embodiment, pylori antigen is SEQ ID NO: 44 (A22 ), SEQ ID NO: 39 (C1), SEQ ID NO: 568 (Y124A), SEQ ID NO: 557 (Y261A), SEQ ID NO: No. 254 (c5), SEQ ID NO: 21 (C7), SEQ ID NO: 55 (B2), SEQ ID NO: 61 (Y104B), And at least the sequence selected from the group consisting of SEQ ID NO: 573 (Y128D) Are also antigens containing six consecutive amino acids.   Also, what forms part of the present invention are anti-H. blood for the presence of pylori antibodies Diagnostic kit for use in screening biological fluids such as Qing is there. The kit comprises at least one anti-H. Above, immunoreactive with pylori antibody A substantially purified H. of the type characterized by: pylori antibodies, and anti-antibodies Includes a reporter for detecting binding to the original. Polypeptide antigen is supported on a solid The kit may further comprise an unbound reporter-labeled antibody. It can include a human antibody, where an anti-H. The binding of the pylori antibody to the polypeptide antigen is Reporter-labeled antibody anti-H. It can be detected by binding to the pylori antibody.   According to one embodiment, the kit comprises at least two with different antibody specificities H. Contains the pylori antigen.   In a related aspect, the present invention relates to a method of treating H. in a subject. detection of pylori infection, Or methods of detecting eradication of bacteria in a previously infected subject. This way Has purified a biological fluid sample from a subject of the type described above. pylori po Reacting the antigen with the repeptide antigen and testing the antigen for the presence of bound antibody Testing.   Preferred antigens for use in this method include the following polypeptide sequences or Corresponds to one of the contiguous regions included below: SEQ ID NO: 44 (A22), SEQ ID NO: 39 (C1), SEQ ID NO: 568 (Y124A), SEQ ID NO: 557 (Y261A), SEQ ID NO: 254 (c5), SEQ ID NO: 21 (C7), SEQ ID NO: 55 (B2), SEQ ID NO: 61 (Y104B), and SEQ ID NO: 5 73 (Y128D).   In yet another aspect, the present invention relates to an H. of the above type. pylori polypeptide H. containing antigens pylori vaccine compositions. The antigen may be a mammalian model system (eg, For example, mice or rhesus monkeys are challenged with the peptide and then Feeling pylori Dyeing). characterized by the ability to reduce the level of pylori infection You.   Preferred antigens for use in vaccines are long-lasting antibodies following antimicrobial treatment As shown by this, it is a vaccine that provokes a long-lasting antigenic response. Wakuchi An exemplary antigen for use in a cancer composition is SEQ ID NO: 565 (Y139), SEQ ID NO: No. 575 (Y146B), SEQ ID NO: 555 (Y175A), SEQ ID NO: 44 (A22), SEQ ID NO: 569 (Y1 84A), SEQ ID NO: 578 (Z9A), SEQ ID NO: 557 (Y261A) and SEQ ID NO: 575 (Y146B ).   These and other objects and features of the invention are set forth in the following detailed description. When read in combination with the examples, it is clear enough.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows native blots of Roost pooled serum (H. pylori positive). H. Two-dimensional (2D) sodium dodecyl sulfate polyacrylamide gel of pylori antigen Computers of Western blot of electrophoresis (SDS-PAGE) analysis (high pH condition) It is a can image.   FIG. 2 shows native H.b. sodium dodecyl sulfate of pylori antigen Polyacrylamide gel two-dimensional electrophoresis (SDS-PAGE) analysis (low pH condition) Figure 9 is a computer scan image showing estanblots;   FIG. 3 is a schematic diagram of the amino acid translation of ORF3 of clone Y104-1.asm (299 amino acids). Is shown. The region of the sequence shown in the figure corresponds to Y expressed in E. coli strain XL1Blue. Confirmed by amino acid sequence analysis of the 104-l.asm protein, and (i) pylori 3 6 kD protein (indicated by underline), (ii) "Spot 15" peptide (indicated by square), And (iii) corresponding to the peptide encoded by clone d7 (in parentheses) Do;   FIG. In vivo processing pathway and book of the 36 kD protein of pylori FIG. 2 is a schematic diagram showing the relationship with the “Spot 15” antigen disclosed in the specification;   FIG. For "Spot 15" antigen isolated from pylori (ATCC 43504) Reverse-phase HPLC peptide profile, which contains a number of identifiable proteins Is illustrated.   FIG. pylori 36 kD protein amino acid sequence, underlined 28 kD protein Shows the protein region.   FIG. proteome (p) used to identify some natural antigens of pylori FIG. 2 is a schematic diagram of a method of (roteome).   FIG. Different claws for pylori immunopositive serum panel Is a graph summarizing the susceptibility ratio of the components;   FIG. 9 shows the approximate location of the immunogenic cluster regions that form one aspect of the present invention. H. pylori genome diagram;   FIGS. 10 to 63 show clusters (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (23 ), (25), (2 7), (28), (29), (30), (32), (33), (35), (36), (37), (38), (39), (40), (41) , (42), (43) , (44), (45), (46), (47), (48), (49), (50), (51), (53), (54), (58), (59), ( 61), (62), ( 68) and (69) are linear maps showing the relative positions of the immunogenic subclones in;   FIGS. 64A-D show a summary of the immunogenic clone clusters of the present invention, wherein (1) Raster number, (ii) clones defining the start and end regions of each cluster, H . Includes the coordinates of the cluster consensus region in the pylori genome and expression data. MU;   FIG. 65 shows H. influenza for various serum panels. pylori recombinant antigen specificity comparison It is a graph showing sensitivity. Sensitivity values refer to various golds as described in Example 6B. Calculated using standards. ;   Figure 66 shows H. for various serum panels calculated against the indicated gold standard. pylor i is a graph showing the comparative specificity of the recombinant antigens; and   67A and 67B show (i) the immunopositive claw forming the basis of the present invention. (Ii) their corresponding cluster numbers (each clone within the H. pylori genome) And the relative position of the star star), and the relative (iii) sensitivity and relative ( iv) Provide a summary in the form of a table of specificity values.Detailed description of the invention I.Definition   The following terms used in the present invention have the meanings indicated below:   The polypeptide sequence or fragment corresponds to the fragment from which it is derived. Having the same amino acid residue sequence as the region to be Is “derived from” the fragment.   A polynucleotide sequence or fragment is a fragment of a fragment from which it is derived. Another polynucleotide sequence if it has the same sequence of nucleic acid residues as the corresponding region Or "derived from" the fragment.   The first polynucleotide fragment is the first fragment or its complement. Under selective (stringent) hybridization conditions, If the fragment can form a double-stranded polynucleotide hybrid, "Can selectively hybridize" to the polynucleotide fragment of First And the second fragment is typically at least 15 nucleotides in length; Preferably it is at least 18-20 nucleotides in length. Selective (stringent Hybridization conditions are used herein at about 45 ° C. at about 1.1 M salt. Defined as hybridization, followed by at least one wash with 0.3 M salt at 37 ° C. Is defined. Such conditions typically involve no more than about 25-30% base pair misuma. Switch.   Two or more polynucleotide or polypeptide fragments The fragments are linked to each other using a computer program such as ALIGN. When correspondingly aligned, the nucleotide base or amino acid residue is Are identical in at least a certain percentage of all base or residue positions, respectively. In some cases, it has at least a predetermined percentage of "sequence identity." (this The ALIGN program is a FASTA version 1.7 package software for the sequence comparison program. G., Pearson and Lipman, 1988; found in Pearson, 1990).   As used herein, “H. pylori polynucleotide” is H. pylori polynucleotide. pylori genome And a polynucleotide sequence derived from the variant thereof. Disclosed herein Type of H. pylori polynucleotides are described in pylori polypeptide antigen Where the antigen obtained is H. For immunoreactivity with pylori positive antiserum Is characterized by: Generally, the H. of the present invention. pylori polynucleotides At least about 18 nucleotides in length (ie, encoding a 6 peptide antigen) ). In an alternative embodiment, H. The pylori polynucleotide has at least about 2 It is 4 nucleotides long (ie, encodes an 8-peptide antigen). Yet another In embodiments, H. A pylori polynucleotide has at least about 30 nucleotides. Is the length. In some cases, H. pylori polynucleotide is about 45-75 nucleotides It is in the range of the length of the nucleotide, but may of course be longer. Polynucle of the present invention Otides can be derived from natural or synthetic sources, or Can be prepared. The polynucleotide sequence may be the polynucleotide of interest, H. described in If it can express the pylori antigen, it is a naturally occurring sequence Obtained or mutated to a particular naturally occurring sequence (of one or more bases) Substitutions, deletions, insertions, and inversions). Polynucleotide The sequence is, if necessary, an expression control sequence located adjacent to the coding region. (Typically from heterogeneous sources).   The nucleotide sequence described herein has the essential nature as defined above. It is meant to include variants having identical "sequence identity". Essentially Nucleotide sequences having the same sequence identity are typically selected (stringent). (Hybrid) hybridization conditions under selective hybridization conditions I can do it. That is, the nucleic acid fragment is pylori polynucleotide sequence or Is a variant thereof (eg, which hybridizes to an H. pylori polynucleotide, Hybridizes to polypeptides from other members of the elicobacter family Stringent hybridization and washing conditions If specific hybridization is possible under the conditions, pylori polynucleotide It is thought that hybridization can be selectively carried out.   "H. pylori antigenic polypeptide" refers to a polypeptide, or a region or region thereof. Include immunoreactive variants of that portion, as long as the variant is immunogenic Means that. Suitable variants are described in identical to the sequence of the pylori polypeptide Defined as any polypeptide having a sequence (ie, sharing sequence identity). Is determined.   For example, H. an antigenic poly which is essentially identical to a pylori polypeptide antigen The peptides are (i) H. selectively hybridizes to the sequence of pylori or its variants Or (ii) encoded by the nucleic acid. By Pylori or its variants Is coded.   Sequence comparisons can also be performed using, for example, the local alignment program LALIGN. Can be used for the purpose of determining "polypeptide homology" . In making such a determination, the polypeptide sequence is typically selected H. For the pylori amino acid sequence or any variant thereof, LALIG N programs with 1 ktup, default parameters and default Compared using PAM.   Optimal alignment longer than about 6-8 amino acids, and more than 70%; Is more preferably any polypeptide having 75% to 80% identically aligned amino acids. Peptides are considered "homologous polypeptides". The LALIGN program uses the array ratio Program found in FASTA version 1.7 package software (Pearson et al. 1988; Pearson, 1990; William R .; Pearson, Department of Biological Chemis try, Box 440, Jordan Hall, Charlottesville, VA) . Sequence variations between antigens are described in pylori strain, H. Coding antigens within the pylori genome Location of the gene or gene family to be transduced (ie, Whether they are highly conserved or easily recombined) and so on. Depends on factors.   An immunogenic polypeptide or polypeptide "fragment" can be obtained from (i) H. pylori Encoded by the open reading frame of the polynucleotide, or Or (ii) H. show sequence identity to the pylori polypeptide And H. pylori immunoreactive with a positive sample (eg, serum) Polypeptide. Typically, an immunogenic fragment is a specific antigen, At least about 6-8 amino acids, and preferably at least about 10-12 Contains consecutive amino acid residues.   H. Disclosed herein are immunogenic variants of the pylori polypeptide antigen. Specific H. amino acid substitutions, deletions, and / or Means addition variants and can be obtained by conventional methods (eg, competition assays or two antibody sandwiches). Specific polypeptide antigen, as determined by witch assay) Have substantially the same or increased binding affinity for a given antibody.   Representative H. pylori antigen is a class selected from the group consisting of SEQ ID NOs: 340-468. Consists of at least six consecutive amino acids contained within the star antigen sequence . Here, the antigen is in a substantially pure form and pylori positive anti It is characterized by immunoreactivity with serum.   "Cluster sequence" refers to the H. elegans encoding a highly immunogenic polypeptide. pylor It corresponds to a region in one genome. The cluster sequence has 250 as described herein. It was determined based on the DNA sequence information of the more immunogenic clones. As a result, 69 Unique clusters have been identified, and the antigens according to the present invention have any of the 69 clusters -Is an antigen encoded by a continuous series of nucleotides contained within. Representative antigen coding sequences falling within each of the clusters are summarized in FIGS. 64A-D . The cluster regions are referred to herein as clusters 1-69 and SEQ ID NOs: 340-4. Corresponds to 68.   H. The location of each cluster region in the pylori genome is illustrated pictorially in FIG. It is. In some cases, clusters are more likely than by a single sequence H. It is defined by various regions within the pylori genome. For example, a specific cluster Are separated by an “start” sequence, an “end” sequence, and possibly an intermediate “intermediate” sequence. And thus may correspond to more than one sequence contained in the sequence listing. This If this is the case, the descriptor for the cluster sequence will be the given cluster (eg, start, middle, , End). Typically, the defining cluster sequence is 1 Coding for one or more antigens, and the rest of the sequence for a particular cluster If not provided in white, based on the information provided herein, for example, Given the information provided in Tomb et al., 1997, it can be readily determined. One The cluster sequence defined by the more representative sequences is cluster 1 ( Cluster No. 469, 470), cluster 5 (SEQ ID Nos. 474, 475), cluster 7 (SEQ ID No. 477, end), cluster 15 (SEQ ID NOs: 485, 486), cluster 35 (SEQ ID NOs: 506-50) 8), cluster 40 (SEQ ID NOs: 513, 514), cluster 41 (SEQ ID NO: 515, end), cluster Raster 43 (SEQ ID NOs: 517, 518), cluster 47 (SEQ ID NOs: 522, 523), cluster 49 (SEQ ID NO: 525, end), cluster 58 (SEQ ID NOs: 534, 535), and cluster 5 9 (SEQ ID NOs: 536, 537).   "Substantially purified" and "substantially purified form" Used in the context, and typically includes at least one purification or isolation step. Depending on the process, unrelated or contaminating components (eg, serum, cells, Protein, non-H. pylori polynucleotide). pylori polynu Refers to at least partial purification of a nucleotide or polypeptide. Target compound Or methods and procedures for the isolation or purification of components are described herein. (Eg, SDS-PAGE of H. pylori polypeptide, affinity of fusion protein) Purification, blotting, and recombinant production).   Under optimal conditions, the antigen is H. Present in samples infected with pylori Binds to antibodies, from subjects who have or have not been infected with pylori The majority of liquid samples (greater than about 60-65%, preferably about 70-80%, More preferably, if it does not bind to antibodies present in greater than about 85%), then H. "Specifically immunoreactive" with pylori positive antisera or biological fluid samples It is. Antigens that are “specifically immunoreactive” are specific H. Produced for pylori antigen May be immunoreactive with a monoclonal or polyclonal antibody raised.   By biological fluid is meant any fluid derived from the body of a mammal, especially a human. Representative biological fluids include blood, serum, plasma, urine, feces, mucus, gastric secretions, Contains plaque, or saliva.   "Immunologically effective amount" A simple agent that is effective in treating or preventing pylori infection Amount administered to a mammalian host, either as a single dose or as part of a series Say. The amount depends on the health and physical condition of the subject to be treated, the antibodies of the subject's immune system. Depends on the ability to synthesize, desired degree of protection, vaccine formulation, etc. You. Such amounts are typically within a relatively wide range, and are routine. Can be determined in a row. II.H . Isolation of pylori antigenic sequence   The present invention relates to more than one million individual H. pylori composition obtained from screening In addition, many highly immunogenic H. Based on the identification and isolation of pylori polypeptide. Departure The light antigen is produced recombinantly or pelleted and dissolved. H. either isolated from a mixture of soluble proteins from pylori Is. In addition, as a result of intensive computer analysis efforts, Different sequence information corresponding to all of the immunogenic clones obtained is described in In the genome of pylori Collection of previously unrecognized antigenic cluster regions contained in on) gathered to provide.   Recombinant H. Preparation and identification of pylori antigens, and into clustered families Are described here. 1.Methods for screening recombinant sublibraries   H. of the present invention. pylori antigen, using the conventional screening method described below, It can be obtained from a phage library. Unless otherwise stated, described herein The DNA λ library used is GeneLabs Technologies, Inc. Culture Collectio n, 505 Penobscot Drive, Redwood City, CA, 94063, or Genelabs Diagnost ics PTE LTD Culture Collection, 85 Science Park Drive # 04-01, The Cavend ish, deposited at Singapore Science Park, Singerpore 118259.   The following H. E. coli containing the insert corresponding to the pylori clone. XL-1 Blue MRF plastic Smid, American Type Culture Collection, September 30, 1997, 12301 Deposit received by Parklawn Drive, Rockville MD 20852, and Number assigned: clone dHC1S.11 (ATCC 98525), clone dHG1a2 S.5 (ATCC 98526), clone Y212A (ATCC 98527), clone dHC7.2cd6 (ATCC 96528), clone y175A (ATCC 98529), clone y146B (ATCC 98530), Clone dHA22.8 (ATCC 98531), clone Y184A (ATCC 98532), clone dHB2d1 (AT CC 98533), clone y104B (ATCC 98534). H. pyloriDNA insertion Y library Bacteriophage λ containing American Type Culture on September 3, 1997 Deposit received by collection and assigned a naming number of ATCC 209234 Have been.   The antigen-encoding DNA fragment of the present invention comprises (i) an immunoscreen as described above. And / or (ii) algorithms for identifying potential antigenic regions Use a system (eg, "ANTIGEN", Intelligenetics, Mountain View, CA) It can be identified by computer analysis of the coding sequence. For example, antigen code DN The A fragment is subcloned and the subcloned insert Is then fragmented by partial DNase digestion, resulting in random fragmentation. Generate specific subunits by specific restriction endonuclease digestion. Generate a fragment. Next, the obtained DNA fragment is used as an expression vector. (Eg, the λgt11 vector) and cloned inserts Subject to immunoscreening to provide a map of the pitope.   In addition, DNA fragments of the type as described herein may have overlapping H. py As a probe in hybridization experiments to identify lori sequences These can then be used to identify additional contiguous sets of clones. Can be further used as a probe.   Any of the clone sequences described herein may have λgt10 or “LAMDA ZAPI I "(Stratagene, LA Jolla, CA). Can be used to probe libraries. Specific subflags of known sequence Fragment uses the polymerase chain reaction or possesses such a sequence. Can be isolated after restriction endonuclease cleavage of the vector. The resulting DNA fragment Fragments can be used as radiolabeled probes against any selected library. It is. In particular, the 5 'and 3' end sequences of the clone insert identify additional clones It is useful as a probe for   In addition, the sequence provided by the 5 'end of the cloned insert is the first strand Useful as sequence-specific primers in DNA synthesis reactions (Maniatis et al., 1 982; Scharf et al., 1986). For example, specifically primed H. pyloriD The NA library contains the cloned DNA sequence described herein as a template. It can be prepared by using specific primers from one of the columns. new The second strand, a new DNA, is synthesized using RNase H and DNA polymerase I. . The above procedure is based on the DNA corresponding to the nucleic acid region Identify or produce the molecule. These newly isolated sequences are then: Further, it is used to identify adjacent sequences and the like. New H. pylori sequence is simple After being released, H. Polynucleic acid to identify specific sequences encoding the pylori antigen Nucleotides are cloned and immunoscreened. 2.Recombinant antigen   H. To identify novel and highly antigenic polypeptides useful for detecting pylori , A DNA library, the expression vector λgt11 or “ZAPII” (Stratagene, La J olla, CA; commercially available H. in any of Examples 1). pylori strain (American Thai Culture Collection, Rockville MD; ATCC Accession No. 43504, ATCC Accession No. 4 3526). The polynucleotide sequence was then converted to H by endoscopy. . Pooled sera from 11 patients identified as pylori positive (here Defined as "Roost pool" serum) or 4 defined as "SFA001" Two H. Polypeptides immunoreactive with another pool of pylori immunoreactive serum samples Was selected for expression of the drug. As described in Examples 6A and 6B, and FIG. And 66, or as described in Example 13. pylori It is also possible to screen with other sources of infected samples. Sump Can be an individual sample (ie, from a single subject) or It may be a pooled sample as described in the specification.   Recombinant proteins identified by this approach are described in Infection by pylori Single or combination as substrates in diagnostic tests to detect It provides candidates for polypeptides that can act on them. In addition, corresponding The nucleic acid coding sequence can be used to identify additional H. pylori antigen coding sequences. Acts as a hybridization probe.   H. above. The pylori strain is used to generate a DNA library in a lambda vector. Used (Example 1). H. Other commonly available H. pylori strains include, for example, # 29 H. 995 and J. 170 strains. Includes pylori sample. Or, la The library is H. H. pylori isolated from a sample confirmed positive. py Can be built from lori. In the method described in Example 1, the library comprises: Produced from genomic DNA isolated from pelleted bacteria. Or far Heart isolation pellets bacteria from infected biological specimens (eg, gastric mucosa) Can be used to   Referring to Example 1 The pylori DNA library uses H.pyl as a starting material. It was generated using a DNase digested genomic DNA fragment isolated from ori. "Short To prepare a library of “antigen clones”, the resulting molecules are ndependent Single Primer Ampllfication (SISPA; Reyes et al., 1991) Ligation and expansion in a non-selective manner, followed by expression of the peptide antigen And a suitable vector for screening (for example, λgt11 or ZAPII). It was learned. The library disclosed herein comprises a "short antigen claw". Library ", typically with a prefix beginning with the letter Y or Z. And the "long antigen clone" library is designated library 1 and And Library 2.   ZAPII libraries 1 and 2 are similarly constructed using the following exceptions: I built it. ZAPII libraries 1 and 2 were converted to the longer H. pylori DNA fragment (That is, have not received the Sequence Independent Single Primer Amplification) Genomic DNA digested with EcoRI or HindIII). Library 1 black (Named in uppercase in this specification) was replaced with EcoRI-digested H.E. pylori DNA to λ “ZAPI It is generated by direct ligation to the EcoRI site of the "I" vector. Library Two clones (named in lower case) were Erase pylori genomic DNA using HindIII And then E. coli Klenow enzyme or T4 DNA polymerase enzyme The blunt-ended fragment is then blunt-ended using SISPA Connect to the rimer. Next, the linker-linked DNA was treated with EcoRI. And ligated to the EcoRI site of the "ZAPII" lambda arm.   λgt11 is H. It is a particularly useful expression vector for producing pylori antigen. This Is located 53 base pairs upstream of the translation stop codon of the β-galactosidase gene. It contains a unique EcoRI insertion site. Therefore, the inserted sequence is β-galactosida Expressed as a β-galactosidase gene product N-terminal part of the heterologous peptide, and optionally β-galactosidase peptide Any of the C-terminal regions (where the C-terminal portion Expressed when no codon is included).   λgt11 vectors also reduce viral lysogenicity at permissive temperatures (eg, 32 ° C). Temperature that results in virus lysis at elevated temperatures (eg, 42 ° C.) Produces a temperature-sensitive repressor (cI857). Advantages of λgt11: (1) High efficiency (2) Based on growth of host cells at permissive temperature The ability to select a host cell that has been lysed but not at non-permissive temperatures, and ( 3) Includes the production of recombinant fusion proteins. In addition, fur containing a heterologous insert Di produces an inactive β-galactosidase enzyme, so typically the insert is The phage containing the phages use a colorimetric substrate conversion reaction with β-galactosidase. To be identified.   In addition to the λgt11 vector, a number of E. coli coli expression vector for antigen expression Useful for Alternative microbial hosts suitable for expression include bacilli (eg, B. s. ubtillis), and other Enterobacteraceae (eg, Salmonella, Serratia, And various Pseudomonas species). In such a host, the expression vector The vector typically contains regulatory sequences compatible with the host cell. Other known promos The promoter sequence is expressed in an expression vector (eg, lactose promoter system, In a phage promoter system, or a promoter system from phage λ). If necessary, the insertion of an in-frame Met codon containing the antigen allows the amino terminal Thionine may be provided. Carboxy-terminal extension of antigens can be added to traditional mutagenesis procedures Can be more removed.   In addition, yeast expression systems may be used, including, for example, yeast-derived proteins. Prepro-alpha factor of Saccharomyces cerevisiae used to direct quality expression There is a child (pre-pro-alpha-factor) leader area. The antigen coding sequence is Can be fused to the leader region at the frame. The construct is then typically Strong transcription promoters (such as the alcohol dehydrogenase I promoter) Or under the control of a glycolytic promoter. Following the antigen sequence There is a translation stop codon, followed by a transcription termination signal. Alternatively, the antigen coding sequence Facilitates purification of fusion proteins by affinity chromatography A second protein coding sequence (eg, β-galactosidase ). For constructs used for expression in yeast, A protease cleavage site can be inserted to facilitate separation of the fusion protein components You.   In addition, mammalian cells can be used for expression of the antigens of the invention. Feeding Vectors useful for expression in target cells are typically strong viral promoters. Insertion of the antigen-coding sequence between the promoter and the polyadenylation (polyA) signal Characterized. Optionally, the vector may be a selectable marker gene (e.g., (A selection marker gene that confers biomaterial resistance). Suitable host cells include: Chinese hamster ovary cell (CHO) strain, HeLa cell, myeloma cell, Jurka t cell line and the like. Expression vectors for these cells are Sequence (eg, origin of replication), promoter, enhancer, information processing unit Information processig ites (eg ribosome binding sites, RNA splices) Sing sites, polyadenylation sites and transcription termination sequences).   A DNA sequence is a sequence in which the sequence of interest is operably linked to an expression control sequence (ie, After being ligated (where possible) to be expressed in the host.   Experiments performed in support of the present invention are described herein, and in particular, Preparation of sphage library and H. of the present invention. Insertion encoding pylori antigen Object characterization is described.   a.Representative H. pylori antigen: short antigen clone library.Example 1 , H. pylori (ATCC No. 43504) describes the preparation of a DNA library. You. The library was Immunoscreen using pyloll positive pool serum (Example 2). Anti-H. Present in pooled serum. immunoreacts with pylori antibody A number of lambda clones were identified. The selected immunoreactive clones are Purified and tested again for their immunoreactivity. A clone of normal human serum ( Control, H. pylori negative) was also tested for immunoreactivity .   As described in Examples 3 and 4, a large number of clones were immunoscreened. Identified and further characterized. Further described in Example 3, and Referred to herein as Y family clones and Z family clones Immunoreactive clones include clone Y-104-1, and SEQ ID NOs: 70-230. Includes the corresponding clones summarized in Table 2.   To obtain polypeptide antigens of the invention, and their respective coding sequences The DNA insert of the immunoreactive recombinant λ clone was Primers corresponding to the lambda arm sequence adjacent to the PCR amplification using each immunoreactive clone as template . For the λgt11 clone, the gt11F (SEQ ID NO: 65) and gt11R (SEQ ID NO: 66) Limer was used. For the λZAPII clone, T3 (SEQ ID NO: 67) and T7 (SEQ ID NO: 67) SEQ ID NO: 68) Primers were used.   The resulting amplification product is agarose gel purified, and the primers and other components Eluted from the gel to remove (Ausbel et al., 1988). Then purified The inserted DNA was subjected to direct sequencing. In some cases, the inserted DNA is first Subcloned into a TA cloning vector (Invitrogen, San Diego, CA). And then sequenced. H. Immunoreactivity against pylori positive pool serum The clones shown are identified in Example 3 and, more particularly, in Table 2. .   Sequencing was performed by the manufacturer (Perkin Elmer Applied Biosystems, Foster City, CA). ) According to the recommendations of Perkin Elmer Applied Biosystems model 373A DNA sequenc "DYEDEOXY TERMINATOR CYCLE SEQUENCING" (Sanger et al. (1 997). Sequence data is shown in the attached sequence listing. You.   The sequence for Y family and Z family clones is "GENBANK" , EMBL database, and dbEST (National Library of Medicine) sequence, Comparisons were made at both the nucleic acid and amino acid sequence levels. Search program FASTA, BLAST P, BLASTN, and BLASTX (Altschul et al., 1990) indicate that these sequences have the nucleic acid sequence And amino acid sequences.   The clone was determined to be significantly longer than the predicted coding region of the protein In some cases, genomic clones were digested with restriction enzymes and the resulting Fragment was inserted into an appropriate expression vector. Obtained subclone (specifically deleted DNA fragments) were screened for immunogenicity. H . Clos identified as immunoreactive with pylori positive pooled sera Were plaque purified. A plasmid containing the insert obtained by recovery from phage Rasmid DNA was sequenced as described above and also in Example 4.   b.Representative H. pylori antigen: ZAPII libraries 1 and 2.H. pylori Libraries isolated by immunoscreening using pooled sera of epidemiological reactivity -1 and Library 2 clones include: Library 1: A3, A22 , B2, B9, B17, B23, C1, C3, C7, DH, and library 2: a1, a3, a5, b5, b8c7, c2, c5, c13, d5, d6, d11, d7, e6, f3, f8, f1Lg2, g9, g11, k4. Clone G1a was purchased from Biogenesis, Inc. Monochrome from (Bournemouth, England) Na And isolated by screening using antibody 1G6. About these clones Are shown in the sequence listing herein.   Preparation of subclones of libraries 1 and 2 for preparing the antigen of the present invention Exemplary procedures for isolating and isolating (derived coding sequence and putative proteins) (Including proteinaceous features) are provided above and are described in detail in Example 3. .   c.Clone d7 and H. Its association with the 36 kD protein of pylori.Claw D7, another immunoreactive clone of the invention, is blunt-ended and linked to an A / B linker. Ligated, digested with EcoRI, and produces a β-galactosidase fusion protein This is a HindIII clone subcloned into the λ vector ZAPII. So Is shown as SEQ ID NO: 11. Polynucleotide from this clone And polypeptides represent a preferred embodiment of the invention.   As described in further detail below, this clone is pylori 36Kd It encodes about 70% of the carboxy terminus of the native protein, whereas Lone Y104 encodes the entire 36K protein. Support of the present invention (Example 8 and And 9), based on the results of the sequencing experiments performed in pylori neate A portion of the amino acid sequence of a live 36Kd protein has been determined. 36kD protein The quality (partially encoded by clone d7) was determined by the “Spot 15” Highly antigenic H., referred to as protein. Precursor to pylori protein (Examples 8, 9 described in more detail below). This band is p The majority of ylori-positive samples were Western-positive. Appear to be present and usually not present in normal samples. Therefore, this antigenic tan Parkin is highly H. It appears to be an indicator of infection by pylori. 36kD (sun That is, the apparent molecular weight) in vitro processing of the protein is schematically shown in FIG. Is shown.   As shown in FIG. pylori translation product (299 amino acids 34kD (ie, calculated molecular weight) protein) It is thought to be cleaved at the noic acid position 23. This is H. pylori's "36kD protein" Yields a 31.6 kD cleavage product commonly referred to as Minutes that refer to this protein Differences in terminology terminology result from: 36 kD observed from SDS-PAGE; 34 kD Is calculated from the corresponding DNA sequence (FIG. 6); and 31 kD is residue 23 of SEQ ID NO: 60. Determined from empirical sequence data.   Post-translational modification of the 36 kD protein (ie, acetyl at the amino acid terminus (position 23)) Fragmentation and cleavage of the carboxy terminus) is a "spot 15" antigen with a molecular weight of 28 kD Is generated. Referring to FIG. 4, "X" represents deamidation (ie, asparagine or Or glutamine) or a point where a point mutation has occurred. Further suggested Modifications leading to a minor spot 15 protein are also shown. To spot 15 The corresponding antigenic polypeptide and the sequence encoding this protein (eg, Clones Y-104 and d7) represent one particular preferred embodiment of the present invention. This is due to the strong antigenicity of the peptide. In addition, spot 15 peptide is listed in Table 9 And various H. as shown in Example 10. pylori strain has been detected.   The results discussed above and the results in the examples show a number of novel H. pylori Polypeptide antigens, and polynucleotide sequences encoding such antigens 3 shows the isolation and identification of   d.Cluster analysis and H.E. Identification of antigenic regions within the pylori genome.This specification The antigenic sequences described in this document can be used to identify more than one million different H. pylori of the antigenic composition Determined based on screening. DNA sequencing was performed by H. pylori immunopositive Open readies performed on clones and encoding antigenic proteins Framing frame was identified. Then encodes the antigen thus identified The nucleic acid sequence is inserted into an expression vector and the expression of the desired antigenic protein is confirmed. I accepted. As a result of this study, more than 250 antigenic clone sequences were identified, And is disclosed herein.   In an effort to further correlate sequence information with various immunogenic clones Clones were organized into clusters (1-69). The isolated herein The antigenic polynucleotide fragments are represented by the following clusters (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23), (24), (25), (26), (27), (28), (29), (30), (31), (32), (33), (34), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (50), (51), (52), (53), (54), (55), (56), (57), (58), (59), (60), (61), (62), (63), (64), (65), 69 classes defined herein as (66), (67), (68), and (69). Be mapped to a star. Complete H. of these antigenic clusters in the pylori genome The locations are shown in FIG. As can be observed from FIG. 9, clusters in the genome (And the clones within the cluster) are highly random, Only a small portion of the total nucleotides contained in the body (ie, about 2-3%) Show. Prior to this study, to unique and highly antigenic regions in the pylori genome No comprehensive guidelines are known.   For cluster analysis, all of the immunoclonal sequences were identified by Pearson and Llpman ( 1988) combined with the FASTA database. This data The base was converted to the BLAST database for high speed searching (Altschul et al. , 1990). The sequence of each immune clone then defines a cluster. The database was searched against the database using the program BLASTN. class Is a collection of clones containing sequences identical to the other clones in the group. The sequences in each group or cluster are then stored in different database files. Combined to provide input to IG-Suite (Oxford Molecular) 's GEL program Formatted for GEL then assembles the sequence and has ambiguity A consensus sequence was proposed. For sequences with no ambiguity, by editing the common sequence, Determined for each cluster. H if the cluster contains non-contiguous sequences . pylori genome sequence (TIGR World Wide Web Site) Used for Clusters 1 to 69 correspond to SEQ ID NOs: 469 to 547. Then anti The open reading frame for the primordium is determined from the common sequence of the cluster Was done. The single clone sequence was translated to directly provide the antigenic sequence. K Each class as determined by translation of the raster's open reading frame Antigenic regions contained within the star are provided as SEQ ID NOs: 340-468. FIG. 10- 63 is a cluster (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (23), (25), (27), (28), (29), (30), (32), (33), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (50), (51), (53), (54), (58), (59), (61), (62), (68) and a linear map showing the relative positions of the immunogenic subclones in (69). It is. Clusters not shown in the linear map are one clone (ie, Clusters (22), (24), (26), (31), (34), (52), (55), (56) , (57), (60), (63), (64), (65), (66), (67)) Cluster.   64A-D show clusters 1-69, the clones contained within each cluster, and Hi H. Here is a table that summarizes the coordinates of the common sequence region of each star in the pylori genome .   During the course of the present invention, the complete genomic sequence of H. pylori was reported (Tomb et al., 1997). And this is incorporated herein by reference. H.pylori has 1,667,867 bases It has a paired circular genome and 1590 predicted coding sequences. Visit the TIGR website The genomic sequence reported in Table 1 contains the sequences of the clusters and immunogenic clones of the invention. Used as a reference to report nucleotide positions. Based on the present invention, H. Relatively small number of predicted open reading frames reported for the pylori genome The gene encodes the antigenic protein of the present invention or a cluster region forming the basis thereof. Can be understood.   Each cluster is from the 5 'end of the most upstream clone in the cluster Extends (i.e., extends) to the 3 'end of the most downstream clone in the cluster. Define a continuous DNA sequence. If the spreading sequence is incomplete, it is reported Filled with sequences from the H.pylori genomic sequence (TIGR website). For example, Using the reference to FIG. 10, the sequence defined by cluster 1 is: A clone containing the short (approximately 730 bases) genomic sequence joining the two clone sequences. At the 5 'end of Y92 (SEQ ID NO: 222) and at the 3' end of Y92 (SEQ ID NO: 223). Contains an ending array. The position of each clone in each cluster is The sequence numbers are shown in FIGS.   The present invention provides stringent hybridization in substantially purified form. Under fragmentation conditions, DNA fragment clusters: (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14) , (15), (16), (17), (18), (19), (20), (21), (22), (23) , (24), (25), (26), (27), (28), (29), (30), (31), (32) , (33), (34), (35), (36), (37), (38), (39), (40), (41) , (42), (43), (44), (45), (46), (47), (48), (49), (50) , (51), (52), (53), (54), (55), (56), (57), (58), (59) , (61), (62), (63), (64), (65), (66), (67), (68) and ( 69) An antigen capable of selectively hybridizing to a DNA fragment that extends over one of And a DNA fragment encoding   Preferably, the antigen coding region is a sequence that extends from the cluster to itself. You. Preferred polynucleotides are stringent in substantially purified form. SEQ ID NO: 43 (A22), SEQ ID NO: 38 (C1) under optimal hybridization conditions , SEQ ID NOs: 94, 95 (Y124A), SEQ ID NOs: 169, 172 (Y261A), SEQ ID NO: 253 (c5) SEQ ID NO: 20 (C7), SEQ ID NO: 51, 54 (B2), SEQ ID NO: 60 (Y104B) and sequence No. 98 (Y128D) selectively hybridizes to a DNA sequence selected from the group consisting of A DNA fragment encoding the H. pylori antigen.   Thus, the clusters disclosed herein represent non-licensed H. pylori antigen coding sequences. Reacts with random collection and H.pylori immunopositive samples And the resulting antigens are useful in a variety of diagnostic applications.   3.Isolation and mapping of native antigen   Experiments performed in support of the present invention are based on proteomics apro- Using several different native antigens of H. pylori (Table 6) Identification.   Disclosed herein and utilized for the isolation and identification of more than 20 H. pylori antigens A summary of the proposed proteome methodology is provided in FIG. 7 (right). Referring now to this figure, bacterial proteins are identified by two-dimensional (2D) electrophoresis. Separated. The immunoreactive spots (ie, Western blots) And reactive with pooled sera from patients infected with pylori). Endoproteolytic digestion, chromatography, and Spectrometry (e.g., flight mass spectrometry matrix Assist laser desorption time (MALDI-TOF). Box A Electrospray mass to determine intact protein mass 4 illustrates the use of spectrometry. Box B shows the number and mass of Lys-C peptides. 4 shows the use of MALDI-T0F mass spectrometry for evaluation. Box C The isolated Lys-C peptide is evaluated chromatographically and later sources MALD to provide sequence information by decay (if the peptide is pure)  Shows the use of I-MS. Box D evaluates peptides via collisions that induce dissociation. And electrospray mass spectrometry for sequencing Indicate use. These procedures are demonstrated by experiments performed in support of the present invention. As described, a conventional clonary for deriving genomic data corresponding to H. pylori antigens Alternatively or in parallel with the data obtained from the coding technology. Follow The antigen is highly immunogenic and is described in Examples 7-9, and Can be isolated and characterized.   To obtain soluble H. pylori protein, pelleted H. pylori is typically Typically, it is dissolved in a French press at> 10,000 PSI, followed by centrifugation. U. Alternative lysis methods include mechanical douncing or surfactant destruction. No.   Antigens are generally obtained from whole lysates as follows. H.pylori Fractionation of soluble proteins is performed by SDS-PAGE, preferably by two-dimensional electrophoresis. (O'Farrell, 1975; O'Farrel et al., 1977).   Isoelectric focusing gel separation is the most important method for performing typical two-dimensional electrophoretic separation. Performed first (first dimension gel). For isoelectric focusing, for example, acrylamide / On bisacrylamide gel,CURRENT PROTOC0LS IN PROTEIN SCIENCEYu Knit 10.46, as described in John Wiley and Sons, Inc., New York (1996) Is performed at the appropriate voltage-hours determined. Then the final tube gel The pH gradient is measured by a surface pH electrode. Protein components in the sample mixture Undergoes a first separation by pl values, as shown on the horizontal axis in FIGS. The first dimension separation depends on the nature of the protein to be separated, under acidic conditions (FIG. 2). ) Or under basic conditions (FIG. 1).   Next, a second dimension, size-based polyacrylamide slab gel separation, This is done for further separation of proteins based on molecular weight. Suitable molecular weight mer Kerr is typically used to determine the molecular weight corresponding to the protein to be eluted. May be added to the gel. Detection is typically by Coomassie blue or silver staining. Done. In some cases, silver staining is significantly more sensitive and Silver staining may be preferred as it can be used to detect small amounts of proteins .   FIGS. 1 and 2 show the H. pylori antigen (Roost H. pylo) commonly obtained as described above. ri Using positive and negative serum pools, respectively, Is blotted). The numbers in each figure are H.pylori protein Corresponding to the spot indicating. These tampers, defined by the spot number, Properties of the proteins (including approximate molecular weights and pI values) are summarized in Table 6. Poly Peptides were tested against anti-H.pylori antibodies in Roost pool sera. Were confirmed to be immunoreactive. Identical spots indicated numerically on the gel Sex was determined by various protein sequencing techniques described in Example 9.   Immunoreactive spots were cut from the gel for further identification of these peptides. Extracted, digested, and sequenced. As above, native H.pylori anti The original was analyzed by N-terminal sequencing, liquid chromatography-mass spectrometry, And internal sequence determination by amino acid-specific chemical cleavage followed by Edman sequencing Further characterized by a combination of sequencing methodologies, including (Example 9). Was.   The internal amino acid sequence can be determined using various site-specific cleavage reagents (eg, Aldehyde (OPA) / cyan bromide (CNBr), hydroxylamine, formic acid, and BNP S-skatole), which are cleaved as follows Perform: CNBr (cleaving the C-terminal of methionine), BNPS-skatole (trypto Cleaves the C-terminus of the fan), formic acid (cleaves the Asp-Pro peptide bond), Roxylamine (which cleaves Asn-Gly bonds) and OPA (which are secondary amines and primary And enzymatic reagents are, for example,End protea Ze Lys-C ,Endoproteinase ASP-N,Endoproteinase GLU-C,andG Lipsin And each of these cuts as follows:End protea Ze Lys-C (Cleaving the C-terminal of lysine),Endoproteinase ASP-N(Asparagus Truncates the N-termini of formic acid and cysteic acid),Endoproteinase GLU-C (Cleaves the C-terminus of glutamic acid), andTrypsin(Arginine and Li Cut the C-terminus of the gin).   The N-terminal sequence corresponding to each spot was determined (Table 6). The above, and The two-dimensional blot (FIGS. 1 and 2) utilizes the approach described in ) Were identified as shown in Table 6.   Referring to the low pH blot shown in Figure 2, spots 9, 11, 12, 13, 15, 16 (major and minor) and 17 presented unique antigens. Spot 9 corresponds to the recombinant protein expressed by ORF2 of clone a1 Spot 12 shows the native H. pylori antigen Shows the native antigen corresponding to the encoded antigenic protein.   Looking at the high pH western blot in FIG. 1, spot 9 (major and minor) -) 10, 12, 13 and 15 (major and minor) Show. The relationship between basic spot 15 and clones Y104 and d7 is described above. Discussed and also illustrated in FIG.   Selected Lys-C digested H.pyl corresponding to the positive spot of Wetane The mass spectral profiles of the ori proteins are provided in Tables 11 and 12. You. The mass “fingerprint” of the peptide digest was then Vs. predicted protein from the database (details provided in Example 12). As a basis for comparison, the selected H. pylori antigens described herein Used to further confirm identity.   The proteomic approach described above describes the genetic diversity of H. pylori To analyze acute and chronic infections (particularly gastroduodenal pathology and H. pylo test antigen-antibody responses between ri-related diseases (with components that may be autoimmune) Useful to In addition, two-dimensional gel electric swimming combined with MALDI-TOF MS Kinetic and in situ proteolytic digestion can be used to rapidly identify H. pylori antigens And for rapid screening of preferred vaccines and diagnostic candidates To provide a very sensitive technology for.   4.Expression and purification of antigenic polypeptides and their respective antibodies Preparation of     a.Expression and purification of antigenic polypeptide. Recombinant antigenic peptide of the present invention Means differential precipitation, molecular sieving chromatography, ion exchange chromatography -, Isoelectric focusing, gel electrophoresis and affinity chromatography Can be purified by standard protein purification procedures.   The H. pylori antigen according to the invention has the following cluster antigen sequence: SEQ ID NOs: 340-468 Comprises at least six consecutive amino acids contained within one of the following. For a specific example The H. pylori antigen has the following SEQ ID NOs: SEQ ID NOs: 2, 4, 5, 7, 9, 10, 12, 1 4, 17, 21, 25 to 28, 36, 37, 39, 44, 48, 55, 59, 61, 69, 249, 250, 252, 25 Choose from one of 4, 256, 258, 260-263, 265-269, 323, 324, and 550-554 At least six consecutive amino acids contained within the polypeptide sequence obtain.   Preferably, the H. pylori antigen is a poiy selected from the group consisting of SEQ ID NOs: 555-602. Corresponds to at least six consecutive amino acids contained within the Here, these sequences represent exemplary expressed proteins corresponding to the H. pylori antigen . Still more preferably, the H. pylori antigen has the following sequence: SEQ ID NO: 44 (A22) SEQ ID NO: 39 (C1), SEQ ID NO: 568 (Y124A), SEQ ID NO: 557 (Y261A), SEQ ID NO: 254 (C5), SEQ ID NO: 21 (C7), SEQ ID NO: 55 (B2), SEQ ID NO: 61 (Y104B), SEQ ID NO: No. 573 (Y128D) by at least six consecutive amino acids contained in one of them. Stipulated.   The polynucleotide sequence encoding the antigen of the present invention can be obtained from plasmid p-GEX (implementation Example 5) or various derivatives thereof (pGEX-del65). Plasmi PGEX (Smith et al., 1988) and its derivatives are derived from the protein glutathione-S-tra Cloned insert fused in-frame to a transferase (sj26) Express the polypeptide sequence of the product. One vector construction (plasmid pGEX-hisB In), the amino acid sequence of 6-histidine is replaced with the carboxy terminal of the fusion protein. Introduced to the end.   Various recombinant pGEX plasmids can be transformed with the appropriate E. coli strain and fused. Synthetic protein production was measured using IPTG (isopropyl-thio- Galactopyranoside). Then the solubilized recombination The fusion protein is obtained using Ni-NTA ++ affinity chromatography. It can be purified from cell lysates of induced cultures (Example 5).   The insoluble fusion protein expressed by the plasmid is an immobilized metal 6 M urea by ion affinity chromatography (Porath, 1992) Or 6M guanidium isothiocyanate (both of which are soluble in proteins) (Which is useful for purification). Alternatively, pGEX-GLI or its Insoluble protein expressed in derivatives of the protein removes soluble protein Centrifugation followed by solubilization of insoluble proteins, and ion-exchange chromatography Standard chromatography, such as chromatography or size exclusion chromatography Using Raffy methodology, and combinations of other such methods known in the art. And can be purified.   β-galactosidase fusion protein (eg produced by λgt11 clone The fusion protein is used for affinity chromatography. Or with anti-β-galactosidase antibody bound cell lysate to the surface It can be easily isolated by passage over a solid support.   The present invention provides for the generation of the H. pylori antigen coding sequence and coding region in a suitable host. Including the expression control element enabling the expression, the above λgt11 or pGEX vector Such expression vectors are also included. Control elements are typically promoted Directing transcription, transcription initiation codons, translation and transcription termination sequences, and insertion into vectors. Includes an insertion site for insertion.   DNA encoding the desired antigenic polypeptide can be obtained in a suitable host system. It can be cloned into any number of commercially available vectors to effect expression of the peptide. You. These systems include, but are not limited to: Baculoy Ruth expression (Reilly et al., 1992; Beames et al., 1991; Pharmingen, San Diego, CA; Clo ntech, Palo Alto, CA), vaccinia expression (Earl et al., 1991; Moss et al., 1991), Expression in fungi (Ausubel et al., 1988; Clontech), expression in yeast (Gellissen 1992; Romanos et al., 1992; Goeddel et al., 1990; Guthrie and Fink, 1991), Expression in mammalian cells (Clontech; Gibco-BRL, Ground Island, NY), eg For example, a Chinese hamster ovary (CHO) cell line (Haynes et al., 1983; Lau et al., 198) 4: KaufTnan, 1990). These recombinant polypeptide antigens are directly expressed Or expressed as a fusion protein. The amount of the sequence expressed in the culture medium A number of features can be manipulated in the expression vector, such as leader sequences that promote ligation. You.   Expression of large polypeptide antigens is described in Example 5. Some long anti The original clone sequence is described in Example 5 and shown in Tables 3a and 3b. Cloned into an expression vector and successfully expressed in E. coli .   Expression in a yeast system has the advantage of a commercially available product. Vaccinia and CHO Recombinant protein production by cell lines has the advantage of being a mammalian expression system. Sa In addition, cottonsia virus expression has several advantages, including: (i ) Wide host range; (ii) reliable post-transcriptional modification, processing, foldy , Transport, secretion, and assembly of recombinant proteins; (iii) relatively soluble High level expression of recombinant proteins; and (iv) broad ability to adapt to foreign DNA .   H. pylori polypeptide produced by a recombinantly expressed polypeptide The source is typically isolated from the lysed cells or culture medium. Purification is salty Chromatography, ion exchange chromatography and affinity chromatography. Including, but not limited to, methods known in the art. Immuno-affinity chromatography The chromatography was generated based on the H. pylori antigen identified by the method of the present invention. This can be done using antibodies that are used.   The resulting DNA coding region may be a fusion protein or an isolated polypeptide. As an alternative, it can be expressed recombinantly. In addition, the amino acid sequence is An adult machine (Applied Biosystems, Foster City, CA) or “PIN” technology (Applied Bi osystems) can be readily chemically synthesized.   Antigens obtained by any of these methods can be used for antibody production, diagnostic testing and And vaccine development.   An exemplary amino acid sequence corresponding to the expressed H. pylori antigenic protein is Provided herein as SEQ ID NOs: 555-602.   b.Antibody production. In another aspect, the present invention relates to a polypeptide antigen of the present invention. Specific antibodies. Antigen obtained by any of these methods Can be used directly for the production of antibodies, or Can be combined. Many such carriers are known in the art and are commercially available. (Eg, Pierce, Rockford, IL). Typically, to prepare antibodies A host animal, such as a rabbit or goat, is a purified antigen or fusion protein. Immunized with antigen. The hybrid or fusion protein is a glutachi Other proteins, such as on-S-transferase or β-galactosidase It can be produced using various coding sequences from quality. The serum or plasma of the host Collected at appropriate time intervals, and this serum is Tested on the body. These techniques are compatible with all immunogens described herein. Is equally applicable to sexual sequences.   The gamma globulin fraction or IgG antibody of the immunized animal can be Monium precipitation or DEAE Sephadex chromatography, affinity Known to those skilled in the art for chromatographic or polyclonal antibody production Can be obtained by using other techniques.   Alternatively, the purified antigen or fusion antigen protein is a monoclonal antibody Can be used to produce Here, spleen or phosphorus from the immunized animal The papule is removed and immortalized by methods known to those skilled in the art, or Used to prepare hybridomas. Producing human-derived hybridomas To do so, a human lymphocyte donor is selected. H.pylori infected Can serve as a suitable lymphocyte donor. Lymphocytes from peripheral blood Can be isolated from the sample. Epstein-Barr virus (EBV) is a human lymphocyte Suitable fusion partners can be used to immortalize It can be used to make hybridomas. Virus-specific polypeptide The first in vitro sensitization used was also used to generate human monoclonal antibodies. Can be used. Antibodies secreted by immortalized cells can be, for example, ELISA Or by using Western blotting (Ausubel et al., 1988) Screened to determine clones that secrete sex antibodies.   The purified polyclonal or then directed against the H. pylori antigen Monoclonal antibodies are directed to the presence of the antigen, as described in Harlow et al., 1988. Used in any of a number of standard immunoassay formats to detect Can be One exemplary assay format is an antigen capture sandwich assay . In a typical sandwich assay, antibodies are immobilized on a solid support. Next H. pylori-infected samples (fecal, plaque, gastric biopsy, biopsy samples The culture suspension) is reacted with the immobilized antibody, followed by H. pylori (eg, total lysis). Incubation with different antibodies directed against Reacted with a secondary antibody having a porter label. Detection of the label in the test area 2 shows the presence of H. pylori antigen in test samples. The above assay is Representative of any antigen-based assay based on an antibody prepared as H. For early detection of H. pylori antigen in samples suspected to be infected by pylori Useful.   5.ELISA And protein blot screening   The H. pylori antigen is typically a plaque immune screen as described above. First identified, expressed, and purified (see previously described). Sea urchin). The antigen is then purified by ELISA or protein using the isolated antigenic peptide. Alternatives such as protein blot assays (Western blots) ) Using immunoassays to test for large amounts of antisera suspected of being H. pylori positive And are quickly screened. Then, the antigen polypeptide fusion protein Is usually for β-galactosidase or glutathione-S-transferase. By affinity chromatography on the fusion partner, such as Isolated as described above. Alternatively, the antigen itself is an antibody raised against it (See below).   The general ELISA assay format is as described in Harlow et al., (1988). Can be used for Purified antigen polypeptide or fusion polypeptide containing the antigen of interest Peptides bound to a solid support (eg, a multiwell polystyrene plate) Is done. The biological fluid to be tested (eg, serum) is diluted and added to the well Is done. After sufficient time for the antibody to bind to the immobilized antigen, the serum is Washed away from. Each labeled reporter antibody is labeled with an appropriate Added to the wells: purified antigen polypeptide or fusion polypeptide containing antigen Wells containing antibodies that bind to the peptide are detected by a positive signal You.   One, any, some or each of the polypeptide antigens of the invention The format for a representative protein blot analysis used is shown in Example 6. It is. General protein blotting methods are described by Ausubel et al., (1988). Is described.   In Example 6A, the antigenic protein expressed by clone dHA22.8 (A22) Quality as well as multiple serum samples (both pooled serum and individual samples) Was used for screening. Produced from recombinant clone dHA22.8 A high percentage of serum reacting with the antigen indicates that it is a dominant epitope, And it shows that it is suitable as an H. pylori infection marker. Example 6A The results shown indicate that H. pylori positive antisera from different sources Demonstrates immunoreactivity with the polypeptide antigen. Similar results were obtained with the clone A recombinant protein expressed by dHC1S.11 (C1) is described.   As described in Example 6B, further experiments performed in support of the invention Data from serum panels using both single antigens and combinations of antigens For reliable and general use in the detection of H. pylori infection Preferred antigens include, but are not limited to, the following: A22, C1, Y12 4A, Y261A, c5, C7, B2, Y104B, and Y128D.   These antigens are derived from H. pylori based on favorable sensitivity and selectivity characteristics. It is effective as a serological marker for detecting active infections.   In Example 8, the native protein from H. pylori was in the “Roos” pool It is shown to be immunoreactive with anti-H. Pylori primary antibodies obtained from serum.   The above results indicate that the polypeptide antigens of the present invention are H.pylo Rapid detection of panel sera samples suspected of H. pylori infection for ri detection Demonstrates that it can be screened. A. Production of protective antibodies, vaccines and protective immunity   a. Protective antibodies. Protected antibodies can be identified, for example, using animal model systems (DuB ois et al., 1996). Polyclonal or monoclonal to identify protective antibodies Antibodies are raised against an antigen of the invention, wherein the antigen is cholera toxin (CT). It can be used as a binding immunostimulatory component arm. Then produced in this way Containing infectious H. pylori prior to infection of cell cultures or animals using purified antibodies The inoculum (eg, serum) is pre-treated. Protect cell cultures or animals from infection Assess the performance of a single antibody or antibody mixture. For example, cell cultures and animals In the absence of antigen and / or nucleic acid production acts as a screen . Furthermore, in animals, the absence of H. pylori disease signs, such as the urea respiration test (U Elevated carbon dioxide / ammonia levels in (BT) also indicate the presence of protective antibodies. Show. Urea breathing test ingested¹³C or¹⁴C urea and radioactive carbon dioxide Utilizing the action of the urease enzyme of H. Pylori, which decomposes into ammonia, Measure carbon dioxide.   Animal models for investigating H. Pylori infection include: (i) Gnotobiotic Newborn piglets (easily infected by H. pylori of human origin, but good for short-term studies) (Ii) months (mouse) or years (ferret), colonies A mouse or ferret capable of forming H. pylori, and (iii) H. pylori, Certain domestic cats (DuBois et al., 1996). H. pylori is found in gnotobiotic piglets Ulcers in mice using H.pylori and THE SYDNEY STRAIN Awake and mouse strain SJL. C3H / HZ. DBA, C56BL / b, and Ba1b / C odors Cause gastritis. A rhesus monkey infection model has also been developed (DuBois Et al., 1996).   Alternatively, convalescent sera can be screened for the presence of protective antibodies, These sera were used to identify H. pylori antigens that bind to the antibody. Was. The identified H. pylori antigen is then produced recombinantly or synthetically. The ability of the antigen to produce protective antibodies is tested as described above.   b.vaccine. After the initial screening, you can save either alone or in combination. Antigens identified as capable of producing protective antibodies can be used as vaccines to inoculate test animals. (Described in more detail below). The animals are then infected with H. pylori infection. Arranged. Infection protection is for animals that produce antibodies that protect animals from infection Show the ability of In addition, the use of animal models allows the identification of antigens that activate cellular immunity Enable.   Challenge with bacterial preparations (e.g., infected serum) in animal model studies A protective immune response in response to (i) protects the animal from infection, or (ii) ) Prevent disease development.   A vaccine is prepared from one or more of the immunogenic polypeptides identified herein. Can be made. Many H. pylori polypeptides of the present invention (e.g., spot 15) Present in pooled sera from many confirmed H. pylori positive donors Has been shown to be extremely antigenic (ie, highly reactive) with respect to antibodies I have. In a typical screening method, the intensity of color development is determined by antigen-anti-H. Pylori Shows the strength of antibody interaction (binding affinity).   Proteins expressed by two clones dHA22.8 (A22) and dHC1S.11 (C1) Representative serum paneling results (Example 6) for these recombinant proteins It indicates that both qualities are highly immunogenic. Produced by clone dHA22.8 Proteins were sourced from H. pylori positive pool serum source, Roost pool and SFA  Reacts with antibodies present in both 001 and H. pylori negative samples Did not show any cross-reactivity with the antibodies present. Similar results for the clone The protein produced by dHClS.11 (C1) was observed.   Currently testing antigen reactivity with individual H. pylori positive serum samples The antigenic proteins expressed by each of clones dHA22.8 and dHC1S.11 Protein reacts with anti-H. Pylori antibody in 100% and 95% of the sample, respectively. That is, the ability of the antigen of the present invention to detect H. pylori infection, and 5 shows the ability to provide components for a vaccine.   Preferred antigens for use in vaccine compositions are described in Example 13. Illustrative antigens enhance their potency for a prolonged period of antimicrobial treatment after H. pylori. Maintain a long-lasting antigenic response, as indicated by the sustained presence of the maintaining antibody It is an antigen that can be activated. On this basis, for use in vaccine compositions Particularly preferred antigens are Y139, Y146B, Y175A, and A22, Y184A, Z9A, Y261Ains And Y146B.   Among the only H. pylori peptides disclosed herein, the other peptides are: H. pylori can be identified as useful in such vaccines. Individual trials The test peptide is prepared in a suitable carrier (eg, adjuvant) at a suitable concentration for injection. (Eg, 5-500 mg / ml). One suitable trial for vaccination The test animals are Rheus monkeys as detailed in DuBois (DuBois et al., 1996). animal Is typically in an amount between 0.2 and 2.0 mg / kg body weight, e.g., for oral, intramuscular injection, or Or vaccination by intravenous injection. After an appropriate period (eg, two weeks) Animals can be boosted by a similar route, and typically in the same amount. After a few weeks, the animals are transferred to a known animal, for example as described in DuBois (DuBois et al., 1996) Challenge with H.pylori in style. As an example, inoculated animals can be given specific immunogenicity. To test the potential vaccine effect of the fragment, H.pylori (e.g., H.p. about 10 of ylori⁸~Ten⁹CFU suspension, 1 ml). Chare at H.pylori After administration, the subject was treated with H.pylori-specific blood as described in DuBois (DuBois et al., 1996). By endoscopy, histological examination, microbiological methods, and / or measurement of serum IgG Therefore, it is typically monitored.   The level of infection in the vaccinated animal then assesses the degree of protection. To the level of infection in control animals. H.pylori infection These peptides, which provide a measurable degree of protection, include dHA22.8 (A22), alone or other peptide proteins, such as dHC1S.11 (C1), and spot 15 peptide Either in combination with a Kuching agent is suitable for vaccine use.   The selected peptides are formulated according to known vaccine formulations. Typically, The peptide can be transferred to a carrier protein (eg, keyhole limpet hemocyanin). Or human serum albumin) and / or a suitable adjuvant (eg, (If used, Freund's adjuvant). Vaccines, as described above, are preferred Or at peptide levels in the range of 0.2-2.0 mg / kg, using the conventional route, typically IM or Or by the IV route. If necessary, give one or more booster injections.   The specificity of the putative immunogenic fragment will determine its cross-reactivity with other related bacteria. By testing serum, other fluids, or lymphocytes from the inoculated animal Can be evaluated.   B.Synthetic peptide   Using the coding sequences of the H. pylori polypeptide antigens disclosed herein, Synthetic peptides corresponding to these polypeptides can be produced. Synthetic peptide It can be synthesized or prepared commercially using standard methods and equipment in the art (Appli ed Biosystems, Foster City CA).   Alternatively, the oligonucleotide sequence encoding the peptide may be an oligonucleotide A large coding sequence that can be synthesized directly by standard methods of tide synthesis The plurality of oligonucleotide fragments corresponding to the coding sequence Can be synthesized by a series of cloning steps involving tandem arrays of (Crea, 19 89; Yoshio et al., 1989; Eaton et al., 1988). The oligonucleotide coding sequence is a standard set It can be expressed by replacement procedures (Maniatis et al., 1982; Ausbel et al., 1988).   C. Immunoassay for H. pylori   One use for one, some, many, or each of the antigens herein The efficacy and efficacy of antibodies present in the serum of test subjects infected with H. pylori The use of an antigen as a diagnostic reagent for real detection, and thereby the test subject Provide instructions for infection in Single or combined in such a way A preferred antigen that can be used in either of the combinations is, for example, SEQ ID NO: 44 (A22), SEQ ID NO: 39 (C1), SEQ ID NO: 568 (Y124A), SEQ ID NO: 557 (Y261A), SEQ ID NO: 254 (c5), SEQ ID NO: 21 (C7), SEQ ID NO: 55 (B2), SEQ ID NO: 61 (Y104B), or SEQ ID NO: 573 (Y12 8D) or antigens derived therefrom. Or prefer New antigens, SEQ ID NO: 43 (A22), SEQ ID NO: 38 (C1), SEQ ID NOs: 94, 95 (Y124A), sequence No. 169, 172 (Y261A), SEQ ID NO: 253 (c5), SEQ ID NO: 20 (C7), SEQ ID NO: 51, 54 (B2) SEQ ID NO: 60 (Y104B), or SEQ ID NO: 98 (Y128D), or It is defined taking into account the incoming DNA coding sequence.   Alternatively, detection of antibodies present in the serum of test subjects infected with H. pylori Used to span one of the following DNA fragment clusters: (1), (2), (3), (4), (5), (6), (7) , (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (2O), (21), (22), (23), (24), (25), (26), (27), (28), (29), (30), (31), (32), (33), (34), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (50), (51), (52), (53), (54), (55), (56), (57), (58), (59), (61), (62), (62), (63), (64), (65), (66), (67), (68) and (69) or immunoreactive variants thereof. Preferably, the antigen coding region The region is the spanning sequence itself from the cluster.   Antigens of the invention detect H. pylori, as exemplified by the results of Example 6B Can be used alone or in combination with one another. The antigen of the present invention , Can be combined with diagnostic assays for other infectious agents.   In one diagnostic arrangement, the test sera contains the surface binding obtained by the method of the present invention. Reacts with a solid phase reagent having a combined antigen (eg, "Spot 15" antigen). Exemplary The antigens are A22 and C1, both of which show high sensitivity (Figures 65 and 66, as well as As shown in Example 6B). Bind anti-H.pylori antibody to reagent and wash After removal of unbound serum components by purification, the reagent was combined with bound anti-H. To bind the reporter to the reagent in proportion to the amount of pylori antibody, use a reporter-labeled antibody. Reacts with human antibodies. The reagents are washed again to remove unbound labeled antibody, Then, the amount of the reporter bound to the reagent is determined. Typically, the reporter For example, 5-bromo-4-chloro-3-indoyl-phosphate (BCIP) and nitrobutane Suitable Fluorescent or Chromogenic Substrates for Lutetrazolium (NBT) (Sigma, St. Louis, MO) Is an enzyme detected by incubating the solid phase in the presence of   Solid surface reagents in the above assays are polymerizable beads, dipsticks Protein on solid support materials, such as 96-well plates, or filter materials Prepared by known techniques for attaching quality. In general, these Methods typically involve chemically reactive groups (eg, activated carboxyl groups) on a solid support. Via a free amine group attached to a sil, hydroxyl, or aldehyde group) And non-specific adsorption of the protein to the support or covalent attachment of the protein. Alternatively, streptavidin-coated plates can be combined with biotinylated antigen. Can be used together.   The forming part of the present invention also provides an assay system or key for performing this diagnostic method. It is. The kit generally comprises a surface-bound recombinant antigen (e.g., Table 3a and And 3b, or representative clones summarized in Table 2. Antigen) or native H. pylori antigen (eg, as described above) A support having Table 6, and the antigens identified in FIGS. 1 and 2), and Includes a reporter-labeled anti-human antibody to detect surface-bound anti-H. Pylori antigen antibodies No.   In a second diagnostic arrangement, known as a homogeneous assay, binding to a solid support Antibodies produce some changes in the reaction medium that can be detected directly. This Known general types of homogeneous assays proposed so far include: (a) A pin-labeled reporter, where the antibody that binds to the antigen (B) a fluorescent reporter, which is detected by the change (broadening of the spin cleavage peak) Where binding is detected by a change in fluorescence efficiency or polarization, (c) the enzyme A reporter, where antibody binding triggers an enzyme / substrate interaction, and (d) Posome-associated reporter, where binding is performed by liposome lysis and encapsulated reporter Leads to the release of the rotor. The application of these methods to the protein antigens of the invention According to conventional methods for preparing the assay reagents.   In each of the above assays, the assay method involves testing the protein antigen with a test solid. Reacting with serum from the body and testing the antigen for the presence of bound antibodies Step. The testing step may include subject antibody (e.g., acute, chronic, or convalescent). The labeled anti-human antibody, and solid support as in the first method. Measuring the amount of reporter bound to the carrier, or a second method. Observing the effect of antibodies binding to homogeneous assay reagents as in May be included. Antigen capture assays as described above in Section D.2. Are also contemplated.   The following examples illustrate but are in no way intended to limit the scope of the invention. Absent.                              Materials and methods   1．General procedure   Synthetic oligonucleotide linkers and primers are compatible with commercially available automated oligonucleotides. Prepared using a nucleotide synthesizer. Alternatively, custom designed synthetic oligonucleotides Leotide can be purchased from commercial suppliers.   Standard molecular biology and cloning techniques are described in Ausubel et al., 1988; Sambrook et al., 19 89; and Maniatis et al., 1982, essentially as described previously.   Antiserum for screening and detection of immunoreactive protein antigens and Common procedures involving the use of antibodies are described in Harlow et al., (1988). The procedure was performed as described. Antibody screening of H.pylori genomic library Was performed by plaque immunoblot assay.   Similarly, Western blot assays were performed according to the manufacturer (Abbott, N. Chicago, IL; Genel. abs Diagnostlcs, Singapore) or as described in the art. Using standard techniques known in the art (Harlow et al., 1988).   2.Bacterial strain ATCC accession numbers 43504 (short antigen clone set) and 43526 (library 1 and A DNA library was generated using a commercially available H. pylori strain corresponding to 2). H.pyl Isolation of native protein produced by H. pylori from ori strain ATCC 43504 Used for Escherichia coli strain Y1088 for libraries 1 and 2, Y1089 and XLI-Blue (Stratagene, La Jolla, CA) were used for phage infection. Was the host. E. coli strain XL1-Blue, XLOLR (Stratagene, La Jolla, CA) Used for protein expression of the loaned gene.                                 Example 1 H.pylori Construction of λgt11 and ZAPII DNA libraries 1．Genomic DNA isolation   H.pylori (American Type Culture Collection, Rockville, MD; ATCC accession number 435 04) on a blood agar plate and incubate in a microaerobic environment at room temperature for 7 days. I was vate. Collect cells by scraping bacterial cells from 10 plates, Subsequently, it was washed once with phosphate buffered saline (Dulbecco's, Gibco BRL, Gaithers  burg, MD).   Genomic DNA was prepared (with slight modifications) as described in Ausubel et al., 1988. . Resuspend cell pellet from 5 plates in 510 μl TE (Ausubel et al., 1988) To it, 60 μl of 10% SDS and 30 μl of 20 mg / ml proteinase K were added. Suspension The liquids were mixed and subsequently incubated at 37 ° C. for 4-8 hours. 80 μl C in suspension TAB / NaCl (10% hexadecyltrimethylammonium bromide in 0.7M NaCl) And then mix the resulting solution and incubate at 65 ° C for 10 minutes. I did it. The solution was then extracted with an equal volume of chloroform / isoamyl alcohol. And spun in a microcentrifuge for 5 minutes. Transfer the separated aqueous phase to a new tube And precipitate the DNA by adding 0.6 volumes of isopropanol, followed by And centrifuged. Wash DNA pellet with 70% ethanol and dry briefly under reduced pressure And dissolved in 100 μl of distilled water. Next, the DNA solution was washed with DNase-free Rnas. e (Boehringer Mannheim, Indianapolis, IN) (Ausubel et al., 1988; Maniatis Et al., 1982), selectively degraded any RNA present in the sample.   2.DNase Digestion and DNA amplification   a.Short antigen clone library.H.pylori genomic DNA as above (1988) and Sambrook et al. (1989). ehringer Mannheim). Aliquots of digested DNA can be taken at various time points. Obtained. DNA digests were analyzed by preparative agarose gel electrophoresis. Desired Product bands containing a range of DNA (200-2000 base pairs) were excised from the gel, Then, according to the manufacturer's instructions, the “GENE CLEAN II” kit (Bio 101 Inc., LaJ (olla, CA) or MERMAID kit (Bio 101 Inc., La Jolla, CA) did.   To generate blunt ends, the recovered DNA fragment is E. coli Klenow fragment (Ausubel et al., 1988; Sa mbrook et al., 1989). Incubate the reaction mixture for 30 minutes at room temperature, followed by Extraction with chloroform / chloroform.   The resulting molecules are sequence-independent single plasmids by the method of Reyes (Reyes et al., 1991). Immer amplification (Sequence Independent Singie Primer Amplificatlon) (SISPA; Rey es et al., 1991). The following was added to the blunt-ended DNA: Phosphorylated SISPA (sequence independent single primer amplification) linker AB, SEQ ID NO: 63 and And a double-stranded linker consisting of SEQ ID NO: 64 (SEQ ID NO: 64 corresponds to SEQ ID NO: 63). (In the 3 'to 5' direction to SEQ ID NO: 63 as a sequence partially complementary to) , 2 μl 10 x ligation buffer (0.66 M Tris.Cl pH = 7.6, 50 mM MgCl_Two, 50 mM DTT, 1 mM MA TP) and 1 μl T4 DNA ligase (0.3-0.6 Weiss Units). Typically, DNA and The linker was mixed at a ratio of 1: 100. Incubate the reaction at 14 ° C overnight. Was. The reaction was then incubated at 70 ° C for 3 minutes to inactivate ligase. I did it. The unlinked linker can be attached to a Chromaspin column (Clo netech, Mountain View, CA) or Sephadex G spin column (Pharmacia, Piscataway) , NJ).   The linker-ligated DNA was then amplified by SISPA (Reyes et al., 1991). 1.5mM MgCl_TwoAnd 100 mM of 10 mM Tris-C1 buffer pH 8.3 containing 50 mM KCl (buffer A) μl of the linker-ligated DNA preparation, a 2 μM plate having the sequence shown as SEQ ID NO: 63. Primers, 200 μM each of dATP, dCTP, dGTP, and dTTP, and 2.5 units Amplitaq DNA polymerase (Applied Biosystems Division, Perkin Elmer, Fost er City, CA) was added. Heating the reaction mixture to 94 ° C. for 30 seconds for denaturation, Cool to 50 ° C for 30 seconds for primer annealing, then 72 ° C for 0.5-3 minutes Until primer extension with Taq polymerase. Continuous heating, Amplification reactions, including cooling and polymerase reactions, are performed on Perkin-Elmer Cetus DNA Repeated an additional 25-40 times with the help of the Marcycler (Mullis, 1987; Mullis et al., 1 987: Reyes et al., 1991; Perkin-Elmer Cetus, Norwalk, CT).   After the amplification reaction, the solution was then extracted with phenol / chloroform and doubled Precipitated with an amount of ethanol.   3. DNA cloning into Lambda vector   a. Short antigen clone library. Linkers used in ligation to DNA Contained an EcoRI site that allows for direct insertion of the amplified DNA into the λ vector. (gt11 or ZAP II, Stratagene, La Jolla). Λ BEC like purchasing from manufacturer The digester is digested at the EcoRI end and treated with alkaline phosphatase to terminate the 5 ′ end. The phosphate was removed and prevented self-ligation of the vector. 1.Amplified DNA from B. , Digested with EcoRI, and short nucleotides were removed by gel filtration. Next The digested DNA preparation was then purified using T4 DNA ligase to λgt11 or “ZAPII” (Boehringer Mannheim, Indianapolis, IN). The binding reaction conditions are as follows: As follows: 1 μl vector DNA (Stratagene (La Jolla, CA) 0.5 mg / ml); 0.5 or 3 μl of PCR amplified insert DNA; 0.5 μl of 10 × ligation buffer (0.5 M Tris-HCl, pH = 7.8; 0.1 M Mg Cl_Two; 0.2M DTT, 10mM ATP; 0.5mg / ml bovine serum albumin (BSA)), 0.5μl T4 DNA Ligase (0.3-0.6 Weiss units) and distilled water to a final reaction volume of 5 μl. Ligation reaction , Incubated at 14 ° C overnight (12-18 hours). Connect ligated DNA to λ DNA package Packaged by standard procedures using the packaging system (GIGAPAK, Stratagene, La JolIa) And then plated the various dilutions to determine the titer.   The titer and recombination rate of the DNA-inserted phage library are based on standard X-gal blue / white Determined by the assay (Miller, 1994; Maniatis et al., 1982). Typically, recombination The library titer is 1.5 × 10^Four~ 3 × 10⁶It was in the range of PFU / ml.   The rate of recombination in each library is also large Selection and isolation of the corresponding phage DNA. Can be The polymerase chain reaction (Mullis, 1987; Mullis et al., 1987) Phage DNA isolated as plate, EcoR for DNA molecule as primer Performed using a λ DNA sequence from the λ sequence adjacent to the I insertion site. Then insert The presence or absence of is evident from gel analysis of polymerase chain reaction products .   b.λZAPII library (libraries 1 and 2). λZAPII library (H .pylori sample ATCC No. 43526) was prepared similarly, with the following exceptions: did. The ZAPII library was used for sequence independent single primer amplification (Sequence Indep endent Single Primer Amplification) Made.   Library 1 clone (shown in uppercase herein) contains EcoRI digested H .pylori DNA by direct ligation to the EcoRI site of the λ “ZAPII” vector. Made. The library 2 clones (shown in lowercase) were prepared using H. pylori genomic DNA Digestion with HindIII, blunt-end the HindIII fragment, and place the resulting molecule on top Obtained by ligation to SISPA primer AB as described. Then phosphorus The DNA linked to the car was treated with EcoRI and ligated to the EcoRI site of the ZAPII λ arm.   Unless otherwise indicated, ATCC Accession No. 43504 and ATCC Accession No. 43526 DNA insert gt11 and ZAPII phage library generated from H. pylori sample Lee, Genelabs Technologies Inc. Culture Collection, 505 Penobscot Dri ve, Redwood City, CA, 94063 or Genelabs Diagnostics PTE LTD Culture Co llection, 85 Science Park Drive # 04-01, The Cavendish, Singapore Science  Park, deposited at Singapore 118259.                                 Example 2 Immunoscreening of recombinant libraries   Endoscopy of the λgt11 and ZAPII phage libraries described in 1.C. Pools from 11 patients identified as H. pylori positive using (Referred to herein as "Roost pool" serum), or the four Hs identified as SFA001. of antigens that can be recognized by another pool of .pylori immunopositive serum samples Birth Lives were immunoscreened. λ short antigen clone library and And ZAPII library 2 were immunoscreened against Roost pool sera ZAPII library 1 was immunoscreened against serum pool SFA001 . The λgt11 library was pooled using the E. coli Y1089 bacterial plating strain. Plates while allowing the λZAPII library to be transferred to E. coli XL1-Blu. e Plated for plaque formation using bacterial plating strain.   The fusion protein expressed by the recombinant lambda phage clone Screening was performed using serum antibodies as described in subel et al. (1988).   Approximately 1.5 to 2 × 10^FourPlate with phage did. A nitrocellulose filter was overlaid on the plate overnight. Filter After washing with TBS (10 mM, Tris pH 7.5; 150 mM NaCl), AIB (TBS buffer containing 1% gelatin) Solution), and incubate with primary antibody 100-fold diluted in AIB. I was vate.   After washing with TBS, the filters were washed with a 1: 1000 concentration of alkaline phosphatase. Incubated with a secondary antibody consisting of conjugated goat anti-human IgG. Reactivity Plaques were washed with NBT (nitro blue tetrazolium salt; Sigma Chemical Co., St. Loul s, MO) using a substrate (eg, BCIP (5-bromo-4-chloro-3-indolylline) Acid)). Re-plate positive areas from primary screening And immunoscreened until a pure plaque was obtained. This screen The results of the tanning are shown in Tables 1a and 1b.NA = Not available   All the above sera were used for HelicoBlot 2.0 (Genelabs Diagnostics Western Blot) Product) and positively infected samples using this product's criteria. I found it to be a pull.   SFA001 was used in HelicoBlot 2.0 to produce crude lysates from Helicobacter lysates. 4 persons showing strong reactive band in Western blot format using the digest antigen preparation Is a pool (plasma pack) of sera from all donors.   NAA121 was used in HelicoBlot 2.0 to obtain crude lysates from Helicobacter lysates. No reactive band in Western blot format using digest antigen preparation A pool of sera (plasma packs) from four donors. Example 3 Characterization of immunoreactive λgt11, Z and λZAPIIY library clones   1.Immunoreactive recombinant λ clones (short antigen clone set, H. pylori PCR amplification, purification and cloning of DNA inserts from roning families Y and Z) And sequencing   Representative sequences encoding specific antigens from the H. pylori cloning family The clone was isolated by PCR amplification. Cloned with .ASM extension Sequence was completely sequenced; sequences with the .SEQ extension were partially sequenced did.   Insert the DNA insert of the immunoreactive recombinant λ clone into the EcoRI clonin vector PCR amplification was performed using primers corresponding to the λ arm sequence adjacent to the DNA site. Polymerase chain reaction using each immunoreactive clone as a template Thus, amplification was performed. For the λgt11 clone, gt11F (SEQ ID NO: 65) and And gt11R (SEQ ID NO: 66) primer were used. For λZAPII clones, Imers T3 (SEQ ID NO: 67) and T7 (SEQ ID NO: 68) were used.   The resulting amplified fragment is then purified on an agarose gel and Eluted (Ausubel et al., 1988). PCR products can be transferred to WIZARD PCR PREPS (Promega, Mad ison, WI) or `` CHROMASPIN '' column (Clonetech, Mountain View, CA). To remove primers and other components. Then, the purified insert DNA was directly subjected to sequencing. In some cases, the insert DNA is first Subcloning into a TA cloning vector (Invitrogen, San Diego, CA) It was then sequenced.   Sequencing for DNA inserts was performed using Perkin Elmer Applied Biosystems 373. A DNA sequencer (Perkin Elmer, Applied Biosystems Division, Foster Ci) ty, CA) using the manufacturer's protocol (dideoxy chain terminator Performed according to sequencing methodology, Sanger et al., 1977).   The sequence data is shown in the attached sequence listing. Table 2 below shows the cloning families -Immunogenic proteins corresponding to Y and Z (ie, H. pylori positive blood Recombinant H. pylori nucleic acid encoding a protein that is shown to be reactive with 1 shows a partial overview of the leotide sequence. The EcoRI site of the linker is typically The corresponding sequence shown has been deleted.   Clone Y104-1 (SEQ ID NO: 60) contains the entire d7 clone and has a 36K peptide And all of the spot 15 peptides as shown in FIG.   Production of the expressed antigenic proteins and subsequent purification thereof was performed in Example 5A and And 5B in general. Clone name, expression, purification and paneling A tabulated summary of the data is shown in Table 3b. Overview of immunoreactivity of various recombinant antigens The point is shown in Table 5b. Expression profiles were obtained and H.pyl like "Roost" Confirmed immunoreactivity of different clones against ori-positive pooled sera Was done.   Briefly, amplification products corresponding to specific ORFs are typically cloned into pGEXhisB vectors. And expressed in E. coli. Then, after determining the size of the expression product, Confirm immunoreactivity (eg, using Roost pool serum).   2.Sequence comparison   The sequence was changed to "GENBANK" (version 1998, 1996), EMBL database and dbEST (N ational Library of Medicine) at both the nucleic acid and amino acid levels. Compared. The search programs FASTA, BLASTP, BLASTN, and BLASTX (Altschul et al., 19 90) is the majority of polynucleotide sequences encoding the antigens disclosed herein. Before the H. pylori genome (Tomb et al., 1997) was published, Not be more than 95% identical to any sequence contained in the database Was shown.                                 Example 4 Characterization of immunoreactive lambda library 1 and library 2 clones   1.H.pylori Subcloning, purification, identification, and sequencing for antigen   Purify additional immunopositive clones as described in Example 2 above And analyzed for DNA insert size. And of the above-mentioned third embodiment As described for protein size. ZAPII clone (Stratagene, La Jolla, Rescue from phagemid to plasmid (according to U.S.A.A. protocol); The cut insert DNA is cut out using EcoRI and placed on an agarose gel. To determine the size. Then, slightly longer versions of T3 and T7 (GD60 and GD61 corresponding to SEQ ID NO: 231 and SEQ ID NO: 232, respectively) Used as primer for determination. These libraries are available in the library Lee 1 "series (EcoRI digestion) (ie, clones A3, A22, B2, B9, B17, B23 , C1, C3, C7), and "library 2" series clones (HindIII cut) (I.e. clones a1, a3, a5, b5, b8c7, c2, c5, c13, d5, d6, d7, d11, f11 , E6, f3, f8, g2, g9, g11, and k4); and EcoRI / Xbal cleavage line. It also corresponds to clone G1a from Bally. Many clones contain protein Was determined to be larger than the estimated code region.   Specific antigen clones derived from H. pylori library 1 and library 2 clones The nucleotide sequence was subcloned. Typically, this subclone is Fragmenting the corresponding genomic clone by restriction endonuclease digestion To produce specific subfragments. This is called "GENE CLEAN I I "kit (Bio 101 Inc., La Jolla, CA) or" MERMAID "kit (Bio 101 Inc. , La Jolla, CA). Then, the obtained DNA fragment is Expression vectors (i.e., PB / Bluescript (SK) vectors (Stratagene, La Jolla , CA)). Expression clones were cloned into 0.5 mM IPTG (isopropylthio-β-D-gal For 4 hours at 37 ° C. and whole bacterial cell lysate Electrophoresis. The immunoreactivity of the expressed protein was measured by Roost, SFA001, And pooled sera of NAA001. Alternatively, the first genome `` Erase-a-Base ''^TMUsing the kit (Promega, Wisconsin, U.S.A.) They were subjected to nested deletions. And the resulting smaller nested clones Tested for immunoreactivity, the location of the encoding antigen was located.   The immunoreactive DNA region is then sequenced and the open reading frame The position was determined. Table 3a below, unless indicated as a β-galactoside fusion product And for each of the clones in 3b, the expression of the coding region was at λgt11. The corresponding H.pyl, not by the promoter of the β-galactosidase gene It was determined to be driven by the ori promoter.                          Library 1 clone   2.Clone A3   Subclone A3CON3 derived from a genomic clone having a size of about 6.0 kb, Obtained from nested deletion clones. The corresponding nucleotide sequence (1878 bp) is It is designated herein as No. 16. Open reading frame (ORF) Ocides from 399 to 1743, and 448 amino acids (as counted from the first methionine) (443 amino acids in total). Open reading The protein sequence corresponding to the translation of Lame (ORF) 399-1743 is set forth as SEQ ID NO: 17. Shown herein.   3.Clone A22   The first genome with an insert size of about 2.2 kb from subclone A22210DMIC Produce β-galactosidase fusion protein in E. coli from clones did. This open reading frame is shown in Tables 3a and 3b below. (ORF) spans nucleotides 12-599 of SEQ ID NO: 43, where bases 12-121 Corresponds to the β-galactosidase fusion peptide and bases 122-599 are unique A22 anti- Encodes a native sequence. The translated sequence for the corresponding protein is set forth in SEQ ID NO: 44. As shown.   4.Clone B2   B3C19 was obtained from the 5.0 kb insert in genomic clone B3 as follows. Subclone. Cut out this insert, digest with DNase, Then, treatment with T4 DNA ligase and Klenow enzyme was performed to create blunt ends. Next The blunt-ended short piece of DNA was then kinased with the A / B linker. -(Linker A corresponding to the top strand of AB SISPA linker, SEQ ID NO: 6 3; Linker B corresponding to the bottom strand (bottom strand) of AB SISPA linker, SEQ ID NO: No. 64), PCR amplified with Primer A, and the amplified product was digested with EcoRI . The digested product was then ligated into EcoRI digested lambda vector ZAPII. B3C19 Is shown as SEQ ID NO: 51.   Based on the sequence of B3C19, primers GD77 (5 ′ primer, SEQ ID NO: 52) and G Using D80 (3 'primer, SEQ ID NO: 53) with genomic B2 DNA as template The B3C19 sequence was designed to walk back and forth. Sequence of clone B3C19 Was extended in both the 5 ′ and 3 ′ directions to give a B2 extended clone, B2197780 (SEQ ID NO: 5). 4) occurred.   Translation of the computer-generated protein of the B2197780 sequence is from amino acids 9-25 Generate a corresponding putative 291 amino acid protein with a putative transmembrane segment (Predicted using the "SOAP" program from "PCGENE"). Analysis results Based on the results, the Shine Dalgarno AGGA sequence should be located 8 bp before the ATG codon Was decided. This ATG codon at nucleotide position 278 is the first of this gene It appears to be a "met" amino acid. Protein translated for clone B2 The sequence is shown as SEQ ID NO: 55. The estimated molecular weight of the full size protein is 31 , 682. (i) between amino acids 91 and 92; and (ii) between amino acids 25 and 26 ("P CGENE ''-putative prokaryotic secretory signal sequence) with two potential cleavage sites I do. The pi of this protein is estimated to be 8.3.   5.Clone B9   Subclone B9.4C is the first genomic clone B9 (4.5 kb in. This is a 1.2 kb HindIII fragment obtained from (Sart).   By inducing genomic clone B9, 32 kd And 14kd in Roost pooled serum Produced an immunoreactive protein. B9 is digested with HindIII and some Hin The dIII subfragment was formed. This is added to each pBKS vector by subcloning. I did it. Protein production is induced in the resulting subclones and the corresponding The size of the protein to be used was determined.   Subclone B9.4C (corresponding to the 1.2 kb HindIII subfragment of genomic clone B9) Responded) produced a 32 kd immunoreactive protein. Nuku of subclone B9.4C The reotide sequence is shown as SEQ ID NO: 47; the translated protein sequence (ie, , Nucleotides 230-931 of SEQ ID NO: 47 are shown as SEQ ID NO: 48. SEQ ID NO: 47 Referring to, the AGGA Shine-Dalgarno sequence occurs at nucleotide 218 and is inverted. The first amino acid in the translated protein sequence is the GTG code for valine at position 230. Starts with Based on PCGENE calculation (CHARGPRO program) as above The estimated molecular weight of the resulting protein is 25.2 kd and has a pI of 10.35. Potential The cleavage site is between amino acids 225 and 226.   Based on FASTA sequence identity analysis of both nucleic acids and proteins, clone B9 , Seems to encode the 50A L1 protein of H. pylori.   6.Clone B17   Is B17CON4 (2006 bases) a genomic clone with an insert size of about 4.0kb? And subcloned. The corresponding nucleotide sequence is shown as SEQ ID NO: 24 . Open reading frame (ORF) corresponds to the following region of SEQ ID NO: 24 ORF1 (nucleotides 500 to 700); ORF2 (nucleotides 870 to 1406); ORF3 (nucleotide) And ORF4 (nucleotides 142-705). The corresponding translated tag The protein sequence is referred to herein as SEQ ID NO: 25 (B17ORF4), SEQ ID NO: 26 (B17ORF1), This is shown as SEQ ID NO: 27 (B17ORF2) and SEQ ID NO: 28 (B17ORF3). Nested deletion fruit Based on experiments, the immunoreactive protein appears to correspond to B17ORF4.   As described above, based on computer-generated protein translation analysis, B17ORF4, B17ORF2, and B17ORF3 have 20.9 kd, 20 kd, and 22.2 kd, respectively. Encodes a putative protein having a putative size. Immunoreactivity of this estimated size Genomic proteins are 22 kd and 23 kd doublets.   7.Clone B23   3.5 kb PstI subclone of the first genomic clone B23 (5.5 kb) immunoreactivity (One PstI site is from the vector pBSK). 3. The 5 kb subclone was further sequenced and the immune response presented in SEQ ID NO: 13 The sex coding sequence was determined. The nucleotide 1078 base pair sequence (SEQ ID NO: 13) Over the open frame (bases 3 to 1076). Translated TA The protein sequence is presented as SEQ ID NO: 14.   8.Clone C1   C1CON6V2 was derived from the first genomic clone with an insert size of about 4.0kb. Subcloned. The sequence information obtained from the exo-mung deletion clone has SEQ ID NO: 38 To present. Open reading frame (ORF) at nucleotides 868-1926 extend. Subcloning of antigen coding sequence from C1 clone into expression vector , And corresponding protein product characteristics are provided in Example 5 below. translation The assigned protein sequence is presented herein as SEQ ID NO: 39.   9.Clone C3   Immunoreactivity of the first genomic clone, C3 (2.9kb insert size) Several different subfragments of a genomic clone were used to determine the coding region. Were obtained and sequenced. Immunoreactive clones were EcoRI / Kpn 2.2kb It was determined to be present in the clone. Numerous subcloning experiments and The C3 DNA sequence is presented as SEQ ID NO: 15 based on the fragment.   Ten.Clone C7   Clone C7.2C is an EcoRI / HindIII subclone obtained from genomic clone C7. You. The first genomic C7 clone was an EcoRI clone of approximately 3.8 kb in size. It produces an immunoreactive protein with a molecular weight of approximately 30 kd.   Subclone C7.2C was obtained as follows. Genomic C7 clones using HindIII Was digested into individual fragments. Next, each DNA restriction fragment was Subcloning via any HindIII site into the The fragment was subcloned through the EcoRI / HindIII site. C The corresponding nucleotide sequence (616 bp) of 7.2C is presented as SEQ ID NO: 20 . Subclone C7.2C produces an immunoreactive protein, which is a genomic clone The same size as the protein produced by Bases 1-5 of clone C7.2C The protein sequence translated for 61 is presented herein as SEQ ID NO: 21. You.   11．Clone B8   Clone B8 (SEQ ID NO: 549) is a genomic clone with an insert size of 3.5 kb. Approximately 50 kd of immunoreactive protein on an SDS western blot. Generate. The DNA sequence of B8 overlaps with the DNA sequence of clone C7 (clone C7.2C), It was a fusion protein with β-galactosidase. Clone B8 matched C7 To add an additional 266 amino acids 5 '(upstream) and include the beginning of the gene . However, B8 terminates 64 amino acids before the C-terminus of the gene, which is also Is coded. Based on the above, the complete sequence of the full-length gene was compiled (sequence Number 549). The corresponding protein sequence from the assembled gene sequence is estimated at 48.9 kd The protein encodes and is presented herein as SEQ ID NO: 550.                     Library 2 clone and G1A   12．Clone a1   Clone a1 is a HindIII clone, which has a 28-30 kd immune response on an SDS gel. Produces a sex protein. The corresponding DNA sequence encoding the antigenic protein is 1 It contains 208 nucleotides (SEQ ID NO: 35). Two open reading frames Exists.   ORF1 extends to bases 53-801 of SEQ ID NO: 35 and is a putative protein containing 249 amino acids. Encoding the protein (SEQ ID NO: 36, translated protein). ORF1 is nucleotchi A Shine-Dalgarno sequence ("AGGA" sequence) extending from step 43 to step 46. Protein prediction The measured molecular weight is 27.5 kd, which is the expected protein size based on SDS-PAGE (above). Matches The putative protein for ORF1 is calculated pK 8.42 and And a predicted transmembrane region extending to amino acids 1-19.   The second ORF contained in clone a1 extends from nucleotides 880 to 1206 and Terminates at two HindIII sites. This means that clone a1 is a partial clone. And The predicted size of the partial protein is 109 amino acids (SEQ ID NO: 37) .   13.Clone a3   Immunoreactive clone a3 (not to be confused with clone A3 from library 1) Is a β-galactosidase fusion clone having an insert size of 1975 base pairs It is. The corresponding nucleotide sequence corresponds to SEQ ID NO: 248. Clone 2 Contains one open reading frame (ORF): ORF1 (nucleotides 3-608, SEQ ID NO: 249) and ORF2 (nucleotides 613-1266, SEQ ID NO: 250). Immune response Based on the observed size of the sex protein, i.e. 30 kd, the expected immunoreactivity The current open reading frame is ORF1. The following sections describe a3 protein expression It describes in.   14.Clone a5   Clone a5 is the first HindIII / EcoRI genomic clone (1 kb), which is 25 kd Produces an immunoreactive protein. The corresponding nucleotide sequence is shown as SEQ ID NO: 3. To present. Clone contains two open reading frames (ORF) : ORF1 encoding a β-galactosidase fusion protein (nucleotides 3 to 54) 5, SEQ ID NO: 5) and ORF2 (nucleotides 569-1021, SEQ ID NO: 4). Shine D The elgarno sequence occurs at nucleotides 559-562 of ORF2. Immunoreactive protein Based on the observed size of the quality, i.e. 28 kd, the expected immunoreactive expression open The reading frame is ORF1.   15．Clone b5   Clone b5 contains a double insert with an internal EcoRI site. Individual Insa The sheets are about 0.3 and 0.2 kb long. Matched nucleosomes for clone b5 The peptide sequence corresponds to SEQ ID NO: 6 and has an open reading frame extending from nucleotides 1-414. A frame (ORF). The corresponding protein sequence translation is shown as SEQ ID NO: 7. To present.   16．Clone c2   Clone c2 is a library 2 clone with an insert size of 2077 nucleotides. It is a loan (SEQ ID NO: 251). The size of the immunoreactive protein on the SDS gel is 30kd. There is one ORF extending from nucleotides 3 to 877, which is Has the first possible start codon at position 45. Provides the corresponding protein sequence translation Presented as column number 253. The protein has a predicted pI of 9.54, It has between ~ 24 potential transmembrane regions. The predicted molecular size of this c2 protein is 30.2 kd, which is consistent with the size observed on the SDS gel.   17．Clone c5   Clone c5 is a 650 base pair HindIII / HindIII clone (SEQ ID NO: 253). Clone c5 is a partial clone, synchronized with the β-galactosidase gene And contains a 29 amino acid stretch that extends into pBluescript at the N-terminus. No. The ORF extends to bases 2-604, which has a predicted protein size of 23.5 kd ( SEQ ID NO: 254). The observed protein size on the SDS gel is 30 kd. Estimation The protein has a pi of 9.6.   18．Clone c13   Clone c13 is a HindIII / HindIII clone with an insert size of 1742 bp (SEQ ID NO: 255). It has a size of 55 kd, observed on SDS gels Β-galactosidase fusion protein. The ORF extends from bases 2 to 1420. versus The corresponding protein has a predicted molecular weight of 54.6 kd (SEQ ID NO: 256). SWISS pro d Sequence searches using atabase show that this sequence can be found in yeast and human And has homology to threonyl-tRNA synthetase.   19.Clone d5   Clone d5 is an EcoRI clone, which is approximately 70 kd on SDS-PAGE gel. Generate a responsive protein (β-galactosidase fusion protein). Cloni The size of the inserted insert was determined to be 1795 bases (SEQ ID NO: 58) and It has an ORF extending from nucleotides 1 to 1704. Open reading frame is 568 A putative protein consisting of amino acids (SEQ ID NO: 59) and having a predicted molecular weight of 62.1 kd. Encode protein. The putative protein has a predicted pK value, 5.1, and Primitive determinants are present in the N-terminal region of the protein.   Computer-based analysis using the PROSITE program on PCGENE I went. Based on this study, several signature sites were added to proteins Identified within. This is the site of cAMP and cGMP-dependent protein kinase phosphorylation. And ATP-GTP binding motif A (P-loop).   20.Clone d7   Clone d7 is a HindIII clone and was cloned as described above for Example 4.1. Was blunt-ended, ligated to the A / B linker, digested with EcoRI, and the λ vector -Subcloned into ZAPII. The product is a β-galactosidase fusion protein Quality. The nucleotide sequence is presented as SEQ ID NO: 11.   The derived protein was determined to have a size of 40 kd and 35 kd. SEQ ID NO: 11 The corresponding protein sequence translated therefrom is presented as SEQ ID NO: 12.   twenty one.Clone f3   Clone f3 is an EcoRI clone with an insert size of 2274 nucleotides. (SEQ ID NO: 257). Clone f3 produces a β-galactosidase fusion protein To achieve. The ORF extends from nucleotides 1-1788 and a predicted molecular size of 67 kd (β -Coding for a putative protein having a (excluding galactosidase moiety) (SEQ ID NO: No. 258). The calculated pI of the protein is 9.66.   Sequence searches for both the SWISS pro database and the DNA database can be found in Haemop hilus influenza, E. coli, Bacillus subtilis and Streptococcus pneumoniae Shows 30% homology to the penicillin-binding protein. EMBL DNA database Search yields 56% homology to the penicillin binding protein of Haemophilus influenza Is shown.   The protein size observed on the SDS gel is 25 kd (major band). With a minor band of about 67kd. This size mismatch is probably due to tampa This may be due to cleavage of the protein to produce smaller fragments.   twenty two.Clone f8   F8CON1 is a HindIII subclone, which has a 33-35 kd immune response on SDS gels Produces a sex protein. The DNA sequence corresponding to the f8 subclone is referred to herein as Presented as SEQ ID NO: 1. Cloned DNA subfragment of 1459 bases Contains an open reading frame (ORF) from nucleotides 134 to 1042.   The predicted protein encoded by the f8 subclone contains 303 amino acids and has 3 amino acids. With a predicted molecular weight of 3.9 kd, which is consistent with the predicted protein size. Anticipation The original determinant is the 3 'end of the putative protein (which extends from amino acid 270 to amino acid 275). (Nucleotides 941-958). The putative protein has a pK of 9.75. The corresponding protein sequence based on the translation of SEQ ID NO: 1 is presented as SEQ ID NO: 2 .   W.Clone g2   Clone g2 has an EcoRI / HindIII with an insert size of 2474 nucleotides. It is a clone (SEQ ID NO: 259). Immunoreactive protein observed on SDS gel Is 25kd in size. The clone contains four ORFs. First ORF and Last OR F encodes a partial protein, i.e. the complete ORF is not included in the clone won. ORF1 extends from nucleotides 2-445 and has a predicted protein of 148 amino acids. Encodes the carboxyl terminus of the protein (SEQ ID NO: 260). ORF2 has bases 461 to 1156 And encodes a putative protein with a molecular size of 25.4 kd (SEQ ID NO: 261). ORF 3 extends to bases 1156-1776 and encodes a protein having a size of about 22.2 kd. (SEQ ID NO: 262). ORF4 (nucleotides 1798 to 2472) has a predicted data of 24.4 kd. Encodes the amino terminus of the protein (SEQ ID NO: 263). ORF2 and ORF3 predictions p I is 7.82 and 5.26, respectively. The putative proteins of ORF2 and ORF3 are Contains no predicted transmembrane regions and putative proteins of ORF4 have six predicted transmembrane helicopters. Includes three predicted transmembrane regions with a matrix.   twenty three.Clone g9   Clone g9 has an insert size of 4292 bases. Two subclones K4 and g11 are contained in clone g9. Clone g9 is approximately 85kd immunoreactive Produces protein bands, while k4 has an immune response of 55kd, 46kd, 35kd and 30kd G11 produces a weak immunoreactive band of 22 kd.   The complete sequence of g9 was obtained by walking in the 5 'direction from the C-terminal sequence of g11, and And walking from the N-terminal sequence of k4 in the 3 'direction (SEQ ID NO: 26). Four). Clone g9 has 5 ORFs. ORF1 is in the region extending from bases 2 to 349 (SEQ ID NO: 265). Corresponding prediction of protein The size is 12.3kd. ORF2 extends from base 495 to 1403 (SEQ ID NO: 266), and The corresponding predicted protein has a molecular size of 33.8 kd. "AGGA" Shi after ORF2 followed by the ne-Dalgarno sequence, which is located before the ATG of ORF3 (bases 1418 to 2209) . The predicted molecular size of the putative protein encoded by ORF3 is 29.7 kd ( SEQ ID NO: 267). ORF4 corresponds to bases 2223-3719. Coded by ORF4 The protein has a predicted molecular weight size of 56.8 kd (SEQ ID NO: 268). ORF5 is Nuku Extend to leotides 3133-4236, with a predicted protein molecular size of 19.9 kd ( SEQ ID NO: 269).   twenty four.Clone G1a   The immunoreactive 2.8 kb HindIII fragment of the corresponding genomic EcoRI clone (this Has an insert size of 6.4 kb), treated with exo-mung nuclease, Nested deletion clones were generated for sequencing. 1610 base pairs The DNA sequence of the region was determined (SEQ ID NO: 8). Two open reading frames (ORF) was determined: ORF1 (nucleotides 32-964); and ORF2 (nucleotide). De 1034-1570). The corresponding translated protein sequences are referred to herein as Presented as SEQ ID NO: 9 and SEQ ID NO: 10.   25. Characterization of additional library 2 clones, e6, dll, f11 and d6 Details are provided in the table below. n.d. = Not measured. The underlined numbers for protein size are The size of the protein observed on the screen. Predicted protein size is β-gal Does not contain cuctosidase fusion peptide. n.d. = Not measured. The underlined numbers for protein size are The size of the protein observed on the screen.^* If there are more than two ORFs,^*Indicates an expressed ORG that is immunoreactive You. Example 5A H.pylori Expression of antigen coding region   Different clone families, such as A3, A22, B2, B9, C1, C5, C7 and G1a The resulting amplification product was cloned into the pGEX vector (Pharmacia). Claw The engineered construct was expressed in an E. coli strain (Stratagene, LaJolla, CA).   1.Clone B2   Expression primers GD96 (forward primer, SEQ ID NO: 56) and GD97 (reverse primer) Primer, SEQ ID NO: 57) into the expression vectors pGEXdel65 and pGEXGLI. Introduce NcoI and BamHI sites for B2 fragment subcloning Designed to be. The expression product lacked the predicted transmembrane region.   Based on the insolubility of the expressed protein, only expression products derived from pGEXdel65 Were further purified. Protein was purified using Ni-NTA ++ resin from Qiagen (Chatsworth, CA). And purified by affinity purification chromatography.   Purified proteins were paneled against various sources of H. pylori-infected serum. nel). Serum paneling was performed twice, but different results were obtained. (I) 75% sensitivity (based on 35 serum samples) and (ii) about 30% sensitivity Degree (based on 183 sera). Serum panel is analyzed by affinity chromatography -Using SDS gel electroelution protein after purification .   2.Clone C1   The amplification product from clone C1CON6V2 was purified using primers BF (SEQ ID NO: 40) and BG (SEQ ID NO: 41) and the expression vector as outlined in Table 5 below. Cloned into pGEXdl65. Expressed protein is immunoreactive However, it did cleave beyond the transmembrane region and An epidemiologically reactive band formed. Based on this observation, the expression of smaller proteins A new forward primer, primer BO (SEQ ID NO: 42) Designed as shown in FIG.   The resulting protein was determined to be immunoreactive, and It was formed in much higher yield than the product.   3.Clone B17   Expression primers CA (forward primer, SEQ ID NO: 29) and CB (reverse primer) The B17 ORF4 fragment into the expression vector GEXdelG5. It was designed to introduce NcoI and BamHI sites for cloning.   Expression primer CC (forward primer, SEQ ID NO: 31) and CD (reverse primer) , SEQ ID NO: 32), and subcloning the B17 ORF2 fragment into an expression vector. It was designed to introduce NcoI and BamHI sites for logging.   4.Clone A3   The product amplified from genomic A3 DNA is digested with NcoI / BamHI restriction enzyme and The expression vector was determined using Limer BP (SEQ ID NO: 18) and BQ (SEQ ID NO: 19), respectively. The pGEXd165 poly-His was subcloned into the NcoI and BamHI sites. Protein Quality expression was induced with 0.5 mM IPTG (isopropylthiogalacto-pyranoside) . The expressed protein was of the expected size and was It was confirmed to be immunoreactive. 5.Clone C7   H. PCR amplified product from pylori genomic DNA, A fragment is then generated using primer GD100 (SEQ ID NO: 22) And GD101 (SEQ ID NO: 23), respectively, using the NcoI / B of the expression vector pGEXdel65 It was subcloned into the amHI site. The expressed protein has the expected size And was very insoluble. The protein is Ni-NTA color as described above. Could be easily purified by chromatography. Immunogenic screening Protein is immunoreactive with Roost pooled sera It was confirmed.   6.Clone G1a   The PCR amplification products were ligated with primer BW (ligand) as outlined in Table 5 below. (SEQ ID NO: 33) and BX (SEQ ID NO: 34) to support the expression vector pdel165 polyHis. It was cloned. The expressed protein was confirmed to be immunoreactive. However, the expression product was cleaved internally, and cleavage probably occurred at the transmembrane region.   Expression of additional cloned constructs was performed similarly.   All protein expression products are relative to Roost pool sera, except for dHB9.4.2. It was determined to be immunoreactive.                                 Example 5B H . expressed by pylori clones Y175A, Y212A, and Y146B, Purification of exemplary antigenic proteins   Clones Y104B, Y175A, Y212A, and Y146B were constructed according to the protocol described above. E. E. coli expressed as a recombinant protein. E. Plies for E. coli expression clones The mer sequences are shown in the sequence listing. Purify the recombinant proteins and their immunogens Sex was confirmed according to the following general procedure.   Spin the pellet from the shake flask and then differentially solubilize (differentia l solubilization). Conveniently, the pellet is dissolved in a solution containing PBS / 5mM PMSF. And spun at 4 ° C., 30k for 60 minutes. E of the following times Modification / centrifugation was as follows: (i) 100 mM Tris / 2% Tr iton / 2M urea / 5mM EDTA / 0.5mM DTT (homogenization step) / 60 min, 4 30 ° C, 30k (centrifugation); (ii) PBS pH7.8 / 2M urea / 0.5mM DTT / 60min, 4 ° C, 30k (Iii) PBS pH7.8 / 4M urea / 0.5mM DTT / 60min, 4 ° C, 30K, (iv) PBS pH8.0 / 6M Urea / 0.5 mM DTT / 60 min, 4 ° C., 30 k; (v) PBS pH 8.0 / 6 M urea / 2 M guanidine HCl / 2mM BME / 60min, 4 ° C, 30k.   The supernatant from step (iv) above was then dialyzed against PBS pH 8.0 / 6 M urea / 2 mM BME, Then 5 column volumes of 0.2M NiCl_TwoLoaded, Chelating Sepharose (Pharmaci Chromatographic separation using the prepacked column of a). Buffer Utilize a 10 column volume (C.V.) gradient of A to Buffer B 100%, where Nickel IMAC buffer Solution A contains PBS pH 8.0 / 6 M urea / 2 mM BME and Nickel IMAC Buffer B contains Buffer A and 250 mM imidazole were included.   Pool the appropriate Nickel IMAC fractions and reconstitute in PBS pH 8.0 / 6 M urea / 0.5 mM DTT. Dialysis was performed overnight. The final product was then transferred to a vial and stored at -80 ° C.   Alternatively, a glutathione-sepharose packing column (Sigma) was utilized.   Fractions were typically analyzed by: 1) Pierce Coomassie protein Assay, 2) SDS-PAGE (on 12% gel), 3) H. Use pylori Roost Pool # 3 Western blot to use, 4) GLT H. pylori negative pool, and 5) anti-E.c oli polyclonal antibody. The results of these analyzes indicate that the expressed protein Was confirmed.                                 Example 6A Antigen for antigens purified from clones dHA22.8 (A22) and dHClS.11 (C1) Concentration optimization and small-scale serum paneling   1.Clone dHA22.8   The protein expressed by clone dHA22.8 (corresponding to clone A22) It was isolated and purified as follows.   2000 ml of culture pellet is mixed with 200 ml of buffer B (48 g urea, 1.2 g NaH_TwoPO_Four, 0. 12 g of Tris-HCl and 90 ml of deionized water (adjusted to pH 8.0 and a total volume of 100 ml) )) And sonicated for 10 minutes to effect cell lysis. Next The sonicated mixture was shaken at room temperature for 1 hour and Spin at 00 rpm to remove cell debris. Then, 1 ml of Ni-NTA resin ( Qiagen GmgH, Hilden, Germany) and then mix it for 1-2 hours at room temperature did. Centrifuge the mixture at 5,000 rpm for 15 minutes at 4 ° C to pellet the resin. Release and the supernatant was discarded. The resin was washed with 50 ml of buffer C (48 g urea, 1.2 g NaH_TwoPO_Four , 0.12 g Tris-HCl, and 90 ml deionized water, pH 6.3 and a total volume of 100 ml. )), Centrifuged and the supernatant discarded. Next, dispose of the resin The ram was loaded and the washing step was repeated with the remaining buffer C. Then, tongue The protein is washed with 50 ml of Buffer III (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 250 mM Imidazole). Collect fractions (2 ml) and run on a 12% SDD-PAGE gel analyzed.   The purified protein is then converted to blood using conventional techniques (Ausubel et al., 1988). Screened with clear antibody. Purify the protein with 0.1-20 μg / ml 0.1 M carbonate Slot with buffer, block with 5% skim milk in TBS buffer, and dilute 100-fold Were reacted with H. pylori positive serum and H. pylori negative serum. 36 total blood A serum panel consisting of Qing was used as described above. Positive serum is Roost Pooh # 2 or SFA 001 pool; negative serum from donor pack Was.   After a one hour incubation period, the strips were washed three times with TBS buffer, And alkaline phosphatase bound 1000-fold in 5% skim milk in TBS buffer. Incubate with a secondary anti-human Ig antibody (Promega Biotech, Madison, WI) did. The strips were then washed three times with TBS wash buffer. Immunoreactive tamper Can be converted to a substrate (eg, BCIP (5-bromo-4-chloro-3-indolyl-phosphate) ), And NBT (nitroblue tetrazolium salt (Sigma)). color The color development is due to the very highly antigenic nature of the purified A22 protein. In other words, H. pylori positive serum).   The optimal concentration of antigen was determined to be 2.0 mg / ml. dHA22.8 (A22) expression product, In both pooled serum sources (Roost pool # 2 and SFA 001 pool) Existing anti-H. pylori antibody and 100% tested individual H. pylori positive Reacted with serum samples. Antigen has no detectable cross-reactivity with normal serum won.   2.Clone dHClS.11 (C1)   The protein expressed by clone dHClS.11 (C1) was used for the following protocol. Therefore, it was isolated and purified.   Proteins were differentially differentiated using a series of homogenization / centrifugation steps. Extracted from the culture pellet by solubilization. Pellets into (i) PBS / 1mM PMSF Homogenize and centrifuge at 4 ° C, 19,000 rpm for 30 minutes, then separate the supernatant (Ii) homogenized using 1 M urea / 10 mM Tris (pH 8.0) / 10 mM DTT, Centrifuged as in (i) and the supernatant separated; (iii) 4M urea / 10mM Tri s Homogenized in pH 8.0 / 2 mM BME followed by centrifugation as in (i) And the supernatant is separated; and finally (iv) 6 M urea / 10 mM Tris pH 8.0 / 2 mM BME The pellet was homogenized and subsequently centrifuged as described above. Combined 150 mM NaCl was added to the supernatant. Then, the protein was added to 2 CV (column volume). 0.2M NiCl_TwoSepharose (Pharmacia, Chelating Seph) loaded with immobilized metal using a pre-packed 5 ml column containing arose Fast Flow) Separated from the combined supernatants by adsorption chromatography. protein Using a 50% buffer of buffer A (4 M urea / 10 mM Tris pH 8.0 / 0.2 mM BME / 150 mM NaCl) Using a 20 CV gradient to B (buffer A with 0.5 M imidazole), the column And eluted. Fractions were collected (2 ml) and analyzed on a 12% SDS-PAGE gel.   The optimal concentration for immunoscreening was determined essentially as described above. Up The serum paneling described above was performed using the purified C1 protein expressed by clone dHClS.11. Was repeated.   As in the above example, the expressed protein was pooled H. pyl ori is reactive with both sources of positive serum and control serum (H .pylori negative sample, one sample of pooled serum and 13 individual Sample) did not show any signs of cross-reactivity. In addition, panels 20-35 and As can be seen in AC, the recombinant protein is 95% H. pylori po Showed immunoreactivity with the positive sample.   In summary, for this small panel study, both A22 and Shows acceptability and specificity (> 90-95%). NA = not available                                 Example 6B For an additional serum panel for purified antigen Further serum paneling: selection of preferred antigens   Further serum paneling studies were performed on various H. Sensitivity and specificity of pylori antigen For further testing, another serum panel (USC (University of Southern Calif. ornia) and POW (Prince of Wales Hospital)).   Gold standard tests are run to compute the sensitivity and specificity of each individual antigen. Used as a reference standard for The standards used were histology, CLO testing, and U It was a conventional gold standard test including the BT test. UBT is active in a non-invasive way It is the gold standard now very widely used for the indication of "infection". Histology, CLO (rapid urease test), and UBT report that these tests produce positive results. All show "active infections" because of the dependence on the presence of the germs. Serology is a past Since both infections and current infections are measured, the purpose of this study was to Identify serological markers that correlate with UBT results for use as "markers" And (ii) H. Effective screening for active infection by pylori Was to test both single and multiple antigen combinations.   A.Use of UBT as a gold standard in single antigen screening   Different antigens were analyzed on different panels (geographical origins from different strains of H. pylori). Differences and the exposure of the population to other cross-antigens from other bacteria specific to the area (Which may be a reflection) Use of all single antigen markers tested using UBT as a single reference standard . 1.USC panel.Based on this panel, the best single antigen selection is the antigen Y261A Or it seemed to be Y124A. Both of these antigens are sensitive and specific Overall balance (> 80% for both sensitivity and specificity of Y261, and Y124A 100% sensitivity and 70% specificity). Other preferred antigens (A22 And C1) are 95-100% sensitive in this panel, respectively, and 6 It was 7-77% specific. 2.POW panel.Based on this paneling study, the best single antigen selection is , Y128D12S and Y124A, where the sensitivity and specificity are It was about 75-80% for S and both about 80% for Y124A. Another A preferred antigen is Y261A, which has 70% sensitivity and 80% specificity. Indicated. For this paneling set, preferred clones A22 and It was found to be 5-98% sensitive and about 60% specific.   B.Use other gold standards   1.Roost panel.Only test positive sera, and antigens A22 and , 90% sensitivity. Y124A is about 80% sensitive and Y261A is about 87% Had the sensitivity.   2.UNSW panel  In this paneling set, A22 and C1 are 90-100% or less. The above specificity was shown. Y124 shows 80% sensitivity and 96% specificity, and Y261A showed 80% sensitivity and 100% specificity.   Other gold standards in USC and POW panels, even when UBT is used alone Sensitivity and specificity values do not change significantly when used in combination with Was observed. Thus, the somewhat lower specificity for A22 and C1 May be a reflection of previous exposure to cross-reactive antigens.   C.2-4 antigen combination test   High sensitivity of A22 and C1, and some about specific panels (POW, USC) Multiple serotypes were examined, taking into account their low specificity. 2-4 serotypes can be any At least both or all three specific for a few highly sensitive antigens Based on criteria that need to be positive with respect to .   This "positive for both antigens" criterion is that the selected set of 12 antigens Applied, where 12 antigens have a sensitivity of at least 40-50% for consideration. Selected.   Computed sensitivity and specificity of a positive reference for both antigens And the tables obtained are tested for good performers. further, Combinations of two antigens with high specificity and lower selectivity are used in all two antigen combinations. Performed on the combined matrix, "positive for both antigens or two other antigens. Provides a result indicating "positive for both antigens". Then the final analysis is USC Generally provide good results beyond the board between the POW panel Provides a selection of the antigen present.   D.Result of 2-4 antigen combination test USC About the panel,15 antigens providing about 95% sensitivity and 100% specificity There are gender combinations.   POW About the panel,10 different antigen combinations have more than 70% sensitivity and It has been shown to provide about 80-90% specificity.   Based on these findings, an exemplary combination selection for both panels is: It is as follows:   (a) Both positive C1 and Y124A (for POW and USC panels, 76.2% and 100% sensitivity, and 89.1% and 95.2% specificity). do it   (b) All positive C1 and Y124A and A22 (for POW and USC panels) 71.4% and 94.7% sensitivity, respectively, and 91.3% and 95.2% specificity sex).   For the USC panel, the C1 and c5 antigen combinations are preferred, and the POW panel For antigens, antigen combinations Y261A and Y124A or antigens C7 and B2 are also preferred. It was good.   Therefore, based on these serum paneling data, H. p Preferred antigens for use in detecting ylori infection include the following: But not limited to: A22, C1, Y124A, Y261A, c5, C7, B2, Y104B and Y1 28D.   Representative purified H. in different serum paneling studies. About pylori antigen A summary of the figures for all sensitivity and specificity data is shown in FIGS. 65 and 66.Example 7 H . Purification of 36kD and "Spot 15" antigen produced by pylori   H. pylori (ATCC No. 43504) is grown under appropriate conditions and the cells Collected in phosphate buffered saline. This is followed by repeated centrifugation and cell Debris and other contaminants were removed. Then, the obtained pellets were subjected to> 10,000 PSI And then centrifuged at 10,000 × g for 25 minutes.   Spot 15 (high pH), followed by sequence / structure studies (eg mass spectrometry, pepti Only when enriching soluble H. Pre-fractionation of pylori lysate supernatant went.   Fractionation of the 36 kD protein was performed by dialysis against 10 mM Tris buffer, pH 8.0, 50 mM NaCl, Subsequently, anion exchange resin Resource Q (Pharmacia Biotechnology, Piscataway, NJ ) Was achieved by gradient elution. SDS-PAGE / Western blot analysis Western positive doublet (doublet at 30kD and positive 36kD band) )showed that. Both fractions are very immune when tested against Roost pool sera It was determined to be reactive.   Fractions of spot 15 antigen were performed in a similar manner. Samples were separated by Sephacryl S-10 0 Ion exchange resin (Pharmacia Biotechnology, Piscataway, NJ) (10 mM phosphate buffer) Buffer, 50 mM NaCl, pH 8.0), followed by Resource S cation exchange resin (Phar macia Biotechnology, Piscataway, NJ) . Fractions containing spot 15 antigen analyzed by SDS-PAGE and Western blot And fractions were similarly analyzed with Western blots and Roost pooled sera. Confirmed to be highly antigenic in nature, as indicated by immunoreactivity Was.                                 Example 8 36kD And spot 15 H. pylori antigen characterization   After fractionation of the antigenic protein as described in Example 7 above, the isolated fraction was And two-dimensional electrophoresis performed according to the method of O'Farrell (1975, 1977). Sign I attached. 1.Two-dimensional electrophoresis under low pH conditions   Two-dimensional electrophoresis was performed essentially as described by O'Farrell (1975). Isoelectric focusing was performed at 900 volts-hours with 2% ampholine (BDH, Hofer Scientifi c Instruments, San Francisco, CA) in a glass tube with an internal diameter of 2.0 mm. Was. The final tube gel pH gradient, as measured by a surface pH electrode, was closed (enc losed) pH gradient form.   Buffer O (10% glycerol, 50 mM DTT, 2.3% SDS, and 62.5 mM tris buffer After equilibration for 10 minutes in a liquid (pH 6.8), the tube gel is washed with a concentrating gel (this is a 10% Adhered to the top of acrylamide slab gel (placed on top of 0.75mm thickness) . SDS slab gel electrophoresis was performed at 12.5 mA / gel for 4 hours. Then slab gel Was fixed in a 10% acetic acid-50% methanol solution overnight. The following proteins are molecules As a quantity marker, it was added to agarose in which tube gel was adhered to slab gel: Osin (mysin) (220 kD), phosphorylase A (94 kD), catalase (60 kD), actin ( 43 kD), carbonic anhydrase (29 kD), and lysozyme (14 kD). these Standards were on silver stained (Oakley et al., 1980) 10% acrylamide slab gel. Appears as a horizontal line. Silver stained gel, acid edge on the left Dry between sheets of cellophane paper.   2.Western blot   After slab gel electrophoresis, duplicate gels were transferred to transfer buffer (12.5 mM Tris (pH 8.8), 86 mM glycine, 10% methanol) at 200 mA (about 50 volts / gel) on RVDF paper. Transblot overnight. Blots were run on T cells containing 2% bovine serum albumin (BSA). Block in TBS (Tween-Tris buffered saline) for 2 hours, rinse with TTBS, From Roost pool or negative serum pool diluted 1: 2500 in 1% BSA / TTBS Incubate in primary antibody for 2 hours and rinse in TTBS Antibody-containing solution (anti-human IgG horseradish peroxidase diluted 1: 5000 in TTBS) Z) for one hour. The blot is rinsed with TTBS and ECL (Amersham Corporatio n, Arlington, Heights, IL) and exposed to X-ray film.   Computation of exemplary stained membranes containing H. pylori-derived antigenic proteins as described above FIG. 2 shows a photograph of the preparation of the rotor. `` Normal '' human serum is `` HELICOBLOT 2.0 '' (Genel abs Diagnostics (PTE) Ltd., Singapore) to be H. pylori negative Was confirmed. The identity of the spots indicated numerically on the gel was determined by Determined by protein sequencing (described in Section 9).   3.Two-dimensional electrophoresis under high pH conditions   Two-dimensional electrophoresis adapted for basic protein separation was performed using O'Farrell (O'Farrel 1977).   1.5% pH 3.5-10 ampholin and 0.25% pH 9-11 ampholin (Pharmaci a Biotechnology, Piscataway, NJ) using non-equilibrium pH gradient electrophoresis at 140 volts. For 12 hours. Purified tropomi with low spot molecular weight of 33 kD and pI of 5.2 Osin and purified lysozyme (Me with a molecular weight of 14 kD and a pI of 10.5-11) rk Shape & Dohme, Philadelphia, PA) as an internal pI marker did.   Buffer (10% glycerol, 50 mM DTT, 2.3% SDS, and 62.5 mM tris (pH 6.8) After 10 minutes equilibration in the tube gel, concentrate the gel (this is 10% acrylamide Adhere to the top of the slab gel (located on top of 0.75 mm thick) and Lab gel electrophoresis was performed at 12.5 mA / gel for 4 hours. Slab gel, 10% acetic acid / 50% Fixed overnight in methanol solution. Using the following proteins as molecular weight standards, Added to agarose with tube gel adhered to Bugel: myosin (220 kD), phospho Lylase A (94 kD), catalase (60 kD), actin (43 kD), carbonic anhydride Lase (29 kD), and lysozyme (14 kD). These standards were silver stained (O akley et al., 1980) Appears as a horizontal line on a 10% acrylamide slab gel. Silver stain The gel was dried between sheets of cellophane paper with an acid edge on the left.   Transblot duplicate gels onto PVDF paper and Western blot Was performed as described in 8.1.b above.   Exemplary Western blots containing antigenic proteins from H. pylori as described above FIG. 1 shows a computer-made photograph of the film thus set. Shown numerically on the gel The identity of the spots was determined by protein sequencing (as described in Example 9 below). Determined by                                 Example 9 Western positive spot isolation and protein sequencing   1.N-terminal sequencing   The PVDF blot from Example 8 above was used with Coomassie Brilliant Blue. Stained. Spot corresponding to western positive band with scalpel Excision and routine sequencing techniques (eg, Miller, 1994; Spiecher, 1989) ) Was sequenced directly using a Hewlett-Packard G1005A N-terminal sequencer Was. The sequencing technique used has a high repeat yield (because of the detection limit of about 100-200 fmol). Typically in the range 93-98%).   2.Internal sequencing   Four different methods were used to obtain the internal sequence information of the H. pylori isolated antigen described above. (Allen, 1981): Lys-C peptide map and sequencing, CNBr peptide map and And sequencing, OPA / CHBr peptide maps and sequencing, and Lys-C digests LC-MS / MS sequencing.     a.Cyanogen bromide cutting. Cyanogen bromide (CMBr) cleavage was applied to PVDF membrane in 70% formic acid. (Crimmins and Mische, 1996). Then, the cyanogen bromide digested peptide Was repurified by capillary HPLC and sequenced directly or Either subject to to-phthalaldehyde (OPA) modification was performed.     b.Ortho-phthalaldehyde (OPA) modification. The digested peptide was converted to Bauer (Bauer et al.). , 1984). Use this reagent to repair primary amines Decorating (but not modifying the secondary amine), thereby providing an Edman-type N-terminal sequence Identification and identification of sequences with proline as the N-terminal residue as determined by analysis And silencing of all other sequences.     c.Liquid chromatography / mass spectrometry. Internal sequence information can also be Obtained from analysis based on chromatography / mass spectrometry.   Copper stained gel slices (Nakayama et al., 1996) and proteins transferred to PVDF were First, extract and use Sep-Pak solid phase extraction (Millipore Corporation, Bedford MA) Pre-purification and pretreatment by protease (Lys C) digestion.   Liquid chromatography-mass spectrometry (LC-MS) uses peptide mass technology and To specify internal sequence residues by ion trapping technology (McAtee et al., 1996) On digested proteins. Digest the ABI Model 410 B double syringe Pump system (Applied Biosystems Division, Perkin Elmer, Foster City, CA) On a Vydac C18 reverse phase microbore column (150 mm x 1 mm) I was torographed. Maintain the flow rate at 50 microliters / minute and elute , Using a linear gradient from 0.1% aqueous TFA to 0.1% TFA in acetonitrile. Ca Use the rlo Erba Phoenix 20 CU pump to add methoxyethanol and isopro A mixture of panol (1: 1, v / v) was delivered at a rate of 50 microliters per minute. This was combined with the column eluate in a post-column mixing chamber. Inla Using an in-line flow splitter, the mass spectrometer The flow was limited to about 10 microliters / minute. Detection with the ABI 759 variable wavelength detector Performed immediately after elution from the column at 214 nanometers. Mass spectrometry detection VG BioQ triple quadrupole mass spectrometer with electrospray ion source Achieved after ram solvent addition and flow splitting. The spectrum Recordings were made in positive ion mode using trospray ionization. Instrument calibration By using direct injection analysis of myoglobulin, the range m / z 500-2000 I went there. Spectra were recorded at 1.5 second intervals and using nitrogen dry gas Solvent evaporation was assisted. Capillary voltage is maintained at about 4 kV with a heat source temperature of 60 ° C did.   3.Sequence determination results   Spots shown in Western blots using the above approach (FIGS. 1 and 2) were identified. The identity of the spot (if known) is shown in Table 6 below. Show.   Looking at the low pH blot shown in FIG. 2, spots 9, 11, 12, 13, 15, 16 (me Jar and minor), and 17 indicate unique antigens. Spot 9 is black Native H.pylo corresponding to the recombinant protein expressed by ORF2 ri antigen, whereas spot 12 shows the antigenic tag encoded by clone a5. Shows the native antigen corresponding to the protein.   Looking at the high pH western blot in FIG. 1, spot 9 (measure and Minor), 10, 12, 13, and 15 (major and minor) have unique antigens Show.   As will be apparent from the following results, the N-terminus of spot 15 antigen (high pH) was Is determined later, and thus spot 15 The N-terminal sequence determined for was later shown to be incorrect.                                 Example 10 H.pylori 36kD antigenic protein isolated from various sources Analysis of spot 15 and spot 15 antigens   The above H. pylori spot 15 antigen was used as described in Examples 8 and 9 for H. pylo ri from three different sources. The following H.pylori samples were used: H. pylori, ATCC 43504 (Australian strain); H. pylorl. # 26695 shares And H.pylori, J-170 (both from the Washington University School of Medicine , St. Louis, MO). The corresponding protein isolated from each source is then The quality was analyzed by reversed phase high performance liquid chromatography (HPLC) as described in Example 9. Was analyzed.   Small spots in native Spot 15 protein from various H. pylori strains Differences are observed, especially in the early eluting peak area, and the TIGR and ATCC Spot 15 antigen from the H. pylori source appears to be closely related. small Differences may include slight minor amino acid changes, deamidation, and possibly High molecular weight (eg, lipopolysaccharide or glycosylation ) Included side chain modifications, as suggested by the modifications. 15 spots from J-170 The protein is thought to contain additional high molecular weight modifications, as indicated by the difference in molecular weight. Will be   However, all peaks were not screened against Roost pool sera. When a protein is highly antigenic, it is an amino acid sequence between native proteins. Any minor discrepancies in the columns are detrimental to the high immunoreactivity of the recovered Spot 15 antigen Does not affect.   Based on the above characterization data, spot 15 antigen was generated as shown in FIG. It appears to be a processed product of the putative 36 kD protein.   In situ Lys-C digestion of "spot 15" produced by the above H. pylori strain MALDI-TOF MS comparison of peptides produced from the products was performed. Checking total digest In the data, 26695 strains and 43504 strains were replaced by 43504 strains and J-170 strains or 26 From their 695 and J170 strains (possibly after their translation) (In the modification profile). This data is All strains tested for immunoreactivity to spot 15 with data shown in Amino acid content and protein content among spots 15 of various strains. This suggests that there is a difference in protein modification.   Lys-C digest was also examined by reverse phase HPLC. All pepti derived from total digest Although this was not observed by RP-HPLC, this was due to the The presence of extremely hydrophobic and / or hydrophilic peptides that cannot be detected in the meter It was thought to be due to the presence. Based on the investigation of the RP-HPLC elution profile, Tide mass fingerprints were higher than those indicated by MALDI-TOF MS results. Seems to be more preserved between. Preferentially, MALDI-TOF and other forms of MS (E.g., electrospray, fast atom bombardment) show more accurate determination of structural analysis You. Example 11 2-D Antigen reactivity between various H. pylori strains separated by electrophoresis   Various H. pylori strains were described in Examples 7 and 8 among other H. pylori strains, As shown in FIG. 1, the protein identified in ATCC strain 45304 Examine to determine the extent of preservation.   Exemplary H. pylori strains tested were: Chico (Oroville Community H clinical isolate from ospital), TIGR 26695, 96-212 (Aras from the Aleut tribe) 4655-1 (Gambian child), J170, A-1c (Lithuanian isolate) , This is as an insert near the cag region and as an "X" seg Rus-95 (including Russian immigration to the US) ), # 9 (Peruvian isolate), C-3c (Lithuanian isolate).   These various H. pylori strains were purified and used in Examples 7 and 8 with ATCC No. 4530. Fractionated by 2D electrophoresis and described in And the immune response spots shown in the set of FIGS. It was determined whether the stocks were conserved between strains. The results are summarized in Tables 9 and 10 below I do.   As can be understood from the above, the immunogen isolated from H. pylori ATCC 45304 strain Sex proteins are highly conserved among different strains of H. pylori. Preferred of the present invention One of the antigens, spot 15 antigen (high pH), is the 100% representative strain tested Was observed. Western blot results show that this protein is highly antigenic Sexual properties were further supported.                                 Example 12 H.pylori MALDI-TOF mass spectrometry based on antigen mapping   Coomassie staining or compatible silver staining as described in Example 9. Transfer the gel pieces stained in to a microcentrifuge tube and add with 10 microliters of water. Rehydrated. The gel pieces were then mixed with 500 microliters of 50% acetonitrile / 0.1%. The plate was washed three times with 05M Tris-HCl (pH 8.5) for 20 minutes. Discard the supernatant and wash The pieces were dried in a Speed-Vac concentrator for 30 minutes. 5 microliters of 0.05 Add a solution containing an microgram of Lys-C to the tube and incubate at 32 ° C for 20 hours. I did it. After digestion, gel slices were removed from 30 microliters of 50% acetonitrile / 0.1% T Extracted three times with FA. The supernatant was transferred to a 0.5 ml microcentrifuge tube and dried. Lottery The released peptide was added to 4 microliters of 4-hydroxy-α-cyanocinnamic acid. Re-dissolve, and transfer 0.8 microliters of sample to MALDI sample plate Spotted. MALDI mass spectrometry was then performed on a PerSeptive BioSystems Voyager  Performed on a DE mass spectrometer. Next, the peptide mass peak was analyzed using the MS-FIT program. PIR, NRDB, Genebank, EMBL, TIGR H. pylori, and The Compared to data in Swiss Protein Databases.   Database of mass spectrum profile peaks for specific Lys-C digested proteins The results matched with the source information are summarized in Tables 11 and 12 below. Example 13 Antigens for use in vaccines   Representative antigens described herein were obtained from patients before and after antimicrobial treatment. Evaluated as a vaccine candidate based on Western blot analysis of serum (above) Was.   Briefly, before treatment and 12 months after antimicrobial treatment, To determine the titer of reactive antibodies. Antibodies whose titer remained high for a long time after treatment The antigen corresponding to was determined to be a good vaccine candidate.   Two serum panels, the Gasbarrinni panel and the Greenberg panel, were Used for analysis. Serum from patients is given before and 12 months or 24 months after treatment. Got a month later. Patients tested positive by UBT after treatment were H. pylori Was not included in this analysis due to active infection by   Gasbarrinni panel diagnosed with H. pylori infection by endoscopy and UBT , Obtained from Italian male and female patients between the ages of 18 and 70. 12 before and during antimicrobial treatment After months, serum was collected from the patient.   Greenberg panel with H.pylori infection as confirmed in antibody tests Are also male and female patients in California between the ages of 18 and 70? I got it. Serum was collected from patients before antimicrobial treatment and 24 months after treatment.   Table 13 summarizes the data obtained from the Gasbarrinni panel. Shown in 12 months Table 13 shows the number and percentage of patients that showed high antibody titers to the antigens that failed. Treatment At 12 months, a high percentage of patients had clones Y139, Y146B, Y175A, and A22. Continued to show high antibody titers.   Table 14 summarizes the data obtained from the Greenberg panel. In 24 months, shown The numbers and proportions of patients that showed high antibody titers to the antigens that were tested. 24 months of treatment Later, a high percentage of patients had clones Y184A, Z9A, Y261Ains, and Y146B. High antibody titers.   Antigens that elicit long-lasting antigen responses are considered good vaccine candidates, And based on the results provided in the table below, the following antigens are preferred vaccines Considered to be candidates: Y139, Y146B, Y175A, and A22, Y184A, Z9A, Y 261Ains, and Y146B.   Although the present invention has been described with reference to particular methods and embodiments, various modifications and changes may be made. It is recognized that changes and modifications can be made without departing from the scope of the present invention.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, L S, MW, SD, SZ, UG, ZW), EA (AM, AZ , BY, KG, KZ, MD, RU, TJ, TM), AL , AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, E E, ES, FI, GB, GE, GH, GM, GW, HU , ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, M D, MG, MK, MN, MW, MX, NO, NZ, PL , PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, V N, YU, ZW (72) Inventors Rem, Moon W.             United States California 94065,             Redwood City, Lido Circle             Ten (72) Inventor McCutty, C .; Pee.             United States California 95136,             San Jose, Park Oxford             Place 93

Claims (1)

[Claims] 1. (I) SEQ ID NO: 39;   (Ii) FASTA version 1.7 LALIGN program, 1 ktup, default Parameter and optimal PAM using with default PAM A sequence that, when aligned, is at least 80% identical to SEQ ID NO: 39; To   (Iii) an arrangement of at least 12 consecutive amino acids shown in (i) or (ii). Column; Isolated and substantially purified, comprising an amino acid sequence selected from the group consisting of: H. pylori antigen, wherein the antigen is H. pylori. pylori positive antiserum H. is immunoreactive with pylori antigen. 2. Claims comprising at least 12 contiguous amino acids contained within SEQ ID NO: 39 Item H. pylori antigen. 3. The antigen has a SEQ ID NO: under stringent hybridization conditions. No. 516 (cluster 42) can selectively hybridize to the DNA sequence represented as 2. The method of claim 1, wherein the H.3 is encoded by a polynucleotide. pylori antigen. 4. A contiguous series of nucleotides contained within the sequence represented as SEQ ID NO: 38 (C1) 2. The method of claim 1, wherein the H. reotide is encoded. pylori antigen. 5. Anti-H. Used in screening biological fluids for the presence of pylori antibodies. Diagnostic kit for use,   At least one anti-H. 2. The method of claim 1, wherein the antibody is immunoreactive with a pylori antibody. Substantially purified H. pylori antigen, and   A reporter for detecting the binding of the antibody to the antigen, A diagnostic kit, comprising: 6. The key of claim 5, wherein said polypeptide antigen is bound to a solid support. To 7. H. in the subject A method for detecting pylori infection,   A biological fluid derived from a subject and the purified H. coli according to claim 1. pylori polypep Reacting with a tide antigen; and   Detecting the presence of the antibody bound to the antigen, A method comprising: 8. 3. The H.-method according to claim 1. A vaccine composition comprising a pylori antigen, wherein the vaccine composition comprises An antigen is challenged with the antigen, and then Rhesus monkeys infected with pylori Is H. in mice. An antigen characterized by its ability to reduce the level of pylori infection A vaccine composition. 9. The antigen comprises at least 12 contiguous amino acids of SEQ ID NO: 39, A vaccine composition according to claim 8. 10. The antigen has an amino acid sequence that is at least 80% identical to SEQ ID NO: 39. 9. The vaccine composition according to claim 8, wherein 11. The antigen has the amino acid sequence identified as SEQ ID NO: 39. Item 10. The vaccine composition according to Item 8. 12. 3. The H.-method according to claim 1. A vaccine composition comprising a pylori antigen, wherein The antigen is administered to a subject challenged with the antigen and subjected to antimicrobial treatment. An antigen characterized by its ability to elicit a long-lasting antigenic response, Vaccine composition. 13. The antigen comprises at least 12 contiguous amino acids of SEQ ID NO: 39, A vaccine composition according to claim 12. 14． An amino acid sequence wherein said antigen is at least 80% identical to SEQ ID NO: 39 13. The vaccine composition of claim 12, having a row. 15. The antigen has the amino acid sequence identified as SEQ ID NO: 39. Item 13. The vaccine composition according to Item 12. 16. 2. The method of claim 1, having the amino acid sequence identified as SEQ ID NO: 39. H. pylori antigen. 17． Having an amino acid sequence that is at least 80% identical to SEQ ID NO: 39, 3. The H.-method according to claim 1. pylori antigen.