细粒棘球绦虫重组CTxB-Eg95(TP)融合蛋白的原核表达

目的构建CTxB-Eg95(TP)融合基因原核表达载体,并诱导表达CTxB-Eg95(TP)融合蛋白。方法采用PCR技术分别克隆CTxB基因和141bp的Eg95基因片段序列Eg95(TP)并鉴定。分别将两个基因片段定向克隆到大肠杆菌表达载体pET28a(+)中,构建pET-CTxB-Eg95(TP)重组质粒。将鉴定为阳性的重组质粒转化E.coliBL21(DE3)感受态细胞,经IPTG诱导后,进行SDS-PAGE电泳鉴定。结果测序结果表明已成功构建CTxB-Eg95(TP)融合基因的原核表达载体,SDS-PAGE电泳结果显示,转染pET-CTxB-Eg95(TP)的E.coliBL21(DE3)菌经IPTG诱导可表达大小约23.5kDa的特异蛋白条带,与预期结果一致。结论CTxB-Eg95(TP)融合基因获得了高水平的表达,为Eg95的表位研究和应用于口服疫苗奠定了基础。

Prokaryotic expression of the recombinant fusion protein CTxB-EG95(TP) from Echinococcus granulosus

To construct a recombinant prokaryotic expression vector for gene encoding fusion protein CTxB-Eg95(TP),the sequences of cholera toxin B subunit(CTxB)gene and the gene for Eg95 of 141 bps were cloned respectively and identified by PCR.These two gene fragments were inserted into a prokaryotic vector pET28a(+)to construct the recombinant plasmid pET-CTxB-Eg95(TP).Then,the recombinant plasmid identified to be positive was transfected into susceptible cells of E.coli BL21(DE3),and the expressed protein obtained after IPTG induction was identified by SDS-PAGE electrophoresis.As demonstrated by the results of sequencing,it was indicated that the prokaryotic expression vector for fusion gene pCTxB-Eg95(TP)had been successfully constructed and the result in SDS-PAGE elctrophoresis revealed the presence of specific bands with molecular mass of 23.5 kDa could be observed in the expressed product from E.coli BL21(DE3)cells trasfected with plasmid pET-CTxB-Eg95(TP)after induction with IPTG.The molecular mass of the special protein was just the same as the expected result of the fusion protein CTxB-Eg95(TP).From these observations,it is concluded that the fusion protein CTxB-Eg95(TP)has already been expressed,and the results obtained in the present study can provide a basis for the development of the oral vaccine for Eg95 in the future.