禽白血病病毒衣壳蛋白p27基因的原核表达及多克隆抗体的制备  

Expression of Capsid Protein p27 Gene of Avian Leucosis Virus and Preparation of Multiclonal Antibodies Against Recombinant p27 Protein

作　　者：李德龙[1,2] 刘在斯[1] 贠炳岭[1] 吴关[1] 高玉龙[1] 秦立廷[1] 祁小乐[1] 王永强[1] 高宏雷[1] 刘思当[2] 王笑梅[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150001 [2]山东农业大学,山东泰安271000

出　　处：《中国家禽》2012年第9期13-16,共4页China Poultry

基　　金：现代农业肉鸡产业技术体系建设(nycytx-42-G3-01);国家自然科学基金(31072146);哈尔滨市科技攻关计划项目(2010AA6AN034)

摘　　要：从J亚群禽白血病病毒(ALV-J)HB09JY03株感染的DF-1细胞的冻融裂解液中提取DNA,通过PCR方法扩增出约720bp的gag-p27基因片段,克隆至pMD18-T载体。鉴定正确后,将该片段克隆至pET-30a(+)原核表达载体,经IPTG诱导表达。表达产物经His Trap TMHP纯化后,测蛋白浓度达到9mg/mL。用兔抗自然p27蛋白阳性血清进行Western blot检测,表明重组p27蛋白有抗原反应活性。免疫新西兰大白兔后,产生的抗体与禽白血病全病毒抗原可以发生特异性反应。所制备的重组p27蛋白和多克隆抗体将在ALV的抗原分析、血清学诊断等方面有着重要的应用价值。DNA was extracted from DF-1 cell lysate infected with subgroup J of avian leucosis virus（ALV） and the gag-p27 gene was amplified by PCR.Then the gene was cloned into plasmid pMD18-T and sequenced.After identified exactly,the whole coding fragment of p27 gene was cloned into prokaryotic expression vector pET-30a（＋） and induced with IPTG.After purification with His TrapTM HP,the density of p27 recombinant protein was up to 9 mg/mL.The immune reactivity of recombinant p27 protein was identified with positive antiserum against nature p27 protein specifically by Western-blot.Multiclonal antibodies against recombinant p27 protein was obtained by subcutaneous injection of rabbit,which is specific to ALV antigen and it would be very useful as a tool for antigenic analysis and serological diagnosis of ALV.

关 键 词：禽白血病 P27基因 原核表达 多克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]