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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 a fusion protein expression vector of His-tagged methionine resistance-related protein FemA of Staphylococcus aureus and express it in Escherichia coli, which will lay the foundation for further study on the biological function and clinical application of femA gene. Methods According to the sequence of femA gene of Staphylococcus aureus in GenBank, Primer Premier 5.0 was used to design PCR primers and BamHI and SalI restriction sites were added to 5 ’of the primers. Staphylococcus aureus genomic DNA was used as template to amplify femA Gene fragment. The target DNA fragment and plasmid pQE30 were double-digested, ligated and transformed into Escherichia coli DH5α. The positive clones were identified by PCR, double enzyme digestion and sequencing. The pQE30-femA recombinant plasmid was transformed into Escherichia coli JM109 and induced by isopropyl-β-D-thiogalactoside (IPTG). His-femA fusion protein was induced by SDS-PAGE and Western blot analysis Protein validation. Results The recombinant plasmid pQE30-femA was successfully constructed by PCR, double enzyme digestion and sequence analysis. The recombinant plasmid pQE30-femA was transformed into Escherichia coli JM109 and induced by IPTG. SDS-PAGE and Western blot showed that the recombinant plasmid pQE30- The protein expression of target protein accounted for 27.5% of total cellular protein at 4 h after analysis by Bandscan software. Conclusion The His-FemA prokaryotic expression vector (pQE30-femA) was successfully constructed and highly expressed in Escherichia coli.