猪FcγRⅢ的原核表达及多克隆抗体的制备  

Prokaryotic Expression of Porcine FcγRⅢ and Production of Its Polyclonal Antibody

作　　者：徐红运[1] 夏平安[1] 张凤华[1] 赵琳[1] 范旭[1] 刘明莉[1] 刘玉松[1] 张志远[1] 崔保安[1] 

机构地区：[1]河南农业大学牧医工程学院,郑州450002

出　　处：《中国生物工程杂志》2010年第6期117-121,共5页China Biotechnology

基　　金：河南省重点攻关项目(92102110018)

摘　　要：目的:构建猪FcγRIII基因的原核表达载体,诱导表达重组蛋白,制备鼠抗猪FcγRIII抗血清。方法:从质粒pTG19-T-FcγRIII中用PCR方法克隆到编码完整猪FcγRIII蛋白分子的基因片段,将其插入到原核表达载体pET-32a中,构建了猪FcγRIII原核表达载体pET-FcγRIII,转化大肠杆菌BL21(DE3),IPTG诱导蛋白表达,经尿素洗涤纯化后,以纯化后的融合蛋白FcγRIII-His为抗原免疫小鼠,获得抗血清。Western blotting、ELISA法鉴定获得的抗血清,ELISA结果显示抗体效价为1∶16000,具有高度特异性,免疫印迹结果显示制备的多抗可以与重组猪FcγRIII蛋白特异性结合。结果:成功构建猪FcγRIII原核表达载体,纯化到融合蛋白FcγRIII-His,用纯化的融合蛋白免疫小鼠制备了多克隆抗体,Western blotting、ELISA法证实多克隆抗体制备成功。结论:成功获得了猪FcγRIII多克隆抗体,为进一步研究猪FcγRIII蛋白的功能奠定了基础。Objective：To construct a prokaryotic plasmid expressing the recombinant proteins of porcine FcγRIII and prepare the polyclonal antibodies of mouse anti porcine FcγRⅢ.Methods The fragment of gene encoding porcine FcγRIII was amplified from pTG19-T-FcγRIII plasmid by PCR.The fragment was then inserted into the prokaryotic expression vector pET-32a to construct the recombinant plasmid pET-FcγRIII,and expressed in E.coli BL21（DE3）.After induction with IPTG,the fusion protein was obtained,and then purified with Urea by washing.A polyclonal antibody,analyzed by Western blotting and ELISA staining,was developed by immunizing mouse with the purified recombinant protein.Results：The recombinant plasmid was constructed successfully ;the fusion protein was successfully expressed and purified.Western blotting and ELISA staining demonstrated that the polyclonal antibody was obtained by immunizing mouse with the purified recombinant protein.The results of ELISA and Western blotting indicated that the prepared polyclonal antibody had high titer and specificity.Conclusion：The preparation of the polyclonal antibody against porcine FcγRIII lays a foundation for the further study on the function of porcine FcγRIII protein.

关 键 词：猪FcγRⅢ 原核表达 蛋白纯化 多克隆抗体 

分 类 号：Q819[生物学—生物工程]