HTERT启动子调控的NK4基因重组腺病毒表达系统的构建与鉴定  

Construction and identification of hTERT promoter-based NK4-expressing adenovirus

作　　者：黄桂填[1] 肖焕擎[2] 曾山崎[2] 徐波[2] 蔡文松[2] 李书华[3] 

机构地区：[1]广州市第一人民医院急诊外科 [2]广州市第一人民医院普外科 [3]广州医学院病理教研室,广东广州510180

出　　处：《广州医学院学报》2011年第2期102-105,111,共5页Academic Journal of Guangzhou Medical College

基　　金：广东省科技计划项目(2008B030301005);广州市医药卫生科技项目(2009-YB-017)

摘　　要：目的：构建靶向结肠癌细胞的人端粒酶逆转录酶HTERT启动子调控的NK4基因重组腺病毒并对该表达体系进行鉴定。方法：根据GenBank中的HTERT启动子（HTERTp）序列设计引物，以人胚肾293细胞提取基因组DNA为模板，PCR扩增HTERTp，并将HTERTp序列克隆入pDC312质粒中，构建成HTERTp调控的腺病毒质粒pSG—HTERTp；NK4 cDNA酶切并回收克隆至pSG—HTERTp载体中，构建成pSG—HTERTp／pA—NK4穿梭质粒，脂质体法在293细胞中包装重组腺病毒；噬斑分析法筛选单克隆重组腺病毒；RT-PCR检测NK4基因的转录；Western Blot检测NK4蛋白的表达。结果：成功构建出成功构建出HTERT启动子调控的NK4基因重组腺病毒，滴度为4．5×10^9空斑形成单位（pfu）／mL，且PCR扩增及western bolt电泳均呈阳性表达。结论：成功构建出HTERT启动子调控的NK4基因重组腺病毒，为进一步研究NK4基因的作用机制打下实验基础。Objective： To construct and identify hTERT promoter-based NK4-expressing adenovirus that targets colorectal carcinoma cells. Methods： The sequence of HTERT promoter（HTERTp） retrieved from GenBank was PCR-amplified using specific primers and HEK 293 genome DNA（ as the template）,and was then cloned into pDC312 to generate the HTERTp-based adenovirus plasmid pSG-HTERTp. NK4 gene was digested by cDNA-ase and clone into the pSG-HTERTp vector to construct a shuttle plasmid pAV- hTERTp-NK4, which was packed into the recombinant adenovirus by liposome. Monoclonal Ad- hTERTp-NK4 was screened by plaque assay; The transcription of NK4 was detected with RT-PCR. Western blotting was employed to detect the expression of the Ad- hTERTp-NK4 in colorectal carcinoma cells HCT116. Results： Ad-hTERTp-NK4 was generated successfully and its titer was 4.5×10^9 pfu/ml. RT-PCR showed that NK cDNA could be amplified from transfected cells, and western blot showed that Ad- hTERTp-NK4 expressed NK4 proteins in HCT116 cells. Conclusion： The successful development of hTERT promoter-based NK4-expressing adenovirus provided a basis for n further experimental study on anti-tumor effects of NK4.

关 键 词：人端粒酶逆转录酶启动 NK4基因 腺病毒 

分 类 号：R735[医药卫生—肿瘤]