鸭CD8α基因启动子区的克隆及荧光素酶报告基因重组体的构建

摘要

旨在了解分化群α基因可能的调控序列及其转录调控机制.本研究利用基因组步移技术扩增鸭CD8α基因的启动子区序列,使用在线软件进行序列分析；分别将鸭肝炎易感组和抗性组的CD8α基因启动子区定向亚克隆至荧光素酶表达载体pGL3-Basic中,利用酶切与测序技术进行鉴定;并瞬时转染细胞,采用荧光素酶报告基因系统检测启动子载体的活性.结果,扩增出一条长度为2 480 bp的片段(包含第一外显子56 bp,启动子区2 424 bp).经序列分析,鸭CD8α基因启动子区具有典型的TATA-box、GC-box和CAAT-box,其转录起始位点位于翻译起始密码子ATG上游-406 bp处,且发现了CdxA、Nkx-2、GATA-1、SRY等41个潜在转录因子结合位点.经酶切与测序鉴定,成功构建了鸭CD8α基因荧光素酶报告基因重组体.荧光素酶报告基因检测系统显示,构建的鸭肝炎易感组和抗性组的报告基因启动子载体具有相当的活性.研究结果为进一步探讨CD8α基因的转录调控奠定了基础.%This experiment was conducted to explore the potential regulatory sequences of duck CD8α gene and the mechanism of transcription regulation. The CD8α gene promoter was cloned by genome walking, the sequence of the promoter was analyzed by online software of bioinforma-tics, and then the promoters from DHV-resistant and DHV-susceptible duck were subcloned into the luciferase expression vector pGL3-Basic directly, and their luciferase activity was detected. The DNA sequence of 2 480 bp was amplified including 56 bp in exonl and 2 424 bp of promoter region. There were TATA-box, GC-box, CAAT-box and transcriptional start site in the sequence. 41 transcriptional factor binding sites including CdxA, Nkx-2, GATA-1, SRY, and so on, were detected. The luciferase reporter gene recombinant pGL3- CD8