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摘要：透明质酸酶（HAase)是一种蛋白水解酶，通过作用于β-1,3糖苷键或β-1,4糖苷键来降解透明质酸，能特异性的分解透明质酸(HA)。除此之外，透明质酸酶也具有药物扩散剂的功能，它通过暂时降低人体细胞间质的粘度，而使皮下输液、渗出液还有血液的扩散速度加快，而使促进吸收。本研究通过升温诱导表达重组透明质酸酶，经SDS-PAGE验证后对重组菌进行扩大培养。然后收集菌体并破碎，提取包涵体且对其进行复性。复性后对获得的粗酶进行盐析及透析，得到一定程度的纯化。而后通过降解透明质酸获得其最适酶解条件，包括温度、pH，并且测定反应过程中透明质酸分子量的变化。升温诱导实验和SDS-PAGE实验结果表明，重组蛋白大小约为96kDa。纯化获得的粗酶品通过DNS法测酶活，结果表明其最适反应条件pH5.5，37℃；用粗酶液降解透明质酸75min后，透明质酸的分子量由160万Da降至1万Da。38485
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毕业论文关键词：透明质酸 ；透明质酸酶；包涵体复性；酶活力
Study on the Inclusion body refolding and the conditions of enzymatic hydrolysis of recombinant hyaluronidase
Abstract:Hyaluronidase(HAase) is a proteolytic enzyme which can break down hyaluronic acid specifically by acting on the β-1,3 or β-1,4 glycosidic bonds.Besides,it is also an important drug diffusion agent which can reduce the viscosity of human body’s cell interstitial temporarily to accelerate the diffusion of subcutaneous infusion,exudate and blood.In this study,recombinant hyaluronidase was expressed by temperature inducing and expanding the recombinant strains after the SDS-PAGE verification.Then thalluses were collected and broken to extract the inclusions and refolding them.After refolding,the crude enzyme was subjected to salting out and dialysis to obtain a certain degree of purification.Then we will get the optimum hydrolysis conditions include the temperature and pH by degrading hyaluronic acid,and determining the change of hyaluronic acid’s molecular weight during the reaction process.Temperature inducing, experiments and SDS-PAGE results showed that the molecular weight of recombinant protein is 96kDa.The enzyme activity of the purified crude enzyme products was detected by DNS assay and the results showed that the optimum reaction conditions was pH5.5,37 ℃.The molecular weight of hyaluronic acid dropped from 1.6 million to 10,000Da after degrading by crude recombinant HAase for 75min.
KeyWords：Hyaluronic acid;Hyaluronidase;Inclusion body refolding;Enzyme activity
目录
1 前言 1
1.1 透明质酸酶概述 1
1.1.1 透明质酸酶的分类 1
1.1.2 透明质酸酶的来源 2
1.2 透明质酸酶的临床应用 4
1.2.1 透明质酸酶的生物学功能 4
1.2.2 HAase与肿瘤 5
1.2.3 局部麻醉 5
1.2.4 白内障手术 5
1.3 包涵体介绍 5
1.3.1 包涵体的理化性质 5
1.3.2 使用大肠杆菌工程菌表达基因重组蛋白质 6
1.3.3 包涵体蛋白的分离和纯化 7
1.3.4 包涵体蛋白的溶解变性 7
1.3.5 包涵体蛋白复性的方法 8
1.4 本课题主要研究内容及立题意义 8
2 材料和方法 9
2.1 材料与试剂 9
2.1.1 菌种 9
2.1.2 实验材料 9
2.1.3 主要仪器和设备 10
2.1.4 实验试剂 10
2.2 实验方法 11
2.2.1 重组HAase在大肠杆菌中的诱导表达 11