本文關(guān)鍵詞： 葡糖基轉(zhuǎn)移酶催化區(qū) 液相-SELEX 隨機(jī)ssDNA文庫 適配體 凝膠阻滯　出處：《蘭州理工大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 葡糖基轉(zhuǎn)移酶(Glucosyltransferase, GTF)是變形鏈球菌合成的重要致齲因子,催化蔗糖合成包括葡聚糖在內(nèi)的多種胞外多糖。GTF結(jié)構(gòu)包括催化區(qū)(Catalytic, CAT)和葡聚糖結(jié)合區(qū)(Glucan binding, GB或GLU)兩個功能區(qū)段,其中催化區(qū)(CAT)是它的重要功能區(qū)段。 本實驗首先利用基因工程的方法在大腸桿菌中分別誘導(dǎo)表達(dá)了帶NusA的rNusA-CAT融合蛋白和NusA對照蛋白。菌體經(jīng)超聲破碎后,重組蛋白存在于菌體超聲上清液中。利用載體上C端的His標(biāo)簽對重組蛋白進(jìn)行了金屬螯合層析純化。蒽酮-硫酸法證明重組rNusA-CAT蛋白具有良好的催化活性。 由于傳統(tǒng)SELEX技術(shù)篩選靶分子時,首先需要將靶分子進(jìn)行純化和固定,因而以天然構(gòu)象存在于液相中的非純化靶蛋白不能通過傳統(tǒng)的方法進(jìn)行篩選。為解決這一問題,本研究將凝膠阻滯(EMSA)的基本原理引入消減SELEX篩選過程,初步建立了一種基于凝膠阻滯的新篩選方法,實現(xiàn)了非純化靶標(biāo)蛋白的液相篩選(液相-SELEX技術(shù))。實驗采用隨機(jī)區(qū)為35個堿基、全長78nt的ssDNA文庫,以表達(dá)rNusA-CAT蛋白的未純化上清為目的靶,以表達(dá)NusA蛋白的未純化上清為消減靶,利用液相-SELEX技術(shù)進(jìn)行了十輪的篩選。放射性同位素檢測發(fā)現(xiàn)：隨著篩選輪次的增加,ssDNA文庫逐漸富集,富集的ssDNA配基可特異的識別rNusA-CAT蛋白,而不識別NusA蛋白。該結(jié)果的取得為今后GTF功能抑制劑的獲得和齲齒病防治奠定基礎(chǔ)。 液相-SELEX技術(shù)的建立實現(xiàn)了非純化的、天然構(gòu)象蛋白的篩選,為今后以病人血清、唾液、體液等作為篩選靶標(biāo),快速、有效地獲得疾病的血清或體液標(biāo)志物及分子探針提供一種新的技術(shù)方法,從而為疾病的診斷和治療帶來新的思路。
[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.
【學(xué)位授予單位】：蘭州理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R378

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本文編號：1539180