H3N2亚型猪流感病毒NS1基因的克隆表达及间接ELISA检测方法的建立

Cloning and expression of swine influenza virus subtype H3N2 and establishment of an indirect ELISA

采用RT-PCR方法成功扩增了H3N2亚型猪流感病毒(Swine influenza virus,SIV)四川分离株(A/Swine/Sichuan/01/2006)的NS1基因,将其克隆于原核表达载体pET-32a(+)中,构建了重组质粒pET32-NS1。将该质粒转化进大肠杆菌Rosetta^(TM),经终浓度为0.5 mmol/L的IPTG诱导表达后,通过SDS-PAGE电泳表明融合的NS1蛋白得到了大量的表达,该融合蛋白的相对分子质量约为43 500。Western-blotting结果显示该蛋白能与阳性血清发生特异性反应具有很好的免疫反应原性。结果表明,所建立的ELISA方法与几种常见猪群传染病病原无交叉反应,具有较好的特异性、敏感性和重复性,可进一步优化应用于临床SIV的检测。

The NS1 gene of H3N2 subtype swine influenza virus was expressed in prokaryotic expression system to obtain recombinant protein which was used to develop an effective diagnostic method.The NS1 gene was amplified by reverse transcriptase polymerase chain reaction（RT-PCR） and cloned into a prokaryotic expression vector,pET- 32a（＋）,and then expressed in E.coil Rosetta^（IM） after induced by 0.5 mmol/L Isopropyl β-D-thiogalactoside （IPTG）.The recombinant protein was purified and identified its antigencity by western-blotting.Based on the western -blotting test,an indirect enzyme-linked immunosorbent assay（ELISA） was constructed to develop a H3N2 subtype swine influenza virus detection kit.The recombinant protein was about 43 500.The result of Western-blotting indicated that the recombinant had specifically immunologic reactionogenicity with postive serum.The result of ELISA showed that it has good specificity,sensitivity and reproducibility without cross reaction to some kinds of common infection disease pathogeny in pigs and was prospective to develop an efficient detection kit for to detection of SIV in clinic.