【作者】 张任飞；

【导师】 杨致邦；

【作者基本信息】 重庆医科大学 ， 病原生物学， 2007， 硕士

【摘要】 目的:构建H.pylori细胞空泡毒素(VacA)毒性片段与霍乱毒素B亚单位(CtxB)融合基因的原核表达载体,并诱导表达,以获得重组蛋白,鉴定其免疫原性,为制备防治H.pylori感染的口服疫苗奠定基础。方法:1)用PCR扩增出vacA目的基因片段,构建原核表达质粒pQE30-vacA。2)用PCR扩增出ctxB目的基因片段,克隆至pQE30-vacA质粒vacA的基因上游,构建含双基因的表达质粒pQE-vctB。3) pQE-vctB转化E.coli DH5α,IPTG诱导表达重组蛋白VCTB,Western blotting分析抗原性,镍离子柱纯化。4)重组蛋白VCTB口服免疫小鼠,ELISA检测小鼠血清特异性IgG、小肠冲洗液IgA,以鉴定其免疫原性。结果:经测序vctB融合基因由1092bp组成,为编码364个氨基酸残基的多肽。重组蛋白VCTB经SDS-PAGE分析相对分子量(Mr)约为40KD,表达量占全菌的20%以上,亲和层析后可获得纯度为92%以上的蛋白。Western blotting分析显示能分别与VacA抗血清和CT抗血清反应。ELISA检测显示,免疫小鼠的血清特异性抗体IgG,肠粘液IgA显著高于VacA对照组(P<0.01 )。结论:vacA和ctxB融合基因原核表达质粒构建成功,转化E.coli DH5α表达菌获得了重组蛋白VCTB,表达量较高,纯度较高,有良好的抗原性和免疫原性,口服免疫小鼠可明显提高其免疫效果,产生较高水平的IgA,可用于制备防治H.pylori感染的口服疫苗。更多还原

【Abstract】 5 Objective: To construct and express the fusion gene of H.pylori vacA and cholera toxin subunit B (ctxB), To explore the immune response with the conjugated antigen of VacA-CtxB, which would lay a foundation for prophylaxis and therapy of H.pylori infection.Methods: A recombinant strain which could express bivalent antigen of VacA and CtxB subunit was constructed.1) vacA gene was amplified by PCR and cloned into plasmid pQE30which was plasmid pQE30-vacA.2) ctxB gene was amplified by PCR and cloned into plasmid pQE30-vacA, which was plasmid pQE-vctB.3) translated into E.coli strain DH5 a to express VCTB fusion protein. Its immunogenicity was analyzed by Western blotting.4) After purification,to fedm ice by oral immunization. antibody(IgG and IgA) in the delected by ELISA assay.Results: vctB fusion gene was sequenced as 1092bp, the fusion protein encoded polypeptides of 364 amino acid residues. The molecular weight was 40kD analysiced by SDS-PAGE.The levels of soluble expression product was about 20% of total cell protein.After affinity chromatography,the purity of fusion protein was above 92%.Western blotting analysis of confirmed that fusion protein could be specicifically recognized by the serum of anti-VacA and anti-CT. The specific antibody response in mice immunized with VCTB was determined by ELISA. The results showed that the levels of specific antibody were higher than those of controls.Conclusion:The successful construction and expression fusion gene of Helicobacter pylori vacA and cholera toxin subunit B(ctxB). Experiment indicated that the oral immunization with VCTB induces effective mucosal immune responece and produced higher levels IgA. The recombinant fusion protein VCTB can be used as an effective oral vaccine for prevention infection of H.pylori.更多还原