【作者】 周海文； 周曾同； 杨雯君； 刘德莉； 刘伟； 曹谊林；

【Author】 ZHOU Hai-wen,ZHOU Zeng-tong.YANG Wen-jun,LIU De-li,LIU Wei,CAO Yin-lin. (Department of Oral Medicine,Ninth People’s Hospital, Shanghai Second Medical University. Shanghai 200011;Department of Oral and Maxillofacial Surgery, Ninth People’s Hospital, Shanghai Second Medical University. Shanghai 200011; Tissue Engineering Center of Shanghai Second Medical University, Tissue Engineering Key Laboratory of Shanghai. Shanghai 200011,China)

【机构】 上海第二医科大学附属第九人民医院口腔内科； 上海第二医科大学口腔医学院口腔颌面外科； 上海第二医科大学组织工程研究中心上海市组织工程重点实验室；

【摘要】 目的:构建具绿色荧光蛋白报道系统的端粒酶基因真核质粒载体,并研究其在293细胞中的表达情况,为基因转染、延长细胞寿命奠定基础。方法:酶切含人端粒酶逆转录酶(hTRT)基因的pGRN145质粒获取hTRT片段,插入经酶切及磷酸化处理的绿色荧光蛋白载体pEGFP-C1,形成8.1Kb质粒,经HindⅢ、NotⅠ酶切电泳筛选、鉴定、测序并转染293细胞检测绿色荧光蛋白表达及外源性端粒酶活性。结果:酶切电泳分析,得到3.4Kb及4.7Kb大小的2个片段;测序结果接头两端序列正确;质粒在293细胞中正确表达,测得细胞外源性端粒酶表达。结论:端粒酶基因pEGFP-hTRT质粒成功构建,并可在真核细胞中表达,可用于研究组织工程种子细胞的衰老问题。更多还原

【Abstract】 PURPOSE: To construct a plasmid which has a reporter gene for exploring the role of human telomerase reverse transcriptase (hTRT) in in-vitro cell cultivation. METHODS: hTERT was cut by restricted enzyme from plasmid pGRN145 and inserted to plasmid pEGFP-C1 (enhanced green fluorescent protein,EGFP). RESULTS: Restricted enzyme analysis and DNA sequencing showed that the sequence of the pEGFP -hTRT transgenic plasmid was correct. CONCLUSIONS: The recombinant vector pEGFP-hTRT has been successfully constructed, and it could be used as a transgenic plasmid in generating immortalized cell lines.更多还原