甲氧西林耐药金黄色葡萄球菌临床分离株青霉素结合蛋白2a的克隆表达及鉴定

Clone, expression and identification of penicillin binding protein 2a of methieillin-resistant Staphylococcus aureus isolated from patients

目的 应用基因重组技术对编码甲氧西林耐药金黄色葡萄球菌（MRSA）临床分离株青霉素结合蛋白2a（PBP2a）的mecA基因片段进行克隆、表达。方法 从临床样本巾分离鉴定出MRSA，根据基因文库收录的mecA基因编码序列，针对编码PBP2a第25～668位氨基酸的mecA基因片段设计引物，扩增目的基因片段，克隆至pQE30载体，经酶切鉴定、测序后，再转化E．coliM15（pREP4）。采用1mmol／L的异丙硫半乳糖苷（IPTG）进行诱导表达及鉴定。结果重组表达质粒pQE30-mecA构建成功，基因测序结果显示，扩增的mecA基因DNA片段全长为1932bp，其巾有9个碱基突变。IPTG诱导后6h，聚丙烯酰胺凝胶电泳显示，M15（pQE30-mecA）出现一条相对分子质量约74×10^3的蛋白条带，且一部分以可溶性形式存在；M15（pQE30）未出现特异性蛋白条带。经诱导表达和鉴定证实，表达出的可溶性目的蛋白为PBP2a。结论 利用本技术可成功表达MRSA临床分离株中的可溶性PBP2a。

Objeetive To clone, express and identify the mecA fragment which encoded penicillin binding protein 2a （PBP2a） from methicillin-resistant staphylococcus aureus （MRSA） isolated from patients by gene recombination method. Methods According to the sequence of mecA gene recorded in GenBank, the primer ofmecA fragment which encoded amino acids 25 - 668 of PBP2a was designed. Then the mecA fragment was amplified by PCR and cloned into pQE30 plasmid. After being identified by enzyme digestion and sequencing, the recombinant plasmid was transferred into E. coli M15 [ pREP4] , and then its expression was induced by 1mmol/L lsopropy-β-D-Thiogalactoside （IPTG）. The expression product was analyzed by SDS-PAGE, protein sequencing and mass spectroscopy. Results The recombinant pQE30-mecA had been successfully constructed. The result of sequencing showed that the mecA fragment had 1932 bases ,in- cluding 9 bases undergoing mutation. After being induced for 6 hours by IPTG, the soluble protein in M15 （ pQE30- mecA ） , with a relative molecular weight of 74 × 10^3 , was found by SDS-PAGE. The soluble protein had been confirmed to be PBP2a after identification. Conclusion The soluble PBP2a of MRSA isolated from patients is expressed successfully by gene recombinant technology.