猪流行性腹泻病毒N蛋白的真核表达及其亚细胞定位的初步分析

Eukaryotic Expression and Subcellular Localization Preliminary Analysis of Porcine Epidemic Diarrhea Virus Nucleocapsid Protein

以构建的含有编码猪流行性腹泻病毒CV777株核衣壳蛋白(N)基因的阳性质粒pMDT-18-N为模板,用含有KpnⅠ和XhoⅠ酶切位点的上、下游引物扩增获得N基因,该PCR产物经KpnⅠ和XhoⅠ双酶切后定向克隆到经相同双酶切的真核表达载体pcDNA3.1(+)中,经过酶切鉴定和测序鉴定,将构建的重组质粒命名为pcD-NA3.1(+)-N,且未发现碱基的缺失和插入。用脂质体法将pcDNA3.1(+)-N转染Vero E6细胞,在48 h后,以针对猪流行性腹泻病毒核衣壳蛋白的鼠源`多抗血清进行Western blot、免疫荧光检测,并运用共聚焦显微镜分析N蛋白的亚细胞定位。结果表明N基因能在真核细胞Vero E6中正确表达,共聚焦分析结果表明N蛋白在细胞质和细胞核中定位。

Using positive plasmid pMDT-18-N which contained the gene encoding nucleocapsid protein of porcine epidemic diarrhea virus strain CV777 as template, the N gene was amplified by the upper/lower primers each with the internal sites of Kpn Ⅰ and Xho Ⅰ , respectively. By digestion with restriction enzymes Kpn Ⅰ and Xho Ⅰ , the PCR product was then subcloned into eukaryotic expression vector pcDNA3, 1（＋） that digested with the same enzymes. After restriction enzyme digestion and DNA sequencing identification, the constructed recombinant plasmid was named pcDNA3.1 （＋）-N, deletion and insertion were not found in its sequence. The pcDNA3. 1（＋）-N was transiently transfected into Vero E6 cells, and the expression of N gene was detected by Western blot and indirect immunofluorescence assay with mouse antiserum against N protein. N protein subcellular localization were analyzed by confoeal microscopy. Results verified that N gene could be expressed successfully in Vero E6 cells, and the N protein localizes both in the cytoplasm and the nucleus.