【作者】 张文娟；

【导师】 曾令文；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 转录因子,也被称为序列特异性DNA结合蛋白,含有一个或多个DNA结合结构域(DBDs)。它们定位于细胞质中,在正常细胞功能状态下或受到特定因子诱导后,从细胞质进入细胞核,与基因组中的顺式作用元件诸如启动子、增强子、衰减子等发生特异性识别结合,组成基因转录机器,进而实现其调控基因表达的功能。因此,检测分析转录因子的表达及活化程度对我们了解细胞功能具有至关重要的作用。目前常用的检测技术主要建立在DNA和蛋白质相互作用这一层次,即运用含有顺式作用元件的DNA为探针,来分析转录因子的DNA结合活性。本课题“基于微孔板核酸杂交酶联免疫吸附法检测DNA结合蛋白”技术巧妙的利用在高严谨度洗脱条件下转录因子蛋白对与之特异性结合的双链核酸序列的保护作用,将对转录因子的检测转换为对相应的特异核酸序列的检测。该方法不需要复杂的仪器、繁琐的操作步骤和特异性抗体就可以对转录因子的表达及活化程度进行定量研究,而且可在较短时间内完成对转录因子的检测。我们以检测转录因子c-Jun的活性为模型来对该检测方法进行评价,在较短的时间内该方法可以准确的检测到3.5ng的转录因子c-Jun纯蛋白和0.625ug的Hela细胞核粗提取物,具有较高的灵敏度。而且该检测方法可以用于任何一种转录因子的检测。鉴于“基于微孔板核酸杂交酶联免疫吸附法”的快速、简便、高灵敏度和选择性,该检测方法可以应用到需要对转录因子的DNA结合活性进行定量,有效,大规模检测的领域和科学研究中,诸如细胞分子生物学中对多种处理的刺激应答筛选方面,进化生物学研究方面,环境监测方面和药物筛选方面等等。更多还原

【Abstract】 Transcription factors, also known as sequence-specific DNA binding proteins,contain one or more DNA binding domains (DBDs). They are located in cytoplasm. In physiological condition or induced by a specific stimulus, transcription factors would be transported into the nucleus from cytoplasm, and then bind to trans-acting factors such as promoters, enhancers or attenuators, thereby up-regulating or down-regulating transcription of adjacent gene. Therefore, the determination of DNA binding activities of transcription factors is pivotal to our understanding of cellular functions. Currently used methods are all based on the interactions between DNA binding proteins and their counterparts, that is to say, use the trans-acting factors as probe to detect the DNA-binding activities of transcription factors. Because transcription factors can prevent specific double-stranded nucleic acids from being eluted to single-stranded in high stringency condition, herein, we detect the protected double-stranded nucleic acids to determine the DNA-binding activities of transcription factors indirectly. Microplate-nucleotide hybridization based ELISA-like assay did not require complex instruments and complicated procedure, and could be applied to multiplex detection without the usage of specific antibody in short time. We have used the method to demonstrate the DNA-binding activity of c-Jun, a well-known model of inductive transcriptional regulatory responses. But the method is easily adaptable for the study of any transcription factor. This allows rapid and accurate detection of as low as 3.5ng of purified c-Jun protein or 0.625ug crude cell nucleus extract. Owing to the rapidness, ease, high sensitivity and selectivity of the method, the microplate-nucleotide hybridization based ELISA-like assay may be used in various applications and research fields where quantitative, cost-effective and large-scale measurements of the DNA-binding activities of transcription factors are required, including screening of responses in multiple treatments in cellular and molecular biology, evolutionary biology, environmental monitoring, and drug discovery.更多还原