【摘要】：隨著人類基因組計劃的完成,生命科學研究已進入了后基因組時代。以細胞內(nèi)全部蛋白質(zhì)的存在及其活動方式為研究對象的蛋白質(zhì)組學(Proteomics),是后基因組時代生命科學研究的核心內(nèi)容之一。細胞內(nèi)蛋白質(zhì)間的相互作用、相互協(xié)調(diào)是進行一切代謝活動的基礎,因此揭示蛋白質(zhì)之間以及蛋白質(zhì)與多肽之間的相互作用關系,建立相互作用關系的網(wǎng)絡圖,不僅對闡明生命在生理或病理條件下的變化機制有重要意義,而且將為尋找疾病分子標記和藥物靶標篩選提供依據(jù)。各種研究蛋白質(zhì)相互作用的方法由此應運而生,如噬菌體展示技術,酵母雙雜交技術,蛋白質(zhì)芯片技術等。然而無論是哪種研究蛋白質(zhì)相互作用的方法最基礎的一步是建立高通量的表達文庫。目前運用較多的文庫包括人工合成的隨機肽庫、cDNA文庫以及基因組片段文庫等。人工合成的隨機肽庫由于其完全隨機性,不能完全反應基因組的遺傳信息;cDNA文庫僅僅只反應特定時期的細胞基因表達水平。相反,基因組片段文庫則可以覆蓋無論高表達還是低表達的所有基因信息,其產(chǎn)生的蛋白多肽片段由于易于空間折疊從而能夠產(chǎn)生有活性的小肽,是研究蛋白多肽之間相互作用的較好方法。本研究的目的就是利用有別于傳統(tǒng)的物理方法(超聲波、攪拌剪力等)或酶切法的一種新方法——RD-PCR技術來構(gòu)建基因組片段表達文庫,從而為研究多肽與蛋白質(zhì)之間相互作用提供一種新的技術平臺。 限制性顯示技術(restriction display, RD)是本實驗室發(fā)明的分子生物學新技術,其在基因表達差異分析、DNA芯片探針設計和樣品處理等方面應用比較廣泛。其基本原理是利用限制性核酸內(nèi)切酶消化基因組DNA或mRNA體外逆轉(zhuǎn)錄的雙鏈cDNA產(chǎn)生限制性片段,將片段與互補的接頭連接,接頭根據(jù)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.
【學位授予單位】：第一軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R346

【引證文獻】

相關碩士學位論文 前1條

1 肖靜;人巨細胞病毒基因組表達文庫的構(gòu)建及其應用于病毒編碼蛋白相互作用的研究[D];暨南大學;2011年

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本文編號：2319408