文章摘要
夏 伟,白 杨,叶哲伟,曾甫清,邬 喻.大鼠pEGFP-PLZF真核载体的构建及在精原细胞中的表达[J].南京医科大学学报,2007,(8):831~834842
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
投稿时间:2007-01-12  
DOI:10.7655
中文关键词: 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
英文关键词: PLZF  recombinant plasmid  green fluorescent protein  spermatogonia
基金项目:国家自然科学基金资助项目(30371424)
作者单位
夏 伟 华中科技大学同济医学院协和医院泌尿外科,湖北 武汉 430022 
白 杨 华中科技大学同济医学院协和医院泌尿外科,湖北 武汉 430022 
叶哲伟 华中科技大学同济医学院协和医院骨科,湖北 武汉 430022 
曾甫清 华中科技大学同济医学院协和医院泌尿外科,湖北 武汉 430022 
邬 喻 华中科技大学同济医学院协和医院泌尿外科,湖北 武汉 430022 
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中文摘要:
      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?
英文摘要:
      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 EcoRⅠ and SalⅠ and 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 EcoRⅠ and SalⅠ overnight 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.
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