哺乳动物细胞西尼罗病毒样颗粒表达、纯化及其鉴定

Expression,purification and identification of West Nile virus-like particles in mammalian cells

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 express prM-E protein of West Nile virus in mammalian cells,which can assemble and form virus-like particles for further immunological diagnostic studies. Methods Virus-like particles of West Nile virus were expressed in 293T cell tranfeeted with the recombinant PreM-E plasmid, which followed with identification tests with transmission electron mieroseope（TEM）, western-blotting（WB）, immunofluorescence assay （IFA）and ELISA. Results Recombinant subviral particles, aboutS0 nm in diameter, were observed by TEM in the supernatant of transfected cells, which indicated that the transfected cells can secrete virus-like particles （VLPs） of WNV. The results of Western-blot, IFA and ELISA imply the recombinant proteins retained immunoreaetivity similar to those of native virus proteins Conclusion West Nile virus-like particles expressed in mammalian ee,lls retains good antigenicity,and lay a basis for further study on the immunoassay of West Nile virus infection and development of specific diagnostic reagents.