大鼠PTHrP亚基因的克隆及其真核反应质粒的构建  

Cloning PTHrP sub-genes and construction of eukaryotic responsive plasmids in rats

作　　者：张树威[1] 陈安民[1] 郭风劲[1] 谢柏臻[1] 祁军[1] 王江[1] 易磊[1] 李昆朋[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院骨科,湖北武汉430030

出　　处：《医学研究生学报》2008年第11期1128-1131,共4页Journal of Medical Postgraduates

基　　金：国家自然科学基金资助项目(批准号:30571872);教育部博士点基金资助项目[批准号:(2005)216]

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。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 extraeted 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-DH5a, 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 eukaryotie 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.

关 键 词：骨骺干细胞 甲状旁腺相关肽 反应质粒 PTHrP亚基因 

分 类 号：Q785[生物学—分子生物学]