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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 purify and identify the penicillin binding protein 2a (PBP2a) transpeptidase region by prokaryotic expression of methicillin-resistant Staphylococcus aureus (MRSA). Methods The mec Af gene fragment encoding the PBP2a transpeptidase region was amplified by PCR and cloned into the prokaryotic expression vector pGEX-6p-1. The recombinant plasmid pGEX-6p-mec Af was constructed and double digested with Bam HI and EcoRI RⅠ). After identification by sequencing, the recombinant plasmid was transformed into competent E. coli BL21 and expressed by SDS-PAGE and Western blot after purified by protein affinity chromatography. Results The recombinant plasmid pGEX-6p-mec Af was confirmed by double enzyme digestion (Bam HⅠand Eco RⅠ) and sequencing. The relative molecular mass of the expressed product was about 63 000 and the expression level was about 14.5% of the total bacterial protein. , Purity of 90% after purification. Conclusion The prokaryotic expression plasmid of PBP2a transpeptidase region was successfully constructed. The target protein was highly expressed in E. coli, which laid the foundation for the preparation of monoclonal antibody and rapid detection of MRSA.