禽网状内皮组织增生病病毒囊膜蛋白基因的克隆及其原核表达

Cloning and Prokaryotic Expressing of the Envelope Glycoprotein Gene of Avian Reticuloendotheliosis Virus

以本实验室保存的REV毒株为模板,采用RT_PCR技术扩增了REV env部分基因,长为939 bp,并进行亚克隆到pMD18_T质粒载体上,连接、转化、鉴定后得到阳性重组质粒pET_env,将目的片段进行序列分析,结果表明,该REV的env基因与已发表的SNV株、中国HA9901株以及A株同源性均达94%以上,推导的氨基酸同源性为94%以上。将该基因片段与原核表达载体pET_32a重组,并将重组质粒转化至宿主菌BL21中,用IPTG诱导表达,对表达的蛋白用SDS_PAGE电泳,免疫印迹和薄层凝胶扫描分析,表达产物与理论推理一致;免疫印迹结果证明,表达的env蛋白可被REV阳性血清所识别。

The viral sub - genome mRNA of Avian Reticuloendotheliosis Virus was amplified with RT-PCR. A DNA fragment was amplified which contains a part of env gene, and the size of the DNA fragment about 939 bp. The PCR products were purified and then cloned into plasmid pMD18-T, the recombinant plasmid were designated pMD-env and analyzed by endonecleoase digestion from proper inserts. The sequence analysis of the insert fragment in the recombinant pMD-env indicated that env shared more than 94% with SNV strain, HA9901 strain, and A strain. The amino acid sequence homology was above 94%. The env gene was subcloned into a prokaryotic expressing vector, pET-32a. Molecular cloning of env gene from REV provides a basis for the studies on protein expressing and molecular characteristic, and the constructed recombinant pET-env can be used in further protein expressing.