FMDV 3D基因真核表达质粒的构建、表达及对Ⅰ型IFN信号通路的作用

Construction and Expression of Eukaryotic Expression Plasmid of FMDV 3D Gene and Its Effect on Type Ⅰ IFN Signaling Pathway

本研究旨在构建口蹄疫病毒(Foot-and-mouth disease virus,FMDV)3D基因真核表达质粒,并探究其对Ⅰ型干扰素(interferon,IFN)信号通路的作用。根据GenBank序列合成3D基因并将其插入真核表达载体pXJ41构建真核表达质粒pXJ41-Myc-3D,经PCR、双酶切及测序鉴定正确后分别转染HEK-293T细胞和PK-15细胞,Western blotting及间接免疫荧光试验(indirect immunofluorescence assay,IFA)检测3D蛋白在细胞内的表达及定位。通过双荧光素酶报告基因(Luciferase)、Real-time PCR、TCID50等试验检测HEK-293T细胞中过表达3D蛋白对水疱性口炎病毒(Versicular stomatitis virus,VSV)诱导的Ⅰ型IFN信号通路的影响。结果显示,真核表达质粒pXJ41-Myc-3D构建成功;3D蛋白在HEK-293T细胞中表达,大小约为55 kDa,主要定位在细胞核中;3D蛋白抑制了VSV诱导的IFN-β启动子活性和IFN-β mRNA水平,促进了VSV的复制。本研究为深入探究3D蛋白抑制Ⅰ型IFN信号通路的作用机制奠定了基础。

This study aimed to construct the eukaryotic expression plasmid of 3D gene of foot-and-mouth disease virus(FMDV) and explore its effects on type Ⅰ interferon(IFN) signaling pathway.The plasmid pXJ41-Myc-3D was constructed after 3D gene was synthesized according to the GenBank sequence and inserted into the eukaryotic expression vector pXJ41,which was transfected to HEK-293T and PK-15 cells after identified by PCR,double digestion and nucleotide sequencing.The expression and cellular localization of 3D protein were detected by Western blotting and indirect immunofluorescence assay(IFA).The effects of 3D overexpression in HEK-293T cells on type Ⅰ IFN signaling pathway induced by versicular stomatitis virus(VSV) were further assayed by dual luciferase reporter assays,Real-time PCR and TCID_(50).The results above showed that the eukaryotic expression plasmid pXJ41-Myc-3D was constructed successfully,the 3D protein could be expressed in HEK-293T cells with the size about 55 kDa and mainly localized in the nucleus,3D protein could inhibit the promoter activity and mRNA level of IFN-β induced by VSV and promote replications of the virus.This study laid a foundation for further exploring the mechanism of 3D protein inhibiting type Ⅰ IFN signaling pathway.