NPM1 K263 SUMO化位点在PEDV N蛋白与NPM1共定位中的作用
发布时间：2019-01-07 10:58
【摘要】：为阐明细胞核仁蛋白NPM1与猪流行性腹泻病毒(PEDV)核衣壳蛋白(N蛋白)共定位的关键位点。本实验通过设计突变引物,利用重叠延伸PCR方法对NPM1氨基酸位点T199、K230和K263进行定点突变,分别将其克隆至真核表达载体pDsRed2-N1中,获得重组质粒pDsRed2-NPM1(T199A)、pDsRed2-NPM1(K230R)、pDsRed2-NPM1(K263R)。随后分别将pDsRed2-NPM1(WT)、pDsRed2-NPM1(T199A)、pDsRed2-NPM1(K230R)、pDsRed2-NPM1(K263R)与重组质粒pAcGFP-N共转染VeroE6细胞。共聚焦结果显示NPM1(T199A)、NPM1(K230R)、野生型NPM1均与N蛋白共定位于细胞的核仁中。虽然NPM1(K263R)与N蛋白具有共定位现象,但两者共定位于细胞核中。该实验结果为进一步研究核仁蛋白NPM1参与PEDV复制的分子机制奠定了基础。
[Abstract]:To elucidate the key loci of nucleolus protein (NPM1) co-localization with porcine epidemic diarrhea virus (PEDV) nucleocapsid protein (N protein). In this experiment, the mutated primers were designed and the NPM1 amino acid sites T199K230 and K263 were mutated by overlapping extension PCR, and then cloned into the eukaryotic expression vector pDsRed2-N1 to obtain the recombinant plasmid pDsRed2-NPM1 (T199A). PDsRed2-NPM1 (K230R), pDsRed2-NPM1 (K263R). Then pDsRed2-NPM1 (WT), pDsRed2-NPM1 (T199A), pDsRed2-NPM1 (K230R), pDsRed2-NPM1 (K263R) and recombinant plasmid pAcGFP-N were co-transfected into VeroE6 cells. Confocal results showed that NPM1 (T199A), NPM1 (K230R) and wild-type NPM1 were colocated with N protein in nucleoli. Although NPM1 (K263R) and N protein are colocalized, they are co-located in the nucleus. The results laid a foundation for further study on the molecular mechanism of nucleolar protein NPM1 involved in PEDV replication.
【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/猪消化道传染病创新团队;
【基金】：国家自然基金优秀青年基金项目(312220541)
【分类号】：S852.651
[Abstract]:To elucidate the key loci of nucleolus protein (NPM1) co-localization with porcine epidemic diarrhea virus (PEDV) nucleocapsid protein (N protein). In this experiment, the mutated primers were designed and the NPM1 amino acid sites T199K230 and K263 were mutated by overlapping extension PCR, and then cloned into the eukaryotic expression vector pDsRed2-N1 to obtain the recombinant plasmid pDsRed2-NPM1 (T199A). PDsRed2-NPM1 (K230R), pDsRed2-NPM1 (K263R). Then pDsRed2-NPM1 (WT), pDsRed2-NPM1 (T199A), pDsRed2-NPM1 (K230R), pDsRed2-NPM1 (K263R) and recombinant plasmid pAcGFP-N were co-transfected into VeroE6 cells. Confocal results showed that NPM1 (T199A), NPM1 (K230R) and wild-type NPM1 were colocated with N protein in nucleoli. Although NPM1 (K263R) and N protein are colocalized, they are co-located in the nucleus. The results laid a foundation for further study on the molecular mechanism of nucleolar protein NPM1 involved in PEDV replication.
【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/猪消化道传染病创新团队;
【基金】：国家自然基金优秀青年基金项目(312220541)
【分类号】：S852.651
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