The Experimental Study On Multicomponent Fusion Vaccine Of Helicobacter Pylori Outer Membrane Protein

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.One of the key work of vaccine developing is to screen for the effective vaccine candidate proteins.At present,various approaches have been followed in the development of vaccines against H.pylori,most of which based on the use of selected antigens known to be involved in the pathogenesis of the infection,such as urease,the vacuolating cytotoxin(VacA),heat shock proteins(Hsp),the cytotoxin-associated antigen(CagA), neutrophil-activating protein(Nap).Outer membrane proteins of gram-negative bacteria had been proved to be of high immunocompetence to induce strong specific immunoreaction. As the targets for attacking of the immunocytes and antibodies,they can mediate immunoreaction to kill the bacteria more directly,and play an important role in immune protection.Omp 18 and UreB are outer membrane protein of H.pylori,Which encoding gene show highly conservative.But none of the antigens was capable of inducing protective immune response as strong as expected.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 omp18 of H.pylori were amplified from H.pylori CQ9806 chromosome DNA by PCR techniques,and the PCR product was cloned into prokaryotic expression vector pQE-30 by restriction endonucleases.The bioinformatics software DNAssist and GenBank were employed to analyze the diversity of the omp18 gene.The recombinant vectors were transformed and expression in E.coli M15,under induction of IPTG.The recombinant protein purified with AKTA-explore 100 system.The expression products were analyzed by Tris-Tricine.The purified r Omp18 was used in combination with Freuds complete adjuvant to immunize the rabbits.Western blot and immunodiffusion assay determined the immunity of r OMP 18.2,We constructed the fusion gene LO and UOL including OMP18,LTB and ureB414 gene by S OE PCR(splicing by overlap extension),setting ureB414 gene before ompl8 and LTB gene and a linker PQDPP was introduced between them.Then the LO and UOL gene were constructed into the expression plasmid pQE-30 respectively.The fusion protein was expressed in E.coli M 15 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.3,144 eight-week-old BALB/c mice were divided into six groups randomly,then immunised orally with 150μg for PBS group,multi-subunit inner adjuvant fusion protein group(UreB414-OMP18-LTB,UreB414-LTB),mono-subunit inner adjuvant fusion protein group(LTB-OMP18),mix proteins group(rOMP18+UreB414-LTB)and mono-subunit fusion protein without adjuvant group(OMP 18) respectively on 0,7,14,28 days.10 days after last immunization,the samples including serum,supernatants from extracted gastric and intestinal mucosal of 12 mices in every group were collected.The level of IgG,IgA and sIgA after immuneization were assessed by ELISA.The left 12 mice were challenged with10⁸ CFU H.pylori.30 days after challenge,Stomachs were cultured with to H.pylori culture and special PCR was 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 ompl8 had been cloned into the plasmid pQE-30 correctly.The length of the omp18 gene was 540 bp.The homology of the strains in nucleotide acid was 99%.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 pQE30-omp18 was 20kDa.The expression product of recombinant protein accounted for 20%of total bacterial protein.The purity of rompl8 was up to 85%after Q Sepharose^TM High Performance and HK 26/60 Superdex-75 chromatography.The titer of rabbit antiserum immunized with romp18 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 LO and UOL.The different gene was linked by PQDPP linker.The LO and UOL gene was cloned into plasmid pET-28a and named pLO and pUOL.Positive plasmid pLO and pUOL were transformed in E.coli BL21(DE3).After induced with IPTG at 37,the rLOand rUOL℃protein was expressed and confirmed by SDS-PAGE and west-bloting,showing an approximate molecular weight 31KD and 43KD of target protein,and the expressing ratio of fusion protein was indicated as 25%and 20%of total bacterial protein by UVP scan.After affinity chromatography,we got the fusion protein with purity of more than 85%.The fusion proteins have binding activety with GM1 ganglioside.3.The level of serologic specific IgG,IgA and the level of slgA in duodenum assessed by ELISA indicated that fusion protein immunized groups have a significant high level compared with PBS control group(p＜0.001),and significant difference were detected between subunit protein groups(p＞0.05),and no significant difference were detected between multicompotent protein mixed groups(p＞0.05).After challenge with H.pylori,protection rates in the fusion protein groups were as follows:67%(8/12) in rLO group,83%(10/12) in rUOL group.The protection rates in the groups immunized with rOMP18 + UreB414-LTB showed no marked difference(p＞0.05),but the protection rates had significantly difference with than rOMP18 groups(p＜0.05),and have a significant high level compared with PBS control group(p＜0.001). Conclusion:1,The Helicobacter pylori omp18 gene was cloned successfully in this study. Sequencing and sequence analysis were done and brought up the results that the omp18 of H.pylori SS1 shared high homology with gene of GenBank strain.The expressed and purified recombinant protein rOMP18 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 protein rLO and rUOL were constructed and expressed successfully.The fusion proteins retains an immunoreactive of every component involved in it as well as a binding activity with GM1 ganglioside,which supplies a basis for the study on immune protection ability and immune mechanism.3,The specific antibody level and immune protection effect prove that the to fusion protein rLO and rUOL of animal model is safe and has good protection effect.This establishes a basis for developing on H.pylori vaccine.