猪干扰素诱导跨膜蛋白3的克隆表达及多克隆抗体制备

Proteins 3 and Preparation of the Polyclonal Antibody

为原核表达猪干扰素诱导跨膜蛋白3(swine interferon-induced transmembrane protein 3,SwIFITM3)并制备其多克隆抗体(polyclonal antibody,pAb),从干扰素刺激的猪外周血淋巴细胞中克隆到SwIFITM3编码基因,然后采用大肠埃希氏菌表达系统表达并纯化目的蛋白免疫小鼠制备其pAb;并以制备的pAb为一抗用Western blot检测了猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)感染猪肺泡巨噬细胞(porcine alveolar macrophages,PAMs)中SwIFITM3表达情况。结果显示,获得了438 bp的SwIFITM3基因,将获得的目的基因插入pColdⅠ载体构建重组质粒转入E.coli BL21(DE3)感受态细胞,用0.2 mmol/L IPTG在16℃诱导24 h后获得了大小约19 ku可溶性表达的目的蛋白,以Anti-His单克隆抗体为一抗进行Western blot检测,获得了与预期大小一致的印迹条带;用纯化的目的蛋白腹腔注射免疫小鼠3次,采用间接ELISA方法检测,小鼠血清中SwIFITM3抗体效价最高达1∶1024000;以制备的pAb为一抗的Western blot检测显示PRRSV感染导致PAMs中SwIFITM3蛋白表达显著降低。试验结果为猪IFITM3的生物学功能及相关研究积累了材料。

To express swine interferon-induced transmembrane protein 3(SwIFITM3)with E.coli and prepare its polyclonal antibody(pAb),the SwIFITM3 gene was cloned from swine peripheral blood lymphocytes stimulated by interferon,and the target protein was expressed using E.coli expression system.Mice were immunized with purified SwIFITM3 recombinant protein to prepare pAb.The expression of SwIFITM3 in PRRSV-infected porcine alveolar macrophages(PAMs)was determined by the Western blot method with the pAb as the primary antibody.The results showed that the gene encoding SwIFITM3 is 438 bp.Then the target gene was inserted into the pColdⅠvector to construct recombinant plasmid which was transferred into E.coli BL21(DE3)competent cells.After induction 24 h at 16℃with 0.2 mmol/L IPTG,the soluble expression target protein with a size of 19 ku was obtained.Western blot detection was performed with anti-His monoclonal antibody as the primary antibody,and bands with the expected size were obtained.The titer of the SwIFITM3 antibody in the serum of immunized mice reached 1∶1024000 by indirect ELISA.PRRSV infection resulted in significantly reduced SwIFITM3 protein expression in PAMs.These results accumulate material for the biological function of SwIFITM3 and the related research.