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

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。

The study of the relationship between the expression of C-Myc protein and the replication of Nervous necrosis virus in GF-1 cells treated with

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.