猪IgGⅡB类Fc受体剪接异构体胞外区及结构域的原核表达与抗体制备  

Prokaryotic expression of functional extracellular domain and structural domains of porcine FcγRIIB by alternative splicing and preparation of polyclonal antibodies

作　　者：刘肖萍[1] 张志远[1] 刘玉松[1] 赵琳[1] 范旭[1] 徐红运[1] 张凤华[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：《河南农业大学学报》2011年第3期307-312,共6页Journal of Henan Agricultural University

基　　金：河南省重点攻关项目(92102110018)

摘　　要：根据GenBank中猪IgGⅡB类Fc受体剪切异构体基因序列(FJ608551),利用Prim er 5.0软件设计合成3对特异性引物,从质粒pTG19-T-swFcγRIIB1中用PCR方法扩增编码胞外区、EC1及EC2结构域蛋白分子基因片段,PCR产物经KpnⅠ和EcoRⅠ双酶切后将其插入到原核表达载体pET-32 a中,构建了3种猪FcγRIIB剪接异构体原核表达载体pET-EY,pET-AY,pET-BY,转化大肠杆菌BL21,在IPTG诱导下成功表达了相对分子质量约为36,29,27 kD融合蛋白表达,纯化后的融合蛋白分别免疫小鼠制备鼠源血清.ELISA结果显示抗体效价为1∶5 120,1∶5 120,1∶10 240.免疫印迹结果显示制备的多克隆抗体可以与融合蛋白特异性结合.Acording to the mRNA sequence（FJ608551） of the Fc gamma receptorⅡB（swFcγRIIB） by alternative splicing in GenBank,three pairs of primers were designed using Primer 5.0 software.The fragments of gene encoding extracellular domain,EC1 domain and EC2 domain were amplified from pTG19-T-swFcγRIIB1 plasmid by PCR.The PCR products were digested with KpnⅠand EcoRⅠ.The fragments were then inserted into the prokaryotic expression vector pET-32a to construct the recombinant plasmid pET-EY,pET-AY,pET-BY and expressed in E.coli.BL21.After induction with IPTG,we obtained the fusion proteins,which molecular mass are about 36,29,27 kD respectively.The polyclonal antibodies were obtained by immunizing mouse with the three purified fusion proteins and analyzed by Western blotting and ELISA.The results of ELISA showed that the antibody titers were 1∶ 5 120,1∶5 120 and 1∶10 240.Western blotting results indicated that the prepared polyclonal antibodies could bind to fusion proteins specifically.

关 键 词：猪 FCΓRIIB 原核表达 蛋白纯化 多克隆抗体 

分 类 号：S852.2[农业科学—基础兽医学]