罗非鱼AT2-R抑制罗非鱼湖病毒复制的初步研究  

Inhibition of TiLV Replication by AT2-R in Nile Tilapia Oreochromis niloticus

作　　者：文静 郑树城[2] 柯紫姗 王英英[2] 李莹莹[2] 莫绪兵 张德锋[2] 尹纪元 周文礼 王庆[2] WEN Jing;ZHENG Shucheng;KE Zishan;WANG Yingying;LI Yingying;MO Xubing;ZHANG Defeng;YIN Jiyuan;ZHOU Wenli;WANG Qing(College of Fisheries,Tianjin Agricultural University,Tianjin 300384,China;Guangdong Provincial Key Laboratory of Aquatic Animal Immunology and Sustainable Aquaculture,Key Laboratory of Fishery Drug Development,Ministry of Agriculture and Rural Affairs,Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510380,China)

机构地区：[1]天津农学院水产学院,天津300384 [2]中国水产科学研究院珠江水产研究所,农业农村部渔用药物创制重点实验室,广东省水产动物免疫与绿色养殖重点实验室,广东广州510380

出　　处：《水产科学》2024年第5期683-693,共11页Fisheries Science

基　　金：国家重点研发计划项目(2019YFD0900101);广东省重点领域研发计划项目(2022B1111030001);广东省现代农业产业技术体系创新团队建设专项资金资助项目(2022KJ150).

摘　　要：为研究罗非鱼血管紧张素Ⅱ2型受体(AT2-R)在罗非鱼湖病毒(TiLV)感染过程中的功能,通过RT-PCR技术克隆得到体质量(20±5)g罗非鱼AT2-R(OnAT2-R)基因完整的开放阅读框序列,并运用qRT-PCR分析OnAT2-R基因的组织分布及时相表达,同时构建重组真核表达载体pEYFPOnAT2-R,在罗非鱼脑细胞中进行亚细胞定位分析,最后通过罗非鱼脑细胞过表达OnAT2-R基因后感染罗非鱼湖病毒,利用qRT-PCR方法检测TiLV-S8片段的相对表达量。试验结果显示,OnAT2-R基因开放阅读框序列全长1338bp,编码445个氨基酸,具有1个7次跨膜区域。在罗非鱼被检测的各个组织中,OnAT2-R基因均有分布,肝脏的表达量最高,其次是肌肉和皮肤,脾脏和胃的表达量最低。健康罗非鱼感染罗非鱼湖病毒后,OnAT2-R基因在肝脏中的表达量先增后减,在肌肉中的表达量先增后减再增。成功构建了pEYFP-OnAT2-R,亚细胞定位结果显示OnAT2-R主要定位于细胞膜上。在罗非鱼脑细胞中过表达OnAT2-R基因后感染罗非鱼湖病毒,发现TiLV-S8节段的mRNA水平显著降低。试验结果表明,OnAT2-R是一个潜在的定位于细胞膜且具有抑制罗非鱼湖病毒复制作用的细胞表面分子受体,这为后续深入研究罗非鱼湖病毒感染机理及罗非鱼抗病毒机制提供理论参考。In order to explore the function of type-2 angiotensinⅡreceptor(AT2-R)of Nile tilapia Oreochromis niloticus in the process of tilapia lake virus(TiLV)infection,the complete open reading frame(ORF)sequence of AT2-R gene of Nile tilapia AT2-R(OnAT2-R)was cloned by reverse transcription PCR(RT-PCR)and the tissue distribution and phase expression were analyzed in OnAT2-R by real-time fluorescence quantitative PCR(qRT-PCR).Meanwhile,the recombinant eukaryotic expression vector pEYFP-OnAT2-R was constructed for subcellular localization analysis in tilapia brain(TiB)cells which were infected with TiLV after overexpression of OnAT2-R gene,and the relative expression of TiLV-S8 segment was detected by qRT-PCR.The results showed that the OnAT2-R gene ORF sequence of the Nile tilapia was found to be 1338 bp in length,encoding 445 amino acids with a 7-transmembrane region.The OnAT2-R gene was found in all tissues examined in the Nile tilapia,with the maximal expression level in liver,followed by muscle and skin,and the minimal expression level in spleen and stomach.The relative expression level of OnAT2-R gene in the liver was shown to be increased first and then decreased in the Nile tilapia with TiLV infection,while the relative expression of OnAT2-R gene in the muscle was increased and then decreased,followed by increased again.Furthermore,the pEYFP-OnAT2-R was successfully constructed and the subcellular localization analysis showed that OnAT2-R protein was mainly localized on the cell membrane.The mRNA level of TiLV-S8 segment was significantly reduced in TiB cells overexpressed with OnAT2-R gene followed by TiLV infection.The finding indicates that OnAT2-R is a potential cell surface molecular receptor located on the cell membrane and has the effect on inhibition of TiLV replication,which will provide theoretical references with further in-depth research on pathogenesis of TiLV infection and antiviral mechanism of tilapia.

关 键 词：罗非鱼 罗非鱼湖病毒 血管紧张素Ⅱ2型受体 G蛋白偶联受体 

分 类 号：Q959.4[生物学—动物学]