为建立稳定表达乙型脑炎病毒(JEV) NS1蛋白的真核细胞系,本研究将编码JEV NS1蛋白的人工合成基因克隆到真核表达载体pCAGG-TK-neo中,构建了重组质粒pCAGG-opti-NS1.重组质粒经脂质体转染RK-13细胞,以含G418的选择性培养基选择培养,经细胞克隆纯化,以间接免疫荧光试验(IEA)筛选表达目的基因的细胞.结果表明,转染的RK-13细胞经G418加压及IFA筛选后,获得表达JEV NS1蛋白的阳性RK-13细胞系.经RT-PCR、western blot和IFA鉴定,该细胞系在传代至第15代后仍然可以稳定表达NS1蛋白.本实验获得了能够稳定表达JEV NS1蛋白的细胞系,为进一步开展JEV相关的研究奠定了基础.%Japanese encephalitis virus (JEV) is an important mosquito-borne virus for human and animal, to generate cell line stably expressing NS1 protein of JEV, RK-13 cells was transfected with the recombinant pCAGG-opti-NS1l plasmid, which was constructed by introducing the synthetic JEV NS1 gene into eukaryotic vector pCAGG-TK-neo, and subsequently selected with G418 and indirect immunofluorescence assay (IFA). The results showed that the established cell line was able to express NS1 protein stably up to the fifteenth passages identified by RT- PCR, western blot and IFA. The cell line expressing NS1 protein of JEV would be useful for further study on JEV.
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