胰蛋白酶核酸适配体筛选及胰蛋白酶固定化新方法研究

发布时间：2018-04-20 06:39

本文选题：胰蛋白酶 + 毛细管电泳　；参考：《青岛科技大学》2017年硕士论文

【摘要】：核酸适配体(Aptamer)是指通过指数富集的配基系统进化技术(Systematic Evolution of Ligands by Exponential Enrichment,SELEX)从随机寡聚核苷酸文库中筛选获得的,与靶分子具有高亲和力,高特异性的寡核苷酸序列。用于核算适配体筛选的方法有很多,与其他适配体筛选技术相比,基于毛细管电泳的aptamer筛选(CE-SELEX)技术具有分离高效、快速,样品消耗量低的优点。在线酶解是蛋白质组学研究向通量化、集成化发展的必经之路,固定化酶反应器是实现在线酶解的关键。传统的酶固定化方法操作过程繁琐且对酶活性影响大、再生困难、成本较高。而酶的核酸适配体与酶结合稳定性强、再生能力突出、使用成本低、对酶活性影响小,具有其他酶固定化方法无法比拟的优点。本论文拟利用CE-SELEX筛选获得胰蛋白酶的核酸适配体,并利用筛选获得的适配体固定胰蛋白酶,制备固定化的胰蛋白酶。本论文的主要研究内容包括:(1)设计了适合胰蛋白酶适体筛选的随机寡聚核苷酸文库,优化了文库的扩增条件和次级文库的制备条件;(2)建立了适用于胰蛋白酶适体筛选的毛细管电泳方法,优化了电泳条件并对文库与胰蛋白酶的相互作用进行了表征;(3)以所设计的核苷酸文库和建立的毛细管电泳方法为基础,用基于毛细管电泳的适配体筛选技术(CE-SELEX)筛选获得了胰蛋白酶的核酸适配体;(4)对筛选获得的胰蛋白酶核酸适配体进行化学修饰后,通过化学交联和聚合的方法接枝于氨丙基硅胶颗粒表面,并将接枝有aptamer的硅胶颗粒与胰蛋白酶混合孵育,从而将胰蛋白酶修饰到硅胶颗粒表面,制备获得可用于固定化微酶反应器的填料。本研究经过4个轮次的筛选,随机寡聚核苷酸文库与胰蛋白酶的结合效率达到83.3%,并成功获得了25条与胰蛋白酶具有较高亲和力的适体,并以此为基础制备了固定化的胰蛋白酶,两种固定化方法的酶固载量分别为1.7μg/mg和5.3μg/mg。所用CE-SELEX技术高效、快速,基于核酸适配体的固定化酶稳定性强、再生能力突出、对酶活性影响小,对CE-SELEX法筛选蛋白类靶标的适体及固定化微酶反应器的制备均具有借鉴意义;制备的填料酶固载量较大,可用作固定化微酶反应器的填料。
[Abstract]:Aptamer, an aptamer, is a highly affinity and specific oligonucleotide sequence obtained from a random oligonucleotide library by the exponential enrichment of the ligand systematical Evolution of Ligands by Exponential EnrichmentmentSLEX. Compared with other aptamer screening techniques, CE-SELEX based on capillary electrophoresis (CE) has the advantages of high separation efficiency, fast separation and low sample consumption. On-line enzymatic hydrolysis is the only way for proteomics research to be quantified and integrated. Immobilized enzyme reactor is the key to realize on-line enzymatic hydrolysis. The traditional method of enzyme immobilization is complicated and has great influence on enzyme activity, so it is difficult to regenerate, and the cost is high. However, the aptamer of the enzyme has the advantages of strong stability, outstanding regeneration ability, low cost and little influence on the enzyme activity, which has the advantage that other immobilization methods can not be compared with other enzyme immobilization methods. In this paper, CE-SELEX was used to screen the aptamer of trypsin, and the trypsin was immobilized by the aptamer. In this paper, we designed a random oligonucleotide library suitable for trypsin aptamer screening. The amplification conditions of the library and the preparation condition of the secondary library were optimized. A capillary electrophoresis method suitable for screening aptamers of trypsin was established. The electrophoresis conditions were optimized and the interaction between the library and trypsin was characterized. It was based on the designed nucleotide library and the established capillary electrophoresis method. The aptamer of trypsin was screened by capillary electrophoresis (CE SELEX). The aptamer 4 of trypsin was chemically modified, and the trypsin aptamer was chemically modified. The surface of aminopropyl silica gel was grafted by chemical crosslinking and polymerization, and the grafted silica gel particles with aptamer were mixed with trypsin to modify trypsin onto the surface of silica gel particles. The fillers which can be used in immobilized microenzyme reactor were prepared. After four rounds of screening, the binding efficiency of random oligonucleotide library with trypsin reached 83.3%, and 25 aptamers with high affinity to trypsin were successfully obtained, and the immobilized trypsin was prepared. The enzyme immobilization capacity of the two immobilized methods was 1.7 渭 g/mg and 5.3 渭 g / mg, respectively. The CE-SELEX technique is efficient and rapid. The immobilized enzyme based on nucleic acid aptamer has strong stability and regeneration ability, and has little effect on enzyme activity. It is useful for the screening of aptamer for protein target by CE-SELEX method and the preparation of immobilized microenzyme reactor. The immobilized enzyme can be used as the filler in the immobilized microenzyme reactor.
【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q814.2

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