猪流行性腹泻病毒N基因重组真核质粒构建及其表达

Recombinant eukaryotic plasmid construction of porcine epidemic diarrhea virus of N gene and its expression

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。

In order to investigate the effect of porcine epidemic diarrhea vims N genes encoding proteins in the PEDV infection early diagnosis, a pair of specific primers was designed to amplify N gene according to the full-length ge-nome sequence of N gene of the PEDV CV777 strain in this experiment, then N gene was cloned into the eukaryotic expression vector pPM-C-His to construct a recombinant plasmid named pPM-C-His-A^, the recombinant plasmid was extracted and the intramuscular injection for Kunming mice was carried out to detect the expression level in mice. At the same time, the plasmid was transfected into Vero cells, and PEDV N expression at gene level and protein level was detected, then N protein distribution in cells was detected in vitro by indirect immunofluorescence assay （ IF A ） . The results showed that the recombinant eukaryotic plasmid expressed successfully at gene level and protein level, the serum antibody titer in immunized mice was 1-51 200. Western blot results showed that the immune serum can react with recombinant N protein particularity. The study constructed recombinant eukaryotic expression plasmid of pPM-C-His-A successfully, and detected green fluorescence in Vero cells, which laid the foundation for establishment of diagnostic method of PEDV and research for pathogenic mechanism.