猪Fc受体γ亚单位基因克隆与原核表达

Gene Cloning of Porcine Fc Receptor Gamma Subunit and Its Prokaryotic Expression

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 eDNA through RT-PCR. Recombinant plasmid pET-FeRγ was constructed successfully by inserting the eDNA sequence into pET-32a,and expressed as soluble protein with a molecular weight of about 29 kDa. Following purifi- cation,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.