论文部分内容阅读
目的构建胰岛素诱导基因-2(Insulin induced gene-2,Insig-2)启动子荧光素酶报告基因重组质粒,并检测其转录活性。方法从人基因组DNA中扩增Insig-2基因转录起始位点上游1 389 bp的启动子片段,插入pGL3-basic质粒中,构建重组质粒pGL3-Insig-2,将其与内参质粒pRL-TK瞬时共转染HEK293、HepG2和3T3-L1细胞,采用双荧光素酶法检测Insig-2启动子活性。结果重组质粒pGL3-Insig-2经双酶切和DNA测序,证实构建正确;pGL3-Insig-2质粒在HEK293、HepG2和3T3-L1细胞中均具有启动子活性,分别为阴性对照组(pGL3-basic空载体)的28、40和214倍、阳性对照组(pGL3-SV40)的2.6、5.5和30倍,呈现出强启动子活性。结论成功构建了Insig-2基因启动子荧光素酶报告基因重组质粒,为后续Insig-2基因的深入研究奠定了基础。
Objective To construct the luciferase reporter gene of Insulin induced gene-2 (Insig-2) promoter and test its transcriptional activity. Methods A 1 389 bp upstream fragment of Insig-2 gene was amplified from human genomic DNA and inserted into pGL3-basic plasmid. The recombinant plasmid pGL3-Insig-2 was constructed and ligated with plasmid pRL-TK The HEK293, HepG2 and 3T3-L1 cells were transiently co-transfected and the Insig-2 promoter activity was measured by dual-luciferase assay. Results The recombinant plasmid pGL3-Insig-2 was confirmed by double-digestion and DNA sequencing. The pGL3-Insig-2 plasmid had promoter activity in HEK293, HepG2 and 3T3-L1 cells, which were negative control group (pGL3- basic empty vector), 2.6, 5.5 and 30 fold higher than the positive control group (pGL3-SV40), showing strong promoter activity. Conclusion The construction of the Insig-2 gene promoter luciferase reporter gene recombinant plasmid has laid a foundation for the further study of Insig-2 gene.