细粒棘球蚴eg95基因的克隆以及原核融合表达

Clone and Prokaryotic Fused Expression of Echinococcus granulosus 95 Gene

从青海省羊源细粒棘球蚴提取基因组DNA,PCR扩增eg95基因并且克隆。测序结果表明该基因序列全长1262bp,由2个内含子和3个外显子组成,3个外显子分别为70bp、306bp和95bp。因此,推测其ORF为471bp,编码156个氨基酸。用RT-PCR方法扩增该基因的ORF,经克隆、DNAstar比对分析序列,它与预测的ORF序列100%相同。将目的基因克隆到原核表达载体pGEX-5x-1,构建重组表达质粒pGEX-5x-1-eg95,将其转入表达菌株BL21中进行原核表达。表达产物经过SDS-PAGE电泳分析,得到一条特异的蛋白条带;质谱鉴定证明表达的蛋白确实是eg95抗原蛋白;酶联接免疫吸附剂测定表明抗原抗体发生了特异性反应,进一步证明融合蛋白进行了正确表达,从而为囊型包虫病的研究奠定基础。

The genomic DNA was extracted from the sheep hydatid cyst protoscolices that was isolated from Qinghai Province in China. eg95 gene was amplified using polymerase chain reaction （PCR）and cloned. The sequencing results showed the whole length of eg95 gene is 1 262bp. There are two introns and three extrons,the length of which are 70bp,306bp and 95bp respectively. So the ORF of eg95 was presumed to be 471bp and encode 157AA. The ORF was cloned by using the RT-PCR and cloned into pGEM-T Easy plasmid. The sequences were analyzed by DNAstar and show 100% similarity with the presumed sequence. The ORF was ligased into prokaryotic expression vector pGEX- 5x-1.The recombinant expressed plasmid pGEX-5x-1-eg95 was expressed in procaryotic strain BL21.The specific protein strap was detected by SDS-PAGE and the mass-spectrum results indicated the expressed protein is the eg95 antigen protein. ELISA showed the special reaction and proved the fight expression,thus it could provide base for further study of cystic echinococcosis.