斜带石斑鱼T细胞免疫球蛋白黏蛋白-4(TIM-4)基因表达及重组蛋白制备  

Expression analysis and preparation of recombinant protein of T-cell immunoglobulin and mucin-domain-containing molecule 4(TIM-4)gene from oblique grouper Epinephelus coioides

作　　者：李金泽 汤菊芬[1] 黄瑜[1] 简纪常[1] 施钢 蔡佳[1,2] LI Jinze;TANG Jufen;HUANG Yu;JIAN Jichang;SHI Gang;CAI Jia(College of Fisheries, Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy culture, Guangdong Key Laboratory of Economic Animal Disease Control of Aquatic Products, Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang), Guangdong Ocean University, Zhanjiang 524088, China;Guangxi Key Laboratory of Marine Natural Products and Combinatorial Biosynthesis Chemistry, Guangxi Beibu Gulf Marine Research Center, Guangxi Academy of Sciences, Nanning 530007, 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控制重点实验室,南方海洋科学与工程广东省实验室(湛江),广东湛江524088 [2]广西科学院广西北部湾海洋研究中心,广西海洋天然产物与组合生物合成化学重点实验室,广西南宁530007

出　　处：《大连海洋大学学报》2022年第3期403-410,共8页Journal of Dalian Ocean University

基　　金：国家自然科学基金(U20A2065,32002426,32073006);广西自然科学基金(2020GXNSFAA297243);南方海洋科学与工程广东省实验室(湛江)(湛江湾实验室)自主立项项目(ZJW-2019-06);广东海洋大学“冲一流”专项资金项目(231419013);广东海洋大学“冲一流”与“创新强校工程”科研项目(2019KTSCX056);广西红树林滨海湿地生态保护与可持续利用人才小高地人才资助项目(BGMRC202101)。

摘　　要：为研究斜带石斑鱼Epinephelus coioides T细胞免疫球蛋白黏蛋白-4(T-cell immunoglobulin and mucin-domain-containing molecule 4,TIM-4)在免疫应答过程中的作用,通过聚合酶链式反应(PCR)获得斜带石斑鱼TIM-4的蛋白编码区(GenBank登录号:MZ465580),利用生物信息学分析其结构特征,采用荧光定量PCR(qPCR)分析TIM-4在健康鱼体中的组织分布,以及经LPS和Poly I:C刺激后的表达变化,并构建原核表达载体质粒TIM-4-4T,转化至大肠杆菌BL21(DE3)中进行TIM-4重组蛋白诱导表达。结果表明:斜带石斑鱼TIM-4基因的蛋白编码区片段长度为702 bp,可编码234个氨基酸,含有V-Type(IGV)、BTK和PRY 3个功能结构域;qPCR显示,TIM-4基因在斜带石斑鱼10种组织中均有表达,在胃中的表达量最高(P<0.05),在脾脏中表达量最低;经LPS刺激后,脾脏细胞中的TIM-4表达量显著降低(P<0.05),而经Poly I:C刺激后,脾脏细胞中的TIM-4表达量显著升高(P<0.05);SDS-PAGE电泳显示,TIM-4重组蛋白相对分子质量约为25100,以包涵体形式表达,在0.4 mmol/L IPTG、37℃下诱导6 h时,TIM-4重组蛋白的表达量最大。研究表明,TIM-4基因可能参与了斜带石斑鱼抗感染免疫反应,本研究结果为深入了解TIM-4的生物学功能提供了理论基础。In order to study the role of T-cell immunoglobulin and mucin-domain-containing molecule 4(TIM-4)gene in the immune response of oblique grouper Epinephelus coioides,protein-coding regions of the oblique grouper was obtained using polymerase chain reaction(PCR),and the structural characteristics of its protein coding region were analyzed by bioinformatics.The tissue distribution of TIM-4 gene were detected by real-time fluorescence quantitative PCR(qPCR).The expression profiles of TIM-4 gene was investigated in spleen(GS)cells exposed to lipopolysaccharide(LPS)and polyinosinic acid(Poly I:C)stimulation.The recombinant plasmid TIM-4-4T was constructed,and transformed into Escherichia coli BL21(DE3)for inducing prokaryotic protein expression.The results showed that the protein coding region(GenBank accession number:MZ465580)of TIM-4 was 702 bp,encoding 234 amino acids protein containing three functional domains,a V-Type(IGV)domain,a BTK domain and a PRY domain.qPCR results showed that TIM-4 was expressed in all examined 10 tissues of oblique grouper,with the maximal expression in stomach(P<0.05),and the minimal in spleen.There was significantly lower expression of TIM-4 in GS cells exposed to LPS stimulation,and significantly increase in GS cells exposed to Poly I:C stimulation(P<0.05).The SDS-PAGE analysis revealed that the TIM-4 fusion protein had relative molecular weight of about 25100.The optimal expression TIM-4 recombinant protein were inducing with 0.4 mmol/L IPTG at 37℃for 6 h.The findings indicate that TIM-4 may be involved in anti-infectious immunity of oblique grouper,which provides a theoretical basis for further study on the biological function of TIM-4.

关 键 词：斜带石斑鱼 T细胞免疫球蛋白黏蛋白-4 基因表达 LPS Poly IC 重组蛋白 

分 类 号：S943[农业科学—水产养殖]