一种可特异识别Hmox1增强子的人工转录因子的构建  

Construction of a Hmoxl enhancer specific artificial transcription factor

作　　者：郭洪峰[1] 魏勇[1] 应大君[1] 

机构地区：[1]第三军医大学解剖学教研室重庆市高校直辖市重点实验室,重庆400038

出　　处：《解剖学杂志》2009年第3期333-338,344,F0002,共8页Chinese Journal of Anatomy

基　　金：国家自然科学基金（30672535）.

摘　　要：目的：设计一种可特异识别人血红素加氧酶-1基因（Hmox1）增强子的人T转录因子。方法：以锌指蛋白（ZFP）作为人工转录因子的DNA结合结构域，在人Hmox1增强子上选择一段序列作为锌指蛋白靶序列，提交Zinc finger tools3.0设计得到该锌指蛋白的氨基酸序列，通过Swiss Model同源建模，对设计结果进行分析；将锌指蛋白与效应结构域相连组成完整的人工转录因子，用双荧光素酶报告基因检测系统检测其活性。结果：得到了能特异识别人Hmox1增强子的锌指蛋白氨基酸序列，同源建模结果显示其空间结构稳定，所构建的完整人工转录因子经双荧光素酶报告基因检测系统检测能特异上调报告基因的表达。结论：设计得到的人工转录因子经实验验证能特异识别人Hmox1增强子并有效上调报告基因的表达，为进一步构建可上调细胞核内Hmox1表达的人工转录因子奠定了基础。Objective： To design a Hmox1 enhancer specific artificial transcription factor （ATF）. Methods： Select a sequence from the enhancer of human Hmoxl as target site of the zinc finger protein （ZFP）, which was used as DNA-binding domain of the ATF. Submit the DNA sequence to Zinc finger tools 3.0 to obtain the amino acid sequence of the ZFP, and evaluate the result by homologous modeling through Swiss Model. Combine the ZFP with function domain to form the integrated ATF, and use dual-luciferase reporter assay system to test its activity. Results： The amino acid sequence of the Hmox1 enhancer specific ZFP was obtained. Homologous modeling suggested that its spatial structure was stable and the integrated ATF up-regulated the expression of reporter gene distinctively by dual-luciferase reporter assay. Conclusion： The results of the experiment suggest that the designed ATF is Hmoxl enhancer specific, which can up-regulate the expression of reporter gene effectively. These can be followed by the next step to construct the ATF that can up regulate the expression of endonu-clear Hmox1.

关 键 词：Hmox1 人工转录因子 锌指蛋白 同源建模 双荧光素酶报告基因检测 

分 类 号：R392.12[医药卫生—免疫学] R971.1[医药卫生—基础医学]