家蝇抗菌肽抗菌活性及抗菌机制的初步研究

目的　研究家蝇抗菌肽的抗菌活性及抗菌机制。方法　用损伤感染的方法诱导家蝇幼虫表达抗菌肽 ,通过 Sephadex过滤层析和 HL PC技术纯化提取 ,用平板法和稀释法作抗菌活性试验 ,并用电子扫描技术研究抗菌肽的抗菌机制。结果　家蝇抗菌肽对铜绿假单胞菌、大肠埃希氏菌、耐甲氧西林金黄色葡萄球菌( MRSA)均有明显的抗菌活性。大肠埃希氏菌与 5 0 μg/ml的纯化抗菌肽溶液一起 37℃孵育 6 0 min后 ,大肠埃希氏菌不能存活。经电镜扫描观察发现 ,细菌的细胞膜出现破损和穿孔现象。结论　家蝇抗菌肽具有明显的广谱抗菌活性 ,对阴性杆菌和阳性球菌均有杀伤作用。抗菌肽的抗菌机制是通过破坏细菌的细胞膜而杀伤细菌的     

乳铁素的融合表达及其体外抗菌活性鉴定

为降低乳铁素Lf-cinB表达时对宿主细胞的毒害,提高乳铁素的表达量及纯化效果,合成了牛乳铁蛋白肽基因,克隆至E.coli表达载体pTYB11中,构建了与蛋白质内含子(intein)的C端融合的表达载体pTYB11-cinB。重组质粒转化至E.coliBL21(DE3)中,经IPTG诱导表达,融合蛋白intein-cinB主要以可溶形式存在于胞内。利用载体中intein中的chitin结合域,将融合蛋白通过chitin亲和层析一步纯化,经DTT诱导in-tein的自我切割活性,实现Lf-cinB在亲和柱上的切割与分离,透析冻干后得到了纯度94%的重组Lf-cinB。活性检测结果显示,重组Lf-cinB对多种检测菌均有抗菌活性。研究认为,内含肽在抗菌肽的基因工程中具有重要的应用前景。     

金环蛇抗菌肽Cathelicidin-BF的体外抗菌活性研究

为了研究金环蛇抗菌肽Cathelicidin-BF对临床常见菌株的体外抗菌活性和细菌是否容易对其产生耐药,分别通过琼脂平板稀释法和肉汤稀释法测定Cathelicidin-BF的最低抑菌浓度(MIC)和最低杀菌浓度(MBC)。研究接种量与pH值对其MIC的影响和本品对细菌生长增殖的抑制作用,利用耐受诱导实验研究细菌对本品是否易产生耐药。结果表明金环蛇抗菌肽Cathelicidin-BF对革兰阳性菌和阴性菌都有抑制作用,MIC低至2μg/mL,在用药处理的10 h内未见细菌生长,且在耐药性诱导实验期间,细菌对Cathelicidin-BF没有产生耐药。金环蛇抗菌肽Cathelicidin-BF具有广谱抗菌活性,且与抗生素相比不易产生耐药,提示其可作为治疗感染性疾病的候选药物。     

Cathelicidins抗菌肽家族的研究进展

Cathelicidins是一族在哺乳动物中发现的含有保守的cathelin区域的抗菌肽，是宿主防御系统的重要组成部分，具有广谱且强大的抗菌活性。文章综述了cathelicidins抗菌肽家族的基因组成、结构、抗菌活性、作用机制及构效关系等。     

抗菌肽的结构特征及其与活性的关系

抗菌肽是生物体内一类具有抗菌活性的多肽,是宿主非特异性防御系统的重要组成部分,广泛存在于自然界中,具有广谱的杀菌活性,其独特的生物学活性与其结构特征密切相关.文章简要介绍了抗菌肽的结构及其活性之间的关系,以期为设计新型抗菌肽分子,开发活性更高,抗菌谱更广的抗菌类药物提供理论依据.     

两栖类皮肤抗菌肽的分离纯化与抗菌活性检测技术

两栖类皮肤抗菌肽由于具有广谱、高效的抗菌活性,且不引起微生物耐药性等特点,已成为目前开发新型抗菌药物的理想材料。文章概述了两栖类抗菌肽的主要特点及其分离纯化和抗菌活性检测的主要方法,并对这些技术的原理、方法及其近年来在抗菌肽研究中的进展进行了介绍。同时本文还比较了各种方法的优缺点,对当前研究中所存在的一些问题进行了初步的探讨,以期为两栖类抗菌肽的应用提供理论依据。     

阳离子抗菌肽的研究进展

阳离子抗菌肽是一类具抗微生物活性的小分子多肽，按结构特征可分为α－螺旋肽和β－折叠肽，不同来源或同一抗菌肽的不同结构形式生物活性差别较大，部分抗菌肽之间或抗菌肽与抗生素之间存在协同作用，特别是最近发现一些抗菌肽具有抗内毒素活性。阳离子抗菌肽的结构特征是其发挥作用的重要基础，α－螺旋肽通过其两亲性的α－螺旋上的正电茶与细菌细胞膜磷脂分子的负电荷之间的静电吸引而结合在细菌膜上，并借助于疏水段分子中连接结构的柔性插入到细胞膜中，最终通过膜内分子间的相互位移使抗菌肽分子相互聚集在一起形成离子通道，使细菌失去膜势，不能保持正常的渗透压而死亡。一般认为β－折叠肽也是和细胞膜结合后，结构发生变化而发挥作用。抗菌肽和细胞膜结合及形成离子通道受多种因素的影响，如阳离子抗菌肽的结构、浓度，环境 pH、温度，介质的离子强度。阳离子抗菌肽杀菌力强，抗菌谱广，不良反应少，因此在食品、农业，特别是在医药领域都有很好的应用前景，极可能发展成为一类全新的抗生素。     

抗菌抗癌肽CM4N在大肠杆菌中的融合表达与活性鉴定

为探讨内含肽intein在抗菌肽表达与纯化中的应用,采用递归PCR法合成CM4末端添加Asn的基因CM4N,克隆至E.coli表达载体pTYB11中,构建了与intein的C端融合的表达载体pTYYB11-CM4N.重组质粒转化至E.coli BL21(DE3)中进行IPTG诱导表达,融合蛋白intein-CM4N主要以可溶形式存在于胞内.利用载体中intein中的chitin结合域,将融合蛋白通过chitin亲和层析一步纯化,经DTT诱导intein的自我切割活性,实现CM4N在亲和柱上的切割与分离,透析冻干后得到了纯度95%以上的重组CM4N.活性检测结果显示,重组CM4N具有抗大肠杆菌、沙门氏菌和K562肿瘤细胞的活性.研究认为,内含肽在抗菌肽的基因工程中可能具有重要的应用前景.     

乳源抗菌肽的分离纯化及部分性质的研究

建立酪蛋白酶解产生抗菌肽的最适条件，对酶解产物进行分离。建立抗菌肽活性测定方法，并对酪蛋白源抗菌肽的理化特性予以评价。本实验采用胰蛋白酶在不同时间条件下水解酪蛋白，经凝胶过滤和RP-HPLC分离纯化制备抗菌肽，酶解液及其分离物质对不同菌株的大肠杆菌和金黄色葡萄球菌的抗菌活性进行了测定。结果表明：2，3h胰蛋白酶的酪蛋白水解液均具有抗菌活性，且有良好的热稳定性。酪蛋白和胰蛋白酶本身无抗菌活性。2h水解液对大肠杆菌ATCC25922和金黄色葡萄球菌CMCC26801的最小抑菌浓度（Minimal Inhibitory Concentration, MIC））分别是1．20mg／ml和0．34mg／ml。2h水解液经SephadexG-15分离收集的5^#组分有抗菌活性，RP-HPLC检测有较单一肽吸收峰。说明酪蛋白经酶水解可以产生抗菌活性肽。     

人工合成抗菌肽生物信息学分析及其抑菌活性研究

目的：对溶血性较高的天然抗菌肽Temporin-1Dra进行氨基酸替换,以期获得低毒性、对白色念珠耐药菌株具有抑菌活性的抗菌肽。方法：采用生物信息学相关软件对改造之后的天然抗菌肽Temporin-1Dra进行分析,主要包括抗菌肽形成概率、理化性质、二级结构的预测;测定合成多肽的最低抑菌浓度,杀菌曲线,部分抑菌浓度指数（FIC index）。结果：对天然抗菌肽进行生物信息学分析改造得到多肽[G4、T5、N8K]Temporin-1Dra,对耐氟康唑（FLC）白色念珠菌的MIC值为128 μg·mL-1;MIC范围内溶血率＜5%;3 h内MIC浓度下杀菌效率显著,且呈现浓度依赖性;与FLC联用的FIC index为0.375,与FLC联合用药有协同作用。结论：改造之后的抗菌肽[G4、T5、N8K]Temporin-1Dra（HFLKKLVKLAKKIL-NH2）对白色念珠菌耐药菌株具有抑制作用,体外溶血性低、杀菌效果显著。     

生物活性肽药理活性的研究进展

随着人们生活水平的提高,伴随现代文明而来的各种富裕病如高血压、高血脂、肥胖病、癌症等越来越引起人们的关注,人们的消费观念已从单纯的吃饱吃好向防病治病方向转变.生物活性肽来源很广,目前已经成为世界范围内研究的热点.大量研究已表明,生物活性肽具有多种药理活性,如神经、激素和免疫调节、抗血栓、抗高血压、抗胆固醇、抗细菌病毒、抗癌作用等,是筛选药物的天然药物宝库.本文综述了生物活性肽的药理活性的研究进展.     

HAZ,a novel peptide with broad-spectrum antibacterial activity

ObjectiveThe growing microbial resistance to antibiotics is a global public concern, which creates serious needs for newer antimicrobial agents. Antimicrobial peptides (AMPs) are increasingly exploited in drug development as therapeutic candidates. Here, we aimed to design and characterize a novel peptide with broad spectrum antimicrobial activity.MethodsHybridization and sequence modification approaches were used to design the novel peptide, named HAZ, aiming at optimizing the physicochemical parameters involved in antimicrobial activity. Peptide activities were assessed alone or combined with different selected antibiotics against various sensitive and drug-resistant bacterial strains. In addition, the hemolysis and the cytotoxic activities of HAZ peptide were evaluated on human red blood cells and epithelial adenocarcinoma cells (A549), respectively.ResultsHAZ peptide was sequentially modified to result in favored physicochemical parameters (helicity 95.24 %, hydrophobic ratio 47 %, and net charge of 8 + ). Functional assessment of HAZ revealed significant antimicrobial activity, with MIC values of 15 – 20 µM against tested bacterial strains. It also exhibited biofilm eradication activity at slightly higher concentrations. HAZ-antibiotics combinations exhibited a synergistic action mode that led to dramatic decrease in the MIC values for both HAZ peptide and the antibiotic. Such efficacy was accompanied with minimal hemolytic toxicity on human erythrocytes. Importantly, HAZ displayed promising anticancer activity against human lung cancer cells.ConclusionRationally-designed antimicrobial peptides offer promising alternatives to the current antibiotics for management of infectious diseases. HAZ peptide is a broad-spectrum AMP, and a promising candidate for antimicrobial and anticancer drug development.     

Sortase A酶突变体的原核表达、纯化及活性测定（英文）

本研究采用pET28a载体对金黄色葡萄球菌(Staphylococcus aureus)中的转肽酶A(sortase A,SrtA)截短体SrtA?N25以及突变体m5SrtA?N59、m9SrtA?N59进行了原核发酵表达,并通过镍柱亲和色谱、阳离子交换色谱进行蛋白纯化。随后,通过荧光共振能量转移试验比较了3种酶的催化活性,测定了酶反应动力学常数、筛选了最适反应条件,并进一步探究了不同生化试剂对SrtA酶学活性的影响。结果显示,SrtA?N25、m5SrtA?N59、m9SrtA?N59在大肠埃希菌中均能以可溶形式高效表达,且纯化后得到的高纯度SrtA酶均具有生物学活性。相较于SrtA?N25,m5SrtA?N59、m9SrtA?N59活性分别提高了约68、5 544倍,尤其是m9SrtA?N59的催化活性大大提高。m9SrtA?N59的最适反应温度为30~37℃,最适反应pH值为6.0~7.0,反应体系中加入三(2-羧乙基)膦(TCEP)、二硫苏糖醇(DTT)等还原剂有助于增强SrtA酶催化活性。     

Antimicrobial Activity of 6-Paradol and Related Compounds

The seeds of Aframomum melegueta K. Schum. (Zingiberaceae) yielded 6-paradol (1) and 6-shogaol (2) as the major antimycobacterium agents, based on a bioactivity-guided fractionation. These isolates were found to be active against Mycobacterium chelonei, M. intracellulare, M. smegmatis and M. xenopi (MIC 10-15 µg/ml). Derivatives were prepared in an attempt to define some of the structural parameters needed for antimicrobial activity. The desmethyl derivative of 1 retained the antimycobacterial activity and was found to be more active against Candida albicans than 1 and 2. Gingerone (9), not previously reported in this source, was found to be inactive. The identity of the compounds were confirmed by spectral analyses (UV, IR, NMR, and EIMS) and by comparison with reported data.     

GP73 融合蛋白原核表达及纯化

目的：原核表达并纯化高尔基体糖蛋白-73（GolgiProtein73,GP73）。方法以人肝癌细胞系HepG2总RNA为模板,经RT-PCR扩增GP73基因,克隆至原核表达载体pET-21a（＋）-TRX中,转化大肠杆菌BL21（DE3）,IPTG诱导表达。His-tag磁珠纯化重组蛋白GP73,SDS-PAGE鉴定。结果克隆目的基因的序列正确,未发生碱基突变;重组表达质粒pET21a（＋）-TRX-GP73经双酶切鉴定构建正确;表达的重组蛋白相对分子量为80 kD,与预期相符。结论本实验成功在大肠杆菌BL21（DE3）中表达并纯化了GP73重组蛋白,为后续研究奠定了基础。     

Peptides and proteins with antimicrobial activity

The increase of microbial resistance to antibiotics has led to a continuing search for newer and more effective drugs. Antimicrobial peptides are generally found in animals, plants, and microorganisms and are of great interest to medicine, pharmacology, and the food industry. These peptides are capable of inhibiting pathogenic microorganisms. They can attack parasites, while causing little or no harm to the host cells. The defensins are peptides found in granules in the polymorphonuclear neutrophils (PMNs) and are responsible for the defense of the organism. Several animal defensins, like dermaseptin, antileukoprotease, protegrin, and others, have had their activities and efficacy tested and been shown to be effective against bacteria, fungi, and protists; there are also specific defensins from invertebrates, e.g., drosomycin and heliomicin; from plants, e.g., the types A and B; and the bacteriocins, e.g., acrocin, marcescin, etc. The aim of the present work was to compile a comprehensive bibliographic review of the diverse potentially antimicrobial peptides in an effort to systematize the current knowledge on these substances as a contribution for further researches. The currently available bibliography does not give a holistic approach on this subject. The present work intends to show that the mechanism of defense represented by defensins is promising from the perspective of its application in the treatment of infectious diseases in human, animals and plants.     

Characterization and Recombinant Expression of Terebrid Venom Peptide from Terebra guttata

Venom peptides found in terebrid snails expand the toolbox of active compounds that can be applied to investigate cellular physiology and can be further developed as future therapeutics. However, unlike other predatory organisms, such as snakes, terebrids produce very small quantities of venom, making it difficult to obtain sufficient amounts for biochemical characterization. Here, we describe the first recombinant expression and characterization of terebrid peptide, teretoxin Tgu6.1, from Terebra guttata. Tgu6.1 is a novel forty-four amino acid teretoxin peptide with a VI/VII cysteine framework (C–C–CC–C–C) similar to O, M and I conotoxin superfamilies. A ligation-independent cloning strategy with an ompT protease deficient strain of E. coli was employed to recombinantly produce Tgu6.1. Thioredoxin was introduced in the plasmid to combat disulfide folding and solubility issues. Specifically Histidine-6 tag and Ni-NTA affinity chromatography were applied as a purification method, and enterokinase was used as a specific cleavage protease to effectively produce high yields of folded Tgu6.1 without extra residues to the primary sequence. The recombinantly-expressed Tgu6.1 peptide was bioactive, displaying a paralytic effect when injected into a Nereis virens polychaete bioassay. The recombinant strategy described to express Tgu6.1 can be applied to produce high yields of other disulfide-rich peptides.     

抗栓肽基因的克隆和表达

抗栓肽(Decorsin)是糖蛋白Ⅱb-Ⅲa(GPⅡb-Ⅲa)的强拮抗剂,能抑制血小板聚集。实验通过设计两对引物,经PCR获得抗栓肽的结构基因,并将基因插入原核表达载体pET32a硫氧还蛋白(TrxA)的下游。将重组质粒转化至大肠杆菌BL21(DE3),进行诱导表达,表达产物以可溶形式存在于胞内,占菌体总蛋白的35%。利用表达载体自身所带的His×6纯化标签,可将融合蛋白通过金属螯合亲和层析柱一步纯化,纯化的蛋白质经SDS-PAGE分析达到电泳纯。体外的抑制血小板聚集实验结果表明,融合蛋白具有显著的抑制ADP诱导的血小板聚集活性,抑制常数IC50为500 nmol/L。