ctB和ureⅠ双基因融合表达及其免疫特性分析

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/ure Ⅰ,并初步研究融合蛋白Ct B/Ure Ⅰ的表达特性和免疫特性.方法：PCR从pUC18 ctB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ure Ⅰ双基因原核表达质粒pET32a（＋）ctB/ure Ⅰ,转该质粒于E.coli BL-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-Pro Analizer4分析,重组蛋白免疫BALB/c小鼠.用Western blot和ELISA分析重组蛋白的免疫特性.结果：工程菌含完整的ctB和ure Ⅰ基因,与相对应基因的序列同源性分别为100%.在22℃,1 mmol/L IPTG诱导4 h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%.Western blot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/Ure Ⅰ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Ure Ⅰ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.

Expression and immunogenesity analysis of a recombinant fusion protein of Ⅴ. Cholera ctB and H. pylori ure Ⅰ

AIM: To express fusion protein of the cholera toxin B subunit (ctB) and the urea membrane channel gene (ure I) of H. pylori in E. coli, and analyze its immunogenicity. METHODS: The prokaryotic expression vector pET32a+/ctB/ure I was constructed by inserting ctB gene amplified by PCR into the 5' terminus of ure I gene of expression vector pET32a+/ure I. The fusion gene was verified by endonuclease digestion and sequence analysis. The fusion protein ctB/ure I was expressed in E. coli BL21(DE3), purified by His-HP affinity chromatography, and analyzed by SDS-PAGE, Western blot and Pro-gel analyzer 4.0. The mice were immunized with purified ctB/ure I, and the immunoreactivity with ctB and ure I of the murine sera was analyzed by indirect ELISA. RESULTS: The pET32a+/ctB/ure I expression vector was constructed successfully and confirmed by endonuclease digestion and sequence analysis. The expressed ctB/ure I protein with molecular weight about 58,000 was shown when induced with 1 mmol/L IPTG for 4 h at 22 degrees C, and the protein could react with horse anti-ctB and human anti-ure I sera when detected with Western blot, and the purity of the purified protein was about 94.3%. The sera from mice immunized with purified ctB/ure I protein could react with ctB, ure I, and ctB/ure I when detected with indirect ELISA. CONCLUSION: The fusion protein expression vector pET32a+/ctB/ure I was constructed successfully. The fusion protein ctB/ure I was shown to have immunoreactivity with both anti-ctB and anti-ure I anti-sera, and could evoke production of anti-ctB and anti-ure I antibody in mice. Our work established a good foundation for further study on the new and effective H. pylori vaccines.