耐甲氧西林金黄色葡萄球菌PBP2a的真核表达及其在抗体鉴定中的应用

Eukaryotic Expression of PBP2a in Methiciilin Resistant Staphylococcus Aureus and Application in Antibody Identification

目的利用杆状病毒表达系统表达耐甲氧西林金黄色葡萄球菌青霉素结合蛋白2a（PBP2a）。方法用EcoRI和BamHI双酶切pGEMT-meeA重组质粒DNA，回收目的基因mecA，与pFastBacl质粒连接转化入含有穿梭载体bacmid的大肠埃希菌DHl0Bac中，筛选重组穿梭载体Bacmid-mecA并转染Sf9细胞，获取完整的重组杆状病毒。用免疫荧光（IFA），Westernblot法进行蛋白鉴定。结果成功地获得含mecA基因的重组杆状病毒。杆状病毒感染Sf9细胞形态变化明显，能够表达出与PBP2a多克隆抗体相结合的蛋白。结论利用Bac—to—Bac杆状病毒表达系统成功地表达了PBP2a蛋白。

Objective To express methicilin resistant Staphylococcus aureus penicillin binding protein 2a （PBP2a） with bacu- lovirus expression system. Methods MecA gene was obtained by double digest pGEMT-mecA plasmid using EcoR I and BamH I, and then the gene was inserted into plasmid pFastBacl. Recombinant plasmid was transformed into E. coli DHIOBac containing a shuttle vector,Bacmid. Recombinant shuttle vector bacmid-mecA was obtained by site-specific trans- position and transfected into sf9 cells. The expressed protein was identified by IFA and Western blot analysis. Results The baculovirus which containing mecA gene was expressed successfully. Morphological changes displayed in the transfected sf9 cells assumed that the transfection was successful. IFA and Western blot proved the expressed protein could combine with PBP2a polyclonal antibody. Conclusion The PBP2a protein was expressed successfully by Bac-to-Bac baculovirus expression system.