论文部分内容阅读
为了克隆小鼠酪氨酸羟化酶(tyrosine hydroxylase,TH)启动子,并对其特异性调控能力进行研究,本实验采用重叠延伸PCR高保真扩增出小鼠TH启动子片段,测序正确后,重组构建质粒,并用TH启动子调控EGFP基因的表达。然后分别转染MN-9D细胞(TH+)和ECV细胞(TH-),观察EGFP基因在细胞内表达情况。结果显示:扩增出小鼠TH启动子序列与GenBank报道一致;单酶切和质粒PCR鉴定证实小鼠TH启动子和EGFP基因已经克隆入重组质粒中;小鼠TH启动子能调控EGFP在MN-9D细胞中表达,不能调控EGFP在ECV细胞内表达。初步确定克隆的小鼠TH启动子具有特异性调控目的基因表达的能力。
In order to clone the mouse tyrosine hydroxylase (TH) promoter and study its specific regulation ability, in this experiment, the mouse TH promoter fragment was amplified by overlap extension PCR. After the sequencing was correct The recombinant plasmids were constructed and the TH promoter was used to regulate the expression of EGFP gene. Then transfected MN-9D cells (TH +) and ECV cells (TH-) respectively to observe the expression of EGFP gene in cells. The results showed that the TH promoter sequence amplified from mice was consistent with that reported in GenBank. The TH promoter and EGFP gene were confirmed by restriction enzyme digestion and plasmid PCR. The TH promoter of mouse TH promoter could regulate the expression of EGFP in MN -9D cells can not regulate the expression of EGFP in ECV cells. It is preliminarily confirmed that the cloned mouse TH promoter has the ability to specifically regulate the expression of the target gene.