LRP16基因启动子的亚克隆及表达调控载体的构建

Sub-cloning of LRP16 gene promoter and constructing of expre ssion regulatory vectors

目的 :为深入研究LRP16基因的表达调控机制 ,克隆及亚克隆了LRP16基因的启动子序列 ,构建LRP16基因启动子表达调控载体。方法 :在NCBI的人类基因组数据库中截取并下载LRP16基因转录起始位点 5′侧翼区约 3kb的基因组序列设计PCR扩增引物 ,从健康外周血中扩增获得该片段 ,以此序列为基础进行亚克隆 ,分别获得 6条 5’端不等、3’端平齐的片段 ,最后插入用于表达调控研究的 pGL3 Basic载体。 结果 :获得了 7条长度依次差别约为 4 0 0bp的LRP16启动子克隆 ,分别构建了调控荧光素酶报告基因的真核表达载体。结论 :上述载体的成功构建为信息资源与实验手段的有效结合克隆启动子序列提出了一种模式 。

Objective:To explore the possible regulation m echanism of LRP16 gene expression and to clone LRP16 gene promoter molecule, sub promoter molecules and to construct sub LRP16 gene promoter pGL3 Basic vect ors Methods: A 2 7kb DNA sequence of LRP16 5′ end was obtain ed from NCBI by BLAST software. 7 different long target sequences from a healthy blood donor DNA sample were amplified by PCR amplification, then the products w ere identified by DNA sequencing and nest PCR Insert these 7 identified sub L RP16 promoter sequences into pGL3 Basic vectors. Results: All 7 LRP16 promoter sequences were successfully cloned and 7 sub LRP16 promoter pG L3 Basic vectors were well constructed. Conclusion:A known gene promoter sequence and sub promoter sequence can be freely obtained from NCBI d atabase, and this is very useful for the gene promoter cloning, sub cloning and promoter vector constructing and sub promoter vector constructing