胸膜肺炎放线杆菌4型菌毛亚单位基因apfA特异性片段的克隆和表达

Cloning,Expression and Identification of Type 4 Fimbrial Subunit Gene ApfA Specific Fragment from Actinobacillus pleuropneumoniae

以猪胸膜肺炎放线杆菌血清型7型国内分离株25-4株基因组DNA为模板,PCR扩增其apfA特异性基因片段,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,亚克隆至原核表达载体pGEX-6P-1中,构建重组表达质粒pGEX-apfA,转化到感受态大肠杆菌DE3中,以IPTG进行诱导,进行SDS-PAGE电泳。结果表明,25-4株apfA基因与基因库中标准7型apfA基因的同源性达到98%,所表达的融合蛋白相对分子质量约为42 000,与预测值相符。apfA特异性基因片段的成功克隆和表达为猪胸膜肺炎放线杆菌病致病机理的研究及新型疫苗的研制打下了基础。

The present study was conducted to clone,identify,and express the ApfA specific fragment of Actinobacillus pleuropneumoniae 25-4 strain. The gene encoding for protein ApfA specific fragment was amplified from APP 25-4 chromosomal DNA by using PCR technique. PCR product was cloned,and the strain containing the vectors were selected on LB-plus ampicillin （50 mg/L） plates ,and plasmid DNA was extracted and digested with enzymes. Plasmids containing the right insertion were sequenced to confirm its identity and then retransformed the recombinant into E. coli. BL21 strain. The bacterial lysates prepared from 1 mmol/L IPTG induced cultures were loaded directly onto SDS0-PAGE. Upon induction,the recombinant pGEX-ApfA produced indeed a new protein with an apparent MW of 44 000（containing GST）. In conclusion,we obtained recombinant pGEX-6P-1 expression vector containing ApfA specific fragment,and the protein has been expressed successfully.