耐甲氧西林金黄色葡萄球菌青霉素结合蛋白2a转肽酶区的原核表达及其纯化  

Prokaryotic expression and purification of transpeptidase domain of penicillin binding protein 2a of methicillin-resistant Staphylococcus aureus

作　　者：李冀[1] 杨慧[1] 唐蕊华[1] 李晶[1] 李琦[1] 邵燕[1] 呼延霆[1] 黄庆生[1] 师俊玲[1] 

机构地区：[1]西北工业大学生命学院空间生物实验模拟技术国防重点实验室,陕西西安710072

出　　处：《中国生物制品学杂志》2015年第5期497-500,共4页Chinese Journal of Biologicals

摘　　要：目的原核表达耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)青霉素结合蛋白2a(penicillin binding protein 2a,PBP2a)转肽酶区,并进行纯化及鉴定。方法 PCR扩增编码PBP2a转肽酶区的mec Af基因片段,克隆至原核表达载体p GEX-6p-1中,构建重组表达质粒p GEX-6p-mec Af,经双酶切(Bam HⅠ和Eco RⅠ)及测序鉴定正确后,转化感受态大肠埃希菌(E.coli)BL21进行诱导表达;经蛋白亲和层析柱纯化后,进行SDS-PAGE和Western blot鉴定。结果重组表达质粒p GEX-6p-mec Af经双酶切(Bam HⅠ和Eco RⅠ)及测序鉴定证明构建正确;表达产物的相对分子质量约63 000,表达量约占菌体总蛋白的14.5%,纯化后纯度达90%。结论成功构建了PBP2a转肽酶区的原核表达质粒,目的蛋白在E.coli中获得高效表达,为制备单克隆抗体及建立MRSA快速检测方法奠定了基础。Objective To express the transpeptidase domain of penicillin binding protein 2a（PBP2a） of methicillin-resistant Staphylococcus aureus（MRSA）in prokaryotic cells, and purify and identify the expressed product. Methods The mec Af gene encoding the transpeptidase domain of PBP2 a was amplified by PCR and cloned into prokaryotic expression vector p GEX-6p-1. The constructed recombinant plasmid p GEX-6p-mec Af was identified by restriction analysis（Bam HⅠ and Eco RⅠ）and sequencing, then transformed to E. coli BL21 and induced with IPTG. The expressed product was purified by affinity chromatography, and identified by SDS-PAGE and Western blot. Results Both restriction analysis and sequencing proved that recombinant plasmid p GEX-6p-mec Af was constructed correctly. The expressed product, with a relative molecular mass of about 63 000, contained about 14. 5% of total somatic protein and reached a purity of 90% after purification. Conclusion The prokaryotic expression vector for transpeptidase domain of PBP2 a was constructed correctly,and target protein was highly expressed in E. coli, which laid a foundation of preparation of monoclonal antibody and development of rapid detection method for MRSA.

关 键 词：耐甲氧西林金黄色葡萄球菌 青霉素结合蛋白2A 转肽酶区 原核细胞 基因表达 

分 类 号：R373.11[医药卫生—病原生物学] R186.3[医药卫生—基础医学]