一种活性缺陷型小鼠端粒酶催化亚基过表达慢病毒载体 的构建及功能 .doc

一种活性缺陷型小鼠端粒酶催化亚基过表达慢病毒载体 的构建及功能初步测定 刘梦颖Δ，韩舟Δ，吴海银，陈晨，沈芯如，周海辉，周其冈*，朱东亚* 摘要： 目的：构建含活性缺陷型小鼠端粒酶催化亚基（去除氨基酸702-712的mTERT，命名为mTERTΔ）基因的慢病毒载体，检测其表达和功能。 方法：从先前构建的表达mTERT全长的pDC315-EGFP-mTERT质粒中，通过缺失突变PCR扩增小鼠mTERTΔ基因，构建真核表达载体GV287-EGFP/mTERTΔ。鉴定正确后将目的基因克隆入慢病毒载体pGC-LV，得重组载体pGC-LV/mTERTΔ-EGFP，采用Lipofectamine2000将其转染293T细胞，包装慢病毒颗粒LV-mTERTΔ-EGFP后感染神经干细胞和原代神经元，TRAP-PCR方法检测端粒酶活性，荧光显微镜观察目的片段表达和细胞增殖情况。 结果：成功构建TERTΔ基因片段，测序证明重组慢病毒载体pGC-LV/mTERTΔ-EGFP构建成功。包装慢病毒颗粒LV-mTERTΔ-EGFP可以感染神经干细胞和神经元，端粒酶活性测试证明目的蛋白的端粒酶催化活性缺陷，抑制神经干细胞增殖，抑制内源性mTERT功能。 结论：慢病毒载体LV-mTERT -EGFP构建成功，可以表达无活性mTERT片段。 关键词： 端粒酶， mTERT， 慢病毒， 神经干细胞， EGFP 单位：南京医科大学药学院药理实验室 作者： 刘梦颖：药理学硕士研究生三年级 朱东亚：南京医科大学教授，药学院院长 dyzhu@ 周其冈：南京医科大学副教授 qigangzhou@ 本研究由国家自然基金面上项目资助。 Construction of lentiviral vector carrying a non-activity mTERT and detection of the function LIU Meng-yingΔ, HAN ZhouΔ, Wu Hai-yin, CHEN chen, SHEN Xin-ru, ZHOU Hai-hui, ZHOU Qi-gang*, ZHU Dong-ya* ΔThese authors contributed equally to this study. *Corresponding author: Dr. Dongya Zhu or Dr. Qigang Zhou Department of Pharmacology, Pharmacy College, Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu 210029, E-mail address: dyzhu@ or qigangzhou@ This work was supported by grants from National Natural Science Foundation of China (No.. ［Abstract］ Objective：To construct the lentiviral vector carrying a type of activity defective mTERT（deleting AA702－712, named mTERTΔ）and to detect its expression and function． Methods：mTERTΔ was constructed from our previous plasmid pDC315-EGFP-mTERT which carrying whole gene encoding mTERT by deletion mutant PCR. And then, eukaryotic expression vector of GV287-EGFP/mTERTΔ was constructed．After DNA sequence analysis，mTERTΔ was cloned into lentiviral vector pGC-LV to construct recombinant vector pGC-LV/mTERTΔ-EGFP． pGC-LV/mTERTΔ-EGFP was transfected into 293T cells by Lipofectamine 2000 mediation to package lentiviral particles．The particles were transfected into neuronal stem cells and primary neurons，a