溶藻弧菌PepA蛋白原核表达载体的构建及其乙酰化鉴定  

Construction of Prokaryotic Expression Vector of PepA Protein of Vibrio alginolyticus and Identification of Its Acetylation

作　　者：徐洲 范晨龙 丁燏 XU Zhou;FAN Chen-long;DING Yu(Fisheries College of Guangdong Ocean University,Zhanjiang 524088;Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals,Zhanjiang 524088;Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes,Zhanjiang 524088)

机构地区：[1]广东海洋大学水产学院,湛江524088 [2]广东省水产经济动物病原生物学及流行病学重点实验室,湛江524088 [3]广东省水产经济动物病害控制重点实验室,湛江524088

出　　处：《生物技术通报》2020年第12期75-81,共7页Biotechnology Bulletin

基　　金：广东省自然科学基金项目(2014A030313604)。

摘　　要：旨为构建溶藻弧菌HY9901 PepA蛋白的原核表达载体、优化其表达条件,并分析该蛋白是否存在乙酰化调控。首先设计特异性引物经PCR克隆pepA基因,构建表达载体pET-28a-PepA并将其转入大肠杆菌BL21(DE3),然后用SDS-PAGE和Western blot分析蛋白的表达及乙酰化情况。结果显示,表达菌株可以正确表达重组蛋白(60.7 kD),其最佳表达条件为:体积分数为0.1%的IPTG,37℃诱导5 h;Western blot结果表明,PepA蛋白是乙酰化蛋白,且在体外不存在去乙酰化。The purpose of this study is to construct a prokaryotic expression vector of PepA protein in Vibrio alginolyticus HY9901,to optimize its expression conditions,and to analyze whether or not it is regulated by protein acetylation.Firstly,the pepA gene was cloned via PCR,and an expression vector pET-28a-PepA was constructed and transferred it into Escherichia coli BL21(DE3).Then,the protein expression and acetylation were analyzed by SDS-PAGE and Western blot.The recombinant expression strain correctly expressed the recombinant protein(60.7 kD),and its optimal expression conditions was,induced for 5 h at 37℃by IPTG with a volume fraction of 0.1%.Western blot results showed that PepA protein was acetylated protein,and there was no deacetylation in vitro.

关 键 词：溶藻弧菌 PepA蛋白 表达条件优化 乙酰化 

分 类 号：Q782[生物学—分子生物学] S941.42[农业科学—水产养殖]