鸭病毒性肝炎病毒RNA聚合酶在真核细胞中的表达及亚细胞定位

为研究鸭病毒性肝炎病毒RNA依赖性RNA聚合酶的分子生物学特性及其在细胞中的定位,构建了RNA依赖性RNA聚合酶与增强型绿色荧光蛋白融合表达的真核重组表达质粒;然后用脂质体介导转染MDCK细胞。分别使用荧光显微镜、激光共聚焦显微镜以及4,6-二脒基-2-苯基吲哚(DAPI)染色和蛋白免疫电泳试验等方法检测了RNA依赖性RNA聚合酶在细胞中的表达和亚细胞定位情况。结果表明,RNA依赖性RNA聚合酶能与增强型绿色荧光蛋白一起在MDCK细胞中良好表达,且主要分布于细胞核中,Western-blot分析结果显示,融合蛋白在80ku处出现阳性条带,说明表达的外源蛋白具有免疫活性。表明成功构建了RNA依赖性RNA聚合酶与增强型绿色荧光蛋白融合表达载体,并在真核细胞内实现了表达,通过检测荧光蛋白,发现RNA依赖性RNA聚合酶主要在细胞核中发挥复制酶的功能。

Eukaryotic expression and subcellular localization of RNA-dependent RNA polymerase of duck hepatitis A virus

To study the molecular biological characteristics and subcellular localization of RNA-dependent RNA polymerase(RdRp) of duck hepatitis A virus(DHAV),the recombinant plasmid pEGFP-RdRp containing RdRp fused with the green fluorescent protein(GFP) was constructed.After transfected into MDCK cells,the expression of RdRp was detected with fluorescence microscope and Western-blotting,and the results showed that RdRp fused with GFP was expressed well in the MDCK cells.Besides,the localization of RdRp in MDCK cells was determined with laser confocal microscope.The results indicated that RdRp was mainly distributed in the nucleus of MDCK cells.And the expressed fusion protein with molecular weight of 80 ku could react with GFP antibody in Western-blotting analysis,demonstrating that the fusion protein possessed immunological activity.The fusion expression vector pEGFP-RdRp was successfully constructed and expressed efficiently in eukaryotic cells,which suggesting that RdRp mainly plays replicase function in nucleus of host cells.