牡蛎诺如病毒受体类Lewis抗原合成相关基因CgFUT5的克隆与表达鉴定  

Cloning,expression and identification of CgFUT5 gene associated with Lewis antigen synthesis of Oyster norovirus receptors

作　　者：桂彬彬 曲梦 张蔚然 李明玉 江艳华[1] 姚琳[1] 王联珠[1] GUI Binbin;QU Meng;ZHANG Weiran;LI Mingyu;JIANG Yanhua;YAO Lin;WANG Lianzhu(Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences/Key Laboratory of Testing and Evaluation for Aquatic Product Safety and Quality,Ministry of Agriculture and Rural Affairs,Qingdao 266071,China;School of Food Science and Technology,Dalian Polytechnic University,Dalian 116034,China;College of Food Science and Technology,Shanghai Ocean University,Shanghai 201306,China;College of Food Science and Engineering,Ocean University of China,Qingdao 266003,China)

机构地区：[1]中国水产科学研究院黄海水产研究所/农业农村部水产品质量安全检测与评价重点实验室,山东青岛266071 [2]大连工业大学食品学院,辽宁大连116034 [3]上海海洋大学食品学院,上海201306 [4]中国海洋大学食品科学与工程学院,山东青岛266003

出　　处：《南方水产科学》2023年第6期150-157,共8页South China Fisheries Science

基　　金：国家重点研发计划项目(2017YFC1600703);国家自然科学基金青年科学基金项目(31101883);中国水产科学研究院基本科研业务费专项资金(2023TD76);国家现代农业产业技术体系(CARS-49)。

摘　　要：Lewis抗原被认为是诺如病毒特异性结合受体,作为诺如病毒传播载体,牡蛎中也存在着类Lewis抗原,但牡蛎合成这种碳水化合物的途径尚未阐明。为解析牡蛎中诺如病毒受体类Lewis抗原的合成路径,利用cDNA末端快速扩增(Rapid amplification of cDNA ends,RACE)技术克隆得到太平洋牡蛎(Crassostrea gigas)的CgFUT5基因全序列并进行生物信息学分析,通过实时荧光定量聚合酶链式反应(RT-qPCR)分析其在5种组织中的表达情况。构建原核表达质粒转化大肠杆菌(Escherichia coli)实现异源表达,并通过免疫印迹法(Western blot)鉴定免疫原性。克隆得到了具有1173 bp开放阅读区的CgFUT5基因cDNA序列,系统发育树显示CgFUT5基因与多个物种具有合成Lewis抗原功能的岩藻糖基转移酶基因遗传学关系较近。重组CgFUT5蛋白可在大肠杆菌中过量表达,且表达的重组CgFUT5蛋白与抗人FUT5抗体及抗6×His标签抗体均能特异性结合。研究发现CgFUT5基因在牡蛎鳃组织中大量表达,CgFUT5蛋白与人FUT5蛋白具有相似的免疫原性,推测牡蛎中存在着类Lewis抗原的合成通路,并且调控牡蛎类Lewis抗原合成的基因还具有组织表达差异性。Lewis antigen is regarded as a specific binding receptor for norovirus,and Lewis-like antigen is also present in oysters as a vehicle for norovirus transmission,but the pathway for synthesis of this carbohydrate in oysters has not been elucidated.To clarify the pathway of norovirus receptor-like Lewis antigen synthesis in oysters,we cloned the CgFUT5 gene from Pacific oyster(Crassostrea gigas)genome and analyzed the expression in five tissues.The full sequence of CgFUT5 gene was obtained by rapid amplification of cDNA ends(RACE)and bioinformatically analyzed by real-time quantitative polymerase chain reaction(RT-qPCR).A prokaryotic expression plasmid was constructed to transform Escherichia coli for heterologous expression,and immunogenicity was identified by immunoblotting(Western blot).The cDNA sequence of CgFUT5 gene with 1173 bp open reading region was obtained by cloning,and phylogenetic tree shows that CgFUT5 gene was genetically related to the rockweed glycosyltransferase gene that had the function of synthesizing Lewis antigen in several species.The recombinant CgFUT5 protein could be overexpressed in E.coli,and the expressed recombinant CgFUT5 protein specifically bound to both anti-human FUT5 antibody and anti-6×His tag antibody.To sum up,CgFUT5 gene was successfully cloned and found to be abundantly expressed in oyster gill tissue,and CgFUT5 protein has similar immunogenicity to human FUT5 protein.It is hypothesized that a Lewislike antigen synthesis pathway exists in oysters,and the genes regulating Lewis-like antigen synthesis in oysters also have differential tissue expression.

关 键 词：太平洋牡蛎 CgFUT5基因 克隆 组织表达 原核表达 

分 类 号：TS254.7[轻工技术与工程—水产品加工及贮藏工程]