pHCN4-DsRed Express2慢病毒载体的构建和鉴定  

Construction and identification of pHCN4-DsRed Express2 lentiviral vector

作　　者：李颖[1] 高飞[1] 蔡军[1] 秦选光[1] 

机构地区：[1]首都医科大学附属北京朝阳医院儿科,北京市100020

出　　处：《中国心血管病研究》2010年第8期593-596,共4页Chinese Journal of Cardiovascular Research

摘　　要：目的构建超极化激活环核苷酸门控阳离子通道基因亚型4启动子（HCN4）重组慢病毒载体并进行鉴定。方法采用Gateway克隆技术，通过attBxattP反应，构建穿梭质粒pup—HCN4，而后将pLV／Des2-Puro、pUP—HCN4、pDown—DsRed Express2三个质粒通过attLxattR反应，构建成pLV／EXPN2-Puro-HCN4-DsRed Express2质粒，再转染感受态细胞stb13，孵育扩增，离心纯化。采用PCR方法对重组慢病毒载体进行鉴定。结果基于位点特异性重组原理，应用Gateway克隆技术构建了多基因共表达的pHCN4-DsRed Express2慢病毒载体，通过提取DNA测序，证明该序列与理论值一致。结论应用Gateway克隆技术成功构建了pHCN4-DsRed Express2慢病毒载体。Objective To construct and identification of pHCN4-DsRed Express2 Lentiviral vector. Methods The Gateway Clone Technology was used in our experiment. First,the entry clones pUp-HCN4 were constructed by BP recombination reaction. Secondly, performed an LR recombination reaction among pUP-HCN4, pDOWN-DsRed Eepress 2 and pLV/Des2-Puro to generate expression clones. Finally, plasmids were identified by sequence analysis. Results The sequence analysis confirmed that the HCN4 promoter was successfully inserted into the lentiviral vector. Conclusion The recombinant lentiviral containing HCN4 promoter is successfully constructed by the method of the Gateway Technology.

关 键 词：HCN4 慢病毒载体 Gateway克隆技术 

分 类 号：Q782[生物学—分子生物学] Q785