Production and cell surface display of recombinant anthrax protective antigen on the surface layer of attenuated Bacillus anthracis.

To investigate the surface display of the anthrax protective antigen (PA) on attenuated Bacillus anthracis, a recombinant B. anthracis strain, named AP429 was constructed by integrating into the chromosome a translational fusion harboring the DNA fragments encoding the cell wall-targeting domain of the S-layer protein EA1 and the anthrax PA. Crerecombinase action at the loxP sites excised the antibiotic marker. Western blot analysis, fluorescence-activated cell sorting and immunofluorescence analysis confirmed that PA was successfully expressed on the S-layer of the recombinant antibiotic marker-free strain. Notwithstanding extensive proteolytic degradation of the hybrid protein SLHs-PA, quantitative ELISA revealed that approximately 8.1 × 10(6) molecules of SLHs-PA were gained from each Bacillus cell. Moreover, electron microscopy assay indicated that the typical S-layer structures could be clearly observed from the recombinant strain micrographs.

Association of NOD1 and NOD2 genes polymorphisms with Helicobacter pylori related gastric cancer in a Chinese population.

AIM: To investigate the association between the tag single nucleotide polymorphisms (TagSNPs) of NOD1 and NOD2 and the risk of developing gastric cancer. METHODS: We conducted a hospital-based case-control study including 296 incident gastric cancer patients and 160 gastritis controls. Eight TagSNPs in the NOD1 and NOD2 genes were selected from the Hapmap database using the haploview software and genotyped by the Sequenom MassArray system. The serum levels of anti-Helicobacter pylori (H. pylori) IgG were measured by enzyme-linked immunosorbent assay to indicate H. pylori infection. The odds ratios (OR) and 95% confidence intervals (CI) were calculated by unconditional logistic regression, including sex and age as confounding factors. RESULTS: The NOD1 rs2907749 GG genotype showed a decreased risk for gastric cancer (OR 0.50, 95% CI: 0.26-0.95, P = 0.04) while the rs7789045 TT genotype showed an increased risk (OR 2.14, 95% CI: 1.20-3.82, P = 0.01). An elevated susceptibility to gastric cancer was observed in the subjects with H. pylori infection and the NaOD1 rs7789045 TT genotype (OR 2.05, 95% CI: 1.07-3.94, P = 0.03) or the NOD2 rs7205423 GC genotype (OR 2.52, 95% CI: 1.05-6.04, P = 0.04). Haplotype analysis suggested that the distribution of AGT (rs2907749, rs2075820 and rs7789045) in NOD1 between the cases and control groups was significantly different (P corrected: 0.04), and the diplotype AGT/AGT was associated with an elevated gastric cancer risk (OR 1.98, 95% CI: 1.04-3.79, P = 0.04). The association of the NOD1 rs7789045 TT genotype and the diplotype AGT/AGT was significant with H. pylori-related diffuse-type gastric cancer (OR 3.00, 95% CI: 1.38-6.53, P = 0.01; OR 4.02, 95% CI: 1.61-10.05, P < 0.01, respectively). CONCLUSION: Genetic polymorphisms in NOD1 and NOD2 may interact with H. pylori infection and may play important roles in promoting the development of gastric cancer in the Chinese population.

Removal of antibiotic resistance of live vaccine strain Escherichia coli MM-3 and evaluation of the immunogenicity of the new strain.

MM-3 was a live vaccine strain candidate for protecting neonatal piglets from diarrhea. Designed in the 1980s, a high degree of protection from colibacillosis was afforded to piglets in a challenge study and field trials. However MM-3 had a drawback of carrying the antibiotic resistance gene (chloramphenicol acetyltransferase gene, cat). The introduction of a host-plasmid balanced lethal system into the vaccine was a good idea to solve the problem. The lambda-Red recombination system was adopted in this study to realize the replacement of cat by aspartate-semialdehyde dehydrogenase gene (asd) in the plasmid pMM085. The new plasmid named pMMASD was introduced into an Escherichia coli strain chi6097 and Salmonella typhimurium chi4072 where the asd gene had been knocked out in their chromosomes. Cultured in an Erlenmeyer flask, expression levels of two antigens K88ac fimbriae and heat-labile enterotoxin B subunit (LTB) in cell lysate were similar among MM-3, chi4072(pMMASD) and chi6097(pMMASD). However, chi4072(pMMASD) possessed the more effective secretion mechanism to transport LTB enterotoxin into culture liquid. The relatively higher stability of pMMASD in Salmonella typhimurium chi4072 than that of pMM085 in MM-3 was determined both in vitro in the absence of selective pressure, and in vivo following oral inoculation. Oral immunization of BALB/c mice with chi4072(pMMASD) or chi6097(pMMASD) was sufficient to elicit IgA responses in mucosal tissues as well as systemic IgG antibody responses to the K88 fimbriae, while MM-3 failed to elicit specific antibody responses to K88 fimbriae in mucosal tissues. Among three live strains, only chi4072(pMMASD) could develop strong humoral responses against LTB enterotoxin. The results suggest that chi4072(pMMASD) is expected to be a promising live vaccine strain.

[Study on the safety and immunogenicity of enterotoxigenic Escherichia coli recombinant active vaccine].

OBJECTIVE: To study the safety, immunogenicity on the enterotoxige Escherichia coli (E. coli) recombinant active vaccine FE3 and FE16. METHODS: Toxicity and immunogenicity of the vaccine were determined by experiments on enterotoxigenic E. coli toxicity and immunological experiments on rabbits and mice. RESULTS: The results of an toxicological experiments were negative. The agglutination titer of antibodies against the S. flexneri 2a and enterotoxigenic E. coli plamid antigen were all higher than 1:640 and 1:1280 in the sera of rabbits. IgG in the serum went up remarkably, while sIgA against CFA/I was also decteted in the dejecta of mice immunized with active bacteria either orogastrically or intranasally. Simultaneously, sIgA was not detected in the dejecta of mice immunized with inactive bacteria either orogastrically or intranasally. CONCLUSION: The enterotoxigenic E. coli recombinant active vaccine showed good safety and immunogenicity, inducing both humoral and mucosal immunity in mice.

Construction of a novel Shigella live-vector strain co-expressing CS3 and LTB/STm of enterotoxigenic E.coli.

AIM: To construct and evaluate a polyvalent recombinant vaccine strain Shigella flexneri 2a T32 against enterotoxigenic E.coli (ETEC). METHODS: By using a host-plasmid balanced lethal system based on asd gene, a polyvalent recombinant strain was constructed to highly express CS3 and regularly express fusion enterotoxin of LTB subunit and mutant ST (LTB/STm) in a vaccine strain Shigella flexneri 2a T32 with specific deletion of asd gene. Fimbria CS3 was observed by immunofluorescence and electron microscopy assay. The security of LTB/STm was examined by ileal loop assay and suckling mouse assay. To evaluate this new candidate vaccine, it was compared with a previous vaccine strain in plasmid and protein level, growth assay and immunogenicity in Balb/c mice. RESULTS: The newly constructed vaccine expressed CS3 and grew better than the previously constructed vaccine except for the lower expression of LTB/STm. Serum IgG and mucosal IgA against CS3, LTB, ST, and host lipopolysaccharide (LPS) were produced after immunization of Balb/c mice by oral route with the new strain. The titers were not significantly different from the Balb/c mice with the previous strain. CONCLUSION: This novel candidate diarrheal vaccine can effectively induce serum and mucosal antibody responses against ETEC and Shigella.

Expression of Helicobacter pylori AlpA protein and its immunogenicity.

AIM: To construct a recombinant strain which expresses adhesin AlpA of Helicobacter pylori (H pylori) and to study the immunogenicity of adhesin AlpA. METHODS: Gene Ab, which was amplified from H pylori chromosomal DNA by PCR technique, was sequenced and the biological information was analyzed, and inserted into the Nco I and Not I restriction fragments of the expression vector pET-22b(+) using T4 DNA ligase. The resulting plasmid pET-AlpA was transformed into competent E.coli BL21(DE3) cells using ampicillin resistance for selection. Recombinant strains were incubated in 5 mL LB with 100 mug/mL ampicillin overnight at 37 degrees. Sonication of BL21(DE3)pET-22b(+)/AlpA was analyzed by Western blot to detect AlpA immunogenicity. RESULTS: The gene encoding AlpA protein was amplified by PCR with chromosomal DNA of H pylori Sydney strain (SS1) as templates. It revealed that AlpA DNA fragment amplified by PCR had approximately 1 500 nucleotides, compatible with the previous reports. The recombinant plasmid pET-22b(+)/AB was successfully constructed. DNA sequencing showed one open reading frame with the length of 588 bp. It encoded seven conservative regions that showed good antigenicity and hydrophobicity by Parker and Welling method. Furthermore, INTERNET EXPASY, NNPREDICT and ISREC predicted that it was a porin-like structure consisting of beta-pleated sheets that were embedded in the outer membrane. BLAST analyzed 836 767 protein sequences and found that the similar sequences were all belonging to H pylori OMP sequences. SDS-PAGE and scan analysis showed that the molecular weight of AB was 22.5 ku and recombinant protein amounted to 29% of the total bacterial protein, among which dissolved expression amounted to 21.9% of sonicated supernatant. The rAB purity amounted to 96% through affinity chromatography. Western blot analysis of rAB confirmed that it could be specially recognized by serum form rabbit immunized with AlpA and H pylori infected. CONCLUSION: Adhesin AlpA recombinant protein may be a potential vaccine for control and treatment of H pylori infection.

[Progress on the vaccine for anthrax].

Bacillus anthracis is the causative organism of the potentially fatal disease anthrax, and the used vaccines have some disadvantages. There are new developments appeared for the Bacillus anthracis in recent years, such as anti-PA antibody kills the spore of Bacillus anthracis, mucosal immunization induces immune responses in both systemic and secretory immune compartments, Poly (gamma-D-PGA) protein induce IgG antibodies to the vegetative bacteria, new pathogens were found by genomic analysis. The DNA vaccine and live vector vaccine will be the next generation vaccines for anthrax. It will have a shorter immunization schedule and will be greater protective efficacy than before.

Construction of attenuated Salmonella typhimurium Strain expressing Helicobacter pylori conservative region of adhesin antigen and its immunogenicity.

AIM: To construct a non-resistant and attenuated Salmonella typhimurium (S. typhimurium) strain which expresses conservative region of adhesion AB of Helicobacter pylori (H pylori) and evaluate its immunogenicity. METHODS: The AB gene amplified by PCR was inserted into the expression vector pYA248 containing asd gene and through two transformations introduced into the delta Cya, delta Crp, delta Asd attenuated Salmonella typhimurium strain, constructing balanced lethal attenuated Salmonella typhimurium strains X4072 (pYA248-AB). Bridged ELISA method was used to measure the expression of AB antigen in sonicate and culture supernatant. According to the method described by Meacock, stability of the recombinant was evaluated. Semi-lethal capacity test was used to evaluate the safety of recombinant. The immunogenicity of recombinant was evaluated with animal experiments. RESULTS: The attenuated S. typhimurium X4072 (pYA248-AB) which expresses AB was successfully constructed. Furthermore, bridged ELISA assay showed that the content of AB in recombinant X4072 (pYA248- AB) culture supernatant was higher than that was in thallus lytic liquor. And after recombinant X4072 (pYA248- AB) was cultured for 100 generations without selection pressure, the entire recombinant bacteria selected randomly could grow, and the AB antigen was defected positive by ELISA. The growth curve of the recombinant bacteria showed that the growth states of X4072 (pYA248) and X4072 (pYA248-AB) were basically consistent. The survival rate of C57BL/6 was still 100%, at 30 d after mice taking X4072 (pYA248-AB) 1.0 x 10(10) cfu orally. Oral immunization of mice with X4072 (pYA248-AB) induced a specific immune response. CONCLUSION: In vitro recombinant plasmid appears to be stable and experiments on animals showed that the recombinant strains were safe and immunogenic in vitro, which providing a new live oral vaccine candidate for protection and care of H pylori infection.

Cloning and expression and immunogenicity of Helicobacter pylori BabA2 gene.

AIM: To construct a recombinant strain which expresses BabA of Helicobacter pylori (H pylori) and to study the immunogenicity of BabA. METHODS: BabA2 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+) and expressed in the BL21 (DE3) E.coli strain. Furthermore, BabA immunogenicity was studied by animal test. RESULTS: DNA sequence analysis showed the sequence of BabA2 DNA was the same as the one published by GenBank. The BabA recombinant protein accounted for 34.8% of the total bacterial protein. The serum from H pylori infected patients and Balb/c miced immunized with BabA itself could recognize rBabA. CONCLUSION: BabA recombinant protein may be an potential vaccine for control and treatment of H pylori infection.

Antigen epitope of Helicobacter pylori vacuolating cytotoxin A.

AIM: To construct and select antigen epitopes of vacuolating cytotoxin A (VacA) for nontoxic VacA vaccine against Helicobacter pylori (H pylori ) infection. METHODS: Eleven VacA epitopes were predicted according to VacA antigenic bioinformatics. Three candidates of VacA epitope were constructed through different combined epitopes. The candidate was linked with E. coli heat-labile enterotoxin B (LTB) by a linker of 7 amino acids, and cloned into plasmid pQE-60 in which fusion LTB-VacA epitope was efficiently expressed. To test the antigencity of the candidate, 6 BALB/c mice were treated with the fusion LTB-VacA epitope through intraperitoneal injection. To explore the ability of inhibiting the toxicity of VacA,cantiserum against the candidate was used to counteract VacA that induced HeLa cells to produce cell vacuoles in vitro. RESULTS: Serum IgG against the candidate was induced in the BALB/c mice. In vitro, the three antisera against the candidate efficiently counteracted the toxicity of VacA, and decreased the number of cell vacuoles by 14.17%, 20.20% and 30.41% respectively. CONCLUSION: Two of the three candidates, LZ-VacA1and LZ-VacA2, can be used to further study the mechanism of vacuolating toxicity of VacA, and to construct nontoxic VacA vaccine against H pylori infection.

[Expression of human mu-opioid receptor cDNA in CHO cell].

Opioid receptor, is classified into three subtypes, mu, kappa and delta, with the mu-type receptor plays important roles in opioid analgesia and opioid addiction. The cDNA encoding mu-type receptor was obtained by RT-PCR from human brain RNA and was cloned into pcDNA3.1(+). The resultant recombinant plasmid pcDNAMORs were transfected into CHO cells by liposome. After PCR identification, the positive clone were treated with agonist and antiagonist were tested for their competence of signal transduction. CHO cells that contained mu-opioid receptor in the expression vector pcDNA3.1(+) acquired naloxone-blockable high-affinity specific binding of morphine and DAMGO. The concentration of cAMP in CHO cells transfected with pcDNAMOR was reduced after binding to morphine and DAMGO, and increased after binding naloxone. These results indicate that the mu-type receptor expreesd on the CHO cell has similar biological property as the nature receptor. The availability of these specific cell lines will facilitate the drug development and promote our understanding the mechanism underlying opiate addiction.

Expression of Helicobacter pylori Hsp60 protein and its immunogenicity.

AIM: To express Hsp60 protein of H pylori by a constructed vector and to evaluate its immunogenicity. METHODS: Hsp60 DNA was amplified by PCR and inserted into the prokaryote expression vector pET-22b (+), which was transformed into BL21 (DE3) E.coli strain to express recombinant protein. Immunogenicity of expressed Hsp60 protein was evaluated with animal experiments. RESULTS: DNA sequence analysis showed Hsp60 DNA was the same as GenBank's research. Hsp60 recombinant protein accounted for 27.2% of the total bacterial protein, and could be recognized by the serum from H pylori infected patients and Balb/c mice immunized with Hsp60 itself. CONCLUSION: Hsp60 recombinant protein might become a potential vaccine for controlling and treating H pylori infection.

[Co-expression of CFA/I and CS6 of Enterotoxigenic Escherichia coli ( ETEC ) in Shigella flexneri 2a T32 Derivative Strain FWL01].

Among the known colonization factors of enterotoxigenic Escherichia coli (ETEC), CFA/I and CS6 (the common antigen in the CFA/IV fimbrial antigens ) are two of the most prevalent fimbriae found in clinical isolates but are never expressed by the same wild-type strains. In this study, CFA/I and CS6 of ETEC were co-expressed in Shigella flexneri 2a T32 derivative strain FWL01 by using a host-plasmid lethal balancing system based on asd gene. The results indicate that the recombinant plasmid carrying CFA/I and CS6 could be stably integrated in FWL01. Expression of the two antigens did not interfere the host growth. The results of immunofluorescence analysis showed that CFA/I and CS6 were localized on the surface of the strain FWL01. In Balb/c mice orally immunized with the recombinant strain, the immune responses against CFA/I and CS6 were observed. Those observations show the feasibility of a multivalent vaccine expressing different fimbrial antigens in attenuated Shigella flexneri.

[Cloning and immunogenicity of conservative region of adhesin gene of Helicobacter pylori].

OBJECTIVE: To construct a candidate strain of Helicobacter pylori (Hp) that expresses the proteins of the conservative region of 4 adhesins (BabA2, AlpA, AlpB, and HopZ) and study its immunogenicity. METHODS: The DNA of Hp was extracted. Primers were designed according to the C-terminal structural gene sequence (called CB) of AlpA. The CB gene was amplified by PCR and inserted into the prokaryotic expression vector pET-22b (+) and expressed in BL21 (DE3) strain of Escherichia coli. The product of expression, CB, was purified by affinity chromatography and identified by Western blot analysis. ELISA assay was used to measure the CB-specific antibody in the specimens of serum of 55 Hp infected patients. Rapid urease test (RUT) was used on biopsy specimens collected by gastroscopy as parallel control. RESULTS: A recombinant plasmid pET-22b (+)/CB was constructed with the conservative region of the 4 adhesins. DNA sequencing showed one open reading frame of 588 bp encoding a polypeptide of 195 amino acids. The recombinant CB (rCB) protein, with a molecular weight of 22.5KD, amounted to 29% of the total bacterial protein. The purity of purified rCB was 96%. Western blot analysis showed that the rCB protein could be specifically recognized by the serum from Hp infected patients. The kappa coefficient was 0.76 for evaluation by ELISA and RUT results. CONCLUSION: CB has the potential to be used as a vaccine against Hp infection.

Recombinant Helicobacter pylori catalase.

AIM: To construct a recombinant strain which highly expresses catalase of Helicobacter pylori (H. pylori) and assay the activity of H. pylori catalase. METHODS: The catalase DNA was amplified from H. pylori chromosomal DNA with PCR techniques and inserted into the prokaryotie expression vector pET-22b (+), and then was transformed into the BL21 (DE3) E.coli strain which expressed catalase recombinant protein. The activity of H. pylori catalase was assayed by the Beers and Sizers. RESULTS: DNA sequence analysis showed that the sequence of catalase DNA was the same as GenBank's research. The catalase recombinant protein amounted to 24.4 % of the total bacterial protein after induced with IPTG for 3 hours at 37 degrees and the activity of H. pylori catalase was high in the BL21 (DE3) E.coli strain. CONCLUSION: A clone expressing high activity H. pylori catalase is obtained, laying a good foundation for further studies.

[Construction of the non-resistant attenuated Salmonella typhimurium strain expressing Helicobacter pylori catalase].

OBJECTIVE: To construct a non-resistant attenuated Salmonella typhimurium (S.typhimurium) strain capable of expressing Helicobacter pylori (Hp) catalase. METHODS: After PCR amplification, the gene fragment encoding Hp catalase was inserted into the expression vector pYA248 containing asd gene, and the recombinant vector was then introduced into the host S.typhimurium strain X4072 depleted of genes encoding adenylate cyclase (delta cya), cyclic adenosine monophosphate receptor protein (delta crp) and aspartate-beta-semialdehyde dehydrogenase (delta asd). Bridged enzyme-linked immunosorbent assay (ELISA) was employed to measure the antigenicity of the catalase expressed in the sonicate and culture supernatant. According to Meacock's method and with the assistance of the growth curve, the stability of the recombinant strain was evaluated. A half lethal oral dose test was conducted to evaluate the safety of recombinant strain. RESULTS: S.typhimurium X4072 (pYA248-CAT) with expected capacity was successfully constructed, and bridged ELISA demonstrated higher catalase levels in the culture supernatant than in the sonicate of the recombinant strain X4072 (pYA248-CAT). After the strain was passaged for 100 generations without selection pressure, all the randomly selected colony of the recombinant strain grew well with positive catalase antigenicity as identified by ELISA. The growth curve of the recombinant strain showed comparable growth status of the 2 strains X4072 (pYA248) and X4072 (pYA248-CAT). The survival rate of C57BL/6 mice was 100% 30 d after oral administration of 1.0x10(10) cfu X4072 (pYA248-CAT). CONCLUSION: Non-resistant S. typhimurium vaccine X4072 (pYA248- CAT) is constructed successfully, which is stable in vitro and safe as confirmed by animal experiment. This vaccine provides a new candidate for viable oral vaccine against Hp infection.

[Simultaneous expression of CS3 colonization factor antigen and LT-B/ST fusion enterotoxin antigen of enterotoxigenic Escherichia coli by attenuated Shigella flexneri 2a].

Enterotoxigenic Escherichia coli (ETEC) causes watery dehydrating diarrhea in infants in developing countries, and is the most common cause of travelers diarrhea. It has been known that the colonazition factor antigens (CFAs) and enterotoxins are important virulence factors of ETEC, and these two kinds of proteins should be included in any effective vaccine against ETEC. In this study, a host-plasmid lethal balancing system was constructed based on asd gene in an avirulent strain of S.flexneri to express CS3 antigens and the fusion LT-B/ST enterotoxins of Escherichia coli. Both of these antigens were expressed steadily in the S. flexneri vector without any antibiotic markers. Antibodies against CS3, LT, ST and LPS of Shigella were detected in sera of mice that were immunized with recombinant bacteria either oragastrically (o.g.) or intranasally (i.n.). SIgA against CS3 and enterotoxins were detected simultaneously in feces of mice. This work is helpful for constructing multivalent recombinant vaccine for prevention of bacterial diarrhea.

[Construction of the attenuated Salmonella typhimurium strain expressing Helicobacter pylori conservative region of adhesin antigen].

To construct a non-resistance and attenuated Salmonella typhimurium strain which expresses conservative region of adhesin(AB) of Helicobacter pylori(Hp). The AB gene was amplified by PCR and inserted into the expression vector pYA248 containing asd gene and was introduceded into the delta Cya, delta Crp, delta Asd attenuated Salmonella typhimurium strain by twice transformations, which is a balanced lethal recombinant. Bridged ELISA method was used to measure AB expressed in sonicate and culture supernatant. According to Meacock's way and growth curve, stability of the recombinant is evaluated. Semi-lethal capacity test was used to evaluate the safty of recombinant. Results showed S. typhimurium X4072(pYA248-AB) was constructed successfully, recombinant X4072(pYA248- AB) content of supernatant serum was higher than that of thallus lytic liquor confirmed by bridged ELISA, and after recombinant pYA248- AB cultured 100 generation without selection pressure, all the recombinant germ selected randomly can grow, and the AB antigen was positive by ELISA detection. The growth curve of the recombinant germ showed that the growth state of X4072(pYA248) and X4072(pYA248- AB) were coincidence on the whole, and the survival rate of C57BL/6 was still 100%, 30 days after taking X4072(pYA248- AB) 1.0 x 10(10)cfu. orally. Non-resistance S. typhimurium X4072(pYA248- AB) was constructed successfully. The recombinant plasmid was stable indicated by in vitro experiment. And the recombinant strain was safe confirmed by animal experiment. This live vaccine strain is worthy to be considered as a new live oral vaccine candidate against Hp infection.

Conservative region of the genes encoding four adhesins of Helicobacter pylori: cloning, sequence analysis and biological information analysis.

OBJECTIVE: To clone the conserved regions of the genes encoding the 4 adhesins (BabA, AlpA, AlpB and HopZ) of Helicobacter pylori (H. pylori) and analyze their sequences and biological information, thus facilitating further research in the molecular mechanism and immunogenicity of H. pylori adhesins. METHODS: Common conserved region (designated as CB) was identified from the confirmed sequences (by ANTHEPROT V4.3c software package) of the 4 adhesin proteins. Their DNA sequences were deduced, according to which primers specific to CB were designed for subsequent PCR, and the products were inserted directionally into pET-22b(+) vector to construct recombinant clones of the conserved region. The DNA sequences were determined with the basic local alignment sequence tool (BLAST) and the biological properties analyzed with ANTHEPROT V4.3c software package. RESULTS: The recombinant plasmid containing the CB sequence was constructed. DNA sequencing showed an open reading frame of 588 bp in length, encoding 195 amino acids. The homogencity of conservative region of the 4 adhesion genes was above 50%. The corresponding protein possessed a relative molecular mass (Mr) of 22 500 as predicted by ANTHEPROT V4.3c software prediction, with excellent antigenicity and hydrophobicity. There were 836 767 sequences analyzed with BLAST, in which those with homogencity of 40% with the identified CB sequence were categorized into H. pylori sequences. CONCLUSION: There are conservative regions in the 4 adhesin genes with similar homogencity, suggesting similar molecular basis for adhesion of the adhesins. Biological information analysis indicates that CB has excellent immunogenicity and strict species specificity.

[Study on the cloning, expression and the immunogenicity of helicobacter pylori heat shock protein 60 gene].

OBJECTIVE: To construct a recombinant strain of bacteria expressing heat shock protein of (Hsp) Helicobacter pylori (Hp) and study the immunogenicity of Hsp60. METHODS: PCR amplification of Hsp60 DNA was performed before it was inserted into the prokaryotic expression vector pET-22b(+) to transform BL21(DE3) E.coli strain. Hsp60 expressed by the recombinant E.coli was collected and purified for immunogenicity assessment in mice. RESULTS: DNA sequence analysis showed identical DNA sequence of Hsp60 thus produced to that published in Genbank. Accounting for a ratio of 27.2% among the total protein production in the bacterium, recombinant Hsp60 protein was recognized by the serum from Hp-infected patients and produced corresponding antibody in Balb/c mice in response to immunization. CONCLUSION: Recombinant Hsp60 protein can be used potentially as a vaccine for controlling and treating Hp infection.