海人藻酸受体亚基GluR6真核表达载体的构建及其表达

目的构建海人藻酸受体亚基GluR6的真核细胞表达载体,并在COS-7细胞中表达。方法根据GenBank数据库中GluR6的cDNA序列（Z11548）,分3段分别设计并合成3对引物。从大鼠海马组织中提取总RNA,采用RT-PCR方法获得目的基因片段,分别克隆至pGEM-T载体中。经测序确证后,将该3段cDNA片段依次与pcDNA3.1（＋）载体定向连接。将鉴定正确的重组体以脂质体法转染至COS-7细胞中,免疫印迹法检测GluR6蛋白质表达水平。结果克隆了GluR6的cDNA,并构建了其真核表达载体GluR6-pcDNA3.1,经限制性内切酶酶切鉴定及测序分析证实了其序列的正确性;免疫印迹分析显示,GluR6在COS-7细胞中表达水平较高。结论成功构建GluR6-pcDNA3.1真核表达载体,并且在COS-7细胞中得到高效表达。

Construction and expression of eukaryotic expression vector for kainate receptor subunit GluR6

Objective To construct the eukaryotic expression system for kainate receptor(KA) subunit GluR6,and express the vector in COS-7 cells.Methods Three GluR6 cDNA fragments were cloned by RT-PCR with the total RNA from rat hippocampus as the template,and subsequently cloned into pGEM-T vector,respectively.The three fragments with their sequences confirmed,were then cloned into pcDNA3.1( ) vectors directionally.The right recombinant was transfected to COS-7 cells by Lipofectamine 2000.The GluR6 expression was detected by immunoblot analysis.Results The correct GluR6-pcDNA3.1 cloning was verified by restriction endonuclease digestion and sequencing.The immunoblot analysis indicated that GluR6-pcDNA3.1 was highly expressed in COS-7 cells.Conclusion GluR6-pcDNA3.1 was constructed successfully and could be abundantly expressed in COS-7 cells.