幽门螺杆菌重组VacA蛋白的表达、纯化和鉴定

Expression,Purification and Identification of Recombinant VacA of Helicobacter pylori

建立从幽门螺杆菌空泡毒素A(VacA)原核表达系统pET32a-vacA-E.coliBL21DE3中,表达、纯化和鉴定重组空泡毒素A(rVacA)蛋白的方法.采用不同浓度的IPTG(0.1、0.5和1.0 mmol.L-1)诱导原核表达系统表达rVacA,10%SDS-PAGE检测表达产量,Ni-NTA亲和层析法纯化rVacA,采用HPLC检测其纯度,Western-blotting进行免疫学鉴定.从已构建的VacA原核表达系统中成功表达和纯化了rVacA蛋白,产量约占细菌总蛋白的28.4%,纯度为82.3%.为后续进一步研究该蛋白的生物活性和相关检测试剂盒奠定基础.

The researds is to express, purify and identify the recombinant VacA（rVacA）protein of Helicobacter pylori from prokaryotic expression system pET32a-vacA-E, coliBL21DE3. The expression of recombination protein is induced by different concentrations of IPTG（0.1,0.5 and 1.0 mmol.L^-1 ）and the expressed product is identified by 10% SDS-PAGE. The expressed recombinant protein is purified by Ni-NTA affinity chromatography. The purity and immunogencity of rVacA are detected by HPLC and Western-blotting, respectively. The expression output of rVacA is approximate 28.4% of the total bacterial protein and the purity is about 82.3%. In this study, the rVacA can be expressed and purified successfully in prokaryotic expression system, which establishes a foundation for the further study of the protein activity and the relational detection kits.