猪胸膜肺炎放线杆菌ApxIVA基因特异片段的克隆和表达

以猪胸膜肺炎放线杆菌的血清7型国内分离株25＿4株基因组DNA为模板,PCR方法扩增apxIVA特异片段,PCR产物经纯化后与载体PMD18＿T进行连接、转化,经酶切及序列分析鉴定后,亚克隆到原核表达载体pGEX＿6P＿1中,构建成重组表达质粒pGEX＿apxIVA,转入到大肠杆菌BL21中,以IPTG进行诱导,进行SDS＿PAGE电泳。结果表明,25＿4株apxIVAgene与基因库中标准7型apxIVAgene同源性达97%,所表达的融合蛋白相对分子量为44kD,与实际预测相符。apxIVA毒素特异片段的成功表达为猪胸膜肺炎放线杆菌病的诊断打下基础。

Cloning,expression and identification of ApxIVA specific fragmen of Actinobacillus pleuropneumoniae

The pupose of this study is to clone,identify,and express the secreted protein ApxIVA specific fragment of Actinobacillus pleuropneumoniae 25-4 strain and to play a foundation for diagnosis and immunity.The gene encoding for protein ApxIVA specific fragment was amplified from APP 25-4 chromosomal DNA by using PCR technique.PCR product was cloned, and the strain containning the vectors were selected on LB-plus ampicillin(60ug/ml)plates,and plasmid DNAwas extracted and digested with enzymes.plasmids containing the right insertion were sequenced to confirm its identity and retransformed the combinant into E.coli .BL21 strain.Bacterial lysates prepared from 1mmol/L IPTGinduced cultures wee loaded directly onto SDS-PAGE.Upon induction,the recombinant pGEX-ApxIVAproduced indeed a new protein with an apparent MW of 44 KDa(containing GST).In conclusion,we obtained recombinant pGEX-6p-1 expression vector contaning ApxIV specific fragment.The protein has been expressed successfully.It will establish the detecting of immunity effectiveness.