限制性显示技术在HIV随机多肽文库构建中的应用
发布时间：2018-11-08 19:13
【摘要】：随着人类基因组计划的完成,生命科学研究已进入了后基因组时代。以细胞内全部蛋白质的存在及其活动方式为研究对象的蛋白质组学(Proteomics),是后基因组时代生命科学研究的核心内容之一。细胞内蛋白质间的相互作用、相互协调是进行一切代谢活动的基础,因此揭示蛋白质之间以及蛋白质与多肽之间的相互作用关系,建立相互作用关系的网络图,不仅对阐明生命在生理或病理条件下的变化机制有重要意义,而且将为寻找疾病分子标记和药物靶标筛选提供依据。各种研究蛋白质相互作用的方法由此应运而生,如噬菌体展示技术,酵母双杂交技术,蛋白质芯片技术等。然而无论是哪种研究蛋白质相互作用的方法最基础的一步是建立高通量的表达文库。目前运用较多的文库包括人工合成的随机肽库、cDNA文库以及基因组片段文库等。人工合成的随机肽库由于其完全随机性,不能完全反应基因组的遗传信息;cDNA文库仅仅只反应特定时期的细胞基因表达水平。相反,基因组片段文库则可以覆盖无论高表达还是低表达的所有基因信息,其产生的蛋白多肽片段由于易于空间折叠从而能够产生有活性的小肽,是研究蛋白多肽之间相互作用的较好方法。本研究的目的就是利用有别于传统的物理方法(超声波、搅拌剪力等)或酶切法的一种新方法——RD-PCR技术来构建基因组片段表达文库,从而为研究多肽与蛋白质之间相互作用提供一种新的技术平台。 限制性显示技术(restriction display, RD)是本实验室发明的分子生物学新技术,其在基因表达差异分析、DNA芯片探针设计和样品处理等方面应用比较广泛。其基本原理是利用限制性核酸内切酶消化基因组DNA或mRNA体外逆转录的双链cDNA产生限制性片段,将片段与互补的接头连接,接头根据研究
[Abstract]:With the completion of the human genome project, life science research has entered the post-genome era. Proteomics (Proteomics), which focuses on the existence and activity of all proteins in cells, is one of the core contents of life science research in the post-genomic era. The interaction and coordination of proteins within cells is the basis for all metabolic activities, so it reveals the interactions between proteins and peptides, and establishes a network of interactions. It is important not only to elucidate the mechanism of life change under physiological or pathological conditions, but also to provide a basis for searching for molecular markers of disease and screening of drug targets. Various methods to study protein interaction emerge as the times require, such as phage display technology, yeast two-hybrid technology, protein chip technology and so on. However, the most basic step in any method of studying protein interactions is the establishment of high-throughput expression libraries. At present, more libraries are used, including synthetic random peptide library, cDNA library and genomic fragment library. The synthetic random peptide library can not completely reflect the genetic information of the genome because of its complete randomness, while the cDNA library only reflects the level of gene expression in the cell at a specific stage. In contrast, a genomic fragment library can cover all gene information, both highly expressed and poorly expressed, and the resulting protein polypeptide fragments can produce small, active peptides because they are easily spatially folded. It is a good method to study the interaction between proteins and peptides. The purpose of this study is to construct a genomic fragment expression library by using a new method, RD-PCR, which is different from the traditional physical methods (ultrasonic wave, stirring shear force, etc.) or enzyme digestion. Therefore, it provides a new technical platform for the study of the interaction between peptides and proteins. Restriction display technique (restriction display, RD) is a new molecular biology technique developed in our laboratory. It is widely used in gene expression differential analysis, DNA chip probe design and sample processing. The basic principle is to use restriction endonuclease to digest genomic DNA or double-stranded cDNA reverse transcription of mRNA in vitro to produce restriction fragments, and to connect the fragments with complementary joints.
【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346
本文编号：2319408
[Abstract]:With the completion of the human genome project, life science research has entered the post-genome era. Proteomics (Proteomics), which focuses on the existence and activity of all proteins in cells, is one of the core contents of life science research in the post-genomic era. The interaction and coordination of proteins within cells is the basis for all metabolic activities, so it reveals the interactions between proteins and peptides, and establishes a network of interactions. It is important not only to elucidate the mechanism of life change under physiological or pathological conditions, but also to provide a basis for searching for molecular markers of disease and screening of drug targets. Various methods to study protein interaction emerge as the times require, such as phage display technology, yeast two-hybrid technology, protein chip technology and so on. However, the most basic step in any method of studying protein interactions is the establishment of high-throughput expression libraries. At present, more libraries are used, including synthetic random peptide library, cDNA library and genomic fragment library. The synthetic random peptide library can not completely reflect the genetic information of the genome because of its complete randomness, while the cDNA library only reflects the level of gene expression in the cell at a specific stage. In contrast, a genomic fragment library can cover all gene information, both highly expressed and poorly expressed, and the resulting protein polypeptide fragments can produce small, active peptides because they are easily spatially folded. It is a good method to study the interaction between proteins and peptides. The purpose of this study is to construct a genomic fragment expression library by using a new method, RD-PCR, which is different from the traditional physical methods (ultrasonic wave, stirring shear force, etc.) or enzyme digestion. Therefore, it provides a new technical platform for the study of the interaction between peptides and proteins. Restriction display technique (restriction display, RD) is a new molecular biology technique developed in our laboratory. It is widely used in gene expression differential analysis, DNA chip probe design and sample processing. The basic principle is to use restriction endonuclease to digest genomic DNA or double-stranded cDNA reverse transcription of mRNA in vitro to produce restriction fragments, and to connect the fragments with complementary joints.
【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346
【引证文献】
相关硕士学位论文 前1条
1 肖静;人巨细胞病毒基因组表达文库的构建及其应用于病毒编码蛋白相互作用的研究[D];暨南大学;2011年
，本文编号：2319408
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