南方医科大学学报

南方医科大学学报 ›› 2013, Vol. 33 ›› Issue (07): 956-.

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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

孟越，谢苗苗，林振，袁亮，李威，郝松，杨德鸿   

  • 出版日期:2013-07-20 发布日期:2013-07-20

Establishment of HEK293 cell lines stably expressing human parathyroid hormone receptors

  • Online:2013-07-20 Published:2013-07-20

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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Not Ⅰ双酶切，经T4
DNA 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EcoR
Ⅰ与Not Ⅰ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Not Ⅰ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。

Abstract: 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.