胰岛素诱导基因-2启动子荧光素酶报告基因重组质粒的构建与鉴定

Construction and identification of recombinant plasmid containing insulin induced gene-2 promoter luciferase reporter

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 recombinant plasmid containing human insulin induced gene-2（Insig-2） promoter luciferase reporter,and determine its transcriptional activity.Methods The promoter fragment at a length of 1 389 bp upstream at transcription site of insig-2 gene was amplified by PCR from human genomic DNA and inserted into vector pGL3-basic.The constructed recombinant plasmid pGL3-Insig-2 and internal reference plasmid pRL-TK were transiently co-transfected to HEK293,HepG2 and 3T3-L1 cells,and determined for Insig-2 promoter activity by dual-luciferase reporter assay.Results Restriction analysis and sequencing proved that recombinant plasmid pGL3-Insig-2 was constructed correctly.The recombinant plasmid showed promoter activity in HEK293,HepG2 and 3T3-L1 cells,which were 28,40 and 214 times of those in negative control group（empty vector pGL3-basic） and 2.6,5.5 and 30 times of those in positive control group（pGL3-SV40）,respectively.Conclusion Recombinant plasmid containing Insig-2 promoter luciferase reporter was successfully constructed,which laid a foundation of further study on the gene.