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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 express the recombinant protein of pQE-vctB (fusion gene containing the vacuolar toxin toxic fragment of H.pylori cell and cholera toxin B subunit) in prokaryotic E. coli DH5α to obtain the recombinant protein and identify its antigenicity and immunity For the preparation of oral vaccine against H.pylori infection laid the foundation. Methods (1) pQE-vctB was transformed into E. coli DH5α, induced by IPTG to express recombinant protein VCTB, purified by Western blot and purified by nickel ion column. (2) The recombinant protein VCTB was orally immunized, and the serum specific IgG and small intestinal wash IgA were detected by ELISA to identify its immunogenicity. Results The relative molecular weight (Mr) of the recombinant protein VCTB was about 40kD by SDS-PAGE. The purity of the protein was 92% after affinity chromatography. Western blot analysis showed reaction with VacA antiserum. The results of ELISA showed that the serum IgG and IgA of immunized mice were significantly higher than those of VacA control group (P <0.01). Conclusion The prokaryotic expression plasmid vacA and ctxB fusion gene was transformed into E. coli DH5α to obtain the recombinant protein VCTB. The recombinant protein VCTB was expressed in high purity with high purity and good antigenicity and immunogenicity. Oral immunization could significantly enhance the immunity Effect, resulting in a higher level of IgA, can be used to prepare oral vaccine against H.pylori infection.