4个种属Mx蛋白抑制水疱性口炎病毒的研究

Research of Mx proteins from four species against vesicular stomatitis virus

[目的]本试验旨在研究4个种属(猪、人、小鼠、牛)Mx蛋白抗水疱性口炎病毒(VSV)的活性,以揭示胞质定位Mx蛋白抗VSV活性的保守性。[方法]设计特异性引物,PCR扩增4个种属Mx蛋白的全长基因,构建真核表达质粒;PK-15细胞过表达重组质粒,Western blot或间接免疫荧光试验(IFA)检测4个种属Mx蛋白在PK-15细胞内的表达及分布;过表达Mx蛋白的PK-15细胞感染VSV,8或16 h后收样,分别用RT-q PCR、Western blot、IFA和噬斑减少试验来分析VSV的复制。[结果]成功克隆并表达了4个种属Mx蛋白的全长基因,且Mx蛋白大小正确;虽然猪Mx1蛋白和小鼠Mx2蛋白以棒状形式分布于细胞质中,而猪Mx2蛋白、人Mx A蛋白和牛Mx1蛋白以颗粒形式弥散分布于细胞质中,但4个种属Mx蛋白均能通过抑制VSV-G mRNA的转录、减少VSV-G蛋白的合成及降低子代病毒的滴度来显著抑制VSV的增殖。[结论]4个种属Mx蛋白都能显著抑制VSV在PK-15细胞中的增殖。

[Objectives]The purpose of this research was to investigate the antiviral activity of Mx proteins from four species（ porcine,human,mouse and bovine） against vesicular stomatitis virus（ VSV）,and to reveal the conservatism of antiviral activities of cytoplasmic Mx proteins. [Methods]Specific primers were designed to amplify full-length genes of Mx proteins from four species by PCR and then eukaryotic expressing plasmids were constructed. After PK-15 cells overexpressing the recombinant plasmids,Western blot and indirect immunofluorescence assay（ IFA） was used to detect the expressing activity and distribution of Mx proteins from four species in PK-15 cells. PK-15 cells overexpressing Mx proteins were infected with VSV for 8 or 16 h and harvested. The replication of VSV was analyzed using RT-q PCR,Western blot,IFA and plaque reduction experiment respectively. [Results]The full-length genes of Mx proteins from four species were successfully cloned and expressed,furthermore,the size of Mx proteins was correct. Although porcine Mx1 protein and mouse Mx2 protein were distributed in the cytoplasm in the form of rods,the porcine Mx2 protein,human Mx A protein and bovine Mx1 protein were dispersed in the cytoplasm in the form of granules,all Mx proteins from four species can significantly inhibit the proliferation of VSV by inhibiting the transcription of VSV-G mRNA,reducing the synthesis of VSV-G protein and reducing the titer of progeny virus. [Conclusions]All Mx proteins from four species can significantly inhibit the proliferation of VSV in PK-15 cells.