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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。
To construct the miniature genome of infectious hematopoietic necrosis virus (IHNV HLJ-09) and express rainbow trout IFN, N, P, L, G and NV protein genes of IHNV HLJ-09 strain were amplified by RT-PCR and subcloned into eukaryotic The pCI-N, pCI-P, pCI-L, pCI-G and pCI-NV helper plasmids were constructed in the expression vector pCI. The two end sequences of IHNV genome, the enhanced green fluorescent protein (EGFP) The rainbow trout type I interferon (IFN) gene was cloned into the eukaryotic expression vector pCI to construct the IHNV minigenome pCI-LFGT expressing EGFP and the IHNV minigenome pCI-LFIT expressing IFN. The plasmid pCI-LFIT was transfected into IHNV HLJ -09 strain of EPC cells, real-time fluorescence quantitative PCR method for determination of IHNV G gene RNA in cells. The results showed that the foreign gene could be correctly expressed in the constructed mini-genome whether co-transfected with helper virus or with 5 helper plasmids. The pCI-LFIT plasmid was transfected into the EPC-cell-vaccinated group compared with the control group Nucleic acid is significantly reduced.