小鼠Nestin启动子的克隆及其表达载体的构建

目的:克隆Nestin基因启动子序列,并研究其调控能力。方法:用高保真酶PCR扩增出小鼠Nestin启动子全序列、核心序列,pcDNA3.1质粒双酶切切掉CMV启动子序列,将Nestin基因启动子序列插入到原CMV启动子位置,pEGFP-N1质粒双酶切下EGFP序列,克隆入pcDNA3.1多克隆位点中,重组构建质粒,分别用Nestin启动子全序列和核心序列调控EGFP基因的表达。重组质粒分别转染Nestin+细胞和Nestin-细胞,观察EGFP基因在细胞内的表达情况。结果:成功克隆出Nestin基因启动子全序列和核心序列,经酶切鉴定及测序证实正确。二序列皆能调控EGFP在各种细胞内的表达。结论:Nestin基因启动子全序列和核心序列具有非特异性调控能力。

Cloning of mice nestin promoter and construction of expression vector

Objective Clone nestin promotor and study its regulation. Methods Mice nestin promoter sequences and nestin promoter core sequence amplified by PCR with TaKaRa enzyme. CMV promoter sequence was cut by double-digested pcDNA3.1 plasmid. Nestin protein promoter sequence would be inserted into the original location of CMV promoter. EGFP sequence was cut by double-digested pEGFP-N1 plasmid, nestin promotor and EGFP gene were cloned into the recombined plasmid, recombine and construct plasmid, and EGFP gene was regulated by nestin promoter sequences and nestin promoter core sequence. The recombinant plasmids were transfected into nestin positiveness cells and negativeness cells, then observe the expression of EGFP gene. Results Cloned nestin gene promoter sequences and nestin promoter core sequence. The recombinant vector was identified by restriction enzyme analysis and nucleotide sequencing. The expression of EGFP gene regulated by the two sequences in various cells. Conclusion Nestin promoter sequences and core sequence has non-specific regulation.