【作者】 刘秀红；

【导师】 赖仞；

【作者基本信息】 南京农业大学 ， 动物学， 2008， 博士

【摘要】 大量研究已经证实,两栖类动物皮肤是生物活性物质丰富的资源库。而抗菌肽（Antimicrobial peptides,AMPs）作为机体先天防御系统的重要组成部分,在两栖类皮肤分泌液中含量异常丰富。本研究中,我们以来源于中国云南西双版纳的一种蛙科动物黑带蛙（Rana,nigrovittata）为试验材料,研究其皮肤分泌液中抗菌肽的分子多样性并对其结构和功能进行研究。通过两步分离纯化:Sephadex G-50凝胶过滤层析和反相高压液相层析（RP-HPLC）,我们从黑带蛙皮肤分泌物中初步分离到两种抗菌肽,与大多数蛙科来源的抗菌肽一样,它们C末端均有由二硫键形成的七肽环状结构,并因含有多个碱性氨基酸残基而使整个分子带正电荷。通过同源性比对并根据抗菌肽结构特点,将其分别命名为Nigroain-A1和Rugosin-RN1。为了进一步研究黑带蛙皮肤分泌液中抗菌肽的分子多样性,以对应编码蛙科抗菌肽前体信号肽区域从起始密码子开始的7个氨基酸残基的碱基序列为引物,从构建的单个黑带蛙皮肤cDNA文库中筛选抗菌肽的编码基因,我们共筛选到了199个编码抗菌肽的克隆。其中编码不同抗菌肽前体的cDNA序列共167条（Genebank登录号为EU136401-EU136567）,这167条序列共编码45个新的抗菌肽。根据其分子结构特点将它们归于15个不同的抗菌肽家族,其中有21个抗菌肽分别属于Rugosin-RN、Gaegurin-6-RN、Temporin-RN和Brevinin-2-RN这4个已知的蛙科来源的抗菌肽家族,其余24个与已知的抗菌肽相似性较低,我们将它们归类到11个新的家族,分别命名为Nigroain-A,-B,-C,-D,-E,-F,-G,-H,-I,-J,-L。这些新型抗菌肽大致可分为两类,线性肽和含分子内二硫键的环状肽,且以环状肽居多,这与以前从蛙科动物分离到大量环状肽而线性肽较少的研究结果相一致。根据家族内及家族间抗菌肽序列及结构的分析结果,我们认为,黑带蛙皮肤抗菌肽的分子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15个家族抗菌肽的前体编码单一拷贝的成熟肽,其SPD区域（包括信号肽和前导肽,Signal and Propiece Domain,SPD）都非常保守,而MD区域（成熟肽区域,Maturepeptide,MD）却变化很大,说明这些抗菌肽相关基因形成了一个起源于共同祖先的多基因家族,这也是导致两栖类抗菌肽多样性产生的分子机制之一。对来自黑带蛙皮肤抗菌肽生物功能的分析表明,这些结构多样的抗菌肽,生物功能也表现出多样性。首先它们具有广泛的抗菌谱和高效的抗菌活性,对革兰氏阳性菌、革兰氏阴性菌包括两者耐药菌株和真菌具有很强的杀灭作用;还普遍具有促进肥大细胞脱粒、促或抗组胺释放功能;相当一部分抗菌肽具有对自由基的清除能力和对Fe³⁺的还原力,表现出强烈的抗氧化活性;还发现同时具有丝氨酸蛋白酶抑制剂活性和凝集红细胞活性的抗菌肽;此外,有些抗菌肽还表现出溶血活性。特别是有些抗菌肽类似物已经失去抗菌活性,却表现出促进肥大细胞脱粒或抗组胺释放活性及抗氧化的功能。对这些抗菌肽一级结构的分析表明,氨基酸残基的个别突变对抗菌肽功能的影响非常明显,我们的试验结果显示可以改变抗菌肽抗菌谱的范围,对微生物的敏感程度;可以使溶血活性从强到弱;可以使抗氧化活性由无到有。可以说,单一的突变就可能很大程度上改变抗菌肽的生物活性。我们在黑带蛙皮肤发现一种同时具有抗菌活性,凝集兔血细胞和凝集细菌的功能,以及胰蛋白酶抑制剂活性的小分子多功能活性肽,Nigroain-A1。其仅对革兰氏阳性菌金黄色葡萄球菌（Staphylococcus aureus）具有抗菌活性,但能迅速引起革兰氏阳性、阴性菌及真菌的凝集,对兔红细胞的最小凝集浓度为15μg/ml,对胰蛋白酶的抑制常数为Ki=5.26×10^-6M;而且这种小分子活性肽在黑带蛙皮肤内含量丰富。这种多种活性集于一种小分子肽的现象推测主要是作为两栖类的一种集约型的防御机制。根据Nigroain-A1前体结构与抗菌肽前体序列的相似性以及三种生物活性物质都起到防御保护作用这一功能的相似性,我们推测这类抗菌肽和丝氨酸蛋白酶抑制剂以及凝集素可能来自同一祖先,长期的自然选择使它们共同的祖先基因发生分化或者先后进化出诸多不同的功能。Nigroain-A1这种小分子多肽作为多种生物活性的载体对研究基因形成机制、基因进化、生物活性物质构效关系及研制新型临床治疗药物具有重要意义。更多还原

【Abstract】 Numerous studies have proved that the amphibian skin is a treasure abundant in bioactive substances. Antimicrobial peptides（AMPs） are an important component of the natural defence of most living organisms, and An impressive number of AMPs have been found in amphibian skin secretions. In this thesis, The molecular diversity, structures and functions of antimicrobial peptides from skin secretions of a ranid frog, Rana nigrovittata was studied.Two novel antimicrobial peptides which belong to two different antimicrobial families were isolated from the skin secretions of Rana nigrovittata by a two-step protocol including Sephadex G-50 and RP-HPLC, and they were named Nigroain-A1, Rugosin-RN1,respectively. As most AMPs from ranid frogs, the two peptides with antimicrobial activity share a conserved disulfide-bridged heptapeptide segment at the C-terminal, and are cationic because of several basic amino acid residues.By molecular cloning, 167 cDNA sequences （Genebank mumbers,EU136401-EU136567）of codeing different antimicrobial peptides precursors were obtained from a single individual skin of Rana nigrovittata. All these 167 cDNA sequences encode 45 novel antimicrobial peptides belonging to 15 divergent families, 21 of which belong to four of the known antimicrobial families previously identified in the skins of other species of Ranid frogs: Rugosin-RN,Gaegurin-6-RN,Temporin-RN and Brevinin-2-RN. The other 24 peptides show little structural similarity towards other known antimicrobial peptides and so are classified into 11 new families: Nigroain-A,-B, -C, -D, -E, -F, -G, -H, -I, -J, -L. The cDNA-encoding precursors of the 15 antimicrobial peptide families code for a single copy of the mature antimicrobial peptide and they share similar N-terminal signal and propiece peptide domain, followed by a markedly different C-terminal domain corresponding to the mature AMPs. All this novel antimicrobial peptides can be roughly placed into one of two groups: Linear peptides and Annular peptides contain a single intramolecular disulfide bond, and the latter is predominant, which is consistent with the known antimicrobial families previously identified in the skins of Ranid frogs. Our results suggest that point mutations, as well as insertion, deletion, and "shuffling" of oligonucleotide sequences can be responsible for the patterns of molecular diversification of antimicrobial peptides from skin of Rana nigrovittata. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides suggests that the corresponding genes form a multigene family originating from a common ancestor,which is one of the reason of molecular diversity of amphibian AMPs.These structural diverse peptides exhibit multiple biological function. They have a broad spectrum of antimicrobial properties, exhibiting against Gram-positive and Gram-negative bacteria including their resistant strains, as well as fungi. Most of this novel peptides can induce And quite a few possess intensive antioxidant activities in terms of free radical scavenging activity and reducing power. we also found a muli-functional antimicrobial peptide with serine protease inhibitor and hemagglutination properties. Moreover, some of them are hemolytic peptides. Especially, some antimicrobial peptide-like peptides which have devoid of antimicrobial activity however can provoke mast cell degranulation or have anti-histamine release activity, as well as antioxidant activity. The individual mutation of amini acid residues has conspicuous influence on the antimicrobial, hemolytic and antioxidant properties.A muli-functional peptide from the skin of Rana nigrovittata, Nigroain-A1,which is abundant in the skin secretions of this ranid frog, only against Gram-positive bacterium {Staphylococcus aureus), but its ability to quickly agglutinate Gram-positive, Gram-negative bacteria and fungi was observed. The minimum agglutinative concentration of rabbit red cells is 15μg/ml. The inhibition constant to trypsin is 5.26×10^-6 M. This multi-functional peptide may provide an effective defense against invading microbes and predators in the amphibian Rana nigrovittata. The precursor structural similarity of Nigroain-A1 with other antimicrobial peptides and the functional similarity of three bioactive compounds which are all direct defensive molecules against microorganisms or pests suggest that these three functional molecules might share a common ancestor. Nigroain-A1,as the multi-functional vector, play an important role in studying gene formation and evolution,structure-function relationship of bioactive substances and novel clinical drugs.更多还原