细粒棘球绦虫硫氧还蛋白过氧化物酶基因的表达及抗原性分析

Expression and antigenicity analysis of thioredoxin peroxidase gene from Echinococcus granulosus

目的在原核系统表达并初步鉴定细粒棘球绦虫硫氧还蛋白过氧化物酶(EgTPx)基因重组蛋白的抗原性。方法对已构建的重组质粒pMD-18T-EgTPx进行限制性酶切,获取目的基因片段后连接到表达质粒载体pET30a,构建重组表达质粒pET30a-EgTPx,转化大肠杆菌DH5α。筛选阳性克隆,经限制性酶切分析、PCR及测序鉴定后,转化大肠杆菌BL21(DE3),以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物,对表达产物纯化后用Western-blot方法和ELISA鉴定重组EgTPx蛋白的抗原性。结果构建的重组表达质粒pET30a-EgTPx阳性克隆经PCR与双酶切鉴定,与预期结果一致,测序鉴定无基因突变,开放阅读框正确;含有pET30a-EgTPx重组表达质粒的大肠杆菌BL21(DE3)诱导后得到了高效表达,SDS-PAGE显示表达产物分子量约为27 kDa,而且主要以包涵体形式存在;Western-blot和ELISA结果表明EgTPx重组抗原可以被棘球蚴感染羊阳性血清特异性识别。结论成功构建了pET30a-EgTPx表达质粒,EgTPx重组蛋白得到了高效表达,表达产物具有良好的抗原性。

The thioredoxin peroxidase gene from Echinococcus granulosus （EgTPx） was obtained by restriction digestion of pMD-18T-EgTPx and ligated into the expression vector pET30a. After transformation to E. coli DH5α and identification of the positive clones, pET30a-EgTPx plasmid was transformed to E. coli BL21 （DE3） and induced by IPTG for expression. The expressed produet was analyzed by SDS-PAGE and the antigenicity of the purified recombinant protein was identified by Western-blot and ELISA. It was demonstrated that after restriction digestion, PCR amplification and sequencing analysis, the constructed pET30a-EgTPx showed no gene variation and had the right open reading frame. The recombinant protein was highly expressed as fusion body with the molecular weight of about 27 kDa. The purified recombinant protein could be specifically recognized by sheep serum infected by E. granulosus by western-blot and ELISA.