【作者】 王旭；

【导师】 赖仞；

【作者基本信息】 南京农业大学 ， 动物学， 2008， 硕士

【摘要】 抗菌肽（Antimicrobial peptides,AMPs）是一类小分子多肽（分子量低于10 KDa）,最早因为具有广谱抗菌活性被发现及命名,后来因为不容易诱导微生物产生耐药性而引起世界范围内的广泛关注。随着研究的不断深入,人们发现:抗菌肽是先天免疫最终效应物之一,在防御和消除外来病原体（细菌、真菌、病毒、原虫等）对机体的侵袭和破坏、维持机体正常的生理机能等方面发挥重要的作用,是炎症反应、免疫激活和创伤愈合等过程中的重要活性因子。在植物、动物（昆虫、两栖类、人类）、甚至微生物体内都发现有抗菌肽（样）的存在。抗感染多肽（Anti-infective peptides）概念的提出,对抗菌肽在名称上进行了功能性的修正。两栖类皮肤分泌液是抵御微生物侵染的第一道防御屏障,自1987年Zasloff等人从非洲爪蟾皮肤中分离到Magainin以来,各国学者对两栖类皮肤分泌液的研究越来越广泛和深入起来。滇蛙（Rana pleuraden）,是我国特有种之一,主要分布在我国云南、贵州、四川等地,药用及经济价值尚未被发掘,有广阔的开发利用空间。在本论文中,我们利用生物化学及分子生物学的手段对滇蛙皮肤的抗感染多肽进行了研究。通过两步法（Sephadex G-50和RP-HPLC）对滇蛙皮肤分泌液的成分进行分离纯化,我们得到2种具有抗菌活性的多肽,命名为pleurain-A1和pieurain-A2.Edman降解法测定其全序列,分别是SIITMTKEAKLPQLWKQIACRLYNTC和SIITMTKEAKLPQSWKQIACRLYNTC.运用基质辅助激光解离离子化技术（MALDI-TOF-MS）测定其分子量分别为3048.5 Da和3024.8 Da。使用CLONTECH公司的Creator^TM SMART^TM cDNA Library Construction Kit构建了滇蛙皮肤的cDNA文库,根据蛋白序列比对,以相近的抗茵肽信号肽N端序列设计引物序列,我们从滇蛙皮肤抗感染多肽亚克隆文库中,筛选得到135条由抗菌肽信号肽编码的抗感染多肽cDNA核酸序列（GenBank accession no.:EF613243-EF613259,EF621707-EF621709,EU138029-EU138143）,编码34种多肽,分属于12个抗菌肽家族,pleurain-As（4个）,pleurain-Bs（4个）,pleurain-Cs（2个）,pleurain-Ds（6个）,pleurain-E（1个）,pleurain-F（1个）,pleurain-Gs（9个）,pleurain-Js（2个）,pleurain-K（1个）,Lividin-RP（1个）,pleurain-Ms（2个）,pleurain-N（1个）。对这些抗感染多肽的分子多样性进行研究发现,这12个家族的抗菌肽前体序列的SPD区域（Signal and PropieceDomain,SPD）非常保守,Mega及DAMBE分子遗传分析表明,它们可能起源于同一个祖先基因。对其进行同义突变率及错义突变率检测发现,它们可能经受着不同的选择压力。随机选取10个家族的抗感染多肽进行人工合成,研究其生物学功能多样性发现:pleurain-A1,pleurain-D4,pleurain-G7,pleurain-M1具有广谱的抗微生物活性,pleurain-J1表现出较窄的抗菌谱（抗金黄色葡萄球菌）,而其他家族的肽类物质并不直接具有杀死微生物的能力。同一家族的抗感染多肽pleurain-G1和pleurain-G7表现出明显不同的抗茵活性,pleurain-G1的抗茵肽活性本实验过程中未检测到。此外,我们还发现这10个家族的抗感染多肽普遍具有促进组胺释放、促进肥大细胞脱粒、凝集红细胞、清除自由基等免疫相关生物学活性的一种或几种,预示着抗菌肽（样）的存在可能跟机体免疫反应密切相关。随着对抗菌肽研究的不断深入,人们对机体先天免疫有了更进一步的理解和认识。本研究为寻找新型传统抗生素替代药物——抗菌肽（抗感染类）药物提供了更多的设计模板。更多还原

【Abstract】 Antimicrobial peptides(AMPs) are purified from the skin secrection of amphibians for many years.As one of important molecular factor,AMPs play roles in anti-infective activities,concluding broad-spectrum antimicrobial activity,inflammatory reaction, immunity activated and wound healing.Amphibian skin glands are rich resources for antimicrobial peptides.R.pleuraden is distributed in Southwest of China,Yunnan,Guizhou and Sichuan provinces.In order to investigate possible novel Rana antimicrobial peptides of amphibians living in plateau,we describe the isolation,characterization,cDNA cloning and biological activities of a new family of antimicrobial peptides from skin secretions of Yunnan frog,R. pleuraden.Two purified antimicrobial peptides were named pleurain-A1 and -A2, respectively,and subjected to amino acid sequence analysis by automated Edman degradation.The result showed that they are composed of 26 amino acids with amino acid sequences of SIITMTKEAKLPQLWKQIACRLYNTC(pleurain-A1) and SIITMTKEAKLPQSWKQIACRLYNTC(pleurain-A2),respectively.The seven amino acids at the C-terminus are disulfide-bridged heptapeptide segments.By MALDI-TOF-MS analysis,the observed molecular weights were 3048.5 Da and 3024.8 Da,respectively that matched well with the theoretical molecular weights(3048.6 Da and 3024.6 Da).By cDNA cloning,135 cDNA sequences of anti-infective peptides were obtained from a single individual skin of Yunnanfrog,encoding 34 novel antimicrobial peptides (GenBank accession no.:EF613243-EF613259,EF621707-EF621709,and EU138029-EU138143). These peptides ban be organized into 12 different families named pleurain-As, pleurain-Bs,pleurain-Cs,pleurain-Ds,pleurain-E,pleurain-F,pleurain-Gs,pleurain-Js, pleurain-K,Lividin-RP,pleurain-Ms,pleurain-N,including 11 novel families.By Mega and DAMBE(Data Analysis in Molecular Biology and Evolution),the rate of nucleotide transition(ns)/nucleotide transversion(nv) and synonymous(ds)/nonsynonymous(dn) nucleotide substitutions of precursor,signal and propiece peptide and mature peptides were calculated.The result displayed that they might origin from the same ancestral gene,but undergo different selecting pressures.Diversity biological activities assay showed that the synthesized 10 families anti-infective peptides,pleuraln-A1,pleurain-D4,pleurain-G7,pleurain-M1 have broad-spectrum antimicrobial activity,which pleurain-J1 narrow.Some anti-infective peptides have one or several of histamine release,mass cell degranulation,erythrocyte hemagglutination,antioxidant activities,and these activities have relationship with immune response.This work has its contribution to augment the number of currently reported amphibian anti-infective peptides.更多还原