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以乙型脑炎病毒SA14-14-2疫苗株全长基因组克隆质粒pBR-JTF为模板,通过PCR分别扩增prM-E及C-prM-E基因片段,构建表达乙型脑炎病毒结构蛋白的真核表达质粒pCJE-ME及pCJE-CME。将这2种重组质粒用脂质体法转染BHK-21细胞后,质粒pCJE-ME表现明显的细胞毒性,转染细胞不能存活;质粒pCJE-cME可导致筛选到表达JEV结构蛋白的稳定细胞系,这种稳定表达JEV结构蛋白的细胞系通过PCR扩增细胞系基因组、ELISA、Western Blot、间接免疫荧光等方法得到鉴定。研究结果表明在C蛋白存在下,乙型脑炎病毒C-prM-E蛋白可以在BHK细胞中稳定表达,为研制JEV新型复制子颗粒疫苗提供了便利工具。
The prM-E and C-prM-E gene fragments were amplified by PCR from the full-length genomic clone plasmid pBR-JTF of Japanese encephalitis virus SA14-14-2 vaccine strain to construct the recombinant protein expressing Japanese encephalitis virus structural protein The eukaryotic expression plasmids pCJE-ME and pCJE-CME. Plasmid pCJE-ME showed obvious cytotoxicity after transfection of these two recombinant plasmids into BHK-21 cells. The transfected cells could not survive. The plasmid pCJE-cME could lead to the selection of stable cells expressing JEV structural protein This cell line stably expressing JEV structural protein was identified by PCR amplification of cell line genome, ELISA, Western Blot, indirect immunofluorescence and the like. The results show that JEV C-prM-E protein can be stably expressed in BHK cells in the presence of protein C, which provides a convenient tool for the development of JEV novel replicon particle vaccine.