Construction of eukaryotic expressing vector of artificial zinc finger protein gene and its expression

魏勇; 应大君; 朱楚洪; 侯春丽; 崔晓萍

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Construction of eukaryotic expressing vector of artificial zinc finger protein gene and its expression

更新时间：2026-01-09

- Construction of eukaryotic expressing vector of artificial zinc finger protein gene and its expression
- Journal of Army Medical University Vol. 30, Issue 2, Pages: 134-137(2008)
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  CLC： R341;R394-33
- Published：2008
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魏勇,应大君,朱楚洪,侯春丽,崔晓萍.人工锌指蛋白真核表达载体的构建及表达[J].陆军军医大学学报(原第三军医大学学报),2008,30(02):134-137.

　WEI Yong,YING Da-jun,ZHU Chu-hong,HOU Chun-li,CUI Xiao-ping.Construction of eukaryotic expressing vector of artificial zinc finger protein gene and its expression[J].J Amry Med Univ (J Third Mil Med Univ),2008,30(02):134-137.
魏勇,应大君,朱楚洪,侯春丽,崔晓萍.人工锌指蛋白真核表达载体的构建及表达[J].陆军军医大学学报(原第三军医大学学报),2008,30(02):134-137. DOI：

　WEI Yong,YING Da-jun,ZHU Chu-hong,HOU Chun-li,CUI Xiao-ping.Construction of eukaryotic expressing vector of artificial zinc finger protein gene and its expression[J].J Amry Med Univ (J Third Mil Med Univ),2008,30(02):134-137. DOI：

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.

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