碱性成纤维细胞生长因子基因真核表达载体的构建  

Construction of mammalian expressing vector pEGFP-C3-bFGF

作　　者：伊海英[1] 孙晓艳[2] 付小兵[2] 任明姬[1] 张广田[1] 高瑗 

机构地区：[1]内蒙古医学院,内蒙古呼和浩特010059 [2]解放军总医院第一附属医院全军创伤修复重点实验室,北京100037 [3]总后勤部管理保障局北京郑常庄干休所,北京100039

出　　处：《感染．炎症．修复》2008年第3期174-176,共3页Infection Inflammation Repair

基　　金：国家重点基础研究发展规划项目(2005CB522603)

摘　　要：目的:构建碱性成纤维细胞生长因子(bFGF)基因真核表达载体pEGFP-C3-bFGF,为基因水平研究bFGF在表皮细胞去分化过程中的调控机制提供实验参考。方法:参考分子克隆技术,采用聚合酶链反应(PCR)的方法从PBR322-bFGF质粒中扩增bFGF,同时在其两端引入HindⅢ和EcoRⅠ限制性内切酶酶切位点,将bF-GFcDNA定向插入pEGFP-C3的多克隆位点中,构建真核表达质粒pEGFP-C3-bFGF,并对重组子进行DNA序列测定。结果:电泳初步鉴定获得的连接产物为重组质粒pEGFP-C3-bFGF;对重组子中含有bFGF基因的一段进行测序,测序结果经Vector NTI软件分析,证实获得bFGF基因。结论:将bFGF插入pEGFP-C3的C末端,可以提高bFGF在真核细胞中的表达,而且不影响其目的蛋白的结构和功能,对研究bFGF调控表皮细胞去分化的分子机制具有重要的意义。Objective： To construct a recombinant plasmid carrying human basic fibroblast growth factor （bFGF） to transfect and induce dedifferentiation of epidermal cells in vitro, for exploring the possible signaling pathway involved in the reversion process of epidermal cells. Methods： All experimental methods were performed according to the standard protocols of molecular clone technology. The bFGF cDNA was amplified by polymerase chain reaction （PCR） from PBR322-bFGF with HindⅢ and EcoR Ⅰ at both sides, and it was subsequently subcloned into the multiple cloning site of pEGFP-C3 to obtain the recombinant plasmid pEGFP-C3-bFGF, followed by the identification of DNA sequencing analysis. Results： DNA sequencing analysis indicated that the recombinant plasmid pEGFP-C3-bFGF possessed the same open reading frame as pEGFP and perfect potential to translate and express in epidermal cells. Conclusion： bFGF is cloned into the C terminus of pEGFP-C3. Under the control of enhancer and promoter of pEGFP-C3, expression of bFGF can be up-regulated without influencing the structure and function of its target protein. Therefore, it is highly significant in the study of molecular mechanism of modulation of dedifferentiation of epidermal cells by bFGF.

关 键 词：碱性成纤维细胞生长因子 基因表达 载体构建 聚合酶链反应 

分 类 号：R644[医药卫生—外科学] R382.31[医药卫生—临床医学]