研究G偶联蛋白受体G2A功能特定细胞模型的建立

Establishment of Specific Cell Model for Functional Study to Proton-sensing G Protein Coupled Receptor G2A

通过克隆人质子感知受体G2A基因、构建G2A基因的表达载体并瞬时转染293T细胞,建立G2A受体调控机理及其功能研究的转基因细胞模型,并检测G2A介导的细胞应答.从人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cells,HUVECs)中提取总RNA,反转录为cDNA为模板,设计一对针对G2A基因的引物,克隆不含终止密码子的G2A基因并亚克隆到pMD-19T载体.将鉴定出的阳性质粒和表达载体pEGFP-N3用HindⅢ和BamHⅠ双酶切回收后连接获得阳性重组质粒pEGFP-N3-G2A,并测序鉴定;重组载体以lipofectamine2000介导转染293T细胞.Real time PCR法检测外源基因在293T细胞中的表达;ELISA检测转染与未转染G2A基因的细胞内三磷酸肌醇(Inositol-1,4,5-triphosphosate,IP3)的含量.测序和酶切证实了克隆到G2A基因并获得pEGFP-N3-G2A表达载体;荧光成像与Real time PCR证实在293T细胞中过表达G2A基因;ELISA检测在酸性刺激下转基因细胞内IP3含量(应答)明显升高,而脂质配体LPC则抑制其IP3的积累,本研究为G2A受体介导信号机制及其功能研究提供一个真核细胞模型.

The G2A expression vector with cloned Homo proton-sensing receptor G2A was tran- siently transfected into 293T cells to study the expression and function of G2A. A pair of special primer was designed according to complete CDS sequence of G2A. The target gene G2A without stop codon was amplified from cDNA which was reverse transcribed from HUVECs＂ total RNA by PCR and subcloned into pMD19-T vector, pMD-19T-G2A and expression vector pEGFP-N3 were digested by Hind]]l and BamH I . G2A was ligated into pEGFP-N3 N3-G2A. Then the recombinant plasmids were transfected The exogenous G2A expression in 293T cells was ana of IP3 in transgenic and non-transgenic cells was ana yzed yzed pEGFP-N3-G2A expression vector was constructed successf to get the recombinant plasmid pEGFP- into 293T cells with lipofectamine2000. by Real time PCR and the concentration by ELISA. The results showed that： 1） ully;2） fluorescence microscope photog- raphy and Real time PCR confirmed that G2A was over-expressed in 293T cells^3） ELISA assay re- vealed that the concentration of IP3 in transgenic cells was significantly higher than that in non- transgenic cells under acid stimulation, while it＇s lipid ligand LPC suppressed IP3 accumulation by a- cidic pH. The study provides a specific eukaryotic cell model for clarifying G2A receptor mediated signaling mechanism and its function.