谷氨酰胺对斜带石斑鱼GF-1细胞中C-Myc蛋白的表达与神经坏死病毒复制的影响  

Relationship between the expression of C-Myc protein and the replication of nervous necrosis virus in GF-1 cells treated with glutamine

作　　者：沈海洋 卢志杰 秦真东 赵丽娟 李亚男 刘春 杨光 叶成凯 潘淦 林蠡 SHEN Haiyang;LU Zhijie;QIN Zhendong;ZHAO Lijuan;LI Yanan;LIU Chun;YANG Guang;YE Chengkai;PAN Gan;LIN Li(Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture,College of Life Science,South China Normal University,Guangzhou 510631,China;Guangdong Provincial Water Environment and Aquatic Products Security Engineering Technology Research Center,Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding,College of Animal Science&Tecgnology,Zhongkai University of Agriculture and Engineering,Guangzhou 510225,China)

机构地区：[1]华南师范大学生命科学学院,广东省水产健康安全养殖重点实验室,广东广州510631 [2]仲恺农业工程学院动物科技学院,广东省水环境与水产品安全工程技术研究中心,广州市水产病害与水禽养殖重点实验室,广东广州510225

出　　处：《水产学报》2020年第9期1385-1396,共12页Journal of Fisheries of China

基　　金：国家自然科学基金(31872606);广东省海洋与渔业局基金(GDME-2018C006,D21822202);中国-东盟海上合作基金(CAMC-2018F);广东省现代农业产业技术体系创新团队建设专项(2019KJ141)。

摘　　要：为探讨C-Myc表达、谷氨酰胺代谢和神经坏死病毒复制三者之间的关系,本研究首先克隆了斜带石斑鱼鳍条细胞(GF-1)中的C-Myc基因(GF-1-C-Myc),结果显示GF-1-CMyc基因cDNA全长814 bp,开放阅读框(ORF)为285 bp,编码95个氨基酸(aa),有亮氨酸拉链结构域与螺旋-环-螺旋(HLH)结构域。实验表达和纯化了GF-1-C-Myc蛋白,并制备其多克隆抗体。采用实时定量PCR技术(qRT-PCR)与免疫印迹法(WB)检测了GF-1-C-Myc基因的表达和神经坏死病毒的复制。结果显示,缺乏谷氨酰胺会同时抑制GF-1-C-Myc基因的表达和神经坏死病毒(NNV)的复制,添加谷氨酰胺可同时促进GF-1-C-Myc的表达和NNV的复制;此外,NNV感染可上调GF-1-C-Myc基因的表达,并显著消耗GF-1细胞培养液中的谷氨酰胺。研究表明,GF-1-C-Myc基因可调控宿主谷氨酰胺代谢,从而有利于神经坏死病毒的复制。本结果为防控NNV的感染提供了参考。In order to investigate the relationships among the C-Myc expression,glutamine metabolism and replication of NNV,C-Myc gene(GF-1-C-Myc)from Epinephelus coioides fin cell(grouper fin cells,GF-1)was cloned.The full length of GF-1-C-Myc gene cDNA was 814 bp with 285 bp ORF,encoding 95 amino acid(aa)with leucine zipper domain and helix-ring-helix(HLH)domain.GF-1-C-Myc protein was expressed and purified,and its polyclonal antibody was generated.The expressions of GF-1-C-Myc gene and the replication of NNV were monitored by real-time quantitative PCR(qRT-PCR)and immunoblotting(WB).The results showed that lack of glutamine could inhibit both the expression of GF-1-C-Myc gene and replication of NNV,while addition of glutamine could promote both the expression of GF-1-C-Myc gene and replication of NNV.In addition,the expression of GF-1-C-Myc gene was up-regulated in GF-1 cells infected with NNV,and the glutamine in the medium was significantly consumed.Taken together,GF-1-C-Myc gene was involved in the regulation of glutamine metabolism in the cell,subsequently facilitated the replication of NNV.Our results will shed a new light on the prevention and control of NNV infection.

关 键 词：斜带石斑鱼 C-MYC基因 GF-1细胞 神经坏死病毒 感染 谷氨酰胺 

分 类 号：Q785[生物学—分子生物学] S941.41[农业科学—水产养殖]