载脂蛋白M基因启动子的克隆及荧光素酶报告基因载体的构建

Cloning of ApoM gene promoter and constructing of luciferase report gene vector

目的为深入研究载脂蛋白M(ApoM)基因的表达调控机制,对ApoM基因启动子序列进行克隆,并构建不同长度启动子荧光素酶报告基因载体。方法在NCBI人类基因组数据库中截取并下载ApoM基因转录起始位点5’侧翼区约2kb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pGL3-Basic表达载体。结果获得了6条长度差别约为200bpApoM启动子片段,并构建了不同长度的pGL3-ApoM真核表达载体。结论上述载体的成功构建及序列分析为进一步研究ApoM基因的启动子活性及基因表达调控奠定了基础。

Objective To construct luciferase report gene vector for ApoM gene promoter in order to provide foundation for further study on the possible regulation mechanism of ApoM gene expression. Methods A 2 kb DNA sequence of ApoM 5＇ end was obtained from NCBI by BLAST software. 6 different long target sequences from a heahhy blood donor DNA sample were amplified by PCR amplification,then the products were identified by DNA sequencing. The ApoM promoter subclone fragments were inserted into the pGLB-basic vector. Results The 6 identified ID4 promoter sequences with an interval of approximate 200 bp were successfully cloned and 6 sub-ApoM promoter- pGL3 basic recombinants were constructed. Conclusion These results may make an important basis for the further study of ApoM promoter activity and regulation of gene expression.