透明质酸酶编码基因的克隆及序列分析
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
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