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

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。

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

This study constructed and identified the minigenome of infectious haemopoietic necrosis virus, which express-ing rainbow trout IFN-Ⅰ.First, the N, P, L, G and NV protein genes of IHNV HLJ-09 were subcloned into the pCI eu-karyotic 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 typeⅠIFN genes, which were cloned into the eukaryotic expression vector pCI to build pCI-LFIT and pCI-LFGT.Transfect pCI-LFGT 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 ex-press 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.