猪脑心肌炎病毒VR-129 B株VP2重组蛋白的构建表达及其免疫活性分析

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(P0.001);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。

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

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.