作　　者： ; ; ; ; ; ;

机构地区： 南方医科大学基础医学院基因工程研究所

出　　处： 《微生物学通报》 2011年第8期1249-1255,共7页

摘　　要： 利用原核表达系统表达人源抑菌蛋白Reg3A,经包涵体的复性和纯化获得有体外抑菌功能的活性抑菌蛋白,并对其体外抑菌功能进行初步研究。构建Reg3A原核表达载体PET-32a-Reg3A转化补充稀缺tRNA基因的表达菌株大肠杆菌BL21-Codonplus,阳性重组子采用诱导培养基诱导5h后,采用超声破碎的方法提取包涵体蛋白,经包涵体蛋白的纯化和透析复性后通过Ni-NTA亲和层析交换柱,获得纯度达95%的蛋白质。Western blot鉴定显示在15 kD处有特异性条带。使用纯化后的蛋白进一步进行抑菌圈实验和抑菌活性实验,对获得蛋白的体外抑菌活性进行评估,从而为进一步进行Reg3A蛋白功能的评估及应用奠定基础。 In order to express and characterize the biological function of protein Reg3A, the partial coding sequence without signal peptide of Reg3A gene were sub-cloned into vector of pET-32a to construct recombinant prokaryotic expression vector pET-32a-Reg3A. After induction with IPTG, the strain of E. coli BL21-Codonplus was transformed successfully with recombinant constructs, which was found to be expressing the recombinant protein in high yield and existed in the form of inclusion bodies. The inclusion body dissolved in urea was refolded into natural conformation after dialysis. The expressed protein was purified by Ni-NTA column. The purified protein, about 95% purity, was confirmed by Western blot. It was further demonstrated that the recombinant protein could effectively inhibited growth of Gram positive bacteria, which indicated that our recombinant Reg3A retained antibacterial activity. The cloning and expression of the Reg3A proteins provide basis for further charac-terization of the Reg3A biological function.

关 键 词： 抑菌蛋白 包涵体复性 抑菌活性

领　　域： [生物学]