KPNB1和Ran蛋白共同介导新城疫病毒基质蛋白的入核转运

发布时间：2018-03-20 00:02

本文选题：新城疫病毒　切入点：基质蛋白　出处：《微生物学报》2017年01期 　论文类型：期刊论文

【摘要】：【目的】鉴定与新城疫病毒(Newcastle disease virus,NDV)基质蛋白(matrix protein,M)入核相关的细胞蛋白,以阐明NDV M蛋白细胞核定位的分子机制。【方法】从鸡胚成纤维细胞中分别克隆核转运受体蛋白KPNA1 KPNA6和KPNB1基因,将其构建到真核表达载体,并与表达NDV M蛋白的重组真核表达载体分别共转染HEK-293T细胞,通过免疫共沉淀方法鉴定与NDV M蛋白相互作用的核转运受体蛋白。另外,将M蛋白与Ran蛋白突变体或与M蛋白互作的核转运受体蛋白缺失体分别共表达,通过荧光共定位确定M蛋白入核转运相关的细胞蛋白。【结果】构建的重组真核表达载体在HEK-293T细胞中能够正确表达;通过间接免疫荧光观察发现,重组蛋白中除Myc-KPNA2蛋白定位在细胞质外,其它核转运受体蛋白均与M蛋白表现出相同的细胞核定位。免疫共沉淀试验结果表明,M蛋白与KPNA1蛋白和KPNB1蛋白均存在相互作用。进一步通过荧光共定位观察发现,M蛋白与KPNA1蛋白缺失体(DN-KPNA1)共表达不改变M蛋白的细胞核定位,而与KPNB1蛋白缺失体(DN-KPNB1)共表达后导致M蛋白变为细胞质定位,说明M蛋白入核转运需要KPNB1蛋白的参与。另外,将M蛋白与Ran蛋白突变体Ran-Q69L共表达,荧光观察发现M蛋白同样由细胞核定位变为细胞质定位,说明M蛋白入核转运还需要Ran蛋白的辅助。【结论】KPNB1和Ran蛋白共同介导NDV M蛋白的入核转运,其过程是KPNB1蛋白首先和M蛋白发生相互作用并形成复合物,然后通过Ran蛋白的辅助作用完成入核转运。
[Abstract]:[objective] to identify the cellular proteins associated with the entry of Newcastle disease virus (NDV) matrix protein into the nucleus of Newcastle disease virus (NDV). To elucidate the molecular mechanism of nuclear localization of NDV M protein. [methods] the nuclear transport receptor protein KPNA1 KPNA6 and KPNB1 genes were cloned from chicken embryo fibroblasts and constructed into eukaryotic expression vector. The recombinant eukaryotic expression vector expressing NDV M protein was cotransfected into HEK-293T cells, and the nuclear transport receptor proteins interacting with NDV M protein were identified by immunoprecipitation. The nuclear transport receptor protein deletions of M protein and Ran protein mutant or M protein interacting with M protein were co-expressed, respectively. [results] the constructed recombinant eukaryotic expression vector could be correctly expressed in HEK-293T cells. The recombinant protein was located in the cytoplasm except the Myc-KPNA2 protein. Other nuclear transport receptor proteins all showed the same nuclear localization as M protein. The results of immunoprecipitation test showed that the M protein interacted with KPNA1 protein and KPNB1 protein. The nuclear localization of M protein was not changed by co-expression of M protein and DN-KPNA1. However, co-expression with KPNB1 deletion DN-KPNB1 resulted in the transformation of M protein into cytoplasm, indicating that KPNB1 protein was involved in the nuclear transport of M protein. In addition, M protein was co-expressed with Ran mutant Ran-Q69L. Fluorescence observation showed that M protein also changed from nuclear localization to cytoplasmic localization, indicating that M protein transport into the nucleus also needed the assistance of Ran protein. [conclusion] KPNB1 and Ran protein co-mediate the nuclear transport of NDV M protein. The process is that the KPNB1 protein first interacts with M protein and forms a complex, and then transports into the nucleus through the assistance of Ran protein.
【作者单位】：贵州大学高原山地动物遗传育种与繁殖省部共建教育部重点实验室;扬州大学农业部畜禽传染病学重点开放实验室;江苏省动物重要疫病与人兽共患病防控协同创新中心;
【基金】：国家自然科学基金(31502074) 教育部“促进与美大地区科研合作与高层次人才培养项目”(教外司美[2014]2029号) 公益性行业(农业)科研专项(201303033) 贵州大学引进人才科研项目(贵大人基合字(2014)10号)~~
【分类号】：S852.65

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