猪脑心肌炎病毒VR-129B株VP2重组蛋白的构建表达及其免疫活性分析

Construction and Immune Activitys of VP2 Recombinant Protein of Porcine Encephalomyocarditis Virus VR-129B Strain

为获得大量猪脑心肌炎VP2基因及蛋白研究的细胞模型,构建pDC315-EMCV-VP2真核表达载体,且将其转染到293T细胞,筛选出阳性质粒进行克隆。EMCV VR-129B株VP2基因序列参照GenBank(登录号:X74312),利用RT—PCR方法扩增VP2的全基因序列,将其与质粒pDC315经NheI和XhoI双酶切后连接,构建pDC315-EMCV-VP2重组表达质粒,并将其转染至293T细胞。使用荧光定量PCR方法观察pDC315-EMCV-VP2真核表达载体在细胞中的表达情况。双酶切PCR结果获得大小为780 bp的基因片段,测序结果显示与GenBank上已经公布的同名基因(登录号:X74312)序列片段同源性为100%,重组质粒构建成功。实时荧光定量PCR(QPCR)结果显示,重组质粒转染组与空质粒组之间相比,EMCV-VP2基因的表达量显著上调(P<0.001);在荧光显微镜下观察转染细胞,转染成功部分出现较亮绿色荧光,EMCV-VP2基因表达稳定。从转染细胞中提取重组蛋白与EMCV阳性血清和阴性血清进行ELisa反应,具有较好免疫活性。

In order to obtain a large of cell models of VP2 gene and protein research in pig brain myocarditis,We establish pC315-EMCV-VP2 eukaryotic expression vector,and transfect it into 293T cells,and screen positive plasmid for cloning.The VP2 gene sequence of EMCV VR-129B strain was ligated with GenBank(accession number:X74312).The whole gene sequence of VP2 was amplified by RT-PCR and ligated with plasmid pDC315 by NheI and XhoI to establish pC315-EMCV-VP2 and was transfected into 293T cells.Fluorescence microscopy and real-time PCR were used to observe the expression of pDC315-EMCV-VP2 eukaryotic expression vector in cells.A 780 bp gene fragment was obtained by double enzyme digestion.The sequencing results showed that the homology with the sequence gene of the same name(accession number:X74312)published in GenBank was 100%,and the recombinant plasmid is successfully constructed.Real-time quantitative PCR is used to detect the expression of EMCV-VP2 gene.The expression of EMCV-VP2 gene is significantly up-regulated the recombinant plasmid transfected group in comparing with the untransfected empty cell group(P<0.01).Transfection is observed under fluorescence microscope.The cells show brighter green fluorescence and the expression of EMCV-VP2 gene is stable.The recombinant protein has an ELisa reaction with EMCV-positive serum and negative serum,and has good immunological activity.