金黄色葡萄球菌附属基因调节子的表达与纯化

Prokaryotic expression and purification of recombination accessory gene regulator of staphylococcus aureus

目的构建金黄色葡萄球菌附属基因调节子(accessory gene regulator C,agrC)的原核表达质粒pET28α(+)-agrC,并诱导该基因在原核细胞中表达与纯化。方法提取金黄色葡萄球(简称金葡菌)菌基因组DNA,PCR扩增agrC基因的目的片断全长,插入表达质粒载体pET28α(+)中,构建pET28α(+)-agrC重组子,经双酶切和测序证实后,转化表达宿主大肠杆菌DH5α,异丙基--βD硫代半乳糖(IPTG)诱导重组蛋白表达,His标签磁株纯化重组蛋白,SDS-PAGE和Western blot检测鉴定蛋白表达。结果成功构建了金葡菌pET28α(+)-agrC大肠杆菌表达重组子,实现了该蛋白在大肠杆菌中的高表达,分离纯化的产物纯度达到90%。结论agrC的成功表达和分离纯化,为该蛋白的生物功能研究和筛选出与该蛋白特异性结合的小肽奠定了基础,从而为深入研究agrC的作用机制并设计针对agrC作用的抗菌药物提供了有力依据。

Objective To contribute Staphylococcus aureus pET28α（＋）-agrC and induce expression and purify in prokaryocyte. Methods The DNA of Staphylococcus aureus was amplified by PCR and cloned into expression vector pET28α（＋） ,and they were confirmed by restriction endonuclease digestion and sequence analysis. The verified recombinant was transformed into E. coli DH5α. After inducing with isopropyl-β-D-thiogalaetopyranoside（IPTG）, the recombinant protein was purified via His Tag magnetic stock. The recombinant proteins were analyzed with SDS-PAGE and Western blot. Results The agrC was successfully constructed and the recombinant agrC protein was expressed in E. coli at a high level. And the purified protein had reached at 90%. Conclusion The successful expression and purify of the agrC will be helpful for the study about bilolgical function and bolting the small peptide which can combine to it specificity,and also have provided evidence for a further study on the mechanism of action of agrC and designing a antibiosis medicine which aim directly at this protein.