细粒棘球绦虫重组pET30a-EgA31疫苗的构建和诱导表达

Construction of the Recombinant Plasimid pET30a-EgA31 of Echinococcus Granulosus and Its Induced Expression in E. coli

克隆细粒棘球绦虫EgA31抗原基因,构建pET30a-EgA31原核表达载体,并在大肠埃希菌宿主系统中表达EgA31重组蛋白,对表达产物进行SDS-PAGE分析。从细粒棘球绦虫成虫组织中提取总RNA,反转录生成cDNA,以此cDNA为模板RT-PCR克隆获得EgA31抗原基因,将其克隆至pUCm-T载体,测序确定其正确性。利用定向克隆技术将EgA31抗原基因片段克隆至原核表达质粒pET30a上,转化E.coliDH5α,根据选择标记的卡那霉素抗性基因筛选到阳性克隆,通过PCR分析和酶切鉴定筛选出阳性克隆。IPTG初步诱导和表达pET30a-EgA31重组蛋白,SDS-PAGE电泳检测,并经凝胶图像分析确定目的蛋白的表达水平。测序表明选取的pET30a-EgA31阳性克隆均为正确连接EgA31抗原基因的重组质粒。经IPTG诱导后重组蛋白得到成功表达,在相对分子量约为31kDa处有表达条带,表达量约占菌体总蛋白质的26%。成功克隆并构建了pET30a-EgA31原核表达质粒,初步诱导表达出EgA31重组蛋白,为进一步研究其免疫特性奠定了基础。

This paper reports our work in cloning EgA31 antigen gene and constructing prokaryotic expression plasmid pET30α-EgA31, then expressing the recombinant protein EgA31 in E. coli and making analyses by SDS-PAGE. Total RNA was extracted from adult worms of Echinococcus granulosus, the eDNA encoding EgA31 antigen was amplified by RT-PCR from the Eg adult eDNA.The acquired EgA31 eDNA was cloned into pUCm-T vector to construct the recombinant plasmid pUCm-T/EgA31. Prokaryotic expressed plasmid pET30α was chosen as a vector for construction of the vaccine, called pET30α-EgA31. The recombinant plasimid pET30α-EgA31 was transformed into E. coli DH5α and the positive clones were screened by Kanamycin, identified by restriction endonuclease analysis and PCR amplification. The fusion protein EgA31 was expressed by induction with IPTG and was detected by SDS-PAGE. Sequencing results show the same results as the reported data for EgA31 eDNA. Positive clone was the exact recombinant plasmid. The recombinant protein pET30α-EgA31 could be expressed in E. coli BL21, with its relative molecule mass of expressed product of about 31 kDa, and the expression product accounting for 26% of total bacterial protein. The recombinant plasmid pET30α-EgA31 is successfully constructed and the recombinant protein EgA31 is expressed. The results obtained provide a foundation for research on its immune characteristics in the future.