耐甲氧西林金黄色葡萄球菌PBP2a转肽酶区的克隆表达及纯化鉴定

目的:对编码耐甲氧西林金黄色葡萄球菌(MRSA)青霉素结合蛋白2a(PBP2a)转肽酶区的mecA基因片段进行克隆、表达、纯化及鉴定。方法:根据基因文库登录的mecA基因的编码序列,设计合成了一对寡核苷酸引物,应用PCR技术从MRSA基因组DNA中扩增获得编码PBP2a转肽酶区的DNA片段,将此目的基因片段克隆至pET-His载体,经酶切鉴定、测序正确后,转化E.coliBL21(DE3)plysS;用IPTG进行诱导表达后,利用Ni2 亲和层析技术从表达蛋白中纯化目的蛋白;对表达的蛋白以MRSA胶乳凝集试剂盒进行鉴定。结果: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。结论:获得了高纯度的PBP2a转肽酶区蛋白,为其进一步研究奠定了基础。

Prokaryotic Expression and Purification of the Transpeptidase Domain of PBP2a of Methicillin Resistant Staphylococcus aureus

Objective: To clone the mecA fragment which encodes the transpeptidase domain of penicillin binding protein 2a(PBP2a) of methicillin resistant Staphylococcus aureus(MRSA),to express and purify the transpeptidase domain of PBP2a.Methods: According to the sequence of mecA gene published in GenBank,primers were designed and synthesized.The mecA fragment which encodes the transpeptidase domain of PBP2a was amplified from the genomic DNA of MRSA by PCR,and then the objective gene fragment was cloned into pET-His plasmid.After being identified by enzyme digestion and sequencing,the recombinant plasmid was transformed into E.coliBL21(DE3)plysS.The expression was induced by IPTG and the expressed product was purified by Ni2 affinity chromatography analyzed by MRSA-screen latex agglutination test.Results: The corresponding prokaryotic expression vector was successfully constructed,and the transpeptidase domain of PBP2a was successfully expressed and purified.Conclusion: the transpeptidase domain of PBP2a with high purity was obtained,which established the foundation for further investigation.