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题名	无指盘臭蛙皮肤抗感染多肽组学研究
作者	李建许
学位类别	博士
答辩日期	2007-06
授予单位	中国科学院研究生院
授予地点	北京
导师	赖仞
关键词	两栖类 抗菌肽 多肽组学 机制
其他题名	Anti-infective Peptidomics of Odorrana Grahami skin
学位专业	动物学
中文摘要	在过去的一个多世纪里，两栖类动物皮肤分泌液作为它们的第一道防御屏障引起了研究者们极大的兴趣，同时也开展了相关的许对研究，到目前为止，已从中分离鉴定出了百余种的活性物质。无指盘臭蛙是我国的一种特有两栖动物，初步的活性检测发现，无指盘臭蛙皮肤分泌液具有很强的抗菌，溶血以及蛋白酶抑制剂活性。 　　在本论文中，我们利用多肽组学与基因组学的方法对无指盘臭蛙皮肤的抗感染多肽组进行了研究。 　　通过三步分离纯化过程：一步Sephadex G-50分子筛和两步反相高压液相（RP-HPLC）的方法，从无指盘臭蛙皮肤分泌液中分离纯化得到了21条新的抗菌肽，它们分别属于17个不同的抗菌肽家族，其中8个分别属于已知的5个抗菌肽家族，它们是：Brevinin-1E(2个)、Brevinin-2E（1个）、Esculentin-1（1个）、Esculentin-2（1个）和Nigrocin（3个）抗菌肽家族。另外的13个抗菌肽与已发现的抗菌肽表现出较低的相似性，我们将它们归类到12种新的抗菌肽家族，它们是：Odorranain-A (1个)、Odorranain-B(1个)、 Odorranain-C(1个)、 Odorranain-G (1个)、Odorranain-H (2个)、 Odorranain-J (1个)、Odorranain-L (1个)、Odorranain-M (1个)、 Odorranain-N (1个)、 Odorranain-O (1个)、Odorranain-Q(1个)、 Odorranain-T(1个)。 　　从单个无指盘臭蛙皮肤里面，我们克隆得到了372条抗菌肽序列，它们编码107条新的抗菌肽，这一发现使得目前发现的两栖类抗菌肽的数目几乎增加了1倍。这也是目前发现的抗菌肽最为丰富的物种。这107条抗菌肽分别属于30个不同的抗菌肽家族，其中有24个为新的抗菌肽家族。这些抗菌肽的多样性可能是通过点突变、碱基的插入或删除、结构域的穿梭以及拼接等多种机制形成的。这些抗菌肽多样性的形成可能与无指盘臭蛙生活环境中微生物的组成有关。30个家族抗菌肽前体序列的SPD区域（包括信号肽和前导肽序列，Signal and Propiece Domain, SPD）非常保守，表明它们可能起源于同一个祖先基因。对7个抗菌肽家族的非同义碱基替代率Dn与同义碱基替代率Ds进行检测发现，它们可能经受着不同选择压力的作用。 　无指盘臭蛙皮肤抗菌肽在二级结构和功能上都表现出丰富的多样性。在一个两栖类个体里面发现如此丰富的抗菌肽甚是让人惊讶。这一发现也使得我们不得不重新认识先天性免疫在两栖类动物防御系统中的重要性，对两栖类生态环境的分子基础以及那种认为一种两栖类只需要20-30种抗菌肽就足以抵御环境中的微生物的看法重新审视。我们的研究还显示：无指盘臭蛙抗菌肽之间还存在着协同效用。 我们对无指盘臭蛙皮肤抗菌肽的去极化作用进行了研究，发现所检测的7个抗菌肽都可使金黄色葡萄球菌发生去极化，但是它们使细菌发生去极化的能力不同。对12种抗菌肽的抗菌机制研究发现，它们通过多种不同的机制发挥作用：有些在细菌内形成片层样的囊泡状结构，有些导致细胞质壁的分离，有些在细菌的膜上形成穿孔，有些则导致了细菌染色质的固缩。总之，这些研究为设计新型的抗菌肽提供了有用的参考资料。
英文摘要	During the past century, numerous researches have been carried on the amphibian skin secretions for they form the first line of host defense. To date, hundreds of bioactive substances have been isolated from them. Odorrana grahami is a specific species of China. Preliminary tests showed that the skin secretions of Odorrana grahami had strong antimicrobial, hemolytic, and protease inhibitory activities. In this thesis, peptidomics and genomics analyses were used to study the anti-infection array of peptides of Odorrana grahami skin. 21 novel antimicrobial peptides were isolated from the skin secretions of O. grahami by a three-step protocol including one step Sephadex G-50 and two steps of RP-HPLC, and they belong to 17 different antimicrobial families. Among the 21 novel antimicrobial peptides, 8 of which belong to five of the known antimicrobial families previously identified in the skins of other species of Ranid frogs: Brevinin-1E (2 peptides), Brevinin-2E (1 peptide), Esculentin-1 (1 peptide), Esculentin-2 (1 peptide), and Nigrocin (3 peptides). The other 13 peptides show little structural similarity towards other known antimicrobial peptides and so are classified into 12 new families: Odorranain-A (1 peptide), Odorranain-B (1 peptide), Odorranain-C (1 peptide), Odorranain-G (1 peptide), Odorranain-H (2 peptides), Odorranain-J (1 peptide), Odorranain-L (1 peptide), Odorranain-M (1 peptide), Odorranain-N (1 peptide), Odorranain-O (1 peptide), Odorranain-Q (1 peptide), Odorranain-T (1 peptide). By cDNA cloning, 372 cDNA sequences of antimicrobial peptides were obtained from a single individual skin of this frog, encoding 107 novel antimicrobial peptides. This contribution almost doubles the number of currently reported amphibian antimicrobial peptides. These peptides can be organized into 30 divergent families, including 24 novel families. The diversity in peptide coding cDNA sequences is, to our knowledge, the most extreme yet described for any animal. The patterns of　diversification, suggest that point mutations, as well as insertion, deletion, and “shuffling” of oligonucleotide sequences have been responsible. The diversity of antimicrobial peptides may have resulted from the diversity of microorganisms. The precursors of the 30 antimicrobial peptide families share similar signal and propiece peptide sequences, which show that they might origin from the same ancestral gene. The rate of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions were calculated in seven antimicrobial peptide families, and they displayed different nucleotide substitution patterns, indicating they might undergo different selecting pressures. These diverse peptides exhibit both diverse secondary structure and “host-defense” properties. Such extreme antimicrobial peptide diversity in a single amphibian species is amazing. This makes us have to reconsider the strong capability of innate immunity and molecular genetics of amphibian ecological diversification and doubt the general opinion that 20-30 different antimicrobial peptides can protect an animal because of the relatively wide specificity of the peptide antibiotics. Our studied also show synergy exists among the skin antimicrobial peptides of O.grahami. The cytoplasmic-membrane depolarization activities were determined on 7 antimicrobial peptides from different families. The results demonstrated that all the peptides studied here could depolarize the cytoplasmic membrane of S. aureus but with different potential. Their antimicrobial mechanisms were investigated. They exert their antimicrobial functions by various means, including forming lamellar mesosome-like structures, peeling off the cell walls, forming pores, and inducing DNA condensation. In conclusion, with respect to the development of antibiotics, these peptides provide potential new templates to further explore.
语种	中文
公开日期	2010-10-14
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/152453/6126]
专题	昆明动物研究所_动物毒素室
推荐引用方式 GB/T 7714	李建许. 无指盘臭蛙皮肤抗感染多肽组学研究[D]. 北京. 中国科学院研究生院. 2007.

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