人ID4基因表达调控启动子及其亚克隆荧光素酶报道重组子的构建  

Cloning of ID4 Gene Expression Regulation Promoter and Subcloning of Recombinant ID4 Promoter Luciferase Reporter

作　　者：李薇[1] 黄玉[1,2,3] 杨波[2] 迟小华[4] 刘丽宏[4] 张峰[3] 严江伟[3] 卢学春[2] 

机构地区：[1]吉林大学第一医院肿瘤中心,吉林长春130021 [2]解放军总医院老年血液科,北京100853 [3]中国科学院北京基因组研究所,北京100029 [4]解放军第二炮兵总医院药剂科,北京100800

出　　处：《中国实验血液学杂志》2010年第2期421-426,共6页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目(编号30873086);国家自然科学基金资助项目(编号30772597)

摘　　要：本研究旨在克隆ID4基因启动子及上游调控序列,并构建一系列启动子亚克隆-pGL3Basic荧光素酶报道重组子,以探讨ID4基因表达调控机制。方法与结果:以ID4基因cDNA全长作为探针,在NCBI的人类基因组数据库中搜索并下载ID4基因转录起始位点上游5′侧翼区约2242bp及下游5′非翻译区212bp的基因组序列;利用TESS和Genomax等在线启动子分析软件进行启动子及上游调控元件的特征分析,据此设计PCR引物,采用分段扩增法,获得了2条长度分别为1 829bp和784bp的产物片段,分别插入pGEM-T载体,转化至TOP10感受态大肠杆菌,筛选阳性菌落;继之先后应用KpnI/NheI、KpnI/EcoRI对获得的2个pGEMT重组载体及pGL3Basic荧光素酶报道重组载体进行双酶切,用T4DNA连接酶连接,转化至TOP10感受态大肠杆菌,筛选阳性菌落,成功构建了人ID4基因启动子-pGL3Basic荧光素酶报道重组子;经KpnI/NheI双酶切和测序鉴定,获得了与GenBank相应序列一致、长度为2 459bp的目的片段;以此片段为模板,相隔400bp左右设计了5对3′端平齐、5′端不同的PCR引物,进行半巢式PCR扩增,获得5条长度分别为2 112bp、1 703bp、1 290bp、784bp和496bp片段,回收纯化后分别插入pGEM-T载体,转化至TOP10感受态大肠杆菌,筛选阳性菌落;继之用KpnI/NheI对获得的5个pGEMT重组载体及pGL3Basic荧光素酶报道载体双酶切,T4DNA连接酶连接,转化至TOP10感受态大肠杆菌,筛选阳性菌落,经KpnI/NheI双酶切和测序鉴定。结论:成功克隆了长度为2.5kb的ID4基因启动子及上游表达调控序列,构建了一系列ID4启动子亚克隆-pGL3-Basic荧光素酶报道重组子。The present study was aimed to clone ID4 gene promoter and upstream regulatory region, and to construct a series of recombinant promoter-luciferase reporter for exploring the mechanism of/D4 gene expression regulation. Methods and results . the upstream 5＇ flanking sequence of 2 242 bp from transcriptional start site （ TSS ） and downstream 5＇ non-coding region of 212 bp on ID4 gene were searched out and downloaded from human genome databank of NCBI using whole length of ID4 gene cDNA as a probe; On-line promoter analysis softwares, including TESS and Genomax, were employed to analyze the characteristics of ID4 gene promoter and upstream regulatory elements. Then, based on the analytic results, PCR primers were designed and synthesized. Segmental amplification method was adopted to obtain two fragments of 1 829 bp and 784 bp. The two fragments were inserted into the plasmid pGEM-T, transformed into TOP10 competent E. coll. , and positive recombinants were screened respectively. Subsequently, restriction enzymes KpnI/NheI and KpnI/EcoRI were used to digest the above-mentioned two plasmids pGEM-T and pGL3, and ligation was completed by T4 DNA ligase. After transformation to TOP10 competent E. coli. and screening of positive colonies, the basic recombinant ID4 gene promoter-pGL3 was successfully constructed. KpnI/NheI double digestion and sequencing showed that the target fragment was 2 459 bp and consistent with the corresponding sequence of GenBank; Using the 2 459 bp fragment as a template, 5 pairs of primers with identical 3＇ terminus and different 5＇ terminus were designed and synthesized for half-nest PCR amplification. 5 fragments with an interval of approximate 400 bp each other, i.e. 2 112 bp, 1 703 bp, 1 290 bp, 784 bp and 496 bp, were produced and inserted into pGEM-T after recovery and purification for transformation to TOP10 competent E. coll. and screening of positive colonies. After that, KpnI/ NheI was used to digest the above-mentioned five pGEM-T recombinant plasmids and pGL3 basic vecto

关 键 词：ID4基因 启动子 荧光素酶报道重组子 聚合酶链式反应 

分 类 号：R730.23[医药卫生—肿瘤]