Objective To construction and identification of mini-DNA replicon vector on the basis of Dengue virus.Methods Based on Dengue virus type 4 infectious clone p4,a cassette with the deletion of PrM-E gene and most of non-structure protein gene was constructed through OL-PCR.Then we constructed the mini-DNA micro replicon vector pcDEN-△ prME by inserting the cassette into the pCDNA3.1 (+),an eukaryotic expression vector.Results To identify and verify the packaging function of the mini replicon vector,the qualitative EGFP report gene and quantitative luciferase gene was respectively cloned into pcDEN-△prME,constrcuting engineering vector pcDEN-△prME-EGFP and pcDEN-△prME-GLUC.Transfection experiment results show that the mini replicon vector could successfully express EGFP reporter gene and sea renal luciferase report gene.Conclusion mini-DNA replicon vector pcDEN-△prME constructed in this study can express exogenous genes successfully,which only contains Dengue virus necessary cis element and NS5 key sequence missing most of the structure of genes at the same time,making the vector in simplifying experiments and greatly improve the capacity of the viral vector.This study provides a powerful tool for further research on Flavivirus antiviral drug screening and new vaccine development,and provides a new technical platform for molecular regulation mechanism of viral replication and translation.%目的 构建登革病毒质粒型微复制子载体,并对其功能进行鉴定.方法 以登革病毒4型感染性cDNA克隆p4为分子基础,通过融合PCR方法构建删除病毒结构基因和大部分非结构基因的表达盒,并将其克隆入真核表达载体pCDNA3.1(+)的CMV启动子下游以构建包含病毒复制和翻译必需顺式作用元件和NS5关键序列的质粒型微复制子载体pcDEN-△prME.结果 为验证微复制子载体的包装功能,将定性的绿色荧光蛋白报告基因和定量的海肾荧光素酶基因分别克隆至pcDEN-△prME构建工程载体pcDEN-△prME-EGFP和pcDEN-△prME-GLUC.转染实验结果显示,微复制子载体可成功表达绿色荧光蛋白报告基因及海肾荧光素酶报告基因.结论 本研究所构建的DNA型微复制子载体pcDEN-△prME在保留病毒必要的顺式作用元件和NS5部分关键序列的同时缺失了大部分非结构基因,使得该载体在简化实验操作的同时大大提高了病毒载体的容量,为深入研究以登革为代表黄病毒抗病毒药物筛选和新型疫苗研发提供了有力工具,更为病毒复制及翻译的分子调控机制提供了新的技术平台.
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