溶藻弧菌磷酸甲基嘧啶合酶(ThiC)的原核表达及其乙酰化修饰鉴定  

Prokaryotic expression and acetylation identification of phosphomethylpyrimidine synthase(ThiC)in Vibrio alginolyticus

作　　者：王旭东 范晨龙 丁燏[1] WANG Xudong;FAN Chenlong;DING Yu(College of Fisheries,Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemilogy for Aquatic Economic Animals,Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes, Guangdong Ocean University, Zhanjiang 524088, China)

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

出　　处：《大连海洋大学学报》2022年第3期435-440,共6页Journal of Dalian Ocean University

基　　金：广东省自然科学基金(2014A030313604,2022A1515012203);广东海洋大学研究生教育创新计划资助项目(201724)。

摘　　要：为研究溶藻弧菌Vibrio alginolyticus磷酸甲基嘧啶合酶(phosphomethylpyrimidine synthase,ThiC)是否存在乙酰化修饰,以溶藻弧菌HY9901基因组为模板,PCR扩增ThiC基因,构建pET-28a-ThiC原核表达载体,在大肠杆菌BL21中诱导表达,并优化其表达条件,然后用SDS-PAGE和Western blot分析蛋白的表达及乙酰化情况。结果表明:克隆获得的溶藻弧菌ThiC基因长度为1941 bp;重组蛋白成功在大肠杆菌BL21中表达,相对分子质量为72500,其最佳诱导条件为IPTG 0.1 mmol/L、温度37℃、诱导时间3 h;经Western blot检测显示,ThiC蛋白具有乙酰化修饰,但在体外不能通过乙酰化酶途径去乙酰化。研究表明,溶藻弧菌ThiC原核表达载体构建成功,该蛋白存在乙酰化修饰,但体外不能去乙酰化,本研究结果为溶藻弧菌的维生素合成过程中酶的翻译后调控机制研究提供了科学参考。To understand whether there is acetylation of phosphomethylpyrimidine synthase(ThiC)in pathogenic Vibrio alginolyticus,the target gene ThiC was amplified via PCR basing on the genome of V.alginolyticus HY 9901.The prokaryotic expression vector pET-28a-ThiC were constructed,which was successfully expressed in Escherichia coli BL21 by induction,and its expression conditions were optimized.Then,the protein expression and acetylation level were analyzed by SDS-PAGE and Western blot.The results showed that the ThiC gene had length of 1941 bp,and that the recombinant Escherichia coli BL21 was expressed as the recombinant protein(72500).The optimal expression of recombinant protein was under conditions of 0.1 mmol/L IPTG for 3 hours at 37℃.Western blot results revealed that ThiC was acetylated protein,and there was no deacetylation in vitro.In conclusion,the recombinant expression strain for V.alginolyticus ThiC was successfully constructed,ThiC was acetylated protein,and there was no deacetylation in vitro.The findings lay the foundation for studying the post-translational regulation mechanism of vitamin nutrition of V.alginolyticus.

关 键 词：溶藻弧菌 磷酸甲基嘧啶合酶 原核表达 乙酰化 

分 类 号：S917.4[农业科学—水产科学]