耐甲氧西林金黄色葡萄球菌PBP2a 382～443片段的原核表达及鉴定

目的对编码耐甲氧西林金黄色葡萄球菌(MRSA)及青霉素结合蛋白2a(PBP2a)382~443位氨基酸的mecA基因片段进行克隆、表达及鉴定。方法根据基因文库登录的mecA基因的编码序列,针对编码PBP2a382~443位氨基酸的mecA基因片段,设计合成4条寡核苷酸片段,再将4条片段人工拼接成目的基因片段,然后克隆至PET-His载体,经酶切鉴定、测序正确后,转化E.coliBL21(DE3)plysS,用IPTG进行诱导表达,并对表达的蛋白以MRSA胶乳凝集试剂盒进行鉴定。结果构建了相应的PET-His克隆,经诱导表达和鉴定,证实成功表达出目的蛋白。结论成功表达出PBP2a382~443片段,为其进一步的纯化和应用奠定了基础。

Prokaryotic expression and identification of amino acids 382-443 of PBP2a of methicillin resistant staphylococcus aureus

Objective To clone and express the mecA fragment which encodes amino acids 382-443 of penicillin binding protein 2a(PBP2a) of methicillin resistant staphylococcus aureus(MRSA) and to identify the expressed product.Methods According to the sequence of mecA gene published in GeneBank,four oligonucleotide fragments were designed,synthesized and spliced into the objective gene fragment by PCR,and then the objective fragment was cloned into PET-His plasmid.After being identified by enzyme digestion and sequencing,the recombinant plasmid was transformed into E.coli BL21(DE3)plysS.The expression was induced by IPTG and the expressed product was analyzed by MRSA-screen latex agglutination test.Results The corresponding PET-His clone has been successfully constructed and the objective protein was expressed successfully.Conclusion The protein of amino acids 382-443 of PBP2a is successfully expressed,which establishes the foundation for its purification and application.