猪流行性腹泻病毒N基因重组真核质粒构建及其表达

发布时间：2018-10-20 17:16

【摘要】：为探明猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)N基因编码的蛋白对PEDV感染早期诊断的作用,根据PEDV CV777株N基因全序列设计一对特异性引物以扩增N基因,定向插入到pPM-CHis真核表达载体中构建重组表达质粒pPM-C-His-N,将重组质粒肌肉注射昆明系小鼠,以检测其在小鼠体内的表达水平;将pPM-C-His-N转染至Vero细胞,分别在基因水平和蛋白水平对N基因的表达进行检测,并采用间接免疫荧光试验(indirect immunofluorescence assay,IFA)检测N蛋白在细胞中的表达分布情况。结果表明,重组质粒在基因水平和蛋白水平均成功表达,免疫小鼠血清中抗体的效价为1∶51 200,Western-blot结果显示,免疫血清能与重组N蛋白发生特异性反应,说明实验成功构建了重组真核表达质粒pPM-C-His-N,且在Vero细胞内检测到了绿色荧光,为PEDV诊断方法的建立及致病机制的研究奠定基础。
[Abstract]:The recombinant expression plasmid pPM-C-His-N, was inserted into the eukaryotic expression vector of pPM-CHis. The recombinant plasmid was injected intramuscularly into Kunming mice to detect its expression level in mice. PPM-C-His-N was transfected into Vero cells. The expression of N gene was detected at gene level and protein level, and the distribution of N protein in cells was detected by indirect immunofluorescence assay (indirect immunofluorescence assay,IFA). The results showed that the recombinant plasmid was successfully expressed at the gene level and protein level. The titer of antibody in the serum of immunized mice was 1:51 200m Western-blot. The results showed that the immunized serum could react specifically with the recombinant N protein. The results showed that the recombinant eukaryotic expression plasmid pPM-C-His-N, was successfully constructed and the green fluorescence was detected in Vero cells, which laid a foundation for the establishment of PEDV diagnostic method and the study of pathogenesis.
【作者单位】：安徽农业大学动物科技学院;
【基金】：安徽省自然科学基金(1708085MC83) 现代农业产业技术体系建设专项资金资助项目(CARS-36-生猪) 安徽省生猪产业技术体系资金资助项目
【分类号】：S852.651

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