本文選題：胰蛋白酶 + 毛細(xì)管電泳　； 參考：《青島科技大學(xué)》2017年碩士論文

【摘要】：核酸適配體(Aptamer)是指通過(guò)指數(shù)富集的配基系統(tǒng)進(jìn)化技術(shù)(Systematic Evolution of Ligands by Exponential Enrichment,SELEX)從隨機(jī)寡聚核苷酸文庫(kù)中篩選獲得的,與靶分子具有高親和力,高特異性的寡核苷酸序列。用于核算適配體篩選的方法有很多,與其他適配體篩選技術(shù)相比,基于毛細(xì)管電泳的aptamer篩選(CE-SELEX)技術(shù)具有分離高效、快速,樣品消耗量低的優(yōu)點(diǎn)。在線酶解是蛋白質(zhì)組學(xué)研究向通量化、集成化發(fā)展的必經(jīng)之路,固定化酶反應(yīng)器是實(shí)現(xiàn)在線酶解的關(guān)鍵。傳統(tǒng)的酶固定化方法操作過(guò)程繁瑣且對(duì)酶活性影響大、再生困難、成本較高。而酶的核酸適配體與酶結(jié)合穩(wěn)定性強(qiáng)、再生能力突出、使用成本低、對(duì)酶活性影響小,具有其他酶固定化方法無(wú)法比擬的優(yōu)點(diǎn)。本論文擬利用CE-SELEX篩選獲得胰蛋白酶的核酸適配體,并利用篩選獲得的適配體固定胰蛋白酶,制備固定化的胰蛋白酶。本論文的主要研究?jī)?nèi)容包括:(1)設(shè)計(jì)了適合胰蛋白酶適體篩選的隨機(jī)寡聚核苷酸文庫(kù),優(yōu)化了文庫(kù)的擴(kuò)增條件和次級(jí)文庫(kù)的制備條件;(2)建立了適用于胰蛋白酶適體篩選的毛細(xì)管電泳方法,優(yōu)化了電泳條件并對(duì)文庫(kù)與胰蛋白酶的相互作用進(jìn)行了表征;(3)以所設(shè)計(jì)的核苷酸文庫(kù)和建立的毛細(xì)管電泳方法為基礎(chǔ),用基于毛細(xì)管電泳的適配體篩選技術(shù)(CE-SELEX)篩選獲得了胰蛋白酶的核酸適配體;(4)對(duì)篩選獲得的胰蛋白酶核酸適配體進(jìn)行化學(xué)修飾后,通過(guò)化學(xué)交聯(lián)和聚合的方法接枝于氨丙基硅膠顆粒表面,并將接枝有aptamer的硅膠顆粒與胰蛋白酶混合孵育,從而將胰蛋白酶修飾到硅膠顆粒表面,制備獲得可用于固定化微酶反應(yīng)器的填料。本研究經(jīng)過(guò)4個(gè)輪次的篩選,隨機(jī)寡聚核苷酸文庫(kù)與胰蛋白酶的結(jié)合效率達(dá)到83.3%,并成功獲得了25條與胰蛋白酶具有較高親和力的適體,并以此為基礎(chǔ)制備了固定化的胰蛋白酶,兩種固定化方法的酶固載量分別為1.7μg/mg和5.3μg/mg。所用CE-SELEX技術(shù)高效、快速,基于核酸適配體的固定化酶穩(wěn)定性強(qiáng)、再生能力突出、對(duì)酶活性影響小,對(duì)CE-SELEX法篩選蛋白類(lèi)靶標(biāo)的適體及固定化微酶反應(yīng)器的制備均具有借鑒意義;制備的填料酶固載量較大,可用作固定化微酶反應(yīng)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.
【學(xué)位授予單位】：青島科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q814.2

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