【摘要】：目的 PCR擴(kuò)增人端粒酶催化亞單位(hTERT)的核心啟動(dòng)子序列,將其克隆入熒光素酶報(bào)告載體pGL3Basic和含有SV40增強(qiáng)子的pGL3Enhancer中,以比較hTERT啟動(dòng)子與hTERT啟動(dòng)子聯(lián)合病毒SV40增強(qiáng)子即hTERT-SV40嵌合啟動(dòng)子系統(tǒng)在端粒酶陽性的惡性腫瘤細(xì)胞株和端粒酶陰性的人胚肺成纖維細(xì)胞株中轉(zhuǎn)錄活性和特異性的差異,試圖通過SV40增強(qiáng)子來增強(qiáng)hTERT啟動(dòng)子的轉(zhuǎn)錄活性,同時(shí)不改變其腫瘤特異性,以便為惡性腫瘤的靶向性治療提供初步的理論基礎(chǔ)和實(shí)驗(yàn)依據(jù)。 方法 以人基因組DNA為模板,應(yīng)用PCR方法擴(kuò)增端粒酶逆轉(zhuǎn)錄酶基因5’端上游長260bp的核心啟動(dòng)子片段,后將其亞克隆入pGEM-T Easy載體,構(gòu)建載體pGEM-hTP,其經(jīng)酶切和DNA測序無誤后,使用限制性內(nèi)切酶Kpn Ⅰ和Bgl Ⅱ雙酶切pGEM-hTP載體,得到hTERT核心啟動(dòng)子片段,將其與同樣經(jīng)Kpn Ⅰ和Bgl Ⅱ雙酶切消化的熒光素酶報(bào)告載體pGL3-Basic和pGL3-Ehancer進(jìn)行連接,構(gòu)建pGL3-hTP和pGL3hTP-SV40重組質(zhì)粒;使用磷酸鈣轉(zhuǎn)染系統(tǒng),將pGL3-hTP、pGL3-hTP-SV40、pGL3-Control和pGL3-Basic質(zhì)粒分別與pRL-TK共同轉(zhuǎn)染HT-29、SW620、MGC-803等端粒酶陽性的惡性腫瘤細(xì)胞株和人胚肺成纖維正常細(xì)胞株MRC5中,其中pGL3-Basic為陰性對照,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，

本文編號(hào)：2385404