NRF-1基因真核慢病毒表达载体的构建及表达

Construction and expression of a recombinant lentivirus expression vector of nuclear respiratory factor 1 gene

目的:构建核呼吸因子-1(NRF-1)基因慢病毒表达载体并包装成重组慢病毒颗粒,感染大鼠心肌细胞H9c2(2-1)后,筛选出稳定上调表达NRF-1的大鼠心肌细胞H9c2(2-1)。方法:利用lipofectAMINE 2000试剂将pLenti6.3-NRF1-IRES2-EGFP质粒与两种包装质粒在293T细胞中包装成重组慢病毒,收集病毒并测定滴度。用重组慢病毒感染大鼠心肌细胞H9c2(2-1),然后用杀稻瘟素(blasticidin)筛选转染阳性细胞。用PCR法鉴定靶细胞基因组中NRF-1基因序列,用QPCR测定转染阳性靶细胞中NRF-1基因表达量。结果:收集的重组慢病毒滴度为5.5×107TU/ml。转染阳性靶细胞在荧光显微镜下可见绿色荧光。PCR结果显示,慢病毒携带NRF-1基因序列已插入靶细胞基因组。QPCR结果显示,转染阳性靶细胞中NRF-1基因的表达量是对照组的2.25倍。结论:本研究成功地构建NRF-1基因重组慢病毒表达载体,并在大鼠心肌细胞H9c2(2-1)中表达,为进一步上调NRF-1基因表达后心肌细胞能量代谢的研究提供了实验支持,并为心力衰竭能量代谢的基因治疗奠定了基础。

AIM： To obtain high expression of gene NRF-1 in rat cardiomyocyte cell line H9c2 （2-1） by examining the package of NRF-1 lentiviral particles and the effect of transfection to H9c2 （2-1）. METHODS： The plasmid of pInti6.3-NRFI-IRES2-EGFP and two packaging plasmids （pLP1, pLP2 and pLP/VSVG） were packaged into recombinant lentivirus in 293T cells by lipofectamine 2000. The virus was collected and the virus titer was measured. Rat cardiomyocyte cell line H9c2（2-1 ） was infected with the recombinant lentivirus and positive cells were screened with blasticidin antibiotics. The expression of NRF-1 in H9c2（2-1 ） cell was identified by fluorescence microscope, PCR and QPCR. RESULTS： The titer of recombinant lentivirus was up to 5.5 x 10^7 TU/ml and the green fluorescence was detected in transfection positive target cells using fluorescence microscope. PCR showed that the sequence of recom binant lentivirus was inserted into the target cell genome and QPCR results demonstrated that the expres sion levels of NRF-1 in positive target cells were 2.25 times higher than those in negative H9c2 ceils. CONCLUSION： In this study, expression of gene NRF-1 in rat cardiomyocyte H9c2 （2-1） has beensuccessfully upregulated, which provides experimental support for further research on myocardial energy metabolism and even the basis for gene therapy of heart failure energy metabolism.