猪繁殖与呼吸综合征病毒orf7和orf5双基因的原核表达研究

Expression of the Combined orf7 and orf5 Genes of Porcine Reproductive and Respiratory Syndrome Virus in E.coli.

根据PRRSV已知序列设计两对引物,采用RT-PCR方法分别扩增orf7和orf5基因片段。利用EcoRI、SpeI和HindⅢ位点将orf7和orf5基因片段依次克隆到pMD-18T载体,构建成重组质粒pMD18NE,并比较所克隆基因序列的同源性。将串联的orf7和orf5基因亚克隆到原核表达载体pGEX-KG,构建重组原核融合表达质粒pGEX-KGNE,并转化BL21,SDS-PAGE和Western-blot分析表明:orf7和orf5基因与GST获得了融合表达,并且表达的融合蛋白GST-NE具有免疫学反应活性,这为猪繁殖与呼吸综合征血清学诊断方法的建立及疫苗研究打下基础。

According to the nucleotide sequence of Porcine reproductive and respiratory syndrome virus (PRRSV), two pairs of primers were designed and used to amplified the orf7 and orf5 genes by RT-PCR, respectively. By using the EcoRI、SpeI and Hind Ⅲ sites, both genes were cloned into pMD-18T vector in turn, then sequencing and comparing the homologies of the cloning genes. The combined orf7 and orf5 genes was subcloned into a prokaryotic expressing vector pGEX-KG, the recombinant plasmid named pGEX-KGNE was constructed and transformed into E.coli BL21.The results of SDS-PAGE and Western-blot indicated that the combined genes of orf7 and orf5 and Glutathion-S-transferase (GST) was expressed in E.coli BL21 as a fusion protein GST-NE about 64kDa in molecular weight, and this fusion protein showed reactive activity immunologically. This study lays a foundation for the development of the serodiagnostic methods and vaccines for PRRSV.