真核表达载体pEGFP-N1-ASIC2a的构建及功能验证

Construction of rat pEGFP-N1-ASIC2a eukaryotic expression vector and its functional verification

摘要:

目的  构建含大鼠ASIC2a全长互补脱氧核糖核酸（cDNA）的绿色荧光蛋白真核表达质粒pEGFP-N1-ASIC2a并转染至HEK293细胞，观察其表达和分布情况。方法  设计、合成ASIC2a基因引物，利用聚合酶链反应（PCR）技术，以现有质粒pcDNA3.1-ASIC2a为模板扩增出含有限制性内切酶（Xho Ⅰ与EcoRⅠ）酶切位点的ASIC2a基因片段与载体pEGFP-N1酶切后连接形成重组质粒pEGFP-N1-ASIC2a，并瞬时转染至HEK293细胞中，利用细胞膜片钳方法观察其表达电流的情况。为进一步明确其亚细胞定位，将质粒EGFP-N1-ASIC2a、pDsRed-Monomer-Mem和pDsRed2-ER共同转染至HEK293细胞中。结果  PCR扩增得到正确的ASIC2a基因片段，酶切鉴定和测序证实获得重组质粒pEGFP-N1-ASIC2a，并将其瞬转至HEK293细胞后，成功记录到ASIC2a同聚体电流。然后利用共转染的方法观察到，pEGFP-N1-ASIC2a主要在HEK293细胞内质网表达，细胞膜表达较少。结论  重组绿色荧光蛋白真核表达载体pEGFP-N1-ASIC2a构建成功，且在HEK293细胞中主要表达于内质网，为下一步研究ASIC2a的功能，特别是为ASIC2a在缺血性神经元损伤中的作用及其机制的研究奠定基础。

Abstract:

Objective To construct the eukaryotic green fluorescent protein expression vector pEGFP-N1-ASIC2a, and to observe its expression and distribution in the HEK293 cells. Methods ASIC2a gene primers were designed and synthesised. ASIC2a gene sequence with Xho Ⅰ, EcoR Ⅰ restriction enzyme cutting site was amplified from the plasmid pcDNA3.1-ASIC2a by reversed transcription-polymerase chain reaction PCR and inserted into the eukaryotic green fluorescent protein expression vector pEGFP-N1 to form a recombinant plasmid pEGFP-N1-ASIC2a. And the plasmid pEGFP-N1-ASIC2a was transfected into HEK293 cells to observe the protein expression of current situation by cell patch clamp technique. The plasmid pEGFP-N1-ASIC2a, pDsRed-Monomer-Mem, pDsRed2-ER were transfected into the HEK293 cells, and the distribution of the protein was observed. Results The correct ASIC2a gene fragment was amplified by PCR. The successful insertion of ASIC2a into the pEGFP-N1 vector was confirmed by restriction and sequence analysis. The recombinant plasmid pEGFP-N1-ASIC2a was successfully constructed in HEK293 cells. The ASIC2a homopolymer channel current was successfully recorded. And more pEGFP-N1-ASIC2a protein expressed in endoplasmic reticulum than in cell membrane were found. Conclusions The recombinant eukaryotic green fluorescent protein expression vector pEGFP-N1-ASIC2a was successfully constructed, and mainly expressed in the endoplasmic reticulum in HEK293 cells. It will be contributed to the further research on the function of ASIC2a, especially help for the studies on the effect of ASIC2a in ischemic neuronal injury and its mechanism of action.