【摘要】 根据GenBank公布的猪繁殖与呼吸综合症病毒(Porcinereproductiveandrespiratorysyndromevirus,PRRSV)ORF5基因的核苷酸序列设计3对特异性引物,从重组质粒pMD-ORF5中扩增去除ORF5基因中包括全部信号肽在内的N端跨膜区(28residues)和中部跨膜区(60residues)。改造后的ORF5基因分别命名为ORF5-1和ORF5-2。将2个基因片段定向克隆到原核表达载体pET-32a(+),构建重组质粒pET-ORF5-1和pET-ORF5-2,转化大肠杆菌(Escherichiacoli)BL21(DE3)感受态细胞,经IPTG诱导表达和SDS-PAGE电泳分析,ORF5-1和ORF5-2基因获得融合表达,表达量分别为12.2%和39%,证明删除双跨膜区能明显提高ORF5基因的表达量。经Westernblot分析,融合蛋白具有一定的免疫学活性,表明摘除跨膜区对该蛋白的抗原性影响不大。更多还原

【Abstract】 Three pairs of specific primers were designed according to the sequence of Porcine reproductive and respiratory syndrome virus ORF5 gene published on GenBank(accession No.AY737282). Two modified ORF5 gene fragments ORF5-1 and ORF5-2 which deleted the N-terminal transmembrane region (28 residues, including signal peptides) sequence and the middle tansmembrane region (60 residues) sequence of ORF5 gene respectively were obtained from recombinant plasmid pMD-ORF5 by PCR amplification using these primers. ORF5-1 and ORF5-2 gene fragments were inserted into prokaryotic expression vector pET-32 a(+), resulting in the recombinant plasmids pET-ORF5-1 and pET-ORF5-2. After transformed into Escherichia coli BL21(DE3) cells and induced with IPTG, SDS-PAGE analysis revealed that ORF5-1 and ORF5-2 recombinant proteins with a expression level amounted to 12.2% and 39% of the total bacterial proteins respectively . Expression of ORF5 gene has been improved by removing the dual transmembrane regions. Western blotting analysis showed that the recombinant proteins had immunoreactivity and proved that the deleting of transmembrane regions had no significant effect on the antigenicity of the recombinant proteins.更多还原