猪源Viperin基因的克隆、原核表达以及抗体的制备

Cloning,Prokaryotic Expression and Ployclonal Antibody Preparation of Swine Viperin Gene

为研究猪Viperin蛋白在猪体内的抗病毒活性,用IFN-α刺激PK-15细胞24 h,收集细胞提取RNA后,采用RT-PCR方法扩增猪Viperin基因,并连接至p EASY-blunt simple平末端连接测序载体,PCR鉴定后送公司测序正确。采用DNAStar分析猪Viperin蛋白的免疫原性和疏水性,选取抗原性较好的一段基因,采用PCR扩增后,插入pET-28a+大肠杆菌表达载体,在37℃条件下用IPTG诱导含有重组质粒的大肠杆菌BL21,SDS-PAGE分析证明,Viperin重组蛋白以包涵体的形式表达,包涵体蛋白经过纯化后得到目的蛋白,将纯化蛋白与弗式佐剂进行乳化后颈背部皮下注射9日龄的BALB/c小鼠,14 d后加强免疫一次,收集小鼠血清。采用Western Blot、IFA鉴定制备多克隆抗体的特异性,该多克隆抗体可以和阳性sViperin真核表达载体pCI-sVIP等表达的猪源Viperin蛋白进行特异性反应。试验成功克隆了猪Viperin基因,并制备了良好Viperin蛋白的多克隆抗体。

To study the antiviralactivity of swine viperin in pigs,we extracted and cloned the swine Viperin genes from PK-15 cells.PK-15 cells were seeded onto a 24-well plate and treaded with IFN-α.After 24 h,the cells were harvested to extract the total RNA,and to amplify the Viperin gene by RT-PCR.Then the gene was cloned into the pEASY-blunt simple plasmid for sequencing.The antigenicity and hydrophobicity of swine Viperin were analyzed by DNA Star software,and the fragment with good immunogenicity were chosen and amplified,which was inserted into the prokaryotic expression vector pET-28a+.E.coli BL21 containing recombinant plasmid pET-sVIP was induced by IPTG.SDS-PAGE analysis demonstrated that the recombinant protein was expressed in the form of inclusion bodies in E.coli.Then inclusion body protein was purified.The BALB/c mice(9 day old)were immunized two times using the purified recombinants Viperin protein emulsified with equal amounts of Freund′s complete adjuvant.Two weeks after final immunization,the serums were collected and the antibody specificity was identified by Western Blot and IFA assay.The result showed that the swine viperin polyclonal antibody could react with the sViperin protein expressed by eukaryotic expression vector pVAX-sVIP.Preparation of polyclonal antibody laid the foundation for further studying the antiviral activity of swine Viperin.