人尿嘧啶糖基化酶cDNA的克隆及其在食管鳞癌组织中的检测  

Cloning of Human Uracil N-glycosylase and Its Detection in Cancer Tissues by Quantitative RT-PCR

作　　者：鲍洪波[1] 张传宝[1] 王晋芳[1] 周传农[2] 刘芳[2] 赵晓航[2] 钱世钧[1] 

机构地区：[1]中国科学院微生物研究所,北京100080 [2]中国医学科学院肿瘤研究所分子肿瘤学国家重点实验室,北京100021

出　　处：《生物工程学报》2003年第5期561-565,共5页Chinese Journal of Biotechnology

基　　金：国家自然科学基金重大项目资助 (No .3 9990 5 70_4) ;传感技术国家重点实验室项目~~

摘　　要：尿嘧啶糖基化酶是碱基切除修复过程的起始酶 ,对于维护基因稳定具有重要意义。在不同组织及不同细胞周期中 ,该酶的表达水平存在差异。通过反转录PCR克隆了人尿嘧啶糖基化酶的cDNA编码序列 ,进一步以克隆所得的已知UNG基因拷贝数的重组质粒作为定量标准 ,通过实时荧光定量RT_PCR测定了食管癌病人手术切除组织中尿嘧啶糖基化酶的mRNA水平 。The uracil in DNA comes from either the misincorporation of dUTP in place of dTTP or deamination of cytosine. In the latter case, it can result in a GC to AT transition mutation if the uracil is not removed before DNA replication. Base excision repair (BER) is a major pathway for removing DNA lesions arising from endogenous processes as well as those induced by exposure to exogenous chemicals or irradiation. BER is initiated by DNA glycosylases that excise aberrant bases from DNA by cleavage of the N_glycosidic bond linking to the base of its deoxyribose sugar. Uracil N_glycosylase (UNG) is the enzyme responsible for the first step in the BER pathway that specifically removes uracil from DNA. The UNG gene undergoes both temporal and spatial regulation mainly at the level of transcription. Normally cancer cells undergo over_proliferation and up_regulate their UNG during tumorigenesis. In this study we examine the correlation between UNG level and carcinogenesis, and explore the possibility of using UNG as a marker for cancer diagnosis. Human UNG gene was amplified from the total RNA of the human choriocarcinoma cell line, JEG_3, by RT_PCR. After purification, the 942bp full_length UNG cDNA coding sequence was digested with EcoR Ⅰ and Sal Ⅰ, and cloned into the digested pET_21 to construct a recombinant vector, pUNG. The UNG protein was expressed under the control of T7 promoter in E.coli BL21 (DE3) cells induced with IPTG. After ultrasonic treatment, the cell lysate and precipitate were analyzed by SDS_PAGE and a 39kD band was detected. The plasmid was serially diluted at appropriate concentrations and employed as standards in the subsequent quantification. Total RNAs were extracted from 18 pairs of clinical samples, each pair contains a sample of esophageal squamous cell carcinoma (ESCC) tissue and its surrounding normal esophageal epithelia. The copy numbers of UNG mRNA in these RNA samples were determined by real_time quantitative RT_PCR using a Lightcycler (Roche). UNG was prese

关 键 词：人尿嘧啶糖基化酶 CDNA 克隆 食管鳞癌 定量PCR 

分 类 号：R735.1[医药卫生—肿瘤]