N9亚型禽流感病毒NA蛋白在昆虫细胞中的表达与鉴定

Expression and identification of NA protein of N9 subtype avian influenza virus in insect cells

为了表达具有反应原性的N9亚型禽流感病毒(AIV)神经氨酸酶(NA)蛋白,本研究将DK/HuN/S11670/2015(H11N9)株NA基因克隆至杆状病毒转移载体中,构建重组转移载体pFastBacHT A-N9并经PCR和测序鉴定后转化DH10Bac感受态细胞,经PCR鉴定获得了重组杆粒rBacmid-N9,转染昆虫细胞sf21获得重组杆状病毒rBaculovirus-N9。将获得的P3代重组杆状病毒感染sf21细胞后经间接免疫荧光试验(IFA)检测N9蛋白的表达和定位,采用western blot鉴定N9蛋白的反应原性。将杆状病毒表达的重组N9蛋白包被ELISA酶标板,初步建立间接ELISA方法,利用该方法分别检测N9蛋白抗体阳性鸡血清和阴性鸡血清。IFA和western blot结果显示,N9蛋白正确表达且主要锚定在细胞膜上,表达的N9蛋白可以与鼠源N9蛋白单克隆抗体发生特异性反应。间接ELISA方法结果显示,初步建立的间接ELISA方法可以检测鸡血清中的N9抗体。上述结果表明,经杆状病毒—昆虫细胞表达的N9蛋白具有良好的反应原性,为N9 AIV检测试剂盒的研发奠定了基础。

In order to express the reactive neuraminidase(NA)protein of N9 subtype avian influenza virus(AIV),NA gene of the DK/HuN/S11670/2015(H11N9)strain was cloned into a baculovirus transfer vector in this study.The recombinant transfer vector PfastBacHT A-N9 was constructed and transformed into DH10Bac competent cells after PCR and sequencing identification..The recombinant bacmid rBacmid-N9 was obtained by PCR identification,and the recombinant baculovirus-N9 was obtained by its transfection in insect cells sf21.After infecting sf21 cells with the obtained P3 recombinant baculovirus,indirect immunofluorescence assay(IFA)was used to detect the expression and localization of N9 protein,and western blot was used to identify the reactogenicity of the protein.The recombinant N9 protein expressed by baculovirus was coated on an ELISA plate,and an indirect ELISA method was initially established to detect the positive and negative chicken serum of N9 protein antibody.The results of IFA and western blot assay showed that N9 protein was correctly expressed in insect cells and mainly anchored on the cell membrane,and the expressed N9 protein could specifically react with the monoclonal antibody of mouse N9 protein.The results of indirect ELISA showed that the established indirect ELISA method could detect the N9 antibody in chicken serum.The above results showed that the N9 protein expressed by baculovirus has good reactivity,which lays a foundation for the development of N9 AIV detection kits.