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

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。

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

Three structural genes,ORF3,ORF5 and ORF6 of porcine reproductive and respiratory syndrome virus(PRRSV) CH-1a strain were amplified by PCR from pOKCH-1a vector and inserted into the eukaryotic expression vector pCAGGS,respec- tively.The resultant recombinant plasmids,pCAGGS-ORF3,pCAGGS-ORF5,and pCAGGS-ORF6,were purified and transfected 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.