非结构蛋白NS1-ELISA检测禽流感野毒感染抗体

Detection of Virus Infected Poultry on the Basis of Antibody to NS 1 of Influenza Virus by NS1-ELISA

利用RT-PCR技术扩增获得了H5N2亚型禽流感病毒的NS1基因,ORF长度为678 bp。成功构建了重组表达载体pET-NS1,将其转化BL21(DE3),用终浓度1 mmol／I的IPTG诱导表达5 h,经15％的SDS-PAGE分析表明,诱导表达出了分子量大约为28 KD的 NS1融合蛋白,且以包涵体的形式存在,NS1融合蛋白经His trap Hp Kit柱子纯化或用尿素变性复性,获得纯度较高的NS1蛋白,并以此为抗原,建立了检测抗NS1蛋白抗体的ELISA(NS1-ELISA)方法。最佳包被浓度为2．5μg／ml,血清的最佳稀释倍数为1:200, 酶标二抗的最佳工作稀释度为1:5 000。重组NS1蛋白只与AIV感染的血清反应,而不与ND、IB、IBD、EDS76的阳性血清反应。分别检测H9N2、H5N2和H5N1亚型灭活疫苗和H9N2纯化病毒免疫的血清,各5份,其平均OD490值分别为0．225、0．210、0．205和0．184．均为阴性;而对H9N2和H5N2亚型活毒感染10-15 d的血清进行检测,各5份,其平均OD490值分别为0．610和0．619,均为阳性。因此,NS1蛋白能特异地区分野毒感染和灭活疫苗免疫鸡群,可作为一种鉴别诊断标记,但不能区分亚型．具有A型特异性。NS1-ELISA 方法的初步应用,不仅为生产提供了一种快速、特异、敏感的鉴别诊断方法,为进一步组装成试剂盒奠定了基础,也为禽流感的早期诊断、适时监控和净化提供了行之有效的方法。

Nonstructural gene 1 （NS1） of H5N2 subtype avian influenza virus QS strain was amplified by RT-PCR, whoes nucleotide sequences is 678bp in length. The fragment was cloned into the pET-28（a） vector to construct a recombinant expression plasmid. The recombinant expression plasmid was induced by lmmol/L IPTG for 5 hours and analyzed by 15% SDS-PAGE. The result showed that NS1 protein was highly expressed by inclusion body. Its molecular weight was 28KD as expected. The expressed NS1 protein was denatured by SKL, then the denatured protein refolded by slow diluting and dialyzing method. The result indicated that the high pure and active protein was obtained. Based on the purified recombinant protein, an indirect ELISA assay for detection of anti-NS 1 protein antibody was developed. The best concentration of coating antigen was 2.5 g/mL, and the best dilution of serum and HRP labeled goat anti-chicken was 1：200 and 1：5000 respectively. The positive serum of ND, IB, IBD and EDS76 were all negative to NS 1 protein by ELISA assay. The sera of virus-infected chicken and vaccinated chicken were detected. The average OD490 value of the former were 0.592, 0.623 and 0.619, positive. The average OD490 value of the latter were 0.225, 0.210 and 0.205, negative; The result demonstrated that the NS1-ELISA assay can differentiate infected from vaccinated poultry on the basis of antibody to NS 1 protein. So the approach not only afforded a kind of quick, sensitive, specific differentiating diagnostic approach, making a solid foundation of special diagnosis kit, but also can provide an effective method for early diagnosis, real-time detection, controlling and clearing of AIV.