含有海肾萤光素酶报告基因的登革4型病毒亚基因组复制子的构建

Construction of sub-genomic replicon of dengue virus type 4 with insertion of Renilla luciferase reporter gene

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．1uc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21d，在转染后24～96h 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 sub-genomic replicons with Renilla luciferase reporter gene, and investigate the role of conserved sequence and elements in the untranslated region（UTR） of dengue virus type 4 during replication and translation. Methods： A dengue replicon （DENRP） with deletion in prM-E gene was constructed by site-directed mutagenesis PCR technique first. Then the Renilla luciferase reporter gene was inserted into the corresponding region of the DENRP by CHYSEL （cis-acting hydrolase element） technique to construct the dengue replicon with Renilla luciferase reporter gene （DEN-R. Iuc2A-RP）. The replicons were linearized and transcripted in vitro, and the transcription RNA was transfected into BHK-21 cells by electroporation. The replicons were further detected and characterized by RT-PCR, IFA and Renilla luciferase assay system, respectively. Results： Both DENRP and DEN-R. luc-RP could self-replicate and express specific proteins of dengue virus type 4 in the transfected BHK-21 cells. The Renilla luciferase gene could be detected till 21d posttransfection, and the Renilla luciferase signals increased exponentially during 24 -96 h post-transfection,which properly represented the replication and translation efficiency of dengue virus type 4. Conclusion： The replicons constructed help study the regulation mechanisms of replication and translation of dengue virus type 4.