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目的克隆并原核表达猪流感病毒(Swine influenza virus,SIV)非结构蛋白1(Nonstructural protein 1,NS1)基因,为建立人工免疫猪和自然感染猪的鉴别诊断方法奠定基础。方法从 H3N2 SIV河南分离株中提取病毒RNA,RT-PCR扩增NS1基因,克隆至原核表达载体pET-28a(+)中,构建重组表达质粒,转化E.coli BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE和Western blot进行鉴定。结果测序结果表明,扩增的NS1基因与国内外多株SIV NS1基因序列同源性达98%以上,表达的重组蛋白相对分子质量约为27 000,能与SIV感染猪阳性血清发生特异性反应。结论已成功克隆了H3N2 SIV河南分离株NS1基因,并在E.coli BL21(DE3)中表达了重组NS1蛋白,为建立人工免疫猪和自然感染猪的鉴别诊断方法奠定了基础。
Objective To clone and prokaryotic express Nonstructural protein 1 (SIV) gene of swine influenza virus (SIV) and lay a foundation for the differential diagnosis of swine influenza virus (IBV) and naturally infected pigs. Methods RNA was extracted from H3N2 SIV Henan isolates. The NS1 gene was amplified by RT-PCR and cloned into prokaryotic expression vector pET-28a (+). The recombinant plasmid was constructed and transformed into E.coli BL21 (DE3) . The expressed product was identified by SDS-PAGE and Western blot. Results The sequencing results showed that the amplified NS1 gene shared more than 98% identity with the NS1 gene sequences of many domestic and foreign SIV strains and the relative molecular mass of the expressed recombinant protein was about 27 000, which could specifically react with SIV-infected pig-positive sera . Conclusion The NS1 gene of H3N2 SIV isolates was successfully cloned and the recombinant NS1 protein was expressed in E. coli BL21 (DE3), which laid the foundation for the differential diagnosis of artificial immunized pigs and naturally infected pigs.