猪繁殖与呼吸综合征病毒ORF5基因的克隆、表达与免疫印迹

Cloning,Expression and Immunity Blot of ORF5 of PRRSV

将猪繁殖与呼吸综合征病毒ORF5基因克隆入原核表达载体pET-28a(+)的多克隆位点中,经鉴定后得到重组质粒pET-ORF5,将此重组质粒分别转化到受体菌E.coliBL21(DE3)中,并用诱导剂IPTG进行诱导表达,2 h后表达达到高峰。经15%SDS-PAGE电泳检测,表达得到的蛋白大小约为13.75 kD。经Western Blotting分析,表达蛋白能与PRRSV阳性猪血清发生特异性反应,而与阴性血清不反应,具有较好的免疫原性,为研究PRRSV亚单位疫苗奠定了基础。

The ORF5 gene of PRRSV was amplified by PCR with a size of 375 bp. The amplified DNA fragment was cloned into pMD18-T, and sequenced. The result of sequencing showed that the consistency was 98% compared with that of standard strains. Inserted in pET-28a（＋ ）,and transformed in Escherichia coli BL21（DE3）, the fragment was expressed after induced with IPTG. The results of SDS-PAGE and Western Blotting showed that the protein was 13.75 kD in size and specifically reacted with PRRSV-positire sera from pigs, not negative sera. These results become the base of researching and producing novel vaccine and developing effective diagnostic method.