哺乳动物细胞西尼罗病毒样颗粒表达、纯化及其鉴定

    咸庆杰1，2，姜宝芹2，郭雪2，王琳2，杨鹏飞2，张丽萍2，平芮巾2，岳启安1，胡孔新2

    1.潍坊医学院，山东 潍坊 261053；2.中国检验检疫科学研究院

    摘要：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-likeparticles，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（virus like particlesVLPs），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。

    关键词：西尼罗病毒；293T细胞；prM-E蛋白；病毒样颗粒；间接免疫荧光试验

    中图分类号：R512.99　文献标识码：B

    Expression， purification and identificationof West Nile virus-like

    particles in mammalian cells

    XIAN Qing-jie*， JIANG Bao-qin， GUO Xue，WANGLin， YANG Peng-fei，

    ZHANG Li-ping， PING Rui-jin, YUE Qi-'an，HuKong-xin

    *Weifang Medical University, Weifang,Shangdong 261053,China

    Abstract:   Objective  To express prM-E protein of West Nile virus in mammaliancells,which can assemble and form virus-like particles for furtherimmunological diagnostic studies.   Methods  Virus-like particles of West Nile virus were expressed in 293T celltranfected with the recombinant PreM-E plasmid, which followed withidentification tests with transmission electron microscope(TEM),western-blotting(WB), immunofluorescence assay (IFA)andELISA.  Results Recombinant subviral particles, about50 nm indiameter, were observed by TEM in the supernatant of transfectedcells, which indicated that the transfected cells can secretevirus-like particles ( VLPs) of WNV. The results of Western-blot,IFA and ELISA imply the recombinant proteins retainedimmunoreactivity similar to those of native virus proteins Conclusion   West Nile  virus-like particlesexpressed in mammalian ce,lls retains good antigenicity,and lay abasis for further study on the immunoassay of West Nile virusinfection and development of specific diagnostic reagents.

    Key words:   West Nile virus;293Tcell;prM-E;VLPs;IFA

    《中国国境卫生检疫杂志》