抗菌肽活性及天然抗菌肽的改造

抗菌肽具有抗菌谱广、热稳定性强、分子量小及免疫原性小等特点,其杀菌机制独特,病原菌不易产生耐药性,有望开发成新一代肽类抗生素。本文主要综述了影响抗菌肽生物活性的生化性质,即螺旋度、疏水性、两亲性、正电荷数等,并从结构的角度论述了其对抗菌肽抑菌活性的影响。部分抗菌肽具有空间结构不稳定、溶血活性等缺点,限制了其临床应用。因此,对天然抗菌肽的改造也成为目前抗菌肽的研究热点,本文还综述了天然抗菌肽的改造方法。     

海洋酸化对海洋无脊椎动物的影响研究进展

人源二氧化碳(CO2)的大量排放,导致空气中CO2浓度越来越高,其中大约1/4至1/3被海洋吸收。过多CO2在海水中的溶解,除引起海水p H值降低外,还导致海水中碳酸盐平衡体系的变化,即"海洋酸化"现象。很多海洋无脊椎动物不但在海洋生态系统中发挥重要作用,还是重要的水产养殖种,因此具有重要的生态与经济价值。由于海洋无脊椎动物的生活史在海水中完成,因此海洋环境的变化极易对其造成影响。大量研究已证实海洋酸化能对多种海洋无脊椎动物的受精、发育、生物钙化、基因表达等生命活动产生显著影响。综述了近年来海洋酸化对海洋无脊椎动物影响研究的相关报道,归纳了其对海洋无脊椎动物不同生命活动的影响,分析了其生态学效应,探讨了现有研究在方法创新、内容拓展以及机理分析等方面存在的局限与不足,并展望了海洋酸化对海洋无脊椎动物影响研究的发展方向。     

三条新颖短肽筛选及抑菌作用的初报

[目的]为了发现新的农作物病原菌抗菌肽,人工设计并构建了大容量短肽文库,从中筛选并合成96条短肽用于鉴定其对农作物病原菌的抑菌活性.[方法]采用琼脂扩散法,对靶标菌一棉花枯萎病菌(Fusarium f.sp.vasinfecum)、棉花红腐病菌(Fusarium moniliforme)、小麦根腐病菌(Bipolaris sorokiniana)和马铃薯早疫病菌(Alternaria solani)进行抑菌初筛,并测定了有抗菌作用短肽的最小抑菌浓度和抑菌持久性.[结果]得到了A6、D4和F10对上述四种病原真菌抑菌效果较强,抑菌时间较长的抗菌肽,通过与抗菌肽数据库氨基酸序列对比,未见这3条抗菌肽的同源序列.[结论]研制的3条短肽属于新颖抗菌肽,为防治农作物真菌病害提供了新的基因资源.     

迟眼蕈蚊抗菌肽BhSGAMP-1-S在大肠杆菌中的优化表达及其活性分析(英文)

【目的】BhSGAMP-1 是迟眼蕈蚊唾液腺抗菌肽,为了能够更好的了解其分子特性,我们将其表达、纯化并进行了活性测定。【方法】依据大肠杆菌稀有密码子设计并合成了抗菌肽基因 BhSGAMP-1-S,以 pMAL-c2X 作为表达载体在大肠杆菌 TB1 中进行融合表达,融合蛋白通过麦芽糖亲和层析柱进行纯化,获得的融合蛋白经肠激酶切割后,混合物通过分子筛凝胶层析和反相高效液相色谱来获得单体重组抗菌肽 BhSGAMP-1-S,对获得的抗菌肽进行活性测定。【结果】在最优的表达条件下融合蛋白以可溶的形式表达,100 mL 诱导菌液经多步纯化后可得 0. 38 mg 的重组抗菌肽 BhSGAMP-1-S,抑菌活性测定表明所获得的抗菌肽对部分测试革兰氏阳性细菌、革兰氏阴性细菌和真菌有较强的抑菌活性。【结论】本研究第一次成功的在大肠杆菌中诱导表达了修饰合成的抗菌肽 BhSGAMP-1-S,纯化后的抗菌肽具有很好的抑菌活性,这为进一步研究和应用奠定了基础。     

黑水虻抗菌肽的研究进展

黑水虻是近几年昆虫资源研究领域的热点,目前其应用研究主要集中在饲料添加剂和有机垃圾处理等方面.然而,近年来研究发现,黑水虻幼虫自身免疫系统产生的抗菌肽对革兰氏阴性菌、革兰氏阳性菌以及真菌都具有广谱抗菌活性,有着广泛的应用前景.为了促进黑水虻抗菌肽的开发和应用,本文主要综述了黑水虻抗菌肽的结构特征、抑菌活性、作用机制、生...     

抗菌肽对糜烂型OLP患者唾液中真菌的影响

抗菌肽经临床研究,发现具有抗生素不具备的优点,可对糜烂型口腔扁平苔藓(oral lichen planus,OLP)患者唾液中真菌有抑制和杀菌的作用,弥补了抗生素的缺点。通过使用不同浓度、不同种类的抗菌肽,作用于自糜烂型OLP患者口腔唾液标本中提取并经鉴定的菌种,对三种常见菌——白色念珠菌、光滑念珠菌、热带念珠菌进行抑菌试验。结果表明,抗菌肽使用前后的数值比对、菌落生长情况、最小抑菌值有明显变化。发现RISE-AP12对三种常见真菌的抑菌、杀菌效果优于其他的抗菌肽,在临床上有较大应用前景。     

Cu2+胁迫对黑水虻幼虫抗菌肽组分及抑菌活性的影响

研究不同浓度Cu~(2+)胁迫对黑水虻5龄幼虫抗菌肽分离纯化组分及抑菌活性影响,为黑水虻无害化处理粪便技术的有效实施提供有力的理论依据,为其副产品在饲料、食品及医药研发中的应用提供有价值的实验数据。本文在人工饲料中添加不同浓度Cu~(2+)(0、150、1 200 mg/kg)以饲喂黑水虻幼虫,采用金黄色葡萄球菌针刺法诱导5龄幼虫,断头收集血淋巴,高速冷冻离心结合超滤离心制备抗菌肽粗提物;利用RP-HPLC对三组抗菌肽粗提物进行分离纯化,收集各纯化峰对应组分,采用纸片琼脂扩散法测定各纯化峰对应组分的抑菌效果,以阐述Cu~(2+)胁迫对黑水虻抗菌肽的影响。结果表明,经Cu~(2+)胁迫,抗菌肽粗提物分离纯化后各组分的出峰时间及峰面积所占比例均不同,不同处理浓度下各分离组分第2峰面积均大于第1峰面积,其中150-2峰面积最大,为73.31%;分离纯化后所得6个组分对金黄色葡萄球菌、大肠杆菌及白色念珠菌均有抑菌活性,对金黄色葡萄球菌、白色念珠菌的抑菌活性显著高于对大肠杆菌抑菌活性,但其对金黄色葡萄球菌及白色念珠菌的抑菌活性未见显著差异;与其他5个组分相比,组分150-2对金黄色葡萄球菌、白色念珠菌及大肠杆菌抑菌活性最强,抑菌直径分别为27.85±0.74 mm、28.34±0.76 mm、21.97±0.54 mm。由此可见,不同浓度的Cu~(2+)胁迫对黑水虻幼虫抗菌肽组分及抑菌活性均产生显著影响,其中组分150-2抑菌活性最强,具有很好的开发潜能。     

黑水虻抗菌肽诱导条件优化及粗提物活性研究

采用不同诱导方法、不同诱导剂量及不同诱导时间诱导黑水虻Hermetia illucens L.幼虫产生抗菌肽，通过研磨提取法提取幼虫抗菌肽粗提物，并对所提取粗提物进行抑菌活性的比较研究，以优化黑水虻幼虫抗菌肽的诱导条件，为黑水虻抗菌肽的分离纯化提供最佳的实验方法。结果表明，不同龄期黑水虻幼虫产生抗菌肽的抑菌活性不同，最佳诱导龄期为5龄；不同诱导方法产生抗菌肽对大肠杆菌抑菌活性不同，大肠杆菌菌液针刺浸泡60 s，诱导后饲养24 h提取抗菌肽抑菌活性最好，其次为超声波100 W诱导20 min产生抗菌肽抑菌活性，但二者活性相比差异不显著；大肠杆菌菌液、金黄色葡萄球菌菌液注射诱导、金黄色葡萄球菌菌液针刺浸泡诱导及40 w超声波诱导所产生抗菌肽虽有一定的抑菌效果但与前两种方法相比差异显著。     

家蝇幼虫抗菌肽的超声诱导及分离纯化和活性研究

目的:分离纯化家蝇(Musca domestica)幼虫免疫血淋巴中的抗菌肽,研究抗菌肽的性质和抑菌特性.为进一步研究家蝇幼虫抗菌肽的产生及应用提供实验基础.方法:通过超声处理诱导家蝇幼虫产生免疫血淋巴,经沸水浴热变性,结合高速离心,两步CM-Sepharose离子交换层析,冷冻干燥浓缩等步骤,分离得到一种具抗菌活性的蛋白质.用Tricine-SDS-PAGE鉴定抗菌肽的纯度和性质,抑菌试验分析抗菌肽的抑菌特性.结果:300W,50Hz超声处理60s能够诱导家蝇幼虫产生抗菌肽,经分离纯化后Tricine-SDS-PAGE凝胶电泳结果显示为一条带,相对分子量大约为5.8kD.纯化蛋白对阴沟肠杆菌,绿脓杆菌等G-杆菌的抑菌活性较强,而对金黄色葡萄球菌效果不显著.结论:诱导和纯化了一种家蝇幼虫抗菌肽,为此类活性物质的分离纯化提供了有效的方式.     

利用精氨酸和缬氨酸设计新型α-螺旋抗菌肽

摘要：【目的】抗菌肽是生命体的自身免疫系统的重要组成部分。其中两性的α-螺旋抗菌肽在抗菌肽家族中又占有重要的地位,发挥着重要的作用。为了得到具有更高抗菌活性同时具有很低细胞毒性的抗菌肽,根据α-螺旋二级结构衍生出来的螺旋轮模型,设计了一条在疏水一侧含有8个缬氨酸和亲水一侧含有5个精氨酸的新型16残基抗菌肽。【方法】测定了设计得到的新型抗菌肽的最小抑菌浓度、对于红细胞和哺乳动物肾细胞的细胞毒性以及杀菌动力学。 【结果】抗菌活性检测表明,新型抗菌肽VGR16显示了强并快速的杀菌作用,其最小抑菌浓度在16－64     

Antimicrobial peptides from marine invertebrates: challenges and perspectives in marine antimicrobial peptide discovery

The emergence of pathogenic bacteria resistance to conventional antibiotics calls for an increased focus on the purification and characterization of antimicrobials with new mechanisms of actions. Antimicrobial peptides are promising candidates, because their initial interaction with microbes is through binding to lipids. The interference with such a fundamental cell structure is assumed to hamper resistance development. In the present review we discuss antimicrobial peptides isolated from marine invertebrates, emphasizing the isolation and activity of these natural antibiotics. The marine environment is relatively poorly explored in terms of potential pharmaceuticals, and it contains a tremendous species diversity which evolved in close proximity to microorganisms. As invertebrates rely purely on innate immunity, including antimicrobial peptides, to combat infectious agents, it is believed that immune effectors from these animals are efficient and rapid inhibitors of microbial growth.     

海洋细菌抗菌和细胞毒活性的初步研究

从不同海域的生物、海水和海泥中分离海洋细菌,利用琼脂扩散法和MTT法对细菌培养液的乙酸乙酯提取物进行了抗菌和细胞毒活性筛选。比较了活性菌株与来源的相关性．结果表明,在分离的341株海洋细菌中。42株细菌的代谢产物具有抗菌活性,7株具有细胞毒活性,其中来源于海洋无脊椎动物和海藻的活性菌株比例(22％和11％)大于来源于海水和海泥的细菌(7％和5％)．细菌分类鉴定结果显示,具有活性的细菌大部分属于假单胞菌属、发光杆菌属、梭状芽孢杆菌属、交替单胞菌属和黄杆菌属．     

Antimicrobial activity of marine sponge Clathria indica (Dendy, 1889)

Sponges are sessile filter feeders that have developed efficient defense mechanisms against foreign invaders such as viruses, bacteria or eukaryotic organisms. Antimicrobial peptides are known as major components of the innate immune defense system in marine invertebrates. The aim of the present work was to study the antimicrobial properties of the Indian sponge Clathria indica with special reference to the identification of antimicrobial peptides. Crude methanolic extract and its chloroform, n-butanol and aqueous fractions were tested against 16 human pathogens which include eleven bacteria with four of them being multidrug resistant and five pathogenic fungi. All fractions showed effective antibacterial activity against common and multidrug-resistant Salmonella typhi and antifungal activity against C. albicans and C. neoformans. However, they were ineffective against Escherichia coli, Pseudomonas aeruginosa, Streptococcus pyogenes and Staphylococcus aureus. Chloroform fraction being the most potent among the fractions tested on chemical investigation was indicative of the presence of peptides as evidenced by ninhydrin positive spots on TLC and presence of peptide bonds by NMR. Its ESI-MS showed presence of several peptides in the range of m/z 850 to 980. Structure of three peptides has been tentatively assigned by ESI-MS/MS or tandem mass analysis, on the basis of the amino acid sequence established. The results clearly show that the sponge C. indica represent an interesting source of marine invertebrates-derived antimicrobial peptides in the development of new strategies to treat various infectious diseases.     

Antimicrobial activity of marine sponge <Emphasis Type="Italic">Clathria indica</Emphasis> (Dendy, 1889)

Sponges are sessile filter feeders that have developed efficient defense mechanisms against foreign invaders such as viruses, bacteria or eukaryotic organisms. Antimicrobial peptides are known as major components of the innate immune defense system in marine invertebrates. The aim of the present work was to study the antimicrobial properties of the Indian sponge Clathria indica with special reference to the identification of antimicrobial peptides. Crude methanolic extract and its chloroform, n-butanol and aqueous fractions were tested against 16 human pathogens which include eleven bacteria with four of them being multidrug resistant and five pathogenic fungi. All fractions showed effective antibacterial activity against common and multidrug-resistant Salmonella typhi and antifungal activity against C. albicans and C. neoformans. However, they were ineffective against Escherichia coli, Pseudomonas aeruginosa, Streptococcus pyogenes and Staphylococcus aureus. Chloroform fraction being the most potent among the fractions tested on chemical investigation was indicative of the presence of peptides as evidenced by ninhydrin positive spots on TLC and presence of peptide bonds by NMR. Its ESI-MS showed presence of several peptides in the range of m/z 850 to 980. Structure of three peptides has been tentatively assigned by ESI-MS/MS or tandem mass analysis, on the basis of the amino acid sequence established. The results clearly show that the sponge C. indica represent an interesting source of marine invertebrates-derived antimicrobial peptides in the development of new strategies to treat various infectious diseases.     

Molecular evolution of animal antimicrobial peptides: widespread moderate positive selection

An increasing number of studies in both vertebrates and invertebrates show that the evolution of antimicrobial peptides is driven by positive selection. Because these diverse molecules show potential for therapeutic applications, they are currently the targets of much structural and functional research, providing extensive background data for evolutionary studies. In this paper, patterns of molecular evolution in antimicrobial peptide genes are reviewed. Evidence for positive selection on antimicrobial peptides includes an excess of nonsynonymous nucleotide substitutions, an excess of charge-changing amino acid substitutions, nonneutral patterns of allelic variation, and functional assays in vivo and in vitro that show improved antimicrobial effects for derived sequence variants. Positive selection on antimicrobial peptides may be as common as, but perhaps weaker than, selection on the best-known example of adaptively evolving immunity genes, the major histocompatibility complex. Thus, antimicrobial peptides present a useful and underutilized model for the study of adaptive molecular evolution.     

The role of antimicrobial peptides in innate immunity

Production of antimicrobial peptides and proteins is an importantmeans of host defense in eukaryotes. The larger antimicrobialproteins, containing more than 100 amino acids, are often lyticenzymes, nutrient-binding proteins or contain sites that targetspecific microbial macromolecules. The smaller antimicrobialpeptides act largely by disrupting the structure or functionof microbial cell membranes. Hundreds of antimicrobial peptideshave been found in the epithelial layers, phagocytic cells andbody fluids of multicellular animals, from mollusks to humans.Some antimicrobial peptides are produced constitutively, othersare induced in response to infection or inflammation. Studiesof the regulation of antimicrobial peptide synthesis in Drosophilahave been particularly fruitful, and have provided a new paradigmfor the analysis of mammalian host defense responses. It nowappears that the general patterns of antimicrobial responsesof invertebrates have been preserved in vertebrates ("innateimmunity") where they contribute to host defense both independentlyand in complex interplay with adaptive immunity.     

Molecular characterization of two novel antibacterial peptides inducible upon bacterial challenge in an annelid, the leech Theromyzon tessulatum

Two novel antimicrobial peptides named theromacin and theromyzin were isolated and characterized from the coelomic liquid of the leech Theromyzon tessulatum. Theromacin is a 75-amino acid cationic peptide containing 10 cysteine residues arranged in a disulfide array showing no similarities with other known antimicrobial peptides. Theromyzin is an 86-amino acid linear peptide and constitutes the first anionic antimicrobial peptide observed in invertebrates. Both peptides exhibit activity directed against Gram-positive bacteria. Theromacin and theromyzin cDNAs code precursor molecules containing a putative signal sequence directly followed by the mature peptide. The enhancement of theromacin and theromyzin mRNA levels has been observed after blood meal ingestion and upon bacterial challenge. In situ hybridization revealed that both genes are expressed in large fat cells in contact with coelomic cavities. Gene products were immunodetected in large fat cells, in intestinal epithelia, and at the epidermis level. In addition, a rapid release of the peptides into the coelomic liquid was observed after bacterial challenge. The presence of antimicrobial peptide genes in leeches and their expression in a specific tissue functionally resembling the insect fat body provide evidence for the first time of an antibacterial response in a lophotrochozoan comparable to that of holometabola insects.     

Endogenous opioids in marine invertebrates

A study of the presence of opioids in invertebrates was carried out on 11 species of marine organisms, ranging from sponges to tunicates. Delipidized acid-acetone extracts from whole organisms or dissected organs were assayed by receptor binding assays and radioimmuno assays. The extracts from all species tested were found to contain substances capable of competing in both assays with opioid peptides.     

酶法制备海洋活性肽及其功能活性研究进展

海洋生物活性肽(Marine biological active peptide)是从海洋生物中提取的具有优化机体代谢环境、有益于机体健康的一类多肽。酶法制备海洋生物活性肽是目前最常用的制备方法,是通过适当的蛋白酶水解海洋生物蛋白来制备生物活性肽的一种方法。海洋生物活性肽在降血压、抗氧化、抗凝血及抗菌等方面效果显著,对治疗和预防疾病具有巨大潜力。介绍海洋生物活性肽在酶解制备及其生物学功能方面国内外研究进展,为进一步开展海洋活性多肽研究提供参考。