ctB和ure I双基因融合表达及其免疫特性分析  

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

作　　者：王保宁[1,2] 杨晓芳[1] 施桥发[1] 李明远[1,3] 陈翠萍[4] 曹康[1] 李虹[1,3] 

机构地区：[1]四川大学基础医学与法医学院微生物学教研室 [2]兰州生物制品研究所,甘肃兰州730046 [3]四川大学人类疾病生物治疗国家重点实验室,四川成都610041 [4]中国药品生物制品检定所

出　　处：《细胞与分子免疫学杂志》2006年第3期276-279,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家科技部重点项目人类疾病动物模型及防治策略专项资助(No.96-A23-06-02)

摘　　要：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。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 analysis its immunogenesity. METHODS： The prokaryotic expression vector pET32a ＋/ctB/ure Ⅰ was constructed by inserting PCR amplified ctB gene into the 5＇terminus of ure Ⅰ gene of expression vector pET32a ＋/ure Ⅰ. The fusion gene was verified by endonuclease digestion and sequence. The fusion protein ctB/ure Ⅰ was expressed in E. coil 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 mouse sera was analysed by indrect ELISA. RESULTS. The pET32a ＋/ctB/ure Ⅰ expression vector was constructed successfully confirmed by endonuclease digestion and sequense. 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℃, and the protein could react with horse anti-ctB and human anti-ure Ⅰ sera when detected with Western blot, the purity of the purified protein was about 94.3%. The mouse sera 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 evoke antibody production of anti-ctB and anti-ure Ⅰ in mice. Our work established a good foundation for further study on the new and effective H. pylori vaccines.

关 键 词：幽门螺杆菌 霍乱毒素B亚单位 尿素膜通道蛋白 融合表达 免疫特性 

分 类 号：R373[医药卫生—病原生物学]