目的：构建编码耐甲氧西林金黄色葡萄球菌（MRSA）青霉素结合蛋白2a （PBP2a）转肽酶区基因片段的原核表达载体，并表达、纯化及鉴定蛋白。方法从临床标本中分离鉴定M RSA ，设计针对编码 PBP2a转肽酶区基因片段的引物，采用聚合酶链式反应（PCR）扩增目的基因片段，克隆至pET28a（＋）载体，双酶切鉴定并测序，转化大肠杆菌BL21（DE3）plysS株；用0．7 mmol／L异丙基硫代‐β‐D‐半乳糖苷（IPTG）诱导表达后，利用Ni亲和层析技术纯化目的蛋白；蛋白免疫印迹法（WB）鉴定重组蛋白。结果重组表达载体经BamHⅠ、EcoRⅠ酶切，产物在预期大小处出现条带，测序结果显示有两个碱基突变，无移码突变。所表达的PBP2a蛋白经十二烷基硫酸钠‐聚丙烯酰胺凝胶电泳（SDS‐PAGE）和WB鉴定，在相对分子质量38×103处可见一新生蛋白条带。结论成功构建了PBP2a转肽酶区原核表达载体，并获得了高效表达，制备了高纯度的目的蛋白。%Objective To construct the prokaryontic expression vector of the gene fragment which encodes the transpeptidase domain of penicillin binding protein 2a(PBP2a) of methicillin‐resistant Staphylococcus aureus(MRSA) ,and to express ,purify and i‐dentify the objective protein .Methods Strains of MRSA were isolated and identified from clinical samples ,according to the se‐quence of mecA gene recorded in GenBank ,the primers of mecA fragment which encoded the transpeptidase domain of PBP2a was designed .The gene fragment from MRSA was amplified by using polymerase chain reaction(PCR) and cloned into pET28a(+ ) plasmid .After being identified by enzyme digestion and sequencing ,the recombinant plasmid was transformed into the strain of Escherichia coli BL21(DE3)plysS .The expression of transpeptidase domain of PBP2a was induced by 0 .7 mmol/L IPTG ,the ex‐pressed products were purified by using Ni afinity chromatography ,then were analyzed by using Western blot .Results The recom‐binant expression vector was digested by BamHⅠ and EcoRⅠ ,and the products were at the expected size .The result of sequencing showed two bases undergoing mutation ,while there were no frameshift mutations .The expressed protein was identified by using SDS‐PAGE and Western blot ,a new protein band was visible at the relative molecular mass of 38 × 103 .Conclusion The corre‐sponding prokaryotic expression vector is successfully constructed ,and the transpeptidase domain of PBP2a is successfully ex‐pressed and purified .
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