His-FemA融合蛋白表达载体的构建及其在原核细胞中的表达  

Construction of His-FemA fusion protein expression vectors and the expressions in prokaryotic cells

作　　者：邹明祥[1] 李军[1] 邬国军[2] 武文君[3] 张宁洁[1] 刘文恩[1] 范学工[4] 

机构地区：[1]中南大学湘雅医院检验科,湖南长沙410008 [2]中南大学基础医学院微生物学系,湖南长沙410078 [3]河南中医学院第一附属医院检验科,河南郑州450000 [4]中南大学湘雅医院感染病科,湖南长沙410008

出　　处：《中国微生态学杂志》2012年第10期878-882,共5页Chinese Journal of Microecology

基　　金：湖南省自然科学基金资助课题(10JJ5027);中南大学自由探索研究创新基金(2010112001166)

摘　　要：目的构建His标签的金黄色葡萄球菌甲氧西林耐药相关蛋白FemA的融合蛋白表达载体,并在大肠埃希菌中表达,为进一步研究femA基因的生物学功能和临床应用奠定基础。方法根据GenBank中金黄色葡萄球菌femA基因序列,利用Primer Premier 5.0设计PCR引物,并在引物的5'加入BamHI及SalI酶切位点;以金黄色葡萄球菌基因组DNA为模版,PCR扩增出femA基因片段。将目的 DNA片段及质粒pQE30分别进行双酶切、连接并转化大肠埃希菌DH5α;阳性克隆以PCR、双酶切及测序进行鉴定。将鉴定正确的pQE30-femA重组质粒转化大肠埃希菌JM109,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达His-femA融合蛋白;采用SDS-PAGE及Western blot分析对表达蛋白进行验证。结果经PCR、双酶切鉴定及序列测定,证实重组质粒pQE30-femA构建成功;重组质粒pQE30-femA转化大肠埃希菌JM109经IPTG诱导后,SDS-PAGE和Western blot分析显示表达出53 kD目的蛋白;经Bandscan软件分析,目的蛋白质在4 h的表达量占细胞总蛋白的27.5%。结论成功构建了His-FemA原核表达载体(pQE30-femA),并在大肠埃希菌中高效表达。Objective To construct His-tagged FemA fusion protein expression vectors,and express it in E.coli and establish foundation for further studies.Methods PCR primers were designed using Primer Premier 5.0 software according to the sequence of femA gene in GenBank.Two restriction endonuclease recognition sites BamHI and SalI were added to the 5′ end of the primer.Genomic DNA from S.aureus was used as the templete for PCR amplification of femA gene.The obtained femA gene and plasmid pQE30 were double-enzyme digested respectively,ligated and transferred into DH5α.The positive clones were selected and the recombinant plasmid pQE30-femA was identified by PCR,restriction endonuclease analysis and DNA sequencing.Then the identified pQE30-femA was transformed into E.coli JM109 and the target protein expression was induced by IPTG.The expressed product was identified by SDS-PAGE and Western blot.Results Verified by PCR,double-enzyme digested assessment and sequencing,recombinant plamid pQE30-femA was successfully constructed.SDS-PAGE and Western blot analysis revealed that the recombinant plasmid pQE30-femA expressed a 53 kD target protein in E.coli JM109.Bandscan software analysis showed that the expressed target protein at 4 h accounted for about 27.5% of the total bacterial protein.Conclusion The His-tagged femA fusion protein expression vectors（pQE30-femA） was successfully constructed and high-effectively expressed in E.coli.

关 键 词：金黄色葡萄球菌 FEMA基因 原核表达 融合蛋白 

分 类 号：R378.11[医药卫生—病原生物学]