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

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.