| Objective:Helicobacter pylori (H. pylori) is well recognized to be a major risk factor for recurrent gastroduodenal inflammatory diseases. The study aimed to choose the fragment from full UreB protein by bioimformatics analysis and urease inhibition (neutralizing) test, then construct fusion protein vaccine and fusion gene DNA vaccine with HspA, HpaA and UreB fragment. Explore the immune protective mechanism and pave the way for the new H.pylori vaccine through different immune responses and protective efficacy with these vaccines in mice Methods:1. choose the suitably fragment protein from UreB With the surface property analysis, solvent-accessible suface calculateions, secondary structure function region analysis and protein 3D structures.2. The gene encoding selective fragment protein (UreB414) was obtained from H.pylori CQ9803 chromosomal DNA by PCR, and then was cloned into prokaryotic expression vector pET-28a(+) by restriction endonucleases. The recombinant UreB414 protein was expressed in E.coli BL21 induced by IPTG and proved by Tris-trcine PAGE and western blotting. An approximate molecular weight was forecasted by Antheprot V 5.0. Rabbits were immunized by the recombinant UreB414 protein purified with AKTA-explore 100 system. And the serum of rabbits m immunized with the UreB414 protein.was used in Urease inhibition test.3. We constructed the fusion gene hhu including HspA, HpaA and UreB414 gene by SOE PCR (splicing by overlap extension), and then cloned the fusion gene hhu into the expression plasmid pET-28a. The fusion protein was expressed in E.coli BL21 induced by IPTG, confirmed by SDS-PAGE and west-blotting tests, and purified with AKTA-explore 100 system also.4. 160 eight-week-old BALB/c mice were divided into four groups ranomly, and thenimmunized orally with the 150ug fusion protein (HspA-HpaA-UreB414), mix proteins (HspA+HpaA+UreB), single protein (UreB) and PBS combined with 10ug LT at day 0,7,14,28 respectively. 10 day after last immunized, the samples including serum, feces, supematants from extracted gastric and intestinal mucosal of 10 mice in every groups were collected. The level of IgG, IgA, IgG1, IgG2a and slgA after immunization were assessed by ELISA, the amount of IgA-ASC, IgG-ASC was assessed by EISPOT, the level of IL-4 and IFN-7 was deteced by RT-PCR. The left 30 mice were challenged with 108CFU H.pylori. On Day 30 after challenged, Stomachs were removed, cut and washed thoroughly in sterile PBS to remove the food contents. H.pylori culture, rapid urease test (RUT) and special PCR was used to evaluate the immune protection on stomachs samples.5. The fusion gene hhu was subcloned into plasmid vector pCDNA3.1(+). COS-7 cells lines were transfected with the plasmid vector expressed hhu (pCDNA3.1-hhu). 24h later, the fusion protein was determined by RT-PCR. BALB/c mice were intramuscularly immunized with lOOug plasmid pCDNA3.1(+) or pCDNA3.1 -hhu each at day 0,21,42 in different groups. The same sample was assessed as previously described. Results:1. According to protein prediction, 414bp fragment was choiced from full UreB. which contained the major function domain of UreB.2. Recombinant prokaryotic expression vector pET-28a-UreB414 was constructed. The fragment UreB414 has an approximate molecular weight of 15.8KD. the UreB414' purity was over 90% after purification. The antibodies from the rabbit serum immunized with the purity protein UreB414 can inhibit the activity of urease in H.pylori.3. The fusion gene was constructed successfully by SOC PCR. The different gene was linked by PAVPPP linker.4. The fusion gene was cloned into plasmid pET-28a(+). The fusion protein was confirmed by SDS-PAGE and west-bloting. The expressing ratio of fusion protein was 26%. After affinity chromatography and ion-exchange chromatography, we got the fusion protein with purity of more than 85%.5. After immunization, the level of IgG, IgA, IgG1, IgG2a in serum and the level of sIgA in both stomach and duodenum was higher than that of PBS control group (p<0.001). The number of...
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