Snail启动子荧光素酶报告基因载体的构建及其应用  

Construction and application of luciferase reporter gene vector containing Snailpromoter

作　　者：江冠民[1] 李玲玲[2] 张坤水[3] 谢婉莹[1] 张秋桂[1] 杜军[4] 

机构地区：[1]南华大学附属第一医院检验科,湖南衡阳421001 [2]中山大学附属第五医院中心实验室,广东珠海519000 [3]中山大学附属第二医院药剂科,广东广州510120 [4]中山大学药学院微生物与生化药学,广东广州510006

出　　处：《中国现代医学杂志》2013年第31期12-17,共6页China Journal of Modern Medicine

基　　金：湖南省自然科学基金(No:13JJ4078);国家自然科学基金(No:81071712;81272311);国家"973"计划资助项目(No:2011CB935800)

摘　　要：目的构建Snail启动子荧光素酶报告基因载体pGL3-Basic-Snai1-luc并对其进行生物活性的鉴定及其初步的应用研究。方法通过PCR技术从人血液基因组中钓出我们需要的具有启动子活性的Snail片段,双酶切后与双酶切的pGL3-Basic空载体连接成重组体pGL3-Basic-Snai1-luc,通过转化扩增,筛选出阳性克隆,并通过酶切,测序及生物学活性检测鉴定构建好的荧光素酶报告基因载体。结果成功地构建了pGL3-Basic-Snai1-luc荧光素酶报告基因载体,并在CNE2细胞内鉴定了其在TGF-β1的诱导下,能启动荧光素酶的表达。此外,笔者通过Western blotting检测表明TGF-β1能显著性的诱导Snail蛋白的表达,充分证明了我们构建的pGL3-Basic-Snai1-luc是具有生物学功能的。结论具有生物活性的pGL3-Basic-Snai1-luc的构建成功为研究Snai1在EMT过程中的调控机制研究和以snail为靶标的抗肿瘤药物快速高通量的筛选提供了非常重要的研究工具。【Objective】To construct Snail promoter luciferase reporter gene vector pGL3-Basic-Snai1-luc, then i- dentify the biological activity and carry out preliminary application study.【Methods】The gene fragment of promoter sequence of snail was amplified from human blood genomic by PCR and was cloned into empty vector pGL3-Basic to construct luciferase reporter gene vectors pGL3-Basic-Snai1-luc, which was amplified by transformation and i- dentified by restriction enzyme digestion, sequencing, and the biological activity detecting. 【Results】We construct- ed the luciferase reporter vector pGL3-Basic-Snai1-luc successfully, the expression of luciferase could be induced by TGF-β1 in CNE2 cell line. Furthermore, Western blotting analysis showed that snail protein expression could also be induced by TGF-β which demonstrated that the recombinant had biological activity.【Conclusion】The luciferase reporter gene vector pGL3-Basic-Snai1-luc was constructed successfully and the biological activity was identified, which provides a very important tool for studying the role of snail in the regulation process of EMT and supplying a fast, high-throughput screening for anti-tumor drugs which targeting the snail.

关 键 词：上皮细胞间质变 SNAIL 肿瘤转移 启动子 

分 类 号：R733[医药卫生—肿瘤]