猪流感病毒血凝素基因原核表达及其在间接ELISA中的初步应用

利用RT-PCR方法扩增猪流感病毒A/Swine/Henan/11/2005(H1N1)血凝素(HA)基因,克隆于pMD18-T载体进行测序。以pMD18-HA为模板、利用带酶切位点的引物再次扩增HA基因的开放阅读框(ORF),克隆到表达载体pET32a中,经双酶切、测序及PCR鉴定得到阳性重组表达质粒pET-HA,将质粒转化到表达宿主菌BL21(DE3)中,经终浓度为1 mmol/L的IPTG诱导,SDS-PAGE结果显示,HA蛋白获得了高效表达,经Western-blot检测证实表达产物具有良好的免疫学活性,在间接ELISA中的初步应用表明具有良好的抗原反应性。本研究为以重组HA蛋白为抗原建立H1亚型猪流感抗体的ELISA检测方法奠定了基础。

Prokaryotic expression of hemagglutinin gene of swine influenza virus and its primary application in indirect ELISA

Hemagglutinin(HA) gene of A/Swine/Henan/11/2005(H1N1),cloned into pMD18-T vector,was sequenced.Primers with the digestion sites of SacI and HindIII were designed based on the conserved regions of both the 5′-end and 3′end of HA gene.The PCR product of HA gene was cloned into pET32a.The recombinant plasmid was confirmed by PCR and restriction enzyme analysis,then transferred into BL21(DE3) and induced by 1mM IPTG.The result of SDS-PAGE indicated that HA protein was expressed effectively.The immune activity was confirmed by Western-blot.The results of indirect ELISA demonstrated that the recombinant protein has a good antigenic reactivity.This study lay a foundation for detection of the antibody against H1N1 subtype of swine influenza virus using recombinant HA protein as antigen.