金黄色葡萄球菌ClfA基因的克隆及在大肠杆菌中的表达  

Cloning and prokaryotic expression of ClfA of Staphylococcus aureus of cow matitis

作　　者：尹荣兰[1] 杨正涛[1] 张艳晶 刘辉[1] 杨琦[1] 刘珊[1] 张乃生[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《中国兽医学报》2009年第2期150-152,共3页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30771596);教育部博士点基金资助项目(20060183010)

摘　　要：根据GenBank中ClfA基因序列设计特异性引物,以金黄色葡萄球菌基因组DNA为模板,进行PCR扩增,获得了约2 300 bp的DNA片段。将PCR产物克隆至pMD18-T载体中,构建了克隆质粒pMD18-ClfA。用BamHⅠ和EcoRⅠ双酶切pMD18-ClfA和pET28a(+),将纯化的基因ClfA亚克隆至pET28a(+),构建重组质粒pET28a-ClfA,并将其转化至大肠杆菌感受态BL21(DE3)中,经1 mmol/L IPTG诱导和SDS-PAGE分析,在87 000处出现了与目的蛋白一致的外源蛋白带,Western-blotting分析表明,该蛋白具有金黄色葡萄球菌的抗原性。Staphylococcus aureus is an opportunistic pathogen capable of causing mastitis of cow. It is becoming important element for prepare DNA vaccine because of specific adhere to ability. ClfA is main adherence factor of surface protein of Stapthylococcus a ureus, ClfA was designed specificity primer according to GenBank, the gene encoding ClfA was amplified from Staphylococcus aureus chromosomal DNA by PCR technique, and the PCR product was about 2 300 bp DNA segment. The PCR product was cloned into pMD18-T vector successfully and constructed plasmid pMD18- ClfA. pMD18 ClfA and pET28a（＋） were digested by BamH Ⅰ and EcoR Ⅰ double enzymes,then the purfied ClfA gene was subcloned into the expression vector pET28a （＋）, and the prokaryotic expression vector pET28a-ClfA was thus constructed successfully. The reconstructed plasmid pET28a-ClfA was transformed into E. coli BL21（DE3） competent cell. The bacterium was induced by IPTG（1 mmol/L） and analyzed by SDS-PAGE,approximately 87 000 exogenous protein was observed on the SDS-PAGE. Western blotting analysis indicated the protein had antigenic of ClfA. This study were expected to established foundation for development of neotype genetically engineering vaccine to prevent Staphylococcus aureus of cow matitis.

关 键 词：金黄色葡萄球菌 ClfA基因 克隆 原核表达 

分 类 号：S852.611[农业科学—基础兽医学]