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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 eukaryotic expression vector pEGFP-N1-PLZF and provide theoretical reference for studying the regulatory mechanism of zinc finger protein (PLZF) of promyelocytic leukemia in the proliferation and differentiation of spermatogonial stem cells. Methods According to the molecular cloning technique, PLZF was amplified from rat testis by RT-PCR and ligated into eukaryotic expression vector pEGFP-N1 containing enhanced green fluorescent protein (EGFP) reporter gene Construction of recombinant plasmid pEGFP-N1-PLZF. Results The total RNA was extracted from rat testis tissue and the full-length cDNA encoding PLZF gene was obtained by RT-PCR. To construct the eukaryotic expression plasmid of PLZF, EGFP reporter gene which emits green fluorescence was fused with PLZF gene and identified by DNA electrophoresis and DNA sequencing. Conclusion The construction of recombinant plasmid pEGFP-N1-PLZF was successful. In the experiment, green fluorescent EGFP reporter gene was fused to the 3 ’end of PLZF gene. Enhances the expression of PLZF in eukaryotic cells without affecting the structure and function of the target protein, and plays an important role in studying the mechanism of PLZF regulating the proliferation and differentiation of spermatogonial stem cells.