反式二羟环氧苯并(a)芘相关新基因brg的全长克隆  

Full-length Cloning of brg, the Anti-BPDE Related Gene

作　　者：安社娟[1] 陈家[2] 陈华洁[1] 常薇[1] 刘莉莉[2] 赵艳丰[2] 陈学敏[1] 

机构地区：[1]华中科技大学同济医学院公共卫生学院劳动卫生与环境卫生学系 [2]广州医学院化学致癌研究所,广东广州510182

出　　处：《癌变．畸变．突变》2006年第4期302-305,共4页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金资助项目(No.30271111)

摘　　要：背景与目的:在前期研究中发现,16HBE-C细胞表达变化的基因中有未知基因参与,因其与BPDE有关,所以命名为brg(anti-BPDErelatedgene)基因。本研究应用cDNA末端快速扩增法(rapidamplificationofcDNAends,RACE)扩增此未知基因的全长,以进行下一步的基因功能研究。材料与方法:应用cDNA末端快速扩增法(RACE技术),对3'和5'端分别设计了梯度PCR,巢式PCR等优化程序,以获得特异的产物,然后对特异产物直接测序,将测序结果进行生物信息学分析,基因拼接等获得新基因brg全长。结果:3'和5'端均成功获得特异性产物,3'RACE测序为394bp,有明显的PolyA加尾信号,有编码区的终止密码子;5'RACE测序为1017bp,有起始密码子ATG,其中197bp为3'和5'全长共有序列。将3'和5'序列拼接,结果全长1214bp,生物信息学分析brg基因阅读框1032bp,编码344个氨基酸。结论:所得结果与设计一致,说明所采用的技术路线是简便可行的,brg基因可能在反式二羟环氧苯并(a)芘的致癌机制中起重要作用。BACIKGROUNG ＆ AIM： Previous studies showed a gene fragment was identified as being over-expressed in 16HBE-C cells when compared to 16HBE cells, which is named brg （anti-BPDE related gene）. The aim of this study was to obtain the full-length cDNA of brg based on its EST fragment and to provide basic data for the functional research of the novel gene. MATERIAL AND METHODS： Based on the rapid amplification of cDNA ends （RACE） method, gradient and nested PCR protocols were used to obtain full-length cDNA in order to get a discrete and bright band on an agarose gel electrophoresis. The nested PCR products were then sequenced and the bioinformatic analysis was carried out. RESULTS： Full-length cDNA of the gene was successfully cloned by both 3′-end RACE and 5′-end RACE. The cDNA had a total length of 1 214 bp. Sequence analysis of the full-length cDNA of the gene revealed that the cDNA encoded an open reading frame of 1 032 bp; with the deduced protein containing 344 amino acids. CONCLUSION： The general and simple protocol was useful for cloning of full-length gene based on EST fragment. The results suggest that brg gene may be involved in carcinogenesis during anti-BPDE related tumor development.

关 键 词：CDNA末端快速扩增 反式二羟环氧苯并(a)芘 基因 

分 类 号：R994.6[医药卫生—毒理学] R730.231.1[医药卫生—药学]