LRP16基因启动子的克隆及荧光素酶报道重组子的构建

Molecular cloning of LRP16 gene promoter and constructing LRP16 promoter-pGL3-Basic vector

目的 :克隆LRP16基因启动子及构建LRP16基因启动子_pGL3_Basic载体。方法 :①以LRP16基因全长作为探针 ,在NCBI的人类基因组数据库中搜索并下载LRP16基因 5′端 3kb基因组DNA序列。②以健康献血员基因组DNA为模板进行PCR扩增目的片段 ,将分离纯化的PCR产物用T4DNA连接酶与pGEM_TEasy载体相连 ,转入JM10 9感受态化大肠杆菌 ,筛选阳性克隆并测序 ,进行KpnⅠ和BamHⅠ双酶切和巢式PCR鉴定。③将LRP16启动子_pGEM_TEasy载体再次酶切 ,纯化回收鉴定过的目的DNA片段 ,构建LRP16基因启动子_pGL3_Basic载体。结果与结论 :长度为 2 .7kb的LRP16基因启动子克隆成功 ,并构建了LRP16基因启动子_pGL3_Basic载体。充分利用因特网上人类基因组数据库的生物信息资源 ,搜索已知基因的启动子序列 ,可实现快速。

Objective:To clone LRP16 gene promoter and construct LRP16 gene promoter_pGL3_Basic vector.Methods:①A 2.7?kb DNA sequence of LRP16 5′_end was obtained from NCBI by BLAST software.②After the 2.7?kb target sequence from a healthy blood donor DNA sample was amplified by PCR method,the product of PCR was inserted into pGEM_T Easy vector by T4 DNA ligase and the vector was transferred into JM109 E.coli . The positive clone was identified by nest PCR, double digestion with Kpn Ⅰ and Bam HⅠ and DNA sequencing.③The identified target LRP16 promoter DNA sequence was regained from LRP16 promoter_pGEM_T Easy vector by Sac Ⅰ and Hin dⅢ cutting, then was inserted into pGL3_Basic vector to construct the target LRP16 promoter_pGL3_Basic vector.Results and Conclusion:A 2.7?kb LRP16 promoter was successfully cloned and the LRP16 promoter_pGL3_Basic vector was well constructed.A known gene promoter sequence can be freely obtained from NCBI database, which is very useful for the gene promoter cloning and promoter vector construction.