猪繁殖与呼吸综合征病毒结构蛋白基因ORF3、ORF5和ORF6在真核细胞中表达

Eukaryotic expression of porcine reproductive and respiratory syndrome virus structural protein genes ORF3,ORF5 and ORF6

用PCR方法从重组质粒pOKCH-1a扩增出猪繁殖与呼吸综合征病毒结构蛋白基因ORF3、ORF5、ORF6,将它们分别定向克隆到含有鸡β-actin启动子的高效真核表达载体pCAGGS的多克隆位点,经酶切、PCR及测序分析,筛选鉴定出含有ORF3、ORF5、ORF6基因的重组质粒,分别命名为pCAGGS-ORF3、pCAGGS- ORF5、DCAGGS-ORF6。将重组质粒纯化后转染293T细胞,用RT-PCR扩增出了特征性的基因片段,并采用特异性单抗进行间接免疫荧光检测,结果在胞浆内有亮绿色荧光,而在细胞膜上没有见到荧光,这说明表达的蛋白在胞浆内而不在细胞膜上。通过Westerm blot检测确定表达的GP3、GP5和M蛋白的分子量分别为42 Ku、25 Ku和19 Ku。本试验结果为进一步研究PRRSV膜蛋白伪病毒粒子及DNA疫苗奠定了基础。

Three structural genes, ORF3, ORF5 and ORF6 of porcine reproductive and respiratory syndrome virus （PRRSV） CH-la strain were amplified by PCR from pOKCH-1 a vector and inserted into the eukaryotic expression vector pCAGGS, respectively. The resultant recombinant plasmids, pCAGGS-ORF3, pCAGGS-ORF5, and pCAGGS-ORF6, were purified and tmnsfected into 293T cells. Expression of ORF3, ORF5, ORF6 genes of PRRSV were detectable by RT-PCR and indirect immunofluorescence analysis （IFA） at 48 h post transfection. The GP3, GP5 and M proteins were expressed in the cytoplasm rather than cell membrane and have a molecular weight of 42 Ku, 25 Ku, and 19 Ku, respectively. This study laid a foundation for further studies on the PRRSV pseudotype virus particle and DNA vaccines.