【作者】 侯峰；

【导师】 高宏伟；

【作者基本信息】 中国人民解放军军事医学科学院 ， 预防兽医学， 2010， 博士

【摘要】 抗菌肽(antibacterial peptides)是生物免疫系统产生的一类抵抗外界病原体感染的多肽,广泛存在于昆虫、植物、动物及人体内。它除有非特异性的抗细菌、真菌、病毒等病原体外,还有抗肿瘤细胞的作用。特别是对多重耐药菌和肿瘤细胞有杀伤作用,而不破坏人体的正常细胞,是一类具有巨大发展潜力的新型抗菌药,是目前抗病原微生物药物开发研究中的一个热点。两栖类动物皮肤光滑、裸露,没有鳞片、甲壳和毛发的保护,但能分泌具有良好保护作用的皮肤粘液,皮肤粘液中含有大量分子结构特殊、功能复杂多样的抗菌活性物质,抗菌肽是其中最重要的生物活性物质之一,是两栖动物机体先天防御系统的重要组成部分。本研究以光滑爪蟾、中国林蛙,东北林蛙和中华大蟾蜍为实验材料,通过两种诱导方式提取这四种两栖动物的皮肤分泌液,进行了一系列的生物活性测定。结果表明,不同种类的两栖动物皮肤分泌液生物活性并不相同,不同方法诱导所提取的同一类两栖动物的皮肤分泌液的生物活性也有差异。体外抑菌活性检测结果表明,四种两栖动物皮肤分泌物对不同菌株均产生不同程度的抑菌效果,其中以光滑爪蟾的抑菌效果最强,除此之外,光滑爪蟾皮肤分泌物还有抑制和杀伤癌细胞的作用,并具有胰酶抑制剂活性和极其微弱的溶血活性。基于上述结果,以光滑爪蟾为实验材料,通过不同方法分离皮肤分泌物中的抗菌肽,并对其结构和功能做进一步研究。通过四步分离纯化：SephadexG-25凝胶过滤层析、制备型反相高压液相层析、离子交换层析和分析型反相高效液相色谱层析,分离纯化出两种抗菌肽：AMP-1和AMP-2,通过氨基酸N端测序和MALDI-TOF/TOF-MS人工De nove手动拼接两种方法相结合的方式对抗菌肽AMP-1和AMP-2的一级结构进行鉴定。网上搜库序列比对后,确定本实验所获得的两种抗菌肽均为首次发现。对两种新发现的抗菌肽进行最小抑菌浓度(MIC)检测、胰蛋白酶抑制活性检测、溶血活性检测和抗肿瘤活性检测。结果显示：AMP-1和AMP-2对革兰氏阳性菌和革兰氏阴性菌都有抗菌活性,AMP-2还对绿脓杆菌也有较弱的抗菌活性,并且AMP-1和AMP-2对多种耐药菌株也具有不同程度的抗菌活性。由于检测抗菌活性的微生物来源不可能与两栖类动物生存环境的微生物谱系相同,还可能由于我们所检测的微生物与特定抗菌肽的天然靶向微生物存在差异,因此导致我们没有检测到抗菌肽对个别菌株的抗菌活性。除抗菌活性外,AMP-1和AMP-2均有较强的胰蛋白酶抑制活性,同时也表现出了极其微弱的溶血活性。抗肿瘤结果显示：AMP-1并没有杀伤癌细胞的作用,而AMP-2却表现出了很好的抗肿瘤的效果,当样品浓度达到146.6μg/ml时,AMP-2可以杀死81.1%人乳腺癌细胞MCF-7。为了进一步研究这两种抗菌肽的结构与功能,本实验通过ExPASy网站上的二级结构预测软件Jpred对其进行二级结构的预测,通过三级结构预测软件CPHmodels对其进行空间结构预测。以此为基础,通过替换和缺失AMP-1中赖氨酸对其进行结构改造,又得到九条新的抗菌肽。同时在NCBI中进行搜索,找到与AMP-1和AMP-2序列相似的抗菌肽,并利用多肽固相合成的方法合成16条抗菌肽。将合成的多肽通过高效液相色谱法进行纯化,共得到5条纯度在99%以上,8条纯度在95%以上的抗菌肽。以金黄色葡萄球菌为指示菌,检测13条纯品抗菌肽的抗菌活性,结合预测结果推测抗菌肽AMP-1中每个赖氨酸对抗菌肽的结构以及抗菌活性的作用。由于抗菌肽一般都是含有α-螺旋结构的两亲性分子,疏水性及带电荷数对其生物活性影响非常大。因此,单一的氨基酸突变就可能很大程度上改变抗菌肽的空间结构和生物活性。在对抗菌肽AMP-1进行结构改造的过程中,我们又获得了四条新的具有抗菌活性的抗菌肽。为了进一步阐明光滑爪蟾抗菌肽的杀菌机理,将E.coli ATCC25922与S.aureus ATCC25923菌株分别与光滑爪蟾抗菌肽AMP-1和AMP-2共孵育后进行透射电镜观察分析,光滑爪蟾抗菌肽对细菌形态及细菌表面结构的影响。结果显示：处理前两者具有完整的细胞壁和细胞膜,并且其细胞结构完整、表面光滑,能观察到完整的菌毛,细胞表面没有损伤。细胞内的蛋白质和DNA等物质能与染色液中所含有的重金属离子结合而使菌体呈现深黑色。经过抗菌肽AMP-1和AMP-2处理后,E.coli ATCC25922和S.aureus ATCC25923均有部分细胞内容物外泄,细胞出现破损或肿胀,菌体部分或完全破裂,并伴有细胞质稀释的现象,细胞表面存在破损成小孔的地方,细胞膜界限模糊不清,甚至细胞膜完全溶解。由于细胞内容物外渗,细胞质解体,形成空腔,致使被染色的细胞颜色变浅。以上结果表明,抗菌肽AMP-1和AMP-2能够使E.coli ATCC25922、S.aureus ATCC25923菌体细胞壁破裂或者在细胞表面形成孔洞,内容物外泄,最终导致菌体死亡。结论：本实验从光滑爪蟾皮肤分泌物中分离纯化出两种新的抗菌肽AMP-1和AMP-2,两种抗菌肽都具有广谱抗菌活性,胰蛋白酶抑制活性,微弱的溶血活性,AMP-2还具有很好的抗肿瘤活性。结构预测后对抗菌肽AMP-1进行结构改造,又获得四种新的具有抗菌活性的抗菌肽AMP-1-2、AMP-1-3、AMP-1-4和AMP-1-5。通过透射电子显微镜观察,抗菌肽AMP-1和AMP-2对细菌的细胞壁都有不同程度的破坏作用。更多还原

【Abstract】 Antibacterial peptide is a kind of polypeptides that generate by biological immune system to resist to infection caused by heterogeneous pathogens they ubiquitously exist in insects,plants and human bodies. Besides nonspecific resistance to bacterias,fungi and viruses, they still can function anti-tumor-cell.Especially their lethal effect to mutidrug resistant bacteria and tumor cells without destroy the normal cells of human, that is a new pattern of antibacterial with tremendous development potential,and is a hotspot of pharmaprojects that function resistant to pathogenic microorganism. Amphibia animals’skins are smooth and glossy, naked, and no scalinesses, shells or hairs,but they can secrete skin mucilage possessing protective effect. The skin mucilage contain mount of antimicrobial active substances that have special molecular structure and complicated and diverse functions,and antibacterial peptide is one of most important bioactive substances that consist of important parts of connatural defense system of Amphibian and extreme abundance in their skins.Using Xenopus laevis daudin,Rana temporaria chensinensis,Rana dybowskii Gurnther as experiment materials,the research extracted skin secretion four species of Amphibians through two induction methods,and measured a series of bioactivities.Among measured bioactivities, skin secretion of differerent species of Amphibians have different bioactivities,while the same Amphibians skin secretion extracted by different induction methods also have different bioactivities. Results of bacteriostatic activity test in vitro showed that skin secretion of four species of amphibians have didderent bacteriostatic effect to different bacteria strains while Xenopus laevis daudin possesses the strongest effect; Addition to this, skin secretion of Xenopus laevis daudin can also resrain, wound and kill tumor cells;they still have trypsogen inhibitors activity and extremely feeble haemolysis activity.Therefore,we made Xenopus laevis daudin as experiment materials,screened antibacterial peptides from their skin secretion and studied their structures and functions.Using four steps separation and purification:SephadexG-25 gel filtration chromatography, preparation mode of reverse phase,ion exchange chromatography,analysis mode of reversed-phase high-performance liquid chromatography,we separated and purified two antibacterial peptides:AMP-1 and AMP-2, combining amino acid sequencing and MALDI-TOF/TOF-MS methods identitied primary structures of antibacterial peptides AMP-1 and AMP-2.After comparing with sequences that searched database on Internet, it can be identitied that those two antibacterial peptides are newfound antibacterial peptides.Then conducting those two newfound antibacterial peptides with minimal inhibitory concentraton test(MIC); trypsinase inhibitory activity test; haemolysis activity test; anti-tumor activity test.Results displayed:the two antibacterial peptides both showed antibacterial activity to both Gram-Positive Bacteria Gram-Negtive Bacteria, AMP-2 showed faint antibacterial activity to seudomonas Aeruginosa, AMP-1 and AMP-2 manifested different degree of antibacterial activity to drug resistant strains.Because the origin of Microorganisms that used to test antibacterial activity cannot be identical with Microorganisms pedigree that existed in amphibian habitat, therefore, the possible differences of Microorganisms between we’ve tested and specific antibacterial peptides targeted may cuased that we hadn’t tested antibacterial peptides’antibacterial activity to individual strains, in may also be the cause that antibacterial peptides showed low hyposensitivity to individual strains.Besides antibacterial activity, AMP-1 and AMP-2 both showed comparatively strong trypsinase inhibitory activity,meanwhile both showed extemly feeble haemolysis activity.Anti-tumor results showed that:AMP-1 had lethal effect on tumor cells while AMP-2 showed well anti-tumor effect,when sample concentration reached 146.6μg/ml,it can killed breast cancer cells MCF-7 up to 81.1%.In order to futher study structures and functions of the two antibacterial peptides, this study predicted their secondary structures and spatial structures through secondary structure prediction software Jpred and tertiary structure prediction software CPHmodels, reformed AMP-1by replacing and deleting its lysines,and thus gained nine new antibacterial peptides,after searching sequence-similar antibacterial peptides in NCBI, using amine acid solid-phase synthesis methods synthesized fifteen antibacterial peptides,after purifying with high efficiency liquid chromatography,obtaining five antibacterial peptides that purity reached above 99%,and nine above 95% Using Staphylococcus aureus as indicator to test antibacterial activity of fourteen antibacterial peptides, and combined with prediction results to predict the functions of every lysine to structure and antibacterial activity of AMP-1.Because antibacterial peptides generally containsα-helices, hydrophobicity and charge number affect their bioactivity extraordinarily. as far as antibacterial peptide is concerned,the mutation of a single of amine acid is possible to a great extent to change its spatial structure and bioactivity. In the process of reforming antibacterial peptide,we again obtain another four new antibacterial peptides that possessing antibacterial activity.In order to further explain the antibacterial activity mechanism of antibacterial peptides originated from Xenopus laevis, After co-incubating E.coli ATCC25922 and S.aureus ATCC25923 with AMP-land AMP-2 respectively, using transmission electron microscope to observe the effect that antibacterial peptides of Xenopus laevis imposed on bacterial morphous andstructure.Before the treatment, E.coli ATCC25922 had intact cell wall and cell membrance,and moreover had complete cellularity,the surface was smooth and glossy,and can be observed intact pili,the cell surface had no injure. In addition, intracellular proteins can combine the heavy metalions that exist in staining solution,and thus make bacteria as Pitch black. E.coli ATCC25922 treated with AMP-land AMP-2 leaked parts of cell entocyte,and cells would be damaged or swelled, thalli would fracture partly or completely acccompanied with cytoplasm being diluent.As a result of permeate of cell entocyte, cytoplasm fractured and formed hollow cave,therefore made the color of stained cells faded.All above indicated that: AMP-land AMP-2 could make cell wall of E.coli ATCC25922 breakage or form the hole,caused cell entocyte leakage and finally led to the death of bacteria.Conclusions:the study separated and purified two new antibacterial peptides AMP-land AMP-2,both of them had broad spectrum of antibacterial activity, trypsinase inhibitory activity and feeable haemolysis activity, AMP-2 also possessed well anti-tumor activity.After structure perdiction,reformed the structure of antibacterial peptide AMP-1,and obtained four new antibacterial peptides with antibacterial activity:AMP-1-2,AMP-1-3,AMP-1-4 and AMP-1-5. Observed AMP-land AMP-2could to different extent destroy the cell wall.更多还原