大鼠pEGFP-PLZF真核载体的构建及在精原细胞中的表达  

Construciton of pEGFP-N1-PLZF eukaryotic expression vector and its expression in spermatogonia

作　　者：夏伟[1] 白杨[1] 叶哲伟[2] 曾甫清[1] 邬喻[1] 

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

出　　处：《南京医科大学学报（自然科学版）》2007年第8期831-834,842,F0003,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(30371424)

摘　　要：目的:构建真核表达重组质粒pEGFP-N1-PLZF,并了解其在大鼠精原细胞中的融合蛋白表达情况。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早幼粒细胞白血病锌指蛋白(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,并用双酶切、测序进行鉴定;将重组质粒用脂质体转染的方法导入精原细胞中,观察有无荧光的表达及用Western blot检测蛋白表达情况。结果:实验从大鼠睾丸组织中获取了编码plzf基因的全序列cDNA,产生了2 kb的目的插入片段,与pEGFP-N1载体连接后经酶切电泳鉴定及DNA测序证实序列正确;重组质粒转染精原细胞24 h后在荧光显微镜下观察到绿色荧光,并用Western blot检测到106 kD目的蛋白的表达。结论:新构建的重组质粒pEGFP-N1-PLZF通过鉴定,结构正确。转染到精原细胞后能在其中表达,发挥功能,为后续研究奠定了基础,对研究PLZF调控精原细胞增殖和分化机制具有重要的意义。Objective：To construct an eukaryotic expression recombinant plasmid pEGFP-N1-PLZF and get its protein translation in spermatogonia of the neonatal rats in vitro.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 EGFP-N1 vector by DNA recombinant technique.Then a new eukaryotic expression recombinant plasmid pEGFP-N1-PLZF was generated and identified by incision enzyme EcoRⅠand SalⅠand DNA sequencing,pEGFP-N1-PLZF was transfected into spermatogonia by liposome,from which protein were extracted after 24 h for detecting their expression by Western blot analysis.The treated cells were continuously traced by fluorescence microscope.Results：The recombinant plasmid cut by incision enzyme EcoRⅠand SalⅠovernight generated a 2 kb fragment,and DNA sequence of the 2 kb fragment was identical with rat plzf mRNA in genebank.In the cells 24 h after transfected with recombinant plasmid,Western blot analysis showed a 106 kD fusion protein.Green fluorescence could be seen by fluorescence microscope after 24 h.Conclusion：The recombinant plasmid was successfully constructed and was a useful tool to study the proliferation and differention of spermatogonia.

关 键 词：早幼粒细胞白血病锌指蛋白 重组质粒 绿色荧光蛋白 精原细胞 

分 类 号：Q786[生物学—分子生物学]