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

作　　者：张志远[1] 张继希[1] 徐红运[1] 刘肖萍[1] 卢晓艳[1] 段二珍[1] 夏平安[1] 崔保安[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002

出　　处：《华北农学报》2012年第1期35-38,共4页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金项目(31172346)

摘　　要：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.

关 键 词：猪Fc受体γ单位 克隆 原核表达 多克隆抗体 

分 类 号：S828[农业科学—畜牧学]