本文關(guān)鍵詞：IgA親和體隨機(jī)組合噬菌體展示文庫(kù)的構(gòu)建及體外分子進(jìn)化　出處：《安徽醫(yī)科大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

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【摘要】：IgA是人體一種重要的Ig類型，研究開發(fā)高效特異性檢測(cè)和純化IgA的試劑在廣大醫(yī)學(xué)領(lǐng)域中具有非常重要的應(yīng)用價(jià)值。定向分子進(jìn)化技術(shù)是探索研究生物大分子結(jié)構(gòu)與功能的重要手段，同時(shí)也是改造、優(yōu)化生物大分子特性的有效方法。DNA重排和噬菌體展示及篩選是體外定向分子進(jìn)化的重要核心技術(shù)。本研究應(yīng)用定向分子進(jìn)化技術(shù)，將特異性結(jié)合IgA的IgA親和體分子隨機(jī)組合，構(gòu)建噬菌體展示文庫(kù)，以體外分子進(jìn)化的方法篩選出高結(jié)合活性的具有重復(fù)結(jié)構(gòu)的新型IgA親和體分子，，通過比較IgA親和體單結(jié)構(gòu)分子與重復(fù)結(jié)構(gòu)分子的IgA結(jié)合活性的差異，研究IgA親和體結(jié)構(gòu)與功能的關(guān)系，與Ig相互作用的機(jī)制及體外重組進(jìn)化的規(guī)律，為進(jìn)一步應(yīng)用分子進(jìn)化手段改造功能蛋白積累經(jīng)驗(yàn)。本研究分為四個(gè)部分： 一、IgA親和體分子的基因合成 根據(jù)文獻(xiàn)提供的IgA親和體的氨基酸序列，選擇2個(gè)有代表性的IgA親和體序列A1、A2，設(shè)計(jì)引物，以基因合成的方法制備得到兩個(gè)IgA親和體片段，克隆于pMD-18T載體。經(jīng)過序列分析并與文獻(xiàn)所提供的序列進(jìn)行比對(duì)，證明基因合成的IgA親和體序列與文獻(xiàn)中序列完全一致。 二、IgA親和體隨機(jī)組合噬菌體展示文庫(kù)的構(gòu)建 在IgA親和體A1、A2片段兩端引入Kpn Ⅰ酶切位點(diǎn)，3′端引入3個(gè)隨機(jī)連接肽，經(jīng)DNA重排，隨機(jī)組合形成各種不同長(zhǎng)度的片段，插入噬菌粒載體pCANTAB5S的Kpn Ⅰ克隆位點(diǎn)上，展示在噬菌體表面，構(gòu)建噬菌體展示隨機(jī)組合文庫(kù)。文庫(kù)的庫(kù)容量為3.4×10~7，滴度為1.6×10~(12)TU／ml；原代組合文庫(kù)中含79％以上的陽(yáng)性克隆，其中由2個(gè)以上親和體組成的陽(yáng)性克隆占18％；DNA序列分析顯示原代組合文庫(kù)由A1、A2序列隨機(jī)組合而成，各親和體分子之間隨機(jī)連接肽的核苷酸序列也呈隨機(jī)分布。從文庫(kù)的庫(kù)容量、多樣性和隨機(jī)性各方面來看，該文庫(kù)可滿足體外分子進(jìn)化研究的要求。 三、IgA親和體隨機(jī)組合噬菌體展示文庫(kù)的分子進(jìn)化 以人IgA分子為誘餌對(duì)IgA親和體隨機(jī)組合文庫(kù)進(jìn)行4輪親和篩選，在人IgA分子導(dǎo)向的進(jìn)化過程中，IgA親和體隨機(jī)組合文庫(kù)的展示序列由1個(gè)結(jié)構(gòu)分子和2
[Abstract]:IgA is an important type of human Ig, research and development of high specific detection and purification of IgA reagent has very important application value in the field of medicine. Directed molecular evolution technology is an important means to explore the structure and function of biological macromolecules, but also the transformation, optimization characteristics of biological macromolecules and effective method for.DNA rearrangement and phage display and screening is an important core technology of directed molecular evolution. The research and application of directed molecular evolution technology, the specific binding of IgA IgA affibody molecules random combination, construction of phage display library, with the method of molecular evolution in vitro screening of novel IgA with high binding activity of the repetitive structure of affibody molecules. Through the comparison of the IgA single molecular structure affinity and repetitive structure of molecular IgA binding activity differences, relationship between structure and function of IgA affinity, mutual and Ig This study is divided into four parts: the mechanism of action and the law of the evolution of the recombinant in vitro.
1. Gene synthesis of IgA affinity molecules
According to the amino acid sequence provided by the literature IgA Affibodies, selected 2 representative IgA Affibodies sequence A1, A2, primers were designed by gene synthesis technology of preparation of two IgA affibody fragments were cloned into pMD-18T vector. After sequence analysis and reference sequence comparison, proof gene synthesis IgA Affibodies sequences and literature sequencing.
Two, the construction of a random combination of IgA affinity phage display library
In IgA Affibodies A1, A2 fragment ends into Kpn enzyme, the 3 'end of the introduction of 3 random linking peptide, DNA rearrangement, random combination form a variety of different length fragments of Kpn I cloning site into the phagemid vector pCANTAB5S, displayed on the phage surface, constructing phage display random combination library. Library capacity is 3.4 * 10~7, the titer of 1.6 * 10~ (12) TU / ml; primary combinatorial library containing more than 79% positive clones, which is composed of more than 2 positive clones of affinity accounted for 18%; DNA sequence analysis showed that the original generation of combinatorial libraries by A1 A2 sequence random combination as the affibody molecules between the nucleotide sequences of random linking peptides were also randomly distributed. The capacity of library, diversity and randomness of each aspect, the library can meet the requirements of molecular evolution study in vitro.
Three, the molecular evolution of a random combination of IgA affinity phage display library
Using human IgA as bait, we performed 4 rounds of affinity screening on IgA affinity library. In the process of human IgA molecular directed evolution, the display sequence of IgA affinity library was composed of 1 structural molecules and 2.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

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相關(guān)期刊論文 前7條

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本文編號(hào)：1370175