新城疫病毒基质蛋白与禽细胞核磷蛋白B23.1在HEK-293T细胞中的相互作用

Matrix protein of Newcastle disease virus interacts with avian nucleophosmin B23.1 in HEK-293T cells

摘要:

摘要:【目的】探讨新城疫病毒(Newcastle disease virus，NDV)基质(matrix，M)蛋白和禽细胞核磷蛋白B23.1在HEK-293T 细胞中的相互作用。【方法】分别参照GenBank 中NDV JS/5/05/Go 株全基因序列(JN631747)和禽细胞核磷蛋白B23.1 基因序列(NM_205267)，设计、合成扩增M 基因和B23.1 基因的引物，利用RTPCR扩增出M 基因和DF1 细胞的B23.1 基因，分别克隆至真核表达载体获得重组表达质粒pEGFP-M、pCMV-HA-M和pDsRed-B23.1;将pEGFP-M和pDsRed-B23.1共转染HEK-293T细胞，利用荧光显微镜观察M蛋白与B23.1蛋白的共定位;利用免疫共沉淀(Co-IP)技术进一步验证两种蛋白的相互作用。【结果】Western blot结果表明构建的重组质粒在转染的HEK-293T细胞中正确表达;荧光显微镜观察显示M蛋白与B23.1蛋白在核仁具有共定位特征;Co-IP进一步证实两者能发生相互作用。【结论】NDV M蛋白与禽细胞核磷蛋白B23.1存在相互作用，M蛋白可能通过与B23.1蛋白的相互作用进入核仁。

关键词:关键词:新城疫病毒，基质蛋白，核磷蛋白B23.1，蛋白相互作用

Abstract:

Abstract:［Objecive］To identify the interaction between Newcastle disease virus (NDV) matrix (M) protein and avian nucleophosmin B23.1 in HEK-293T cells．［Methods］Specific primers used to amplify M gene and B23.1 gene were designed and synthesized according to JS/5/05/Go whole gene sequence (JN631747) and avian nucleophosmin B23.1 gene sequence (NM_205267)．Viral RNA and cellular RNA were extracted from allantoic fluid of NDV JS/5/05/Go strain and DF1 cells with TRIzol reagent，respectively．The M gene and B23. 1 gene were amplified by RT-PCR and then subcloned into eukaryotic expression vectors to generate the recombinant plasmids pEGFP-M，pCMV-HA-M and pDsRed-B23.1．To investigate the localization features of M protein and B23. 1 protein，we transfected the plasmids pEGFP-M and pDsRed-B23. 1simultaneously into HEK-293T cells and observed the results by fluorescence microscopy．We further confirmed the interaction between the two proteins by co-immunoprecipitation (Co-IP) assays． ［Results］ The fusion proteins were successfully expressed in transfected HEK-293T cells by Western blot analysis．The NDV M protein and avian nucleophosmin B23. 1 showed co-localization features in the nucleolus in co-transfected HEK-293T cells．Furthermore，the binding of M with B23.1 was demonstrated by Co-IP assays．［Conclusion］These results suggested that NDV M protein interacted with avian nucleophosmin B23.1 and the nucleolar localization of M might be regulated via interaction with B23.1.

Key words:Keywords: Newcastle disease virus，matrix protein，nucleophosmin B23. 1，protein-protein interaction