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。
Objective：To construct lentiviral vector carrying activity-defective mTERT（deleting amino acid 702-712,named mTERTΔ） and to detect its expression and function.Methods：Mice mTERTΔ gene was amplified by our previous constructed plasmid pDC315-EGFP-mTERT carrying the whole gene encoding mTERT by deletion mutant PCR.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 Lipofectamine2000 to package lentiviral particle LV-mTERTΔ-EGFP.The particles were transfected into neuronal stem cells and primary neurons.TRAP-PCR was performed to detect telomerase activity,and fluorescence microscopy was performed to observe fragment expression and cell proliferation.Results：TERTΔ gene fragment was successfully constructed,and the analysis of DNA sequence proved that the recombinant lentiviral vector pGC-LV/mTERTΔ-EGFP was successfully constructed.Package of lentiviral particle LV-mTERTΔ-EGFP were transfected into neuronal stem cells and primary neurons.The expressed mTERTΔ detected by telomerase activity test was activity defective,and inhibited neural stem cell proliferation and endogenous mTERT function.Conclusion：The lentiviral vector of LV-mTERTΔ-EGFP was constructed successfully and infected cells could express activity-defective mTERT.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2014年第6期691-698,共8页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然科学基金面上项目(81370033)
国家自然科学基金重点项目(81030023)
