人IP-10启动子的克隆和转录活性的检测

Cloning of human IP-10 promoter and identification of its transcriptional activity

目的克隆干扰素诱导蛋白10(IP-10)5′非编码区(NCR)片段,检测克隆的IP-10启动子驱动的荧光素酶报告基因在LPS激活的人脐静脉内皮细胞(HUVEC)中的转录活性。方法提取人外周血淋巴细胞基因组DNA,以其为模板,利用巢式PCR扩增人IP-10启动子片段,测序正确后克隆入荧光素酶报告基因表达载体pGL3;将重组质粒pGL3/IP-10P导入HUVEC细胞,检测LPS刺激下荧光素酶活性。结果正确克隆出人IP-10启动子(-2028^-1)片段,成功构建了pGL3/IP-10P报告基因表达载体;证实了LPS可诱导HUVEC细胞中IP-10的高表达。结论成功克隆出人的IP-10启动子片段,并利用荧光素报告基因证实了在HUVEC细胞中LPS可诱导IP-10高转录表达,为深入研究LPS诱导IP-10转录表达的调控机制提供了基础。

Objective To clone the 5＇ nor-coding region （NCR） of human interferon-y-inducible protein 10（IP-10）, and to identify the transcriptional activity of IP-10 promoter induced by lipopolysaccharide （LPS） in human umbilical vein endothelial cells （HUVEC）. Methotis Genomic DNA of lympbocytes was isolated from the human blood. With above DNA as the template, the 5NCR of human IP-10 was amplified by nest polymerase chain reaction （PCR） method. Then, the IP-10 promoter was cloned into luciferase reporter vector, pGL3. The recombined vector was transfected into HUVEC, and then the activity of the luciferase was determined after the cells were stimulated by I.PS. Results Human IP-10 promoter was obtained and the pGL3/IP-10 was successfully constructed. Moreover, the activity of luciferase driven by human IP-10 promoter was observed to obviously increase in the HUVEC stimulated by LPS. Conclusion We successfully cloned human IP-10 promoter, constructed luciferase reporter vector driven by the human IP-10 promoter, and confirmed that high transcriptional activity of human IP-10 promoter was induced by LPS in HUVEC. The results supplied an experimental base for the further study of the transcriptional regulation of human IP-10.