大鼠pEGFP-N1-PLZF真核表达载体的构建和意义  

Construciton and meaning of pEGFP-N1-PLZF eukaryotic expression vector

作　　者：夏伟[1] 白杨[1] 曾甫清[1] 王振迪[1] 叶哲伟[2] 

机构地区：[1]华中科技大学同济医学院协和医院泌尿外科,武汉430022 [2]华中科技大学同济医学院协和医院骨科

出　　处：《中国男科学杂志》2007年第8期6-8,13,共4页Chinese Journal of Andrology

基　　金：国家自然科学基金资助项目(30371424)

摘　　要：目的构建真核表达载体pEGFP-N1-PLZF,为基因水平研究早幼粒细胞白血病锌指蛋白(PLZF)在精原干细胞增殖和分化中的调控机制提供理论参考。方法参考分子克隆技术,采用RT-PCR的方法从大鼠睾丸组织中扩增PLZF,将该基因连接克隆到含有增强型绿色荧光蛋白(EGFP)报告基因的真核表达载体pEGFP-N1上,以构建重组质粒pEGFP-N1-PLZF。结果实验从大鼠睾丸组织中提取总RNA,以RT-PCR方法获取编码PLZF基因的全序列cDNA。构建PLZF的cDNA真核表达质粒时将能发出绿色荧光的EGFP报告基因融合在PLZF基因,并经酶切后DNA电泳鉴定及DNA测序证实结果。结论构建重组质粒pEGFP-N1-PLZF成功,实验中将能发出绿色荧光的EGFP报告基因融合在PLZF基因的3’端。提高PLZF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PLZF调控精原干细胞增殖和分化机制具有重要的意义。Objective To construct an eukaryotic expression vector named pEGFP-N1-PLZF for researching the proliferation and differention of spermatogonia. Methods Using the primers of PLZF gene and the total RNA extracted from the tissue of rat testes, RT-PCR was performed, the product was inserted to multiple clone sites of EGFPN1 vector by DNA recombinant techinique. Then a new eukaryotic expression recombinant plasmid named PEGFPN1-PLZF was generated. Results The recombinant plasmid carried PLZF and EGFP gene by restriction enzyme cutting analysis and DNA sequence analysis were succeeded. Conclusion PLZF was cloned into pEGFP-N1 successfully and increasing the translation efficiency of PLZF in mammalian cells without changing the structure and function of its translating protein. It is a useful tool to research the proliferation and differention of spermatogonia.

关 键 词：PLZF 基因表达 绿色荧光蛋白 

分 类 号：E321.1[军事—军事理论]