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.
出处
《中国家禽》
北大核心
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)
关键词
禽白血病
P27基因
原核表达
多克隆抗体
avian leucosis
p27 gene
prokaryotic expression
multiclonal antibodies
