LCRG1基因5′端调控区域的克隆及活性测定  

Cloning of 5'flank upstream regulation region of LCRG1 and the measurement of promoter activity

作　　者：谢海龙[1] 李金花[1] 

机构地区：[1]南华大学肿瘤研究所细胞生物研究室,湖南衡阳421001

出　　处：《中国癌症杂志》2008年第1期20-25,共6页China Oncology

基　　金：国家自然科学基金(No.30572030)

摘　　要：背景与目的:LCRG1基因的调控机制至今不清楚,本研究拟克隆LCRG1基因的转录起始区上游序列,寻找与其表达有关的调控序列。方法:利用生物信息学技术预测LCRG1基因5′端调控区域的启动子活性区域,采用PCR方法从人基因组DNA中扩增LCRG1基因5′端调控区域1.776kb(-1191bp～+585bp)片段,并将其构建到荧光素酶报告基因pGL3basic载体中。与内参照质粒PhRL-SV40共转染COS7细胞,通过双荧光素酶活性检验测定其启动子活性。结果:成功扩增LCRG1基因5′端调控区域1.776kb(-1191bp～+585bp)片段,测序正确;pGL3-1776启动子活性大约为阳性对照组pGL3-control的0.16倍,为阴性组pGL3basic的35倍。结论:成功克隆LCRG1基因5′端调控区域1.776kb(-1191bp～+585bp),该片段具有启动子活性。Background and purpose： The molecular regulation mechanism of the LCRG1 gnen is unclear; The study was designed to clarify the regulatory elements of LCRG1 gene in its 5＇flanking region. Methods： Bioinformatics approaches were adopted and a putative promoter region was found in 5 ＇flank upstream fragment of LCRG1 gene, 5＇flank upstream regulation fragment 1. 776 kb（from - 1 191 bp to ＋ 585 bp）of LCRG1 gene was amplified by PCR using genomic DNA as template, construct was obtained by cloning DNA fragments into pGL3 reporter vector. The construct was then introduced into COS7 cells by Lipofectemin method for transient expression of reporter gene, and luciferase activities was measured by luciferase assay. Results： The sequence of 5＇flank upstream regulation fragment 1. 776 kb（ from -1 191 bp to ＋585 bp）of LCRG1 gene was successfully cloned and proved to be correct by DNA sequencing , the activity of pGL3-1776 was about 0.16-fold higher than that of pGL3-control cotransfection with PRL-TK and 35-fold higher than that of pGL3- basic cotransfection with PRL-TK. Conclusions： 5＇flank upstream regulation region 1. 776 kb（ from - 1 191 bp to ＋ 585 bp） of LCRG1 was cloned successfully, the fragment presented promoter activity.

关 键 词：LCRGl 喉癌 启动子 生物信息学技术 PCR 

分 类 号：R730.231[医药卫生—肿瘤]