霍乱毒素B亚单位与志贺毒素2B亚单位融合表达及抗原性检测  

Clone and express ctb-stx2b fusion gene in Enterohemrrhagic escherichia coli O157 ： H7 Shigeai toxin 2B subunit and V cholera toxin B subunit and the detection of their immunogenicity

作　　者：李振军[1] 孙强正[1] 景怀琦[1] 徐建国[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所传染病预防控制国家重点实验室,北京102206

出　　处：《中华流行病学杂志》2008年第4期378-382,共5页Chinese Journal of Epidemiology

摘　　要：目的克隆表达出血性大肠埃希菌（EHEC）O157：H7志贺毒素2B亚单位（Stx2B）与霍乱毒素B亚单位（CTB）的融合蛋白（CTB-Stx2B），并检测其抗原性和与神经节苷脂（GM1）结合能力。方法设计引物扩增融合蛋白CTB—Stx2B的编码基因ctb-stx2b，T-A克隆测序验证后克隆入原核表达质粒pET30a（＋）C，构建表达质粒pET30a（＋）-ctb-stx2b，转化E．coliBL21（DE3），IPTG诱导表达、纯化，获得目的蛋白CTB-Stx2B，SDS-PAGE和Western-blot检测其抗原性和形成五聚体的能力；GMI-ELISA法检测其与GM1结合能力。结果扩增出约750bp的目的片段，测序鉴定与设计序列一致；原核表达质粒pET30a（＋）-ctb-stx2b转化E．coliBL21（DE3）后，经酶切和PCR扩增鉴定正确；IPTG诱导后SDS-PAGE初步测定CTB-Stx2B单体的相对分子质量（Mr）约为20×10^3；Western-blot检测CTB-Stx2B能与CTB单克隆抗体结合，纯化产物经复性后大多以五聚体形式存在；ELISA检测CTB-Stx2B具有结合GM1活性。结论成功克隆、表达了融合蛋白CTB-Stx2B；表达的蛋白具有良好的CTB抗原性和GM1结合能力。Objective To clone and express the fusion gene encoding Enterohemrrhagic escherichia coli O157：H7 （EHEC O157 ： H7） Shigela toxin 2B subunit （Stx2B） and vibrio cholera toxin B subunit （CTB） as well as to detect the immunogenicity and GM1-binding ability of fusion protein. Methods To design a primer to amplify stx2b gene and ctb-stx2b fusion gene encoding Stx2B and CTB-Stx2B respectively and to clone the genes into express plasmid pET30a（ ＋ ）C in order to construct pET3Oa-ctbstx2b after T-A sequencing was varified, then to transform constructed plasmid into E. coliBL21 （DE3） induced by IPTG and purified by a purify kit and to detect molecular weight and immunogenicity by SDSPAGE and Western-blot. Results The amplified ctb-stx2b fragments appeared to be 750 bp and gene sequence was identical to designed sequence. The prokaryotic expression system pET3Oa-ctb-stx2b/BL21 could express protein weight about Mr 20×10^3 and the expressed protein could react to CTB monoclone anti-body. The fusion protein CTB-Stx2B could bind GM1. Conclusion CTB-Stx2B had successfully been expressed in prokaryotic while the expressed protein had good immunogenicity and GM1-Binding ability. This study provided information on further EHEC O157：H7 vaccine research.

关 键 词：肠出血性大肠埃希菌O157:H7 志贺毒素2B亚单位 霍乱毒素B亚单位 抗原性 

分 类 号：R392[医药卫生—免疫学]