摘要
目的构建含目的基因mecA C末端的重组子并在大肠杆菌M15中进行蛋白表达。方法采用PCR方法扩增mecA C末端DNA,将其与表达载体pQE30连接;再通过PCR及双酶切鉴定连接产物。将含目的基因片段的阳性重组质粒转化大肠杆菌M15后,用1mmol／L的IPTG进行诱导表达。应用Ni-NTA琼脂糖进行亲和层析纯化目的蛋白,并用SDS-PAGE和Western blotting对表达产物进行鉴定。结果成功构建pQE30-mecA重组表达质粒,转化大肠杆菌M15,经IPTG诱导表达,亲和层析纯化获得分子量为39．7kDa的蛋白;Western blotting和肽指纹图谱证实该蛋白为带有多聚组氨酸标签的融合蛋白。结论在大肠杆菌内成功表达具有活性的PBP2a,为后期研究和开发抗MRSA药物奠定了基础。
Objective To construct a recombinant vector containing C-terminal of mecA gene and express it in E. coli M15. Methods C-terminal of mecA DNA was amplified by PCR and inserted into expression vector pQE30. The recombinant was identified by PCR and enzyme digestion. E. coli MI5 was transformed with the posi- tive reconstructed expression vector pQE30-mecA and induced by 1 mmol/L IPTG. The induced protein PBP2a was identified by SDS-PAGE and western blotting. The soluble protein was purified with Ni-NTA agarose. Results Positive recombinant plasmid pQE30-mecA was successfully constructed. The target protein of 39.7kDa with 6-His tag was detected by SDS-PAGE and western blotting and PMF. The target PBP2a has transpeptidase activity. Conclusion The soluble PBP2a of MRSA was successfully expressed and purified in E. coli M15. It paved the way for study of recombinant protein bioactivity and seek for PBP2a inhibitor against MRSA.
出处
《创伤外科杂志》
2007年第1期28-31,共4页
Journal of Traumatic Surgery
