马嘉霖,战盈瑾,周杰,黄鉴涛,闫秀英,简纪常

• 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/水产经济动物病害控制广东普通高校重点实验室

摘要(Abstract)：

【目的】鉴定草鱼（Ctenopharyngodon idella）miR-1260(cid-miR-1260)，分析cid-miR-1260在鱼淋巴囊肿病毒中国株（Lymphocystis disease virus China,LCDV-cn）感染草鱼卵巢细胞系（Grass carp ovary cells,GCO）过程中的时序表达特性，揭示cid-miR-1260对其靶基因znf574和LCDV-cn复制的调控作用。【方法】制备LCDV-cn感染的GCO细胞，通过茎环RT-PCR和测序鉴定cid-miR-1260；采用定量PCR检测cid-miR-1260在LCDV-cn感染GCO过程中的时序表达；应用生物信息学方法预测cid-miR-1260的靶基因，并用双荧光素酶报告基因系统验证靶基因；通过转染cid-miR-1260 mimic和cid-miR-1260 inhibitor使cid-miR-1260过表达和抑制表达，并用定量PCR检测cidmiR-1260、靶基因znf574、LCDV-cn mcp基因的表达；应用Reed-Muench法测定LCDV-cn在GCO细胞中的滴度。【结果】cid-miR-1260与其他已知miR-1260仅有一个碱基差异，且cid-miR-1260在LCDV-cn感染GCO过程中呈先上升后下降的表达变化，72 h时表达量最高（P <0.05）；生物信息学分析表明，znf574为cid-miR-1260的靶基因；重组质粒pmirGLO-znf574-WT与cid-miR-1260 mimic共转染后，荧光素酶活性显著降低（P <0.05），证实znf574为cidmiR-1260的靶基因；cid-miR-1260过表达后，靶基因znf574的表达显著下降（P <0.05）,LCDV-cn mcp的表达和LCDV-cn在GCO细胞中的滴度显著上升（P <0.05）；抑制cid-miR-1260表达后，靶基因znf574的表达量显著上升（P <0.05）,LCDV-cn mcp的表达量和LCDV-cn在GCO细胞中的滴度显著下降（P <0.05）。【结论】LCDV-cn感染GCO细胞后，cid-miR-1260的表达显著上调；在LCDV-cn感染中，cid-miR-1260对其靶基因znf574的表达起负调控作用，且cid-miR-1260表达与LCDV-cn的复制呈正相关，起促LCDV-cn感染作用，而锌指蛋白ZNF574具有抗LCDV-cn作用。本研究为淋巴囊肿病的防治提供新的依据和思路。

关键词(KeyWords)： 鱼淋巴囊肿病毒中国株;草鱼卵巢细胞系;cid-miR-1260;锌指蛋白基因znf574;病毒复制

Abstract:

Keywords:

基金项目(Foundation): 广东省科技特派员专项（GDKTP2021029800）;; 国家自然科学基金（31602199）

作者(Author): 马嘉霖,战盈瑾,周杰,黄鉴涛,闫秀英,简纪常

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