PD-1核心启动子的获得及活性鉴定

PD-1 Core Promoter Acquired and Identification

目的:构建PD-1(programmed death receptor 1)全长启动子及不同截短体的报告基因,并对其转录活性进行检测。方法:通过PCR及双酶切方法,从人全血基因组DNA中获得PD-1基因编码序列,包含不同长度碱基的PD-1启动子序列,分别克隆到报告基因pGL3-Basic载体上,构建8个不同长度PD-1启动子的报告基因;用脂质体转染法将8个报告基因分别转染至Jurkat细胞系;采用双萤光素酶报告基因系统评估PD-1启动子的活性。结果:经PCR方法扩增出大小分别为1650、1450、1250、1128、874、674、474和274 bp的不同长度的PD-1启动子序列,测序正确(与GenBank报道一致),酶切鉴定正确;瞬时转染Jurkat细胞系后经报告基因检测,8个启动子均具有转录活性。结论:构建了PD-1启动子的报告基因,并证实均有转录活性,且以pGL3-1128活性最高,为PD-1的核心启动子,为进一步研究PD-1的转录调控奠定了实验基础。

Objective：To construct the luciferase reporter gene vectors with the whole promoter gene region and different truncated body of PD-1（programmed death receptor 1） and detect the transcriptional activity.Methods：Acquired the PD-1 gene sequences with different length of the promoters from human whole blood genome by PCR and dual-restriction.Subcloned them into luciferase reporter gene vector pGL3-Basic respectively and constructed 8 reporter gene with different length of promoters.Then the recombinant constructs were transfected into Jurkat cells.The promoters activity were evaluated by dual-luciferase reporter assay.Results：The promoter sequence of PD-1 with different length（1650,1450,1250,1128,874,674,474,274 bp） have been amplified by PCR,the sequences were all right（consistent with the sequence showed on GenBank）,and the identification of restriction were right.The 8 promoters all were transcriptional activated after transiently transfected into Jurkat cells.Conclusion：The luciferase reporter gene vectors with PD-1 promoter have been constructed successfully and testi-fied to be activity,and pGL3-1128 had the highest activity.All the results laid the experimental foundation for further study on the transcriptional regulation of PD-1.