pAAV-PEG3-sTRAIL质粒的构建及表达研究  

Construction of pAAV-PEG3-sTRAIL plasmid and sTRAIL expression

作　　者：蒋祥龙[1] 彭炬[1] 潘艺[1] 卢光琇[1] 

机构地区：[1]中南大学生殖与干细胞工程研究所,人类干细胞国家工程研究中心,湖南长沙410078

出　　处：《中国现代医学杂志》2010年第4期481-484,489,共5页China Journal of Modern Medicine

基　　金：973计划(No:2007CB948103);863计划(No:2006AA02A102)课题资助

摘　　要：目的构建pAAV-PEG3-sTRAIL质粒并检测其诱导细胞凋亡的作用。方法从大鼠尾巴中提取gDNA,PCR扩增PEG3启动子,测序后定向替代pAAV载体中的CAG启动子。从培养的NTERA-2细胞中提取总RNA,RT-PCR扩增sTRAIL基因(编码氨基酸序列114-281),测序后定向插入至pAAV-PEG3质粒的多克隆位点。用pAAV-PEG3-sTRAIL质粒FUGENE HD转染人hSF、NTERA-2和hES细胞,用半定量RT-PCR和流式细胞仪分别检测sTRAIL基因的表达和细胞的早期凋亡。结果限制性内切酶酶切和测序分析证实,本文成功地获得了PEG3启动子和sTRAIL基因片断,并将PEG3和sTRAIL准确插入了pAAVL质粒。转染hSF、NTERA-2和人胚胎干细胞(hES)后,检测到sTRAIL基因的表达及其引起的肿瘤细胞NTER-A-2的早期凋亡。结论成功构建了pAAV-PEG3-sTRAIL质粒,并有可能用于肿瘤的基因治疗。ObjectiveTo construct pAAV-PEG3-sTRAIL plasmid and evaluate the apoptotic effect induced by sTRAIL gene driven by PEG3 promoter on hSF, NTERA-2 and hES cells. MethodsTotal RNA and gDNA were extracted from NTERA-2 cells and rat tail, respectively. The sTRAIL gene was obtained from the total RNA by RTPCR and the PEG3 promoter was obtained from the gDNA by PCR. Then the CAG promoter of pAAV was substituted by the PEG3 promoter to construct vector pAAV-PEG3 and the sTRAIL sequence was inserted into the vector pAAV-PEG3 to construct plasmid pAAV-PEG3-sTRAIL. The plasmid was transfected into hSF, NTERA-2 and hES cells with FUGENE HD. Semi-quantitative RT-PCR and FACS were employed to detect sTRAIL expression and early apoptosis of the cells, respectively. ResultsSequencing analysis proved that the sequences of the PCR products were totally consistent with the data in GenBank. The fragments of PEG3 and sTRAIL were cloned into the plasmid pAAV in the right sites and directions. After hSF, NTERA-2 and hES cells were transfected with pAAVPEG3-sTRAIL plasmid, the expressions of sTRAIL gene were detected in the three cell lines, and the early apoptosis was induced by sTRAIL in NTERA-2 cells. ConclusionThe pAAV-PEG3-sTRAIL plasmid has been successfully constructed and it may be useful for gene therapy of tumors.

关 键 词：PEG3 STRAIL 凋亡 胚胎干细胞 NTERA-2 

分 类 号：R321[医药卫生—人体解剖和组织胚胎学]