H3N2 SIV河南分离株NS1基因的克隆及原核表达

Cloning and Prokaryotic Expression of Nonstructural Protein 1 Gene of a H3N2 SIV Isolate from Henan Province,China

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 the nonstructural protein 1（NS1） gene of a swine influenza virus isolate of subtype H3N2（H3N2 SIV） from Henan Province,China,express in prokaryotic cells,and provide a basis for development of a method for differentiation of immunized and naturally infected pigs.Methods Viral RNA was extracted from the Henan isolate of H3N2 SIV,with which NS1 gene was amplified by RT-PCR and cloned into prokaryotic expression vector pET-28a（＋）.The constructed recombinant plasmid was transformed to E.coli BL21（DE3） for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot.Results Sequencing result proved the homologies of more than 98% of amplified NS1 gene to several domestic and abroad SIV strains.The expressed recombinant NS1 protein,with a relative molecular mass of about 27 000,showed specific reaction with the sera of pigs infected with SIV.Conclusion The NS1 gene of Henan isolate of H3N2 SIV was successfully cloned,and recombinant NS1 protein was expressed in E.coli BL21（DE3）,which laid a foundation of development of a method for differentiation of immunized and naturally infected pigs.