摘要
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。
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 direetionally. The right recombinant was transfected to COS - 7 cells by Lipofectamine 2000. The GluR6 expression was detected by immunoblot analysis. Re- sults 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.
出处
《徐州医学院学报》
CAS
2007年第6期354-357,共4页
Acta Academiae Medicinae Xuzhou
基金
江苏省教育厅重大基础研究项目(06KJA31026)
江苏省六大人才高峰项目(06-B-039)
