STGC3基因启动子生物信息学分析及载体构建  

Bioinformatics analysis of s TGC3 gene promoter and construction of dual luciferase report gene expression Vector

作　　者：李素云[1,2] 李佳[3] 王莉莉 钟嘉宏 陈青[3] 熊文芳[3] 曾小微 贺修胜[1] 

机构地区：[1]南华大学肿瘤研究所,湖南衡阳421001 [2]南华大学医学院解剖学教研室 [3]南华大学医学院2013级临床医学专业 [4]湖北省大冶市人民医院病理科

出　　处：《中南医学科学杂志》2017年第1期48-53,共6页Medical Science Journal of Central South China

基　　金：国家自然科学基金资助项目(2011GZK46);湖南省科技厅资助项目(2012TT2020);湖南省大学生研究性学习与创新性实验资助项目(231)

摘　　要：目的运用生物信息学预测并分析鼻咽癌抑癌基因STGC3启动子,构建双荧光素酶报告基因表达载体。方法采用生物信息学软件预测STGC3基因5'端上游调控区5 000 bp序列进行启动子预测与分析,克隆最有可能的启动子,构建萤火虫荧光素酶双报告基因重组质粒,重组质粒经MluⅠ和BglⅡ限制性内切酶酶切及测序鉴定。结果 STGC3基因5'端上游-3 046 bp至-46 bp区域内有启动子活性,在-2 992 bp至-69 bp间启动子分值达0.8以上,其中-845 bp至-795 bp间分值最高,达到1.0。在-1 140 bp和-774 bp处检测到GC盒信号;在-441 bp处检测到CAAT盒信号。在-1 805 bp至-1 705 bp和-900 bp至-684 bp间各有一个Cp G岛;在-2 348 bp和-948 bp处各有一个转录起始位点。经不同长度的片段缺失比对,取最有可能的283 bp(-1 360 bp至-1 077 bp)、281 bp(-934 bp至-653 bp)和571 bp(-500 bp至+72 bp)启动子片段构建p GL3-en283、p GL3-en281及p GL3-en571三种质粒,质粒经酶切测序,酶切片段大小一致,序列正确。结论根据生物物信息学分析结果,成功构建STGC3基因启动子萤火虫荧光素酶报告基因表达载体,为双荧光素酶报告基因检测系统研究该基因启动子活性提供前期基础。Objective Using bioinformatics to predict and analyse the promoter of STGC3 gene,then construct dual luciferase reporter gene expression vector for 5＇ Upstream regulated elements of STGC3 gene. Methods The bioinformatics was used to predict and analyze the promoter region of STGC3 during gene 5＇upstream 5000 bp regulatory region,and then the most likely promoters were cloned.The dual-luciferase reporter gene plasmids were constructed,and these recombinant plasmids were identified by the MluⅠand BglⅡenzyme digestion and DNA sequencing. Results The bioinformatics results show that there are promoter activity from-3 046 bp to-46 bp.The activity value of-2 992 bp to-69 bp is 0.8 and-845 bp to-795 bp is 1.0.There is a GC box in-1 140 bp to-774 bp and a CAAT box in-441 bp.Two Cp G islands were detected in-1805 bp to-1 705 bp and-900 bp to-684 bp.Two transcription start sites（-2 348 bp and-948 bp） were found.The most possible fragments 283bp（-1 360 bp to-1 077 bp）,281 b P（-934 bp to-653 bp） and 571 b P（-500 bp to-72 bp） were chosen to construct plasmids p GL3-en283,p GL3-en281 and p GL3-en571. These plasmids were identified by enzyme digestion and sequencing,the size of the fragment and the sequence was correct. Conclusion Based on the results of bioinformatics analysis,the successful construction of STGC3 gene dual luciferase report gene expression vector,provides a preliminary basis for the dual luciferase report gene detection system of the gene promoter activity.

关 键 词：鼻咽癌 生物信息学 STGC3基因 启动子 

分 类 号：R739.63[医药卫生—肿瘤]