【摘要】 目的:克隆甲状旁腺相关肽(PTHrP)亚基因PTHrP(1-36)、PTHrP(38-94)和PTHrP(107-139)并构建四环素反应性元件调控的反应质粒pTRE-PTHrP(1-36)、pTRE-PTHrP(38-94)和pTRE-PTHrP(107-139),为调控PTHrP亚克隆在软骨前体细胞中的表达打下基础。方法:提取骨骺干细胞的总RNA,以RT-PCR方法获得带有酶切位点的PTHrP(1-36)、PTHrP(38-94)和PTHrP(107-139)基因片段,扩增的DNA片段分别与含潮霉素筛选标记的四环素反应性元件载体pTRE-2Hyg双酶切后连接,转化扩增后对重组质粒进行提取和酶切、测序鉴定。结果:经酶切图谱分析和DNA序列测定证实目的基因已经插入重组质粒。结论:成功克隆出PTHrP亚克隆基因PTHrP(1-36)、PTHrP(38-94)和PTHrP(107-139)并构建了反应质粒pTRE-PTHrP(1-36)、pTRE-PTHrP(38-94)和pTRE-PTHrP(107-139),为进一步精确调控PTHrP亚基因的表达奠定了基础。更多还原

【Abstract】 Objective: To clone rat parathyroid hormone-related peptide(PTHrP) sub-genes and construct the plasmids of the tetracycline(Tet) responsive element,which may regulate and control the expressions of the PTHrP sub-genes.Methods: Total RNA was extracted from precartilaginous stem cells and the PTHrP(1-36),PTHrP(38-94) and PTHrP(107-139) sub-genes were obtained by the RT-PCR method.Then the sub-genes were subcloned into the plasmids of the Tet-responsive element with the selection marker of hygromycin pTRE-2Hyg to construct recombinant eukaryotic responsive plasmids.After transferred into E.coli-DH5α,the clones were amplified and the recombinant plasmids purified and identified by double-enzyme digestion.Results: Double enzyme digestion analysis and sequencing showed that the target sub-genes were cloned into the recombinant plasmids.Conclusion: The eukaryotic responsive plasmids containing the PTHrP(1-36),PTHrP(38-94) and PTHrP(107-139) sub-genes were successfully constructed,which might contribute a lot to the rigorous regulation of the expressions of PTHrP sub-genes.更多还原