大蹼铃蟾新型抗菌肽的研究
文献类型：学位论文
| 作者 | 王婷 |
| 学位类别 | 硕士 |
| 答辩日期 | 2005-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 张云 |
| 关键词 | 大蹼铃蟾 抗菌肤 抗支原体活性 cDNA文库 |
| 中文摘要 | 两栖类动物皮肤裸露，柔软而湿润，体表富含腺体，其皮肤分泌物含有大量的具有特殊分子结构、功能复杂多样的生物活性分子。从两栖类己经分离到的包括血管紧张素样肤，促甲状腺素释放肤，蛙啡肤，舒缓激肤样多肤，速激肤样多肤，雨蛙肤样多肽等14类多肤家族以及生物胺生物碱等。"大蹼铃蟾是两栖类无尾目的一种，是我国的一个特有物种，主要分布于中国西南，其中云南山区种群数量巨大，多生活在污水糖和沟渠中，生活环境富有大量病原体。其皮肤腺体发达，受刺激能大量分泌富含活性物质的粘液，是分离生物活性分子的优良来源。其中抗菌肤作为活性分子的一大类，在大蹼铃蟾皮肤分泌物中不断有新的发现。通过大蹼铃蟾皮肤cDNA文库的构建，并对cDNA文库中的793个长度大于500个碱基的cDNA克隆进行随机测序，得到一类新的大蹼铃蟾抗菌肤，命名为Inaximins。由4个cDNA克隆推测得到这一类的新的抗菌肤，一共可以分为5个不同的种类，分别命名为maximinS1-5。这一类由cD瓜序列推测而来的新型抗菌肤，除了maximinS1只有14个氨基酸残基组成，maXiminS2-5都由18个氨基酸残基。并且从氨基酸残基序列上分析，新的maximinS与以往分离的到的大蹼铃蟾抗菌肤InaX1in和IllaXi"inH都不具有相似性。和东方铃蟾、多彩铃蟾皮肤分泌物中分离到的bobinin和bOInininH相比较，序列上也无同源性。从cDNA的结构上看，其编码的前体蛋白呈现一种全新的排列方式。每个cDNA克隆编码的maxininS前体蛋白都是由两个部分：信号肤加上一组不同maximinS和间隔肤的不同排列。这种奇特的排列组合方式，在东方铃蟾和多彩铃蟾都未曾发现。出现这个排列方式的原因，是由于基因本身结构，或是由于mRNA的可变剪切，或是由于体细胞基因重排，都不得而知，需要进一步的研究除了maximinS1，maximinS2-5，其二级结构分析都显示出一种类似的两亲性的Q螺旋空间结构。为了检测这一类新的大蹼铃蟾的活性，maximinsl和maximinS4用人工合成的方法得到，并对其进行了抗菌肚活性的测定。活性测定的结果显示，相同条件下，maximinS1不具抗菌活性，maximinS4具有显著的抗支原体的活性。这些现象间接证明了，小分子的抗菌肤其功能可能与氨基酸序列无关，而和空间构象由更加密切的联系。由于抗菌肤maximinS是由cDNA序列推导而来，并没有直接通过生化分离方法从大蹼铃蟾皮肤中得到。为了证明maximinS确实会被大蹼铃蟾表达，设计了一系列试验，将大蹼铃蟾皮肤分泌物通过简单的分子筛分离，追踪抗菌活性，并对具有活性的组分用MALDI-TOP质谱仪分析，证明maximinS类抗菌肤确实载大谱铃蟾皮肤分泌物中存在。 |
| 英文摘要 | Amphibian skin glands are rich resources of biologically active compounds, such as biogenic amines, complex alkaloids, or peptides, which are produced by holocrine type serous glands in the integument. These compounds should play different roles in the regulation of physiological functions of the skin and in defense against predators or microorganism. The Chinese red belly toad (Bombina maxim) is an endemic amphibian in the mountainous regaions of southwester China. The toad lives, in very harsh environment such as pools with microorganism-rich mud and that its skin is very "toxic" . The antimicrobial peptides secreted by the toad, which are either constitutive or inducible, play an important role in innative immunity. A novel type of peptide, designated as maximin S, was deduced by random sequencing of 793 clones from a'constructed B. maxima skin cDNA library. The putative primary structure of maximin S peptides can be grouped into five species, in which maximin SI has 14 amino acid residues and the rest of maximin S peptides (maximin S2~5) all have 18 amino acid residues. No obvious similarity was found in their amino acid sequence compared with other known animicrobial maximin peptides and maximin H peptides. Meanwhile, the same situation was found when compareing maximin S with bombinin and bominin H peptides, which were isolation from Bombina oriental is and Bombina variegateu Most of the amphibian antimicrobial peptides so far identified, like bombinin and bominin H precursors, are simply assemblies of .one or two copies of BLP plus a bombinin H peptide. The newly characterized four maximin S precursors are composed of maximin SI and different combination of tandem repeated maximin S2-5. linked by internal peptides. The number of copies and the position of each copy are diverse but not irregular. Excep maximin SI, the predicted secondary structures of maximin S2~S5 show a similar amphipathic a-helical structure. Two deduced maximin S peptideds (SI, S4) were synthesized and their antimicrobial activities were tested. Maximin S4 only had an antibiotic activity against mycoplasma and had no antibacterial or antifungal activity toward tested strains, Maxiniin SI had no activity under the same conditions. Considering the small size of antimicrobial peptides, the activity is more related with the three-dimensional structure than the sequence identity. To investigate whether the predicted maximin S peptides are expressed at the surface of the toad1 s skin, the B. maxima skin secretions were partially purfied and relative antimicrobial fraction were subjected to mass spectrum analysis- MALDI~TOP mass spectrometry analysis indicates that most of the deduced maximin S peptides are expressed |
| 语种 | 中文 |
| 公开日期 | 2010-10-15 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6167]  |
| 专题 | 昆明动物研究所_动物活性蛋白多肽组学 |
| 推荐引用方式 GB/T 7714 | 王婷. 大蹼铃蟾新型抗菌肽的研究[D]. 北京. 中国科学院研究生院. 2005. |
入库方式： OAI收割
来源：昆明动物研究所
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