猪繁殖与呼吸综合征病毒ORF7基因的克隆、高效表达与鉴定

Cloning,expression and identification of porcine reproductive and respiratory syndrome virus ORF7

根据猪繁殖与呼吸综合症病毒(PRRSV)VR2332株ORF7的全长基因序列设计一对特异性引物,应用RT-PCR扩增出完整的ORF7基因,并将PCR产物克隆至pMD-18T载体进行测序分析。双酶切测序后重组质粒pMD-18T-N得到目的片段连接至原核表达载体pET-28a(+)上,构建重组质粒pET-28a(+)-N。再转化至BL21宿主菌,对诱导表达条件(IPTG质量浓度、诱导时间、诱导温度)进行优化。结果显示,在37℃、0.2 mmol/LIPTG诱导5 h可实现重组N蛋白的高效表达。该重组N蛋白经Ni-NTA亲和层析柱纯化后,Western-blotting分析显示,重组的N蛋白与PRRSV阳性血清发生特异性反应,表明表达的重组N蛋白具有较好的反应活性。

A pair of specific primers was designed according to gene sequences ORF7 of PRRSV VR2332 strain to amplify a complete ORF7 gene by RT-PCR.The PCR products were cloned into the vector pMD-18T and sequenced.The double-digested fragment of the sequenced recombinant plasmid pMD-18T-N was obtained and connected to prokaryotic expression vector pET-28a（＋）.Recombinant plasmid pET-28a（＋）-N was converted to the host bacteria BL21 and the conditions（concentrations of IPTG,induction time,induction temperature） were optimized.The results showed that 37 ℃,0.2 mmol/L IPTG induced 5 h can realize high expression of the recombinant protein.Ni-NTA affinity chromatography and Western-blotting analysis showed that expression recombinant N protein can react specifically with PRRSV-positive sera.