摘要
为建立稳定表达脑心肌炎病毒先导蛋白的N_2a细胞系,本研究应用脂质体介导DNA转染法,将表达脑心肌炎病毒(EMCV)先导(L)蛋白的重组质粒pNTAP-B-L转染至N_2a细胞中,通过筛选G418抗性单克隆细胞,建立能够稳定表达融合蛋白TAP tag-L的N_2a细胞系;利用免疫印迹试验(Western blotting)和间接免疫荧光试验(IFA)对融合蛋白TAP tag-L的表达及细胞内定位情况进行鉴定检测。结果表明EMCV的L基因已经整合进N_2a细胞基因组DNA中且L蛋白在获得的阳性N_2a细胞中稳定表达,表达产物TAP tag-L主要分布在细胞浆内。建立的稳定表达L蛋白的N_2a细胞系,为进一步研究先导蛋白功能及EMCV感染过程中与宿主细胞(N_2a)相互作用蛋白的筛选奠定了基础。
We wished to construct a cell line stably expressing the leader protein(L)of the encephalomyocarditis virus(ECMV).Recombinant plasmids pNTAP-L expressing the L protein of the EMCV were transfected into N_2a cells mediated by liposomes.The cell line with stable expression of the fusion protein TAP tag-L gene was selected by the antibiotic G418.Expression and localization of exogenous TAP tag-L were detected by Western blotting and an indirect immunofluorescence assay.Results showed that the TAP tag-L gene was integrated into the genome of N_2a cells,and was expressed stably.Consequently,the N_2a cell line with stable expression of the L protein was established.The present study provides a cell model and potential foundation for future research on the function of the L protein and its interaction with the host cell(N_2a)in EMCV infection.
引文
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