创伤弧菌FrsA蛋白的纯化及生化表征  

Purification and Biochemical Characterization of FrsA from Vibrio vulnificus

作　　者：鲍玲玲 李至敏[2] 王小琴[1] 汤亚兰 许文武 李志敏[1] BAO Lingling;LI Zhimin;WANG Xiaoqin;TANG Yalan;XU Wenwu;LI Zhimin(College of Bioscience and Bioengineering,Jiangxi Agricultural University,Nanchang 330045,China;College of Science,Jiangxi Agricultural University,Nanchang 330045,China)

机构地区：[1]江西农业大学生物科学与工程学院,江西南昌330045 [2]江西农业大学理学院,江西南昌330045

出　　处：《河南农业科学》2018年第11期130-133,共4页Journal of Henan Agricultural Sciences

基　　金：江西省自然科学基金重大项目(20161ACB21012);江西省教育厅科技研究重点项目(GJJ160353);江西农业大学国家级大学生创新创业训练项目(201710410005)

摘　　要：为确定创伤弧菌FrsA蛋白(VvFrsA)在大肠杆菌中的表达条件及体外纯化步骤,并对其进行生化表征分析。通过分子生物学手段将合成的创伤弧菌的FrsA基因(VvFrsA)连接到pET28a载体上,构建pET28a-VvFrsA原核表达载体。将该载体转化至大肠杆菌BL21(DE3)感受态细胞后,经IPTG诱导实现了VvFrsA蛋白的可溶性表达。采用Ni-NTA亲和层析法纯化,得到重组VvFrsA蛋白。酶催化活性研究结果显示,VvFrsA具有酯酶功能,可以催化对硝基苯酚脂肪酸酯的水解,并且其最适底物为对硝基苯酚短链脂肪酸酯。综上,利用大肠杆菌BL21(DE3)菌株成功表达了可溶性VvFrsA蛋白,并且VvFrsA体外具有催化对硝基苯酚脂肪酸酯水解活性。The objective of this study was to identify the expression condition and purification procedures of VvFrsA from Vibro vulnificus,and analyze its biochemical characterization.The FrsA gene from Vibrio vulnificus(VvFrsA)was synthesized and ligated with pET28a vector to construct the pET28a-VvFrsA expression plasmid,which was then transformed into Escherichia coli BL21(DE3)competent cells.The soluble expression of VvFrsA protein was obtained by inducing with IPTG and the recombinant VvFrsA protein was purified by Ni-NTA affinity chromatography.Enzymatic catalytic activity studies indicate that VvFrsA was a type of esterase,which could catalyze the hydrolysis of p-nitrophenol fatty acid ester.Furthermore,the optimal substrate of VvFrsA was p-nitrophenol acetate.In conclusion,recombinant soluble VvFrsA could be expressed in Escherichia coli BL21(DE3)cell culture and VvFrsA displayed hydrolytic activity towards p-nitrophenol fatty acid ester in vitro.

关 键 词：创伤弧菌 FrsA蛋白 蛋白质纯化 生化表征 酯酶 

分 类 号：S917.1[农业科学—水产科学] Q936[生物学—微生物学]