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目的:构建卡波济肉瘤相关疱疹病毒(Kaposi’s sarcoma-associated herpesvirus,KSHV)ORF50启动子系列截短序列克隆,初步寻找HSV-1作用于KSHV ORF50启动子区中特异性应答位点序列。方法:以KSHV基因组为模板,PCR扩增KSHVORF50启动子系列截短序列,克隆至含有虫荧光素酶(Luciferase)报告基因的基本载体pGL-3中,构建含ORF50启动子系列截短序列的重组Luciferase报告质粒。系列重组报告质粒经酶切鉴定和序列测定分析后,分别转染单纯疱疹病毒1型(HSV-1)感染后的非洲绿猴肾细胞(Vero细胞),进行Luciferase活性检测,计算并比较相对Luciferase活性单位(Relative luciferase activity unit,RLU)。结果:成功分离、克隆KSHV ORF50启动子系列截短序列;HSV-1感染随后转染重组质粒p50-95、p50-46和p50-17与转染p50-1500、p50-750、p50-375及p50-185相比,Vero细胞中Luciferase活性显著下降。结论:成功构建了含KSHV ORF50启动子系列截短序列的重组Luciferase报告质粒;HSV-1所对应的、能够主导ORF50启动子活性的最小顺式调控区位于-185 bp和-95 bp之间。
OBJECTIVE: To construct a truncated sequence clone of ORF50 promoter from Kaposi’s sarcoma-associated herpesvirus (KSHV) and to find out the sequence of HSV-1 acting on the specific response site of KSHV ORF50 promoter region. Methods: KSHV genome was used as a template to amplify the truncated sequence of KSHVORF50 promoter and cloned into pGL-3 vector containing Luciferase reporter gene to construct recombinant truncated ORF50 promoter sequence Luciferase reporter plasmid. A series of recombinant reporter plasmids were identified by restriction enzyme digestion and sequence analysis. After transfection of Vero cells infected with HSV-1, Luciferase activity was measured and compared with that of Luciferase Relative luciferase activity unit (RLU). Results: The truncated sequence of KSHV ORF50 promoter was successfully isolated and cloned. The recombinant plasmids p50-95, p50-46 and p50-17 transfected with HSV-1 were transfected into p50-1500, p50-750, p50-375 Luciferase activity was significantly decreased in Vero cells compared to p50-185. CONCLUSION: The recombinant Luciferase reporter plasmid containing the truncated sequence of KSHV ORF50 promoter was successfully constructed. The minimal cis-regulatory region corresponding to HSV-1 that can dominate ORF50 promoter activity is between -185 bp and -95 bp.