甲状旁腺素受体真核表达载体的构建及稳定转染HEK293细胞系的建立  

Establishment of HEK293 cell lines stably expressing human parathyroid hormone receptors

作　　者：孟越[1] 谢苗苗[1] 林振[1] 袁亮[1] 李威[1] 郝松[1] 杨德鸿[1] 

机构地区：[1]南方医科大学南方医院脊柱骨科,广东广州510515

出　　处：《南方医科大学学报》2013年第7期956-961,共6页Journal of Southern Medical University

基　　金：国家自然科学基金(30973061;81272043)~~

摘　　要：目的构建小鼠甲状旁腺素受体(PTHR)及其突变受体(DSEL)全长结构基因真核表达载体,建立稳定高表达PTHR及DSEL的HEK293细胞系,以应用于甲状旁腺素(PTH)模拟肽活性药物筛选及其信号通路的研究。方法通过双酶切、胶回收方法分别纯化目的片段(PTHR、DSEL基因)和质粒pcDNA3.1(+),二者分别由DNA限制性内切酶EcoRⅠ与NotⅠ双酶切,经T4DNA Ligase连接的方法将PTHR、DSEL基因克隆到质粒表达载体pcDNA3.1(+)中,采用测序方法及DNA限制性内切酶EcoRⅠ与NotⅠ双酶切方法鉴定重组质粒,脂质体转染法将重组体pcDNA3.1(+)-PTHR、pcDNA3.1(+)-DSEL及空质粒pcDNA3.1(+)分别转染至HEK293细胞,经G418筛选抗性细胞克隆,、RT-PCR及ELISA检测转染细胞PTHR、DSEL的表达情况。结果重组质粒经基因测序及DNA限制性内切酶EcoRⅠ与NotⅠ双酶切证实质粒表达载体pcDNA3.1(+)-PTHR、pcDNA3.1(+)-DSEL构建正确。重组体经脂质体法转染HEK293 48 h后,经G418筛选3周得到细胞抗性克隆,ELISA检测到PTHR、DSEL基因在重组质粒表达载体pcDNA3.1(+)-PTHR、pcDNA3.1(+)-DSEL转染的HEK293中成功表达。RT-PCR方法检测到PTHR、DSEL基因在转录水平的表达。ELISA方法检测到重组质粒转染的HEK293中,加入甲状旁腺激素刺激后,PLC、cAMP蛋白表达量远高于空质粒pcDNA3.1(+)转染的HEK293中PLC、cAMP的表达。结论成功构建了稳定高表达PTHR、DSEL的HEK293细胞,该稳定转染细胞系的建立为进一步研究甲状旁腺素受体下游信号通道的分子机制以及筛选其模拟肽作用的活性奠定了实验基础。Objective To establish HEK293 cell lines with stable expression of human parathyroid hormone （PTH） receptors. Methods The purified gene fragments of PTH- related peptide receptor （PTHR） and its mutant form （DSEL） were cloned separately into pcDNA3.1（＋） vector after digestion with EcoR I and Not I, and the resulted pcDNA3.1（＋）-PTHR and pcDNA3.1 （＋ ）- DSEL plasmids were verified by restriction enzyme digestion and DNA sequencing. HEK293 cells were transfected with these plasmids and the expression of PTHR and DSEL in the cells were examined by RT-PCR and ELSIA. Results Sequencing and restriction enzyme digestion analysis showed that PTHR and DSEL cDNAs were correctly cloned into pcDNA3.1（＋）vector. After a 48- h transfection of HEK293 cells with the recombinant plasmids and G418 selection, the positive cell clones stably expressing the constucts were obtained, which showed expressions of PTHR and DSEL mRNAs detected by RT- PCR. These positive cells showed high levels of PLC and aAMP production in response to PTH stimulation. Conclusion The HEK293 cell lines with stable expression of PTH1R or DSEL gene established in this study provide useful cell models for studying the physiological functions of PTH peptides.

关 键 词：小鼠PTHR 真核表达 全长结构基因 稳定转染 

分 类 号：R96[医药卫生—药理学]