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目的构建霍乱毒素B亚单位(CtB)和幽门螺杆菌尿素膜通道蛋白(UreI)融合的原核表达质粒pET32a(+)ctB/ureI,并初步研究融合蛋白CtB/UreI的表达特性和免疫特性。方法PCR从pUC18ctB中克隆ctB基因,定向在pET32a(+)/ureI的ureI基因5′端插入ctB基因,构建ctB和ureI双基因原核表达质粒pET32a(+)ctB/ureI,转该质粒于E.coliBL-21(DE3),经酶切和序列分析鉴定工程菌。IPTG诱导表达,HP-His亲和层析纯化,SDS-PAGE和Gel-ProAnalizer4分析,重组蛋白免疫BALB/c小鼠。用Westernblot和ELISA分析重组蛋白的免疫特性。结果工程菌含完整的ctB和ureI基因,与相对应基因的序列同源性分别为100%。在22℃,1mmol/LIPTG诱导4h后,重组蛋白的表达占菌体总蛋白12%,亲和层析纯化后蛋白纯度为94.3%。Westernblot表明重组蛋白分别能与相应的抗体反应,该蛋白免疫小鼠后能产生相应的IgG抗体。结论成功构建了能表达CtB/UreI蛋白的大肠杆菌表达菌株。对融合蛋白表达和纯化后,初步证明了该重组蛋白有CtB和UreI的双特异反应原性和免疫原性,为研究新型幽门螺杆菌疫苗奠定了坚实的基础。
Objective To construct the prokaryotic expression plasmid pET32a (+) ctB / ureI fused with the cholera toxin B subunit (CtB) and the Helicobacter pylori urea membrane channel protein (UreI) and to study the expression and immunogenicity of the fusion protein CtB / UreI. Methods The ctB gene was cloned from pUC18ctB by PCR. The ctB gene was inserted into the 5 'end of the ureI gene of pET32a (+) / ureI. The prokaryotic expression plasmid pET32a (+) ctB / ureI of ctB and ureI was constructed. coliBL-21 (DE3), identified by enzyme digestion and sequence analysis of engineering bacteria. IPTG induced expression, HP-His affinity chromatography purification, SDS-PAGE and Gel-ProAnalizer4 analysis, the recombinant protein was immunized BALB / c mice. The immunological characteristics of the recombinant protein were analyzed by Western blot and ELISA. Results The engineered bacteria contained intact ctB and ureI genes with 100% homology with the corresponding genes respectively. After induced by 1mmol / L IPTG at 22 ℃ for 4h, the recombinant protein expressed 12% of the total bacterial proteins, and the purity of purified protein was 94.3% after affinity chromatography. Western blot showed that the recombinant protein can react with the corresponding antibody, which can produce the corresponding IgG antibody after immunization. Conclusion The recombinant E. coli expressing CtB / UreI protein was successfully constructed. After the fusion protein was expressed and purified, the recombinant protein was preliminarily proved to have the bispecific reactivity and immunogenicity of CtB and UreI, which laid a solid foundation for the study of new H. pylori vaccine.