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摘要：透明质酸酶(Hyaluronidase ，简称HAase)是一种能水解透明质酸的酶（透明质酸为组织基质中具有限制水分及其它细胞外物质扩散作用的粘多糖），是一种重要的药物扩散剂。临床用作药物渗透剂，促进药物的吸收，促进手术及创伤后局部水肿或血肿消散。HAase可以用来将大分子量的透明质酸降解成小分子量的透明质酸，这种降解方式相比于其他物理、化学和机械等方法更加温和且方便可行，安全可靠可大批量生产且水解效率更高等优点。细菌细胞表面展示技术是一项新兴的蛋白质应用技术，其体系由运载蛋白、靶蛋白和宿主菌三者构成，现已被应用于活体疫苗开发、蛋白质及多肽文库筛选、抗体合成、生物传感器研制、全细胞生物催化剂和生物吸附剂等领域。本实验通过设计合适的引物，用PCR的方法从马疫链球菌基因组中扩增得到马疫链球菌透明质酸酶HAase编码基因。将inaQ-N与HAase编码基因融合入大肠杆菌表达载体pTrcHisC，构建inaQ-N-HAase大肠杆菌细胞表面展示体系，通过酶切和PCR验证表明，inaQ-N-HAase融合基因构建成功。24338
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【毕业论文关键词】细菌细胞表面展示；运载蛋白；靶蛋白；大肠杆菌；透明质酸酶
Construction of hyaluronidase diaplayed on the surface of Escherichia coli
Abstract: Hyaluronidase (HAase) is an enzymatic hydrolysis of hyaluronic acid (hyaluronic acid matrix organization with restricted water diffusion foreign substances and other cell components). It is an important drug diffusion agent. HAase can be used to degeste high molecular weight hyaluronic acid hydrolyzed into small molecular weight hyaluronic acid. Compared with physical, chemical and mechanical method, HAase degestion is more practical and mild, safe and reliable to a large production and higher hydrolysis efficiency, etc.
Bacterial cell-surface display of a heterologons protein is a novel biotechnological platform towards various protein-level applications. Generally, this system is composed of carrier protein, passenger protein and bacterial host. This system has been developed to be a promising approach to many biotechnological fields such as live recombinant vaccine development, constructing and screening of protein libraries, biosensor development, whole cell biocatalysis, whole cell bioadsorbence and biodegradation of environmental pollutants. This experiment was carried by the designing of appropriate primers which can amplify hyaluronidase (HAase) encoding gene from Streptococcus zooepidemicus by PCR method. . Then, inaQ-N and HAase encoding sequences were fused and inserted to an expression vector in E. coli. An appropriate of inaQ-N-HAase surface display system in E. coli cells was constructed using InaQ-N as a carrier protein and HAase as a passenger protein. According to the results of PCR amplification and enzyme digestion, the HAase was proved to be inserted into the cell surface display system and a InaQ-N-HAase fusion was successfully constructed.
Key Words：bacterial cell surface display；carrier protein；passenger protein；escherichia coli；hyaluronidase
目 录
1前言 1
1.1透明质酸酶的概述 1
1.1.1酶的结构 1
1.1.2透明质酸酶的分类 1
1.1.3透明质酸与透明质酸酶 2
1.1.4透明质酸酶的应用 3
1.2细菌表面展示体系的概述 3
1.2.1细菌表面展示体系的原理 4
1.2.2细菌表面展示体系的构成 5
1.3运载蛋白的类型与性能 7
1.3.1 细胞膜孔道蛋白 7
1.3.2冰晶核蛋白 7
1.3.3自动转运蛋白 8
1.3.4细胞壁相关蛋白 8
1.4细菌表面展示技术的应用 8