金黄葡萄球菌ClfA活性基因的克隆及原核表达  

Cloning and Prokaryotic Expression of ClfA Active Gene of Stapylococcus aureus

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

机构地区：[1]吉林大学畜牧兽医学院,长春130062

出　　处：《中国生物制品学杂志》2009年第1期38-40,共3页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(30771596)

摘　　要：目的克隆金黄葡萄球菌表面蛋白凝集因子A(ClfA)活性基因,并进行原核表达。方法以金黄葡萄球菌基因组DNA为模板,采用PCR方法扩增ClfA(221-550)活性基因,克隆入载体pET28a(+)中,构建重组表达质粒pET28a-ClfA(221-550),转化感受态大肠杆菌BL21(DE3),IPTG诱导表达,并对表达产物进行SDS-PAGE和Western blot分析。结果PCR扩增出987bp的目的基因片段,重组表达质粒经双酶切证明构建正确。表达产物经SDS-PAGE分析,在相对分子质量约36000处可见目的条带,目的蛋白表达量约占菌体总蛋白的36.76%,且具有良好的反应原性。结论已成功克隆了金黄葡萄球菌ClfA活性基因,并在大肠杆菌BL21(DE3)中表达了目的蛋白。Objective To clone the clumping factor A （ClfA） active gene of Stapylococcus aureus and express in prokaryotic cells. Methods ClfA （221-550） active gene was amplified by PCR using the genomic DNA of Stapylococcus aureus as a template and cloned into vector pET28a （＋）. The constructed recombinant plasmid pET28a-ClfA （221-550） was transformed to competent E.coli BL21 （DE3） for expression under induction of IPTG. The expressed product was identified by SDS-PAGE and Western blot. Results The target gene fragment at a length of 987 bp was amplified by PCR. Restriction analysis proved that recombinant plasmid pET28a- ClfA （221-550） was constructed correctly. The target protein band with a relative molecular mass of about 36 000 was observed on SDS-PAGE profile. The expressed product contained about 36. 76% of total somatic protein and showed good reactogenicity. Conclusion The ClfA active gene of Stapylococcus aureus was successfully cloned and expressed in E. coli BL21 （DE3）.

关 键 词：金黄葡萄球菌 表面蛋白凝集因子A 活性基因 克隆 原核表达 

分 类 号：R378.11[医药卫生—病原生物学] Q785[医药卫生—基础医学]