文章摘要
程 伟,郝婷婷,王子盾,朱小飞,冯宁翰,华立新,秦 娣,卢 春.HSV-1作用于KSHV ORF50启动子区中特异性应答位点序列的初寻[J].南京医科大学学报,2011,(5):595~600
HSV-1作用于KSHV ORF50启动子区中特异性应答位点序列的初寻
The detection of the specific sequence of the promoter region of the KSHV ORF50 acted by HSV-1
投稿时间:2010-11-01  
DOI:10.7655
中文关键词: KSHV  HSV-1  ORF50启动子  虫荧光素酶
英文关键词: KSHV  HSV-1  ORF50 promoter  luciferase
基金项目:国家自然科学基金(30900064);教育部高等学校博士点专项科研基金新教师基金(20093234120004);江苏省高校自然科学基金(09KJB310007);江苏省卫生厅医学重点学科(实验室)开发课题(09hx41);南京医科大学科技发展基金(08NMUM009)
作者单位
程 伟 南京医科大学第一附属医院泌尿外科,江苏 南京 210029 
郝婷婷 南京医科大学微生物学与免疫学系,江苏 南京 210029 
王子盾 南京医科大学第一附属医院泌尿外科,江苏 南京 210029 
朱小飞 南京医科大学微生物学与免疫学系,江苏 南京 210029 
冯宁翰 南京医科大学第一附属医院泌尿外科,江苏 南京 210029 
华立新 南京医科大学第一附属医院泌尿外科,江苏 南京 210029 
秦 娣 南京医科大学微生物学与免疫学系,江苏 南京 210029 
卢 春 南京医科大学微生物学与免疫学系,江苏 南京 210029 
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中文摘要:
      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KSHV ORF50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?
英文摘要:
      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.
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