PRRSV变异株ORF6基因与IL-18共表达真核质粒的构建及其表达

Construction and identification of eukaryotic expression plasmid for IL-18 and ORF6 of porcine reproductive and respiratory syndrome virus

针对白细胞介素18(IL-18)基因和本实验室分离的PRRSVJL/07/SW毒株全基因序列,分别设计了2对特异性引物,扩增出IL-18基因和ORF6基因。将2个目的基因克隆于真核表达载体pEGFP-N1中,成功构建了重组真核质粒pEGFP-ORF6和pEGFP-IL18-ORF6,阳性质粒转染Marc-145细胞进行筛选。通过表达载体在转染细胞中的高效转染,增强型绿色荧光蛋白(EGFP)快速、准确的反映出阳性质粒在细胞质中的正确表达,再通过SDS-PAGE和Western blot鉴定构建的真核表达质粒pEGFP-IL18-ORF6的正确性。结果表明,ORF6基因和IL-18基因在Macr-145细胞中能有效表达。结论,所构建的真核质粒pEGFP-IL18-ORF6结构正确,能够在Marc-145细胞中高效表达,而且表达产物具有特异免疫学反应。

The genes of IL-18 and ORF6 were amplified using two pairs of primers based on the gene of interleukin 18（IL-18） and the sequence of mutant PRRSV JL/07/SW strain which was isolated in our laboratory.The products were cloned into the eukaryotic expression vector pEGFP-N1 to construct the recombinant eukaryotic expression plasmids pEGFP-ORF6 and pEGFP-IL18-ORF6,which were then used to transfer to Marc-145 cells for screening.The enhanced green fluorescent protein（EGFP） could quickly and exactly reflect the expression of plasmids in cell-substance according to the high performance of expression vectors,and then the correctness of pEGFP-IL18-ORF6 was identification by SDS-PAGE and Western-blot.The results indicated the ORF6 gene and IL-18 gene could beexpressed properh in Marc-145.The eukaryotic plasmid pEGFP-IL18-ORF6 which had the correct structure could beexpressed efficiently and the expression product could specially react to the sera of PRRSV.