[1]魏勇,应大君,朱楚洪,等.人工锌指蛋白真核表达载体的构建及表达[J].第三军医大学学报,2008,30(02):134-137.
 WEI Yong,YING Da-jun,ZHU Chu-hong,et al.Construction of eukaryotic expressing vector of artificial zinc finger protein gene and its expression[J].J Third Mil Med Univ,2008,30(02):134-137.
点击复制

人工锌指蛋白真核表达载体的构建及表达(/HTML )
分享到:

《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
30卷
期数:
2008年第02期
页码:
134-137
栏目:
论著
出版日期:
2008-01-30

文章信息/Info

Title:
Construction of eukaryotic expressing vector of artificial zinc finger protein gene and its expression
作者:
魏勇应大君朱楚洪侯春丽崔晓萍
第三军医大学基础医学部解剖学教研室,重庆市生物力学与组织工程重点实验室
Author(s):
WEI Yong YING Da-jun ZHU Chu-hong HOU Chun-li CUI Xiao-ping
Department of Anatomy, College of Medicine, Chongqing Key Laboratory of Biomechanics and Tissue Engineering, Third Military Medical University
关键词:
人工锌指蛋白真核表达载体EGFPCOS-7细胞
Keywords:
artificial zinc finger protein eukaryotic expression vector EGFP COS-7 cells
分类号:
R341;R394-33
文献标志码:
A
摘要:
目的 构建人工锌指蛋白ZFP基因的真核表达载体,检测其在真核细胞的表达情况,从而探讨人工锌指蛋白作为人工转录因子DNA结合域的可能性。  方法 全基因合成人工锌指蛋白(putative zinc finger protein, ZFP)的核酸序列并克隆入pEGFP-N1及pIRES2-EGFP真核表达载体中,构建重组质粒pEGFP-N1/ZFP 及pIRES2-EGFP/ZFP-Flag,通过酶切、菌落PCR及测序来鉴定重组载体的正确性。脂质体DOTAP体外转染重组质粒于COS-7 细胞中,用荧光显微镜、RT-PCR、Western blot 方法鉴定ZFP在该细胞中的表达。  结果 正确构建了pEGFP-N1/ZFP、pIRES2-EGFP/ZFP-flag重组表达质粒,并且在COS-7细胞中成功进行了人工锌指蛋白ZFP的表达。  结论 采用生物信息学手段获得的假定的锌指蛋白基因序列可以在真核细胞内正常表达。
Abstract:
Objective To construct the recombinant plasmid pEGFP-N1/ZFP and pIRES2-EGFP/ZFP-flag and explore the possibility of engineering zinc finger protein (ZFP) as DNA binding domain of artificial transcription factor (ATF).   Methods Full length ZFP gene was synthesized and cloned into pEGFP-N1 and pIRES2-EGFP plasmids to construct the recombinant plasmids pEGFP-N1/ZFP and pIRES2-EGFP/ZFP-flag that were identified by colony PCR and sequencing. Recombinant plasmids were transformed into COS-7 cells by liposome DOTAP. The expressed EGFP was observed under fluorescent microscope and the ZFP expression was detected by RT-PCR and Western blot.   Results Recombinant plasmids pEGFP-N1/ZFP and pIRES2-EGFP/ZFP-flag were constructed correctly, which could express ZFP well in the transformed COS-7 cells.  Conclusion It is feasible that engineered artificial zinc finger proteins by bioinformatics technique can be expressed in eukaryotic cells.

参考文献/References:

魏勇,应大君,朱楚洪,等. 人工锌指蛋白真核表达载体的构建及表达[J].第三军医大学学报,2008,30(2):134-137.

相似文献/References:

[1]王海晶,杨和平,周向东.RNA干扰CA916798基因真核表达载体的构建与鉴定[J].第三军医大学学报,2008,30(01):23.
 WANG Hai-jing,YANG He-ping,ZHOU Xiang-dong.Construction and identification of CA916798 eukaryotic expression vector for RNA interference[J].J Third Mil Med Univ,2008,30(02):23.
[2]曾凡新,董志,傅洁民,等.人β3肾上腺素受体真核表达质粒pcDNA3.1(+)-β3-AR的构建及表达[J].第三军医大学学报,2007,29(21):2070.
 ZENG Fan-xin,DONG Zhi,FU Jie-min,et al.Construction and expression of a eukaryotic β3-adrenoceptor expression vector pcDNA3.1(+)-β3-AR[J].J Third Mil Med Univ,2007,29(02):2070.
[3]周春丽,郝进,唐书谦,等.人CTLA-4Ig融合基因减毒鼠伤寒沙门菌真核表达载体的构建及表达[J].第三军医大学学报,2006,28(03):243.
[4]田坤,钱桂生.人CD40L真核表达体系的建立[J].第三军医大学学报,2007,29(10):963.
 TIAN Kun,QIAN Gui-sheng.Construction of eukaryotic expression system of a cell surface molecule——human CD40 ligand[J].J Third Mil Med Univ,2007,29(02):963.
[5]周琪,梁后杰,阎晓初,等.RNA干扰Neuropilins-2基因真核表达载体的构建及其鉴定[J].第三军医大学学报,2007,29(08):691.
 ZHOU Qi,LIANG Hou-jie,YAN Xiao-chu,et al.Construction and effect of Neuropilins-2 eukaryotic expression vector for RNA interference[J].J Third Mil Med Univ,2007,29(02):691.
[6]滕小春,刘海峰,房殿春,等.NHE-1反义基因对胃癌细胞的酸化作用及其意义[J].第三军医大学学报,2007,29(03):240.
 TENG Xiao-chun,LIU Hai-feng,FANG Dian-chun,et al.Acidification of antisense NHE-1 gene transfection on gastric cancer cell line SGC-7901 and its significance[J].J Third Mil Med Univ,2007,29(02):240.
[7]张炜炜,王涛,艾国平,等.小鼠微小RNA miR-22真核表达载体的构建及功能初步研究[J].第三军医大学学报,2007,29(09):803.
 ZHANG Wei-wei,WANG Tao,AI Guo-ping,et al.Construction of eukaryotic expression vector of mouse microRNAs miR-22[J].J Third Mil Med Univ,2007,29(02):803.
[8]钱春荣,刘昕,刘丽莎,等.PTTG1基因上调表达对A549细胞增殖和凋亡的影响[J].第三军医大学学报,2006,28(09):961.
[9]闫国和,粟永萍,王军平,等.重组真核表达载体pEGFP-N1/PDGF-A的构建及真皮干细胞的转染[J].第三军医大学学报,2005,27(20):2005.
[10]章波,粟永萍,艾国平.ERp29基因的克隆及其在IEC-6细胞中的表达[J].第三军医大学学报,2005,27(18):1830.

更新日期/Last Update: 2008-04-30