HHV-8 ORF50启动子区序列分离、鉴定及在293细胞中启动活性分析

目的：分离和鉴定人类疱疹病毒8型(HHV-8)ORF、50基因上游启动子区序列，评价其在293细胞中启动活性。方法：以佛波酯(TPA)刺激的BCBL-1细胞总DNA为模板，PCR扩增HHV-8 ORE50启动子区序列，克隆进pGL-3基本载体中虫荧光素酶(Luciferase)报告基因上游多克隆位点，分别构建含正反双向ORE50启动子重组报告质粒，并分别转染293细胞，作Lu-ciferase活性检测，计算相对Luciferase活性单位(RLU)。结果：①克隆的HHV-8 ORF50启动子区序列长655碱基(bp)，含多个潜在推测AP1、SP1和ERE等转录因子结合序列；②ORF50启动子与正常pGL-3基本载体相比，其RLU增加了26．3倍；③TPA刺激后ORF50启动子与刺激前相比启动活性增加了4．1倍。结论：HHV-8 ORF50启动子区序列在293细胞中具有较强的启动活性；TPA可以作为一有效的阳性对照刺激物用于进一步鉴定ORF50启动子特性。

Isolation and Identification of Promoter Sequence of Human Herpesvirus 8 Open Reading Frame 50(ORF50)and Detection of Its Promoter Activity in the 293 Cells

Objective: To isolate, clone and identify the promoter sequence which was situated at the upstream of ORF50 of human herpesvirus 8 (HHV-8/and evaluate promoter activity in the 293 cells. Methods: Promoter sequence, which was situated at the upstream of HHV-8 ORF50, was amplified using PCR, total DNA from BCBL-1 cells treated with phorbal esters(TPA)of primary effusion lymphoma(PEL)as a template, and two sets of specific primers with Sac I and Bgl JH restriction enzyme cut sites engineered on the ends to facilitate directional cloning, separately. The PCR product was confirmed by sequence determination and predicted functional analysis, and further cloned into pCL-3 basic vector to construct recombinant luciferase reporter plasmids containing sense and antisense orientations sequences of HHV-8 ORF50 promoter. Then transfection of 293 cells with the ORF50 promoter-driven luciferase construct was performed with stimulation of TPA to induce luciferase gene expression and calculate the relative luciferase activity unit (RLU). Results: A 655 bp HHV-8 ORF50 promoter was cloned and sequenced. Functional analysis of ORF50 promoter disclosed several API and SP1 sites and Ets-relevant elements (ERE) in minimal promoter region. This ORF50 promoter exhibited a strong promoter activity with an increase of 26. 3-fold of RLU in 293 cells when compared with pGL-3 basic vector. Meanwhile the ORF50 promoter-driven luciferase expression was increased 4. 1-fold after treatment with TPA in 293 cells. Conclusion: A 655-bp promoter of HHV-8 ORF50 isolated in this study had a strong basal promoter activity in 293 cells. And TPA as an excellent stimulus can be used as the positive control when ORF50 promoter was further characterized.