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

摘要

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 healthy 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 pGL3-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.%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2 kb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。