粪肠球菌介导的细粒棘球绦虫重组Efs-Eg95-EgA31疫苗的构建、鉴定及表达

Construction,identification and expression of Enterococcus faecalis_based recombinant Efs-Eg95-EgA31 vaccine of Echinococcus granulosus

目的构建粪肠球菌(Efs)为载体的细粒棘球绦虫重组Efs-Eg95-EgA31疫苗,并研究其表达效率。方法采用电穿孔技术将重组质粒pGEX-Eg95-EgA31转化粪肠球菌ATCC47077株,构建rEfs-Eg95-EgA31疫苗,抽提质粒进行PCR扩增鉴定;经IPTG诱导后进行10%SDS-PAGE和Western blot等分析和鉴定表达产物。结果以从rEfs抽提的质粒为模板进行PCR可扩增出约1016 bp的Eg95-EgA31融合基因片段,SDS-PAGE显示表达产物为62.5 ku的重组蛋白,表达蛋白约占菌体总蛋白的11%;Western blot表明重组蛋白能被细粒棘球蚴感染的鼠血清识别。结论成功构建了细粒棘球绦虫rEfs-Eg95-EgA31疫苗,表达的融合蛋白具有特异的抗原性。

Objective To construct an Enterococcus faecalis_based recombinant Efs-Eg95-EgA31 vaccine of Echinococcus granulosus and observe its expression efficiency.Methods The recombinant plasmid pGEX-Eg95-EgA31 was electroporated into Enterococcus faecalis(Efs)ATCC47077 strain to construct rEfs-Eg95-EgA31 vaccine,the plasmid was extracted from rEfs for PCR identification.The rEfs vaccine was expressed through IPTG induction,and the recombinant protein was verified by 10%SDS-PAGE and Western blotting.Results PCR showed that 1016bp Eg95-EgA31 fusion gene was amplified when the extracted plasmid from rEfs as template;10%SDS-PAGE confirmed the Mr of the expressed GST-Eg95-EgA31 was about 62.5 ku,the expressed effficiency was approximately 11%;Western blotting demonstrated that the expressed protein could be recognized by sera from mice infected with hydatid cyst.Conclusions The rEfs-Eg95-EgA31 vaccine was constructed in success,and the expression protein was provided with special antigenicity.