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摘要：低分子量透明质酸具有润滑、保湿等作用，广泛应用于美容及预防和治疗骨关节炎。而透明质酸酶（HAase）可特异性水解透明质酸，生成低分子量透明质酸。本研究通过设计PCR引物，特异性扩增Streptococcus equi ATCC43079基因组中透明质酸酶编码基因，并将该基因连接到pMD18-T载体上，构建pMD18T-HAase重组质粒。将构建的pMD18T-HAase质粒导入制备好的大肠杆菌Escherichia coli DH5α感受态细胞中，获得含HAase编码基因的重组菌。通过DNA测序得到Streptococcus equi ATCC43079中的透明质酸酶编码基因片段长2586bp，利用分析工具得出：该基因编码蛋白质含864个氨基酸，分子量97364Da。根据氨基酸序列分析结果，该基因编码蛋白质的等电点（PI）理论值为6.11。该HAase基因与马疫链球菌6种菌株来源的HAase基因对比结果为98.15%。从序列分析结果可知，本研究成功克隆获得了一个来源于马疫链球菌的透明质酸酶编码基因。19992
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毕业论文关键词： 透明质酸酶；马疫链球菌；克隆；序列分析
Cloning and sequence analysis of hyaluronidase encoding gene
Abstract: Low molecular weight hyaluronic acid has functions of smooth, moisturizing and so on. Therefore, it is widely used in beauty, prevention and treatment of osteoarthritis. Low molecular weight hyaluronic acid can be produced by digestion of hyaluronic acid using hyaluronidase (HAase). In this study，genomic DNA from Streptococcus equi ATCC43079 was extracted，then PCR primers were designed to amplify hyaluronidase encoding gene. The HAase gene was cloned and inserted into the pMD18-T vector to construct pMD18T-HAase plasmid.After transforming the constructed plasmid to E. coil DH5α competent cells，strains were successfully selected by the method of blue-white screening. Finally, selected strains were sent to Sangon for sequencing. According to the analytic tools, the results were as follows：the measured length of encoding hyaluronidase gene in S. equi ATCC43079 strains is 2586bp, encoding 864 amino acids and its MW is 97364D. The theoretical isoelectric point （PI）of this protein is 6.11. Compared with six HAase gene sequences in Streptococcus strains, the results of homology analysis is 98.15% In summary, a new HAase gene from S. equi ATCC43079 was successfully cloned.
Key Words: Hyaluronidase; Streptococcus equi; clone; sequence analysis
目 录
1 前言 1
1.1 透明质酸酶概述 1
1.1.1 透明质酸酶的发现及分类 1
1.1.2 透明质酸酶的降解机制 2
1.1.3 透明质酸酶的结构 2
1.1.4 透明质酸酶的作用 3
1.2 产透明质酸酶的微生物概述 4
1.3 透明质酸酶编码基因概述 4
1.4 基因克隆技术概述 6
1.4.1 PCR技术 6
1.4.2 构建重组质粒 6
1.5本研究主要内容及研究意义 9
1.5.1 研究内容 9
1.5.2 研究意义 10
2 材料与方法 10
2.1 材料与仪器设备 10
2.1.1 菌种及质粒 10
2.1.2 实验试剂 11
2.1.3 主要仪器和设备 11
2.1.4 培养基及试剂的制备 12
2.2 实验方法 12
2.2.1 提取细菌基因组DNA 12
2.2.2 PCR反应 13
2.2.3 凝胶电泳验证及割胶回收 14
2.2.4 质粒抽提 14