幽门螺杆菌重组VacA-CtxB蛋白的原核表达及免疫原性研究

发布时间：2018-05-31 15:00

本文选题：幽门螺杆菌 + 细胞空泡毒素　；参考：《重庆医科大学》2007年硕士论文

【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(P0.01 )。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。
[Abstract]:Objective: to construct the prokaryotic expression vector of the fusion gene of H.pylori cell vacuolating toxin (Vaca) and cholera toxin B subunit (CtxB), and to obtain the recombinant protein and identify its immunogenicity. It lays a foundation for the preparation of oral vaccine for the prevention and treatment of H.pylori infection. Methods: 1) the vacA target gene fragment was amplified by PCR, and the prokaryotic expression plasmid pQE30-vacAwas constructed. 2) ctxB target gene fragment was amplified by PCR and cloned into pQE30-vacA plasmid vacA. The expression plasmid pQE-vctB. 3) the expression of recombinant protein was induced by pQE-vctB transformation into E.coli DH5 伪 -IPTG. The antigenicity of the recombinant protein was analyzed by Western blotting and purified by nickel ion column. 4) the immunogenicity of the recombinant protein VCTB was determined by Elisa. Results: the vctB fusion gene was composed of 1092bp and was a polypeptide encoding 364 amino acid residues. The relative molecular weight of the recombinant protein VCTB was about 40 KD by SDS-PAGE analysis, which accounted for more than 20% of the total bacterial expression. After affinity chromatography, the protein with purity of more than 92% was obtained. Western blotting analysis showed that the recombinant protein could react with VacA antiserum and CT antiserum respectively. Elisa analysis showed that the recombinant protein could react with the antiserum of VacA and CT, respectively. The serum specific antibody IgG and intestinal mucus IgA of immunized mice were significantly higher than those of VacA control group (P0.01). Conclusion the prokaryotic expression plasmid of the fusion gene of Vaca and ctxB was successfully constructed, and the recombinant protein VCTB was obtained by transforming E.coli DH5 伪 expression strain. The recombinant protein VCTB was obtained with high expression quantity, high purity, good antigenicity and immunogenicity. High level of IgA can be used to prepare oral vaccine to prevent and treat H.pylori infection.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

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