应用RT-PCR技术从90日龄健康仔猪肺巨噬细胞中克隆出猪Fcγ亚单位的cDNA序列,并将其亚克隆到原核表达载体pET-32a中,构建重组表达质粒pET-FcRγ,成功表达了分子量约为29 kDa的γ亚单位重组蛋白。将重组蛋白FcRγ-His免疫小鼠,制备获得鼠抗FcRγ-His重组蛋白多克隆抗体,将得到的重组蛋白多克隆抗体采用间接ELISA和Western-blotting试验方法检测,ELISA测定多克隆抗体的效价为1∶8 000。Western-blotting结果表明,制备的鼠抗FcRγ-His重组蛋白多克隆抗体可以与重组γ亚单位蛋白进行特异性结合,从而证明重组蛋白具有较好的免疫原性。成功克隆出猪Fc受体γ亚单位基因并表达,为进一步研究其结构与功能奠定了基础。
mRNA encoding porcine FcR gamma subunit obtained from PAMs of 90-day piglets was transcribed into cDNA through RT-PCR. Recombinant plasmid pET-FcRγ was constructed successfully by inserting the cDNA sequence into pET-32a, and expressed as soluble protein with a molecular weight of about 29 kDa. Following purification, recombinant protein FcRγ-His was used to immune mouse, and polyclonal antibody against it was obtained. Western-blotting indicated that such polyclonal antibody could react specifically with FcRγ-His. Indirect ELISA shows that the titer of the polyclonal antibody is 1∶ 8000, revealing the good immunogenicity of FcRγ-His. All these results provide the foundation for further studying the structure and function of FcR gamma subunit.
[1] Rosenfeld P,Turner P V,MacInnes J I.Evaluation of porcine reproductive and respiratory syndrome virus replication in laboratory rodents[J] Can J Vet Res,2009,73(4):30-90.
[2] Klinge K L,Vaughn E M.Age-dependent resistance to porcine reproductive and respiratory syndrome virus replication in swine[J] Virol J,2009,27(1):67-88.
[3] Cano J P,Dee S A,Murtaugh M P.Infection dynamics and clinical manifestations following experimental inocμLation of gilts at 90 days of gestation with a low dose of porcine reproductive and respiratory syndrome virus[J] Can J Vet Res,2009,73(4):38-79.
[4] 郭宝清,陈章水,刘文兴,等.从疑似PRRS 流产胎儿分离PRRSV 的研究[J] 中国畜禽传染病,1996(2):16-42.
[5] 刘明,张改平,肖治军,等.PRRSV 河南株ORF7 基因的克隆表达及条件优化[J] 河南农业科学,2011,40(1):136-140.
[6] 鄂巍,张改平,罗俊,等.鸡传染性发法氏囊病病毒河南分离heYDVP2 基因高变区基因克降及序列分析[J] 河南农业科学,2011,40(3):149-153.
[7] Delrue I,Delputte P L,Nauwynck H J.Assessing the functionality of viral entry-associated domains of porcine reproductive and respiratory syndrome virus during inactivation procedures,a potential tool to optimize inactivated vaccines[J] Vet Res,2009,40(6):62-126.
[8] Abin M F,Spronk G,Wagner M.Comparative infection efficiency of porcine reproductive and respiratory syndrome virus field isolates on MA104 cells and porcine alveolar macrophages[J] Can J Vet Res.2009,73(3):4-200.
