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目的利用哺乳动物细胞表达含有西尼罗病毒(WNV)prM和E蛋白,形成病毒样颗粒(virus-like particles,VLPs),为西尼罗病毒感染的免疫诊断试剂的研制奠定基础。方法筛选典型西尼罗病毒株,构建重组质粒,转染293T细胞,表达并纯化西尼罗病毒prM-E蛋白,利用透射电镜、免疫印迹试验、间接免疫荧光实验(IFA)和酶链免疫吸附试验(ELISA)对表达产物进行鉴定。结果重组质粒转染细胞后产生病毒样颗粒(viruslike particles VLPs),转染细胞上清纯化物中透射电镜观察到重组蛋白形成的球型颗粒,免疫印迹试验和间接免疫荧光试验表明,表达的病毒样颗粒蛋白能够与抗西尼罗病毒抗体特异结合,具有良好的抗原性;间接ELISA证实,重组蛋白可以作为抗原用于检测患者血清特异性抗体。结论在哺乳动物细胞中表达的西尼罗病毒样颗粒具有良好的抗原性,为西尼罗病毒感染快速特异诊断试剂研制奠定了基础。
Objective To construct virus-like particles (VLPs) containing the prM and E proteins of West Nile virus (WNV) by mammalian cells and lay the foundation for the development of immunodiagnostic reagents for West Nile virus infection. Methods The typical West Nile virus strain was screened and the recombinant plasmids were constructed and transfected into 293T cells. The prM-E protein of West Nile virus was expressed and purified. Transmission electron microscopy, Western blotting, indirect immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay The expression product was identified by ELISA. Results The virus-like particles (VLPs) were produced after transfected with the recombinant plasmids. The purified recombinant plasmids were observed by transmission electron microscopy. The results of immunoblotting and indirect immunofluorescence assay showed that the expressed virus-like particles Granular protein can bind specifically to anti-West Nile virus antibody and has good antigenicity. Indirect ELISA confirmed that the recombinant protein can be used as antigen to detect serum-specific antibody in patients. Conclusion The West Nile virus-like particles expressed in mammalian cells have good antigenicity, which lays the foundation for the rapid and specific diagnosis of West Nile virus infection.