传染性造血器官坏死病毒微型基因组表达干扰素对病毒的抑制效应  

Infectious haemopoietic necrosis virus minigenome expressing IFN and its inhibiting effect on virus

作　　者：王会英[1] 蒋烨[2] 赵丽丽[1] 唐丽杰[1] 乔薪瑗[1] 姜艳平[1] 崔文[1] 李一经[1] 刘敏[2] 

机构地区：[1]东北农业大学动物医学学院,哈尔滨150030 [2]东北农业大学动物科学与技术学院,哈尔滨150030

出　　处：《淡水渔业》2016年第1期3-8,17,共7页Freshwater Fisheries

基　　金：国家自然科学基金(31372568);东北农业大学博士启动基金项目(2012RCB66)

摘　　要：为构建传染性造血器官坏死病毒(IHNV HLJ-09)微型基因组并表达虹鳟IFN,采用RT-PCR扩增IHNV HLJ-09株的N、P、L、G和NV蛋白基因并亚克隆入真核表达载体pCI中,构建辅助质粒pCI-N、pCI-P、pCI-L、pCI-G和pCI-NV;将扩增获得的IHNV基因组两末端序列、增强型绿色荧光蛋白(EGFP)报告基因、虹鳟I型干扰素(IFN)基因克隆到真核表达载体pCI中构建出表达EGFP的IHNV微型基因组pCI-LFGT和表达IFN的IHNV微型基因组pCI-LFIT;将pCI-LFIT质粒转染已接种IHNV HLJ-09毒株的EPC细胞,实时荧光定量PCR法测定细胞中IHNV G基因RNA。结果显示:构建的微型基因组不论与辅助病毒还是与5个辅助质粒共转染,外源基因均能正确表达;pCI-LFIT质粒转染已接种病毒的EPC细胞组与对照组相比其中的病毒核酸显著减少。This study constructed and identified the minigenome of infectious haemopoietic necrosis virus,which expressing rainbow trout IFN-Ⅰ. First,the N,P,L,G and NV protein genes of IHNV HLJ-09 were subcloned into the pCI eukaryotic expression vector by using RT-PCR method,this research constructed pCI-N,pCI-P,pCI-L,PCI-G and pCI-NV support plasmids. Amplifying the both ends of IHNV genome sequences and EGFP reporting genes,rainbow trout type ⅠIFN genes,which were cloned into the eukaryotic expression vector pCI to build pCI-LFIT and pCI-LFGT. Transfect pCILFGT into EPC cell which was infected by IHNV HLJ-09,the real-time PCR assay was used to detect IHNV G gene RNA replication level and total proteins were extracted for Western blot. The reporter genes in the building minigenome could express correctly both with support virus and co-transfect with five support plasmids. The minigenome containing inverted IFN could specifically be expressed in EPC cell lines infected IHNV,the viral replication level was significantly decreased to a certain extent by real-time PCR detection.

关 键 词：传染性造血器官坏死病毒 辅助质粒 虹鳟Ⅰ型干扰素 微型基因组 

分 类 号：S917.4[农业科学—水产科学]