慢病毒Tet-On系统调控Notch1-EGFP融合蛋白在PC12细胞中表达的研究

Construction of inducible lentiviral vector containing human Notch1 and EGFP gene and its expression in PC12 cells

目的:构建Te-t On系统调控的人源性Notch1胞内段(NICD)和EGFP融合蛋白的慢病毒真核表达载体,并探讨Notch1-EGFP在PC12细胞中的表达条件。方法:采用RT-PCR从人胎盘组织中获得NICD的cDNA,利用PCR扩增pEGFP-C1质粒中EGFP的编码序列,两者均定向克隆到pcDNA3.1(+)质粒中组成Notch1-EGFP片段,将该片段进一步酶切回收,并定向克隆到pLVX-Tigh-t puro载体获得pLVX-Notch1-EGFP。将构建好的质粒进行病毒包装并感染PC12细胞,抗性筛选2周。在不同时间点和不同浓度Dox作用下,用荧光显微镜观察EGFP表达,流式细胞术检测Notch1表达。结果:酶切和DNA测序均证实pLVX-Notch1-EGFP质粒构建成功。镜下可见感染后的PC12细胞在500 ng/ml Dox诱导36 h时90%以上细胞均表达EGFP。流式细胞术检测Notch1表达结果显示,随着Dox浓度增加,Notch1阳性细胞百分比逐渐增加;随着Dox诱导时间延长,Notch1阳性细胞百分比逐渐增高,到36 h时达到最高(81.5%)。结论:成功构建携带Notch1-EGFP融合蛋白的Te-t On调控的慢病毒真核表达载体,实现在PC12细胞中调控Notch1-EGFP融合蛋白的表达。

Objective： To construct inducible lentiviral vector containing human Notch1 intracellular domain（NICD） gene and enhanced green fluorescent protein（EGFP）,and to study its expression in PC12 cells.Methods： NICD cDNA was amplified by RT-PCR from human placenta tissue.EGFP gene was amplified by PCR from pEGFP-C1.Both NICD and EGFP were cloned into pcDNA 3.1（＋） plasmid to form pcDNA3.1-Notch1-EGFP.Then the Notch1-EGFP fragment was separated and cloned into pLVX-Tight-puro to form pLVX-Notch1-EGFP.The lentivirus were packaged and harvested,which were used to infect PC12 cells.After antibody selection for 2 weeks, the PC12 cells were induced by doxcycline（Dox）.The expression of Notch1-EGFP was detected by fluorescence microscope and flow cytometry.Results： The recombinant inducible lentiviral vectors（pLVX-Notch1-EGFP） were success fully constructed.The EGFP positive cell percentage was over 90% in transfected PC12 cells after 500 ng/ml Dox induction for 36 h.The expression of Notch1 was posited correlated to the Dox concentration.The expression of Notch1 increased with the duration of Dox induction,which got the peak at 36 h after Dox induction.Conclusion： The recombinant inducible lentiviral vectors containing Notch1 and EGFP gene are successfully constructed,which provides an effective and simple method to regulate the expression of Notch1 in PC12 cells.