液相-SELEX方法的建立及变形链球菌葡糖基转移酶催化区特异适配体的筛选

发布时间：2018-02-26 18:33

本文关键词： 葡糖基转移酶催化区液相-SELEX 随机ssDNA文库适配体凝胶阻滞　出处：《兰州理工大学》2010年硕士论文　论文类型：学位论文

【摘要】： 葡糖基转移酶(Glucosyltransferase, GTF)是变形链球菌合成的重要致龋因子,催化蔗糖合成包括葡聚糖在内的多种胞外多糖。GTF结构包括催化区(Catalytic, CAT)和葡聚糖结合区(Glucan binding, GB或GLU)两个功能区段,其中催化区(CAT)是它的重要功能区段。本实验首先利用基因工程的方法在大肠杆菌中分别诱导表达了带NusA的rNusA-CAT融合蛋白和NusA对照蛋白。菌体经超声破碎后,重组蛋白存在于菌体超声上清液中。利用载体上C端的His标签对重组蛋白进行了金属螯合层析纯化。蒽酮-硫酸法证明重组rNusA-CAT蛋白具有良好的催化活性。由于传统SELEX技术筛选靶分子时,首先需要将靶分子进行纯化和固定,因而以天然构象存在于液相中的非纯化靶蛋白不能通过传统的方法进行筛选。为解决这一问题,本研究将凝胶阻滞(EMSA)的基本原理引入消减SELEX筛选过程,初步建立了一种基于凝胶阻滞的新筛选方法,实现了非纯化靶标蛋白的液相筛选(液相-SELEX技术)。实验采用随机区为35个碱基、全长78nt的ssDNA文库,以表达rNusA-CAT蛋白的未纯化上清为目的靶,以表达NusA蛋白的未纯化上清为消减靶,利用液相-SELEX技术进行了十轮的筛选。放射性同位素检测发现：随着筛选轮次的增加,ssDNA文库逐渐富集,富集的ssDNA配基可特异的识别rNusA-CAT蛋白,而不识别NusA蛋白。该结果的取得为今后GTF功能抑制剂的获得和龋齿病防治奠定基础。液相-SELEX技术的建立实现了非纯化的、天然构象蛋白的筛选,为今后以病人血清、唾液、体液等作为筛选靶标,快速、有效地获得疾病的血清或体液标志物及分子探针提供一种新的技术方法,从而为疾病的诊断和治疗带来新的思路。
[Abstract]:Glucosyltransferase (GTFs) is an important cariogenic factor in the synthesis of Streptococcus mutans. The catalytic region (CAT) is an important functional region. In this experiment, rNusA-CAT fusion protein with NusA and NusA control protein were induced to express in E. coli by genetic engineering. The recombinant protein was purified by metal chelation chromatography using C-terminal His tag. The recombinant rNusA-CAT protein was proved to have good catalytic activity by anthrone sulfuric acid method. In order to solve this problem, unpurified target proteins that exist in liquid phase in natural conformation cannot be screened by traditional SELEX technique, which requires purification and immobilization of target molecules. In this study, the basic principle of gel arrest was introduced into the process of subtractive SELEX screening, and a new screening method based on gel block was established. Liquid phase screening of non-purified target proteins (liquid-SELEX technique) was carried out. A ssDNA library with a random region of 35 bases and a total length of 78 NT was used to express the unpurified supernatant of rNusA-CAT protein, and the unpurified supernatant of NusA protein was used as the subtractive target. Ten rounds of screening were carried out using liquid-SELEX technique. Radioisotope detection showed that the enriched ssDNA ligands could specifically recognize rNusA-CAT proteins with the increasing number of screening rounds. The results laid a foundation for the acquisition of GTF functional inhibitors and the prevention and treatment of dental caries. The establishment of liquid-SELEX technique has achieved the screening of non-purified, natural conformation proteins, which can be used as screening targets in patients' serum, saliva, body fluids and so on. To obtain the serum or body fluid markers and molecular probes of diseases effectively provides a new technical method for the diagnosis and treatment of diseases.
【学位授予单位】：兰州理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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