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目的:构建含有海肾萤光素酶(Renilla luciferase,R.luc)报告基因的登革4型病毒亚基因组复制子,为探讨病毒基因组中保守序列和元件在复制和翻译调控机制中的作用提供新的手段。方法:利用定点诱变PCR技术构建prM-E基因缺失的登革4型病毒亚基因组复制子(DENRP),用顺式水解酶元件(cis-acting hydrolase element,CHYSEL)技术将R.luc报告基因插入到DENRP缺失的结构基因区,构建含有R.luc报告基因的复制子(DEN-R.luc2A-RP)。将构建的复制子线性化并体外转录成RNA后,经电穿孔转染BHK-21细胞,通过间接免疫荧光、RT-PCR和R.luc检测技术对复制子进行鉴定。结果:所构建的prM-E基因缺失的复制子和含有海肾萤光素酶报告基因的复制子,均可在BHK-21细胞中自主复制并稳定表达病毒特异蛋白。DEN-R.luc2A-RP可持续表达外源R.luc报告基因21 d,在转染后24~96 h R.luc荧光信号呈线性增强。在该区间进行实时检测可显示复制子复制和翻译水平的变化。结论:获得了两株可在BHK-21细胞中稳定表达的复制子,为进一步研究登革4型病毒复制和翻译机制奠定了基础。
OBJECTIVE: To construct a subgenomic replicon of Dengue 4 virus that contains Renilla luciferase (Rluc) reporter gene and provide a molecular basis for exploring the role of conserved sequences and elements in the viral genome in the regulation of replication and translation New means. Methods: The dengue virus 4 subgenomic replicon (DENRP) with prM-E gene deletion was constructed by site-directed mutagenesis PCR. The gene of R.luc was cloned by using the cis-acting hydrolase element (CHYSEL) Was inserted into the DENRP-deleted structural gene region to construct a replicon containing the R. luc reporter gene (DEN-R.luc2A-RP). The constructed replicon was linearized and transcribed into RNA in vitro, then transfected into BHK-21 cells by electroporation, and the replicons were identified by indirect immunofluorescence, RT-PCR and R. luc assays. Results: The constructed replicon with prM-E gene deletion and the replicon containing Renilla luciferase reporter gene could autonomously replicate and stably express virus-specific protein in BHK-21 cells. DEN-R.luc2A-RP can sustainly express exogenous R.luc reporter gene for 21 days. The fluorescence intensity of R.luc increased linearly from 24 to 96 h after transfection. Real-time detection in this interval shows changes in replicon replication and translation levels. Conclusion: Two replicon stably expressed in BHK-21 cells were obtained, which laid the foundation for further study on the mechanism of replication and translation of Dengue 4 virus.