【摘要】 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。更多还原

【Abstract】 Objective:To construct the recombinant luciferase reporter plasmids containing series of truncated sequences of Kaposi’s sarcoma-associated herpesvirus(KSHV) ORF50 promoter and seek the specific response sites sequences of ORF50 promoter acted by HSV-1.Methods:Series of truncated sequences of KSHV ORF50 promoter were amplified using PCR from KSHV genome.The PCR products were further cloned into pGL3 basic vector to construct the recombinant luciferase reporter plasmids.The recombinant plasmids were confirmed by restriction enzymes digestion and sequence analysis.After infected with HSV-1,Vero cells were transfected with the ORF50 promoter-driven luciferase constructs to induce luciferase gene expression.Relative luciferase activity unit(RLU) was calculated and compared.Results:Series of truncated sequences of KSHV ORF50 promoter were isolated and cloned successfully.The promoter-driven luciferase expression of pGL3-95,pGL3-46 and pGL3-17 were declined significantly in Vero cells infected with HSV-1 when compared with pGL3-1500,pGL3-750,pGL3-375 and pGL3-185.Conclusion:The recombinant luciferase reporter plasmids containing series of truncated sequences of KSHV ORF50 promoter were constructed successfully.The specific response sites sequences of ORF50 promoter acted by HSV-1 are between-185 bp and-95 bp.更多还原