表达绿色荧光蛋白的复制缺陷型西尼罗病毒的构建

发布时间：2018-10-31 12:44

【摘要】：为构建表达绿色荧光蛋白(GFP)的复制缺陷型西尼罗病毒(WNV),本研究参考NY99株WNV基因组序列,分段合成除结构蛋白基因外的其它基因组序列,以及插入缺失的结构蛋白基因位置的gfp报告基因序列,并按WNV基因组顺序克隆于pCI-neo载体中,构建含有GFP基因的WNV复制子重组质粒pWNVrep-GFP;同时构建表达WNVC蛋白的重组质粒pCAG-WNV-C。将pWNVrep-GFP和pCAG-WNV-C共转染稳定表达WNVprM-E蛋白的W-92细胞系进行WNV粒子包装及病毒粒子拯救。结果显示转染细胞能够表达GFP,将转染细胞培养液接种BHK-21细胞后,能感染细胞并且表达GFP,westernblot可以检测到分子质量为42ku的NS1蛋白条带。然而,感染BHK-21细胞的培养液不能再感染BHK-21细胞,表明拯救的病毒只具有一次感染能力,为复制缺陷型病毒。WNV阳性血清能中和拯救病毒对BHK-21细胞的感染,该复制缺陷型病毒为WNV中和抗体检测和疫苗评价提供了相关的技术平台。
[Abstract]:In order to construct the replication-deficient West Nile virus (WNV),) (WNV), expressing green fluorescent protein (GFP), the genomic sequence of WNV strain of NY99 strain was studied, and other genomic sequences except structural protein gene were synthesized. The gfp reporter gene sequence of the deleted structural protein gene was inserted and cloned into the pCI-neo vector according to the WNV genomic sequence. The WNV replicon recombinant plasmid pWNVrep-GFP; containing the GFP gene was constructed. Construction of Recombinant plasmid pCAG-WNV-C. expressing WNVC protein at the same time PWNVrep-GFP and pCAG-WNV-C were co-transfected into W-92 cell line, which expressed WNVprM-E protein stably. WNV particle packaging and virus particle rescue were carried out. The results showed that the transfected cells could express GFP, after inoculating the transfected cells with BHK-21 cells, and the expression of GFP,westernblot could detect the NS1 protein bands with molecular weight of 42ku. However, the culture medium infected with BHK-21 cells can no longer infect BHK-21 cells, indicating that the rescued virus has the ability to infect BHK-21 cells only once. WNV positive serum can neutralize the infection of the rescued virus to BHK-21 cells. The replication-deficient virus provides a technical platform for WNV neutralizing antibody detection and vaccine evaluation.
【作者单位】：中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室;
【基金】：国家重点研发计划项目(2016YFD0500403)
【分类号】：S852.65

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