溶藻弧菌Ⅲ型分泌系统C-环组分VscQ的原核表达及免疫原性  

Prokaryotic Expression and Immunogenicity of the Type Ⅲ Secretion System C-ring Component Protein VscQ from Vibrio alginolyticus

作　　者：曾福源 伍艳清 邱明生 武沛文[1,2,3] 周诗慧 王俊霖 简纪常 谢妙 庞欢瑛[1,2,3] ZENG Fuyuan(Shenzhen Institute of Guangdong Ocean University,Shenzhen 510000,China;Fisheries College of Guangdong Ocean University,Zhanjiang 524088,China;Guangdong Provincial Key Laboratory of Pathogenic Biology and Epi-demiology for Aquatic Economic Animals,Key Laboratory of Diseases Controlling for Aquatic Economic Animals of Guangdong Higher Education Institutions,Zhanjiang 524088,China)

机构地区：[1]广东海洋大学深圳研究院,广东深圳510000 [2]广东海洋大学水产学院,广东湛江524088 [3]广东省水产经济动物病原生物学及流行病学重点实验室,广东省教育厅水产经济动物病害控制重点实验室,广东湛江524088

出　　处：《安徽农学通报》2020年第18期101-105,共5页Anhui Agricultural Science Bulletin

基　　金：深圳市科技计划项目(JCYJ20190813104207152,JCYJ20170818111629778,JCYJ20190813114409506)。

摘　　要：为探究溶藻弧菌(Vibrio alginolyticus)HY9901菌株Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS)胞质环(C-环)组分VscQ作为疫苗候选抗原的可能性,根据GeneBank上登陆的溶藻弧菌vscQ序列(NO.MN862753),设计1对带酶切位点的特异性引物,PCR扩增vscQ基因,进行序列分析。结果显示,该基因全长969bp,预计分子量35.61kDa。将vscQ基因定向插入原核表达载体pGEX-4T-1中,构建重组表达质粒pGEXvscQ。用异丙基-β-D-硫代半乳糖苷(IPTG)诱导后,能在大肠杆菌(Escherichia coli)BL21(DE3)中表达分子质量约为61.6kDa的VscQ融合蛋白。VscQ蛋白表达的最优条件为:0.4mmol/L IPTG,15℃条件下诱导12h。用纯化后的VscQ融合蛋白免疫白兔,获得高效多克隆抗体,抗体效价为1∶121500。兔抗VscQ血清能与重组VscQ蛋白发生特异性反应,表明T3SS C-环组分VscQ可能是溶藻弧菌的重要保护性抗原之一。To investigate the prokaryotic expression and the possibilities as a vaccine candidate antigen of VscQ from V.alginolyticus HY9901,primers ware designed according to the publicated vscQ gene sequence(NO.MN862753).Followed by digestion,connection,prokaryotic expression,vector pGEX-vscQ was constructed through directional inserting into pGEX-4T-1,and the Gst-VscQ fusion protein with 61.6kDa was successfully expressed in Escherichia coli BL21(DE3).The recombinant protein was highly expressed under induction conditions of exposure with 0.4mmol/L IPTG at 15℃for 12 hours.The purified fusion protein was injected into rabbit to produce anti-VscQ se⁃rum.Western blot analysis revealed that the prepared antiserum specifically reacted to the VscQ fusion protein,which indicated that the VscQ may be one of the important protective antigens of V.alginolyticus.

关 键 词：溶藻弧菌 VscQ Ⅲ型分泌系统 原核表达 免疫原性 

分 类 号：S941.42[农业科学—水产养殖] Q786[农业科学—水产科学]