SV40增强子修饰的hTERT核心启动子靶向转录荧光素酶载体的构建和鉴定
发布时间：2018-12-18 05:16
【摘要】：目的 PCR扩增人端粒酶催化亚单位(hTERT)的核心启动子序列,将其克隆入荧光素酶报告载体pGL3Basic和含有SV40增强子的pGL3Enhancer中,以比较hTERT启动子与hTERT启动子联合病毒SV40增强子即hTERT-SV40嵌合启动子系统在端粒酶阳性的恶性肿瘤细胞株和端粒酶阴性的人胚肺成纤维细胞株中转录活性和特异性的差异,试图通过SV40增强子来增强hTERT启动子的转录活性,同时不改变其肿瘤特异性,以便为恶性肿瘤的靶向性治疗提供初步的理论基础和实验依据。 方法 以人基因组DNA为模板,应用PCR方法扩增端粒酶逆转录酶基因5’端上游长260bp的核心启动子片段,后将其亚克隆入pGEM-T Easy载体,构建载体pGEM-hTP,其经酶切和DNA测序无误后,使用限制性内切酶Kpn Ⅰ和Bgl Ⅱ双酶切pGEM-hTP载体,得到hTERT核心启动子片段,将其与同样经Kpn Ⅰ和Bgl Ⅱ双酶切消化的荧光素酶报告载体pGL3-Basic和pGL3-Ehancer进行连接,构建pGL3-hTP和pGL3hTP-SV40重组质粒;使用磷酸钙转染系统,将pGL3-hTP、pGL3-hTP-SV40、pGL3-Control和pGL3-Basic质粒分别与pRL-TK共同转染HT-29、SW620、MGC-803等端粒酶阳性的恶性肿瘤细胞株和人胚肺成纤维正常细胞株MRC5中,其中pGL3-Basic为阴性对照,pGL3-Control
[Abstract]:Objective to amplify the core promoter sequence of human telomerase catalytic subunit (hTERT) by PCR and clone it into luciferase report vector pGL3Basic and pGL3Enhancer containing SV40 enhancer. To compare the transcriptional activity and specificity of hTERT promoter and hTERT promoter combined with virus SV40 enhancer (hTERT-SV40 chimeric promoter subsystem) in telomerase positive malignant tumor cell line and telomerase negative human embryonic lung fibroblast cell line. This paper attempts to enhance the transcriptional activity of hTERT promoter by SV40 enhancer without changing its tumor specificity so as to provide a preliminary theoretical and experimental basis for the targeted therapy of malignant tumors. Methods using human genomic DNA as template, the core promoter fragment of telomerase reverse transcriptase gene 5'upstream long 260bp was amplified by PCR, then subcloned into pGEM-T Easy vector to construct pGEM-hTP, vector. The pGEM-hTP vector was digested by restriction endonuclease Kpn 鈪，
本文编号：2385404
[Abstract]:Objective to amplify the core promoter sequence of human telomerase catalytic subunit (hTERT) by PCR and clone it into luciferase report vector pGL3Basic and pGL3Enhancer containing SV40 enhancer. To compare the transcriptional activity and specificity of hTERT promoter and hTERT promoter combined with virus SV40 enhancer (hTERT-SV40 chimeric promoter subsystem) in telomerase positive malignant tumor cell line and telomerase negative human embryonic lung fibroblast cell line. This paper attempts to enhance the transcriptional activity of hTERT promoter by SV40 enhancer without changing its tumor specificity so as to provide a preliminary theoretical and experimental basis for the targeted therapy of malignant tumors. Methods using human genomic DNA as template, the core promoter fragment of telomerase reverse transcriptase gene 5'upstream long 260bp was amplified by PCR, then subcloned into pGEM-T Easy vector to construct pGEM-hTP, vector. The pGEM-hTP vector was digested by restriction endonuclease Kpn 鈪，
本文编号：2385404
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