幽门螺杆菌重组vacA-CtxB蛋白的原核表达及免疫学研究  

Prokaryotic expression and immunological study of recombinant vacA-CtxB fusion protein of Helicobacter pylori

作　　者：张任飞[1] 杨致邦[2] 马永能[1] 潘淑[1] 张婧[1] 

机构地区：[1]绵阳市第三人民医院检验科,四川绵阳621000 [2]重庆医科大学基础医学院病原生物学教研室,重庆400016

出　　处：《中国微生态学杂志》2015年第11期1272-1275,共4页Chinese Journal of Microecology

基　　金：四川省卫生厅课题(120316)

摘　　要：目的原核表达幽门螺杆菌pQE-vctB(含H.pylori细胞空泡毒素毒性片段与霍乱毒素B亚单位的融合基因)在E.coil DH5α中诱导表达,以获得重组蛋白,鉴定其抗原性和免疫原性,为制备防治H.pylori感染的口服疫苗奠定基础。方法 (1)pQE-vctB转化E.coli DH5α,IPTG诱导表达重组蛋白VCTB,Western blot分析抗原性,镍离子柱纯化。(2)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。结果重组蛋白VCTB经SDS-PAGE分析相对分子量(Mr)约为40kD,亲和层析后可获得纯度为92%以上的蛋白。Western blot分析显示能与VacA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。Objective To achieve the prokaryotic expression of pQE-vctB（recombinant gene containing VacA cytotoxin segment from H.pylori and cholera toxin B subunit gene）in E.coli DH5 alpha to obtain recombinant protein,identify its antigenicity and immunogenicity,and lay the foundation for the preparation of oral vaccine for prevention of H.pyloriinfection.Methods（1）The pQE-vctB fusion gene was translated into E.coli DH5alpha;VCTB expression was induced by IPTG;Western blotting was used to analyze the antigenicity;nickel ion column was used for purification.（2）The mice were immunized with the recombinant protein of VCTB orally;ELISA was used to detecti the specific IgG in serum and IgA in intestinal washes of the immunized mice in order to identify the immunogenicity.Results SDS-PAGE showed the relative molecular weight of the recombinant VCTB protein was about 40 kD,and the purity was over 92% by affinity chromatography after purification.Western blotting showed it was able to react with VacA.ELISA assay showed that specific serum IgG and intestinal IgA were significantly higher than those of VacA control group（P〈0.01）.Conclusion The recombinant protein VCTB was successfully expressed at high level and purity,and with satisfactory antigenicity and immunogenicity,which can be used for the preparation of oral vaccine for prevention of H.pylori infection.

关 键 词：幽门螺杆菌 细胞空泡毒素 霍乱毒素B亚单位 原核表达 口服疫苗 

分 类 号：R378.9[医药卫生—病原生物学]