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

【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RT-PCR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。

[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.