中国现代医学杂志2017，Vol.27Issue(11)：8-13,6.DOI:10.3969/j.issn.1005-8982.2017.11.002
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
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 XhoⅠ, EcoRⅠ 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 inser tion 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 homopoly- mer 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.关键词
ASIC2a/载体构建/真核表达载体/绿色荧光蛋白Key words
ASIC2a/vector construction/eukaryotic expression vector/green fluorescent protein分类
生物科学引用本文复制引用
李栋,张康,马晓芸,袁维秀,刘晓燕..真核表达载体pEGFP-N1-ASIC2a的构建及功能验证[J].中国现代医学杂志,2017,27(11):8-13,6.基金项目
国家自然科学基金(No:31400917) （No:31400917）
