| Objective:More than half of the population in the world is chronically infected by the gastric pathogen,Helicobacter pylori,which is a major cause of chronic superficial gastritis, chronic active gastritis and peptic ulcer disease,and has a close relation with gastric mucosa associated lymphoid tissue lymphoma and gastric cancer.Current therapies for eradicating H.pylori depend on the use of combined antibiotics.However the high cost, low patient compliance,and increasing of resistant strains make these therapies impractical on a large scale.Therefore,there is an urgent need for the development of less costly and more efficient means to prevent and control H.pylori infection.Ample precedence from previous experiences suggests that vaccination may be an alternative.More recently,a new major virulence factor of H.pylori was identified and termed neutrophil activating protein(HP-NAP) for its ability of inducing adhesion of neutrophils to gastric endothelial cells and production of reactive oxygen radicals.HP-NAP is highly conserved among H.pylori strains.Since H.pylori urease has the following advantages: distribution in the bacterial outer membrane,highly conserved nucleotide and amino acid sequences,granular structure,high molecular weight and strong immunogenicity,the bacterial enzyme is initially selected as an excellent candidate of antigens in H.pylori vaccine.Since the adhesion to human gastric mucosa is the initial step for H.pylori colonization,inhibition of adhesion is thus an effective strategy to prevent H.pylori infection.HpaA has been showed that it was an adhesin presenting on bacterial surface of H.pylori and involved in the attachment to gastric epithelial cell receptors,so it is supposed to be a perfect antigen.Mucosal vaccination necessitates a suitable adjuvant.Escherichia coli heat-labile enterotoxin B subunit(LTB) have been found to be effective adjuvants.Therefore,it is necessary to make more investigation on the gene and the proteins encoded by them.We construct fusion protein vaccines and fusion gene DNA vaccines with these fragments,feed mice with them,observe different immune responses and protective efficacy develop new H.pylori protein vaccine antigens.Methods:1,The gene encoding napA of H.pylori were amplified from H.pylori SS1 chromosome DNA by PCR techniques,and the PCR product was cloned into prokaryotic expression vector pET-11c by restriction endonucleases.The bioinformatics software DNAssist and GenBank were employed to analyze the diversity of the napA gene.The recombinant vectors were transformed and expression in E.coli BL21(DE3),under induction of IPTG.The recombinant protein purified with AKTA-explore 100 system.The expression products were analyzed by Tris-Tricine.The purified rNAP was used in combination with Freuds complete adjuvant to immunize the rabbits.Western blot and immunodiffusion assay determined the immunity of rNAP.2,We constructed the fusion gene NL and NHUL including napA,LTB or hpaA-ureB414-LTB gene by SOE PCR(splicing by overlap extension),setting napA gene before LTB or hpaA-ureB414-LTB fusion gene and a linker PQDPP was introduced between them.Then the NL and NHUL gene were constructed into the expression plasmid pET-22b(+) respectively.The fusion protein was expressed in E.coli BL21 induced by IPTG, confirmed by Tris-Tricine and Western blot.The isoelectric point of target protein was predicted by DNAssist.The fusion proteins were purified with AKTA-explore 100 system. It's combination test was processed with GM1 ganglioside.3,Recombinant engineering bacterium pET28a-hul/BL21 was induced by IPTG,and after washing of inclusion body,the fusion protein was confirmed by Tris-Tricine and Western blot.4,110 eight-week-old BALB/c mice were divided into five groups randomly,then immunised orally with 150μg for PBS group,multi-subunit inner adjuvant fusion protein group(hpaA-ureB414-LTB,napA-hpaA-ureB414-LTB),mono-subunit inner adjuvant fusion protein group(napA-LTB) and mono-subunit fusion protein without adjuvant group(NAP) respectively on 0,7,14,28 days.10 days after last immunization,the samples including serum,supernatants from extracted gastric and intestinal mucosal of 10 mices in every group were collected.The level of IgG,IgA and sIgA after immunization were assessed by ELISA.The left 12 mices were challenged with 108 CFU H.pylori.30 days after challenge, H.pylori culture,rapid urease test(RUT) and special PCR were used to evaluate the immune protection on stomachs samples.Results:1,The results of digestion with restriction DNA enzymes and sequencing of the recombinant plasmid showed that the gene napA had been cloned into the plasmid pET-11c correctly.The length of the napA gene was 432 bp.The homology of the strains in nucleotide acid was 95%-98%.The homogeneity with the H.pylori strains in the amino acids was 100%.Tris-Tricine analysis showed that the relative molecule mass(Mr) of expressed product of pET11c-napA was 17kDa.The expression product of recombinant protein accounted for 50%of total bacterial protein.The purity of rNAP was up to 93.5% after Q SepharoseTM High Performance and HK 26/60 Superdex-75 chromatography.The titer of rabbit antiserum immunized with rNAP was 1:32.Western blotting showed the expressed protein was recognized by positive sera from rabbit infected with H.pylori.2,The results of digestion with restriction DNA enzymes and sequencing of the recombinant plasmids showed the fusion gene was constructed successfully by SOE PCR. The NL and NHUL gene was cloned into plasmid pET-22b(+) successfully.Positive plasmids were transformed in E.coli BL21(DE3).After induced with IPTG at 37℃,the rNL and rNHUL proteins were expressed and confirmed by Tris-Tricine and Western blot, showing approximate molecular weight 29KD and 59KD of target proteins,and the expressing ratio of fusion proteins were respectively indicated as 30%and 25%of total bacterial protein by UVP scan.After affinity chromatography and ion-exchange chromatography,we got the fusion protein with purity of more than 80%.The fusion proteins have binding ability with GM1 ganglioside.3,After induced with IPTG,the rHUL protein was expressed and confirmed by Tris-Tricine and Western blot,showing an approximate molecular weight 42KD.After washing of inclusion body,we got the target fusion protein with purity of more than 80%.4,The level of serologic specific IgG,IgA and the level of sIgA at different mucosal sites assessed by ELISA indicated that multi-subunit fusion protein immunized groups have a significant high level compared with PBS control group and rNAP immunized group(p＜0.001),and significant difference were detected between rNL immunized group (p＜0.05).After challenge with H.pylori,protection rates in the different groups were as follows:rNAP group 16.7%,rNL group 66.7%,rHUL group 83.3%,rNHUL group 90.9%.The protection rates in the groups immunized with multi-subunit fusion protein showed significant difference with than rNAP group(p＜0.05),and have a significant high level compared with PBS control group(p＜0.001).But the protection rates between rHUL and rNHUL showed no significant difference(p＞0.05).Conclusion:1,The Helicobacter pylori napA gene was cloned successfully in this study. Sequencing and sequence analysis were done and brought up the results that the napA of H.pylori SS1 shared high homology with gene of GenBank strain.The expressed and purified recombinant protein rNAP was proved with immunogenicity and competence to induce protective immune response.The gene can be taken as candidate gene in H.pylori vaccine developing.2,The fusion proteins rNL and rNHUL were constructed and expressed successfully. The purified fusion proteins keep GM1 binding ability,immunogenicity and immunoreactivity.3,The fusion protein rHUL has been induced and expressed.After washing inclusion body,the purified protein retains an immunoreactivity.4,The specific antibody level and immune protection effect of animal models prove that the fusion proteins is safe and have good protection effect.This establishes a basis for developing H.pylori vaccine.
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