大鼠pEGFP-N1-PLZF真核表达载体的构建和意义

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）在精原千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。

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

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.