【摘要】 为建立稳定共表达乙型脑炎病毒(JEV)完整M前体蛋白(prM)和E蛋白的BHK-21细胞系,本研究在prM蛋白furin蛋白酶剪切位点编码基因中引入突变,将突变后的prM基因克隆于质粒中构建重组表达质粒pCAG-JEV-prM(R89A)E。将重组质粒转染BHK-21细胞,转染48 h后细胞用含G418的选择性培养基选择培养,进一步经细胞克隆纯化制备表达剪切位点突变的JEV prM-E蛋白的稳定细胞系。经IFA和western blot鉴定表明,该细胞系能表达JEV prM与E蛋白,所表达的prM蛋白未发生剪切;细胞经多次传代后仍能够稳定地共表达prM与E蛋白。该细胞系的建立为研究prM蛋白剪切对JEV粒子形成的影响以及亚单位疫苗的制备奠定了基础。更多还原

【Abstract】 In order to establish the BHK-21 cell lines stably co-expressing prM-E fusion protein of Japanese encephalitis virus(JEV) with a mutant furin site to disable the pre-peptide cleavage of prM,BHK-21 cells was transfected with the recombinant plasmid pCAG-JEV-prM(R89A)E,which was constructed by introducing a point mutation at the cleavage site of prM gene by PCR and cloned into eukaryotic vector pCAG-neo with E gene.Cells stably co-expressing prM-E fusion protein were selected in the present of G418 and identified by indirect immunofluorescence assay and western blot.In addition,the cells displaying high-level protein expression were purified through limiting dilution cloning.Eventfully,the results showed that the cell line was able to express the intact prM of prM-E fusion protein.The established cell line would provide a basis for further study the effect of the cleavage event on mature mechanism of JEV and the development of JEV subunit vaccine.更多还原