重组透明质酸酶包涵体复性及其酶解条件研究
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
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