IgA亲和体随机组合噬菌体展示文库的构建及体外分子进化

发布时间：2018-01-02 17:17

本文关键词：IgA亲和体随机组合噬菌体展示文库的构建及体外分子进化　出处：《安徽医科大学》2006年硕士论文　论文类型：学位论文

【摘要】：IgA是人体一种重要的Ig类型，研究开发高效特异性检测和纯化IgA的试剂在广大医学领域中具有非常重要的应用价值。定向分子进化技术是探索研究生物大分子结构与功能的重要手段，同时也是改造、优化生物大分子特性的有效方法。DNA重排和噬菌体展示及筛选是体外定向分子进化的重要核心技术。本研究应用定向分子进化技术，将特异性结合IgA的IgA亲和体分子随机组合，构建噬菌体展示文库，以体外分子进化的方法筛选出高结合活性的具有重复结构的新型IgA亲和体分子，，通过比较IgA亲和体单结构分子与重复结构分子的IgA结合活性的差异，研究IgA亲和体结构与功能的关系，与Ig相互作用的机制及体外重组进化的规律，为进一步应用分子进化手段改造功能蛋白积累经验。本研究分为四个部分：一、IgA亲和体分子的基因合成根据文献提供的IgA亲和体的氨基酸序列，选择2个有代表性的IgA亲和体序列A1、A2，设计引物，以基因合成的方法制备得到两个IgA亲和体片段，克隆于pMD-18T载体。经过序列分析并与文献所提供的序列进行比对，证明基因合成的IgA亲和体序列与文献中序列完全一致。二、IgA亲和体随机组合噬菌体展示文库的构建在IgA亲和体A1、A2片段两端引入Kpn Ⅰ酶切位点，3′端引入3个随机连接肽，经DNA重排，随机组合形成各种不同长度的片段，插入噬菌粒载体pCANTAB5S的Kpn Ⅰ克隆位点上，展示在噬菌体表面，构建噬菌体展示随机组合文库。文库的库容量为3.4×10~7，滴度为1.6×10~(12)TU／ml；原代组合文库中含79％以上的阳性克隆，其中由2个以上亲和体组成的阳性克隆占18％；DNA序列分析显示原代组合文库由A1、A2序列随机组合而成，各亲和体分子之间随机连接肽的核苷酸序列也呈随机分布。从文库的库容量、多样性和随机性各方面来看，该文库可满足体外分子进化研究的要求。三、IgA亲和体随机组合噬菌体展示文库的分子进化以人IgA分子为诱饵对IgA亲和体随机组合文库进行4轮亲和筛选，在人IgA分子导向的进化过程中，IgA亲和体随机组合文库的展示序列由1个结构分子和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.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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