金黄色葡萄球菌FnBP配体结合区基因的克隆及其原核表达  

Cloning and Prokaryotic Expression of FnBP Ligand Binding Gene of Staphylococcus aureus

作　　者：尹荣兰[1] 杨正涛[1] 张艳晶 刘辉[1] 刘珊[1] 杨琦[1] 曹永国[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：《安徽农业科学》2009年第1期60-61,69,共3页Journal of Anhui Agricultural Sciences

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

摘　　要：[目的]克隆金黄色葡萄球菌FnBP配体结合区基因,并构建原核表达载体,进行原核表达。[方法]设计引物,采用PCR方法扩增FnBP配体结合区基因,T-A克隆后,构建了克隆质粒pMD18-FnBP。用BamH I和EcoR I双酶切pMD18-FnBP和pET28a(+),将纯化的基因FnBP亚克隆至pET28a(+),构建重组表达质粒pET28a-FnBP,并将其转化至大肠杆菌感受态BL21(DE3)中,IPTG诱导表达,并对表达产物进行分析。[结果]PCR扩增出1条约370 bp的目的片段,表达产物经SDS-PAGE分析,在30 kDa处出现了与目的蛋白一致的外源蛋白带,Western blot分析表明该蛋白具有金黄色葡萄球菌的抗原性。[结论]已成功构建了FnBP配体结合区基因,并在原核细胞中表达。[Objective] This study was to clone FnBP ligand binding gene of Staphylococcus aureus and construct a prokaryotic expression vectory, running pmkaryotie expression. [ Method] The gene encoding FnBP ligand binding gene was amplified from Staphylococcus aureus chromosomal DNA by PCR technique. After T-A cloning, plasmid pMD18-FnBP was constructed, pMD18-FnBP and pET28a（ ＋ ）were digested by BamH I mid EcoR I double enzymes, then the puttied FnBP ligand binding gene was subeloned into the expression vector pET28a（ ＋ ）, and the prokaryotic expression vector pET28a- FnBP was thus constructed. The construeted plasmid pET28a-FnBP was transformed into E. coli BI21 （DE3） competent cell. The bacterium was induced by WIG and analyzed by SDS-PAGE and Western blot. [ Result] The gene fragment at the length of 370 bp was amplified by PCR amplification. Approximately 30 kD exogenous protein was observed by SDS-PAGE. Westem blot analysis indicated the protein had antigenic of Staphylococcus aureus. [ Conclusion] The FnBP ligand binding gene of Staphylococcus aureus was successfully cloned and expressed in prokaryotic cells.

关 键 词：金黄色葡萄球菌 FnBP配体结合区 基因克隆 原核表达 

分 类 号：S188[农业科学—农业基础科学]