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|本期目录/Table of Contents|

NRF-1基因真核慢病毒表达载体的构建及表达

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2014年第3期
页码:
280-284
栏目:
基础研究
出版日期:
2014-03-20

文章信息/Info

Title:
Construction and expression of a recombinant lentivirus expression vector of nuclear respiratory factor 1 gene
作者:
宋 熔1; 王程铖2; 3; 王 强3; 王玉姣3; 李君良3; 赵 巍3
(1.中国人民解放军第五医院重症医学科，宁夏 银川 750004；
2.宁夏医科大学检验学院，宁夏 银川 750004；
3.宁夏回族自治区医学科学研究所、宁夏医科大学医学科学技术研究中心，宁夏 银川 750004)
Author(s):
SONG Rong1;  WANG Cheng cheng2; 3;  WANG Qiang3;  WANG Yu jiao3;  LI Jun liang3;  ZHAO Wei3
(1.ICU of PLA 5th Hospital, Yinchuan 750004, Ningxia, China;
2.School of Laboratory Medicine, Ningxia Medical University, Yinchuan 750004, Ningxia, China;
3.Ningxia Institute of Medical Sciences & Research Center of Science and Technology, Ningxia Medical University, Yinchuan 750004, Ningxia, China)
关键词:
NRF-1基因; 慢病毒表达载体; H9c2（2-1）细胞; 心力衰竭
Keywords:
NRF-1 gene;  lentiviral vector;  H9c2(2-1);  heart failure
分类号:
R541.6
DOI:
-
文献标识码:
A
摘要:
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5.5×107 TU/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。
Abstract:
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 pLenti6.3-NRF1-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 5.5×107 TU/ml and the green fluorescence was detected in transfection positive target cells using fluorescence microscope. PCR showed that the sequence of recombinant lentivirus was inserted into the target cell genome and QPCR results demonstrated that the expression levels of NRF-1 in positive target cells were 2.25 times higher than those in negative H9c2 cells. CONCLUSION: In this study, expression of gene NRF1 in rat cardiomyocyte H9c2(2-1) has been successfully upregulated, which provides experimental support for further research on myocardial energy metabolism and even the basis for gene therapy of heart failure energy metabolism.

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备注/Memo

备注/Memo:
收稿日期：2013-07-11
基金项目：宁夏回族自治区自然科学基金项目资助(NZ03680)；国家自然科学基金项目资助(81360042)
通讯作者：赵巍，教授，主要从事分子疫苗及包虫病相关研究 Email:zw-6915@163.com
共同通讯作者：宋熔，副主任医师，主要从事心力衰竭发病机制与防治研究 Email:srlyk@163.com 共同第一作者：王程铖，硕士生 Email:wangchch1119@163.com
更新日期/Last Update: 2014-03-21