幽门螺杆菌cag4蛋白的原核表达、纯化及鉴定

目的:构建幽门螺杆菌cag4蛋白的原核重组体系,表达、纯化融合蛋白。方法:利用PCR技术从幽门螺杆菌NCTC11637中克隆了cag4基因;经T-A克隆,酶切鉴定,构建了原核表达载体pET-28a-cag4;经测序鉴定正确后,转化进入大肠埃希菌BL21(DE3)进行异源表达。利用IPTG体外诱导后,经SDS-PAGE和Western bolt鉴定目的蛋白表达后,采用Ni2+-NTA柱在变性条件下纯化目的蛋白,并对重组蛋白进行透析复性处理。将SDS煮沸法获得的溶壁微球菌肽聚糖掺入SDS-PAGE作为底物,进行酶谱分析。结果:成功扩增了cag4基因基因;重组质粒在大肠埃希菌BL21(DE3)中诱导表达出pET-28a-cag4融合蛋白;优化了pET-28a-cag4原核表达体系的最适条件;Western bolt分析结果显示表达的基因重组蛋白与目的蛋白相符;酶谱分析显示其具有明显的肽聚糖水解活性。结论:幽门螺杆菌cag4蛋白具有溶菌糖基转移酶活性。

Prokaryotic expression,purification and identification of cag4 in Helicobacter pylori

Objective:To construct a recombination prokaryotic system of cag pathogenicity island protein 4(cag4) for expression and purifica-tion of the fusion protein.Methods:PCR technique was used to amplify cag4 gene encoding lytic transglycosylase in CagPAI of helicobacter pylori NCTC11637.By TA cloning and restriction enzyme digested,we constructed prokaryotic expression plasmid pET-28a-cag4 and transformed the plasmid into E.coli BL21(DE3) for heterogenesis expression after sequenced.SDS-PAGE and Western blot we...