His_FemA融合蛋白表达载体的构建及其在原核细胞中的表达

【收稿日期】2012-04-27【基金项目】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)【作者简介】(1971-)，男，副主任技师，医学博士，硕士生导师，

从事细菌耐药性及耐药机制研究，Email :zoumingx-iang@126．com ;范学工，通讯作者，教授，博士生导

师，

Email :xgfan@hotmail．com 文章编号:1005-

376X (2012)10-0878-05【论著】

His-FemA 融合蛋白表达载体的构建及其在原核细胞中的表达

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

(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)

【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BamH I 及Sal I 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53kD 目的蛋白;经Bandscan 软件分析，目的蛋白质在4h 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。

【关键词】金黄色葡萄球菌;femA 基因;原核表达;融合蛋白

【中图分类号】R378．1+1

【文献标识码】A

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

ZOU Ming-xiang 1，

LI Jun 1，WU Guo-jun 2，WU Wen-jun 3，ZHANG Ning-jie 1，LIU Wen-en 1，FAN Xue-gong 4

(1．Department of Clinical Laboratory ，Xiangya Hospital of Central South University ，Changsha 410008，China ;2．Department of Microbiology ，School of Basic Medicine ，Central South University ，Changsha 410078，China ;3．Department of Clinical Laboratory ，the First Affiliated Hospital of Henan University of Traditional Chinese Medicine ，Zhengzhou 450000，China ;4．Department of Infectious Diseases ，Xiangya Hospital of Central South University ，Changsha 410008，China )

【Abstract 】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 5．0software according to the sequence of femA gene in GenBank．Two restriction endonuclease recognition sites BamH I and Sal I 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 pQE30were 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 JM109and the target protein expression was in-duced by IPTG．The expressed product was identified by SDS-PAGE and Western blot．Results Verified by PCR ，double-en-zyme 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 53kD target protein in E．coli JM109．

Bandscan software analysis showed that the expressed target protein at 4h accounted for about 27．5%of the total bacterial pro-tein．Conclusion The His-tagged femA fusion protein expression vectors (pQE30-femA )was successfully constructed and

high-effectively expressed in E．coli ．

【Key words 】Staphylococcus aureus ;FemA gene ;Prokaryotic expression ;Fusion protein

金黄色葡萄球菌是威胁人类健康的重要致病菌，

可引起化脓性感染、食物中毒、表皮剥脱综合征以及血流感染等多种疾病。随着抗菌药物的广泛使用，

金黄色葡萄球菌的耐药现象日趋严重［1，2］

。其中，耐

甲氧西林金黄色葡萄球菌(MRSA )不仅具有严重的多重耐药性，同时具有广泛的流行性，极大地增加了患者和社会的医疗负担，已成为全球严重的公共卫生