溶藻弧菌AphB蛋白的原核表达及其乙酰化验证  

Prokaryotic Expression and Acetylation of AphB Protein of Vibrio alginolyticus

作　　者：万明月 范晨龙 丁燏[1] WAN Mingyue;FAN Chenlong;DING Yu(Fisheries College,Guangdong Ocean University/Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture/Key laboratory of Center for Diseases Control of Aquatic Economic Animals of Guangdong Province,Zhanjiang,Guangdong 524088,China)

机构地区：[1]广东海洋大学水产学院/广东省水产动物病害防控与健康养殖重点实验室/广东省水产经济动物病害防控重点实验室,广东湛江524088

出　　处：《福建农业学报》2022年第10期1250-1255,共6页Fujian Journal of Agricultural Sciences

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

摘　　要：【目的】研究LysR家族转录因子AphB蛋白是否存在乙酰化修饰,为进一步研究乙酰化修饰对该蛋白功能的影响提供理论基础。【方法】以溶藻弧菌(Vibrio alginolyticus)HY9901 LysR家族转录因子AphB为对象,根据GenBank上溶藻弧菌aphB序列(No.WP_005380599.1)设计引物,采用大肠杆菌表达体系异源诱导表达,设置时间、温度和IPTG浓度梯度优化表达条件,最后纯化AphB蛋白,通过抗乙酰赖氨酸特异性抗体验证AphB蛋白的乙酰化修饰程度。【结果】aphB全长约876 bp,37℃时菌液加入0.1 mmol·L^(-1)的IPTG诱导6 h后,AphB蛋白的表达量最高,纯化后的蛋白大小为37.3 kD,AphB自身存在乙酰化修饰位点,但其乙酰化程度在体外不受脱乙酰酶CobB的调控。【结论】初步证明了AphB蛋白是一种乙酰化蛋白且在体外不能被脱乙酰酶CobB脱乙酰化,研究结论丰富了原核生物弧菌中乙酰化修饰的相关理论,为溶藻弧菌毒力基因aphB的翻译后调控机制研究提供了科学参考。【Objective】Prokaryotic expression and acetylation modification of the LysR family transcription factor AphB protein were studied.【Method】Primers of AphB of Vibrio alginolyticus HY9901 were designed based on the GenBank database No:WP_005380599.1.Heterologous induction was carried out by using E.coli expression system with optimized time,temperature,and IPTG concentration gradient.AphB protein was then purified to determine the degree of acetylation modification using anti-acetyl lysine specific antibody.【Result】The full length of aphB gene was approximately 876 bp.After the bacterial solution was induced with 0.1 mmol·L^(-1)of IPTG at 37℃for 6 h,the expression of the fusion protein peaked.The purified fusion protein was 37.3 kD with an acetylation modification site.However,in vitro,the degree of acetylation was not regulated by deacetylase CobB.【Conclusion】This study preliminarily determined AphB protein to be an acetylated protein that could not be enzymatically deacetylated by deacetylase CobB in vitro.The results reinforced the existing theory of acetylation modification in prokaryotic Vibrio and provided a valuable reference for studying the post-translation regulation mechanism of virulence gene aphB of V.alginolyticus.

关 键 词：溶藻弧菌 AphB 大肠杆菌表达体系 赖氨酸乙酰化修饰 

分 类 号：S941.42[农业科学—水产养殖]