家蝇抗菌活性物质研究进展

家蝇Musca domestica,是一种重要的卫生害虫。近年来,对其体内外抗菌活性物质的研究受到了广泛的关注。通过对家蝇抗菌物质的种类、性质,特别是对家蝇抗菌肽的结构特点、生物活性、作用机理与分子生物学研究做了概述,并对家蝇抗菌肽的应用前景进行了讨论。     

链球菌诱导后家蝇幼虫提取物的抗菌活性研究

[目的]为枯草杆菌诱导后的家蝇幼虫提取物在饲料中的应用提供依据。[方法]采用抑菌圈测量法测定链球菌诱导后家蝇幼虫提取物的抗菌活性,分析其对温度、酸碱和离子强度稳定性的影响。[结果]链球菌诱导的家蝇幼虫提取物对细菌的抑菌圈直径为6.55~11.01 mm。诱导组中家蝇幼虫提取物对6种病原菌和工程菌大肠杆菌JM109均有抑制作用,其对革兰氏阳性菌的抑制作用较强。而未诱导组提取物对细菌的抑菌圈直径为6.01~8.01 mm,诱导组的抗菌活性高于未诱导组。抗菌肽样品具有较好的温度稳定性。当pH值为4~10时,抗菌肽样品的抗菌活性均较高,以pH值为8时最高。抗菌肽样品的抗菌活性随离子强度的升高而下降。[结论]抗菌肽的抑菌活性受环境离子强度的影响,与其作用机理有关。     

家蝇幼虫抗菌肽MDL-2干扰细菌功能的初步研究

研究了家蝇幼虫抗菌肽MDL-2对细菌细胞壁的溶解作用、细胞膜渗透性和代谢的影响.抗菌肽MDL-2在抗菌过程中首先与细菌的细胞壁相互作用,使其破裂.菌肽对革兰氏阴性细菌大肠杆菌的细胞壁的作用有浓度依赖性,而对革兰氏阳性细菌金黄色葡萄球菌,MDL-2在较低的浓度时即具有破坏细胞壁的作用;抗菌肽MDL-2与金黄色葡萄球菌的细胞膜作用曲线呈现典型的S型,有较强的协同效应,并随着作用时间的延长细胞的渗透性增强;抗菌肽对细菌的代谢功能有较强的干扰作用,阻碍菌体的生命活动,加速菌体死亡.     

大肠杆菌刺激蝇蛆产生抗菌肽浓度和抑菌活性的测定

[目的]确定大肠杆菌菌液刺激蝇蛆产生浓度高、活性强的抗菌肽时间和蝇蛆饲喂家禽的安全性。[方法]采用不同浓度的大肠杆菌菌液分别与麸皮混合饲喂蝇蛆,于10、24、48 h分别收集存活蝇蛆并对其粗提取抗菌肽,通过考马斯亮蓝法和抑菌试验分别测定各试验组抗菌肽粗提液的浓度和抑菌力,同时用CFU计数法测定细菌培养的家蝇幼虫的细菌含量以确定其作为绿色抗生素的安全性。[结果]浓度为3.0×107cfu/ml的大肠杆菌作用24 h的蝇蛆组产生的抗菌肽浓度最高、活性最强,且蝇蛆体内细菌含量为1.6×105cfu/g,低于国家规定的饲料细菌标准含量最低值(2×106cfu/g)。[结论]为进一步利用动物死尸、畜禽粪便生物转化蝇蛆产生大量安全、浓度高且活性强的蝇蛆抗菌肽作为绿色抗生素运用于禽类养殖业奠定了基础。     

家蝇抗菌肽的提取及其对凡纳滨对虾抗病力的影响

采用超声波诱导的方式筛选出生长状况良好的蝇蛆,通过组织匀浆、乙酸铵浸提、热处理、冻融处理、盐析、透析、蒸馏浓缩等方法提取家蝇抗菌肽,应用分光光度法测定其浓度和提取率,比浊法测定其抗菌活性.在基础饲料中分别添加0、40、80、120 mg/kg家蝇抗菌肽制成4组试验饲料,饲养凡纳滨对虾6周后采用注射法进行白斑综合征病毒攻毒试验,攻毒期为52 h.结果显示,4日龄家蝇繁育的蝇蛆经刺激后生长状况良好,产量最高;提取的家蝇抗菌肽浓度为25.05 mg/mL,提取率为2.36％;提取液稀释11倍后,其对铜绿假单胞菌的抑菌率为21.05％,抗菌活力为24.58 U.与未添加组相比,40、80 mg/kg组累积死亡率显著降低,各添加组的相对保护率分别为42.42％、33.33％和9.09％.结果表明,该提取工艺可保证抗菌肽的提取率,保持其稳定性和抗菌活性;在饲料中添加该抗菌肽,可提高凡纳滨对虾抗白斑综合征病毒的能力.     

一个家蝇抗菌肽的分离纯化

以野生家蝇干燥幼虫为原料,在常规的生化提取基础上,通过反相高效液相色谱(RP-HPLC)法进一步纯化,并结合电洗脱技术,利用灵敏的杀菌活性检测手段,从家蝇抗菌肽总组分中分离纯化出1个20 ku的抗菌肽,对苏云金芽孢杆菌、枯草芽孢杆菌、金黄葡萄球菌均有杀灭作用。     

抗菌肽及其在农业上应用的研究进展

抗菌肽是生物防御系统产生的一类对抗外源性病原体的多肽类物质。抗菌肽对细菌、真菌和病毒等都具有明显的杀伤作用。本文对天然抗菌肽的来源、结构特点和作用机理研究进行了综述,并介绍了抗菌肽在农业上的应用研究进展。     

抗菌肽的生物学功能与作用机制研究进展

抗生素的长期大量使用导致耐药菌和超级细菌在世界范围内陆续出现，对环境安全和公共健康造成了极大的威胁。抗菌肽具有免疫调节，广谱抗细菌、真菌、病毒、寄生虫、癌细胞等多方面的生物学作用。而且抗菌肽的抗菌机制与传统抗生素不同，其主要是通过破坏细菌细胞膜来发挥抗菌活性，因此不易产生耐药性，使其具备成为新一代抗菌药物的潜质，受到农业、畜牧、食品、医药等多领域的广泛关注。文章对国内外抗菌肽的研究进行了梳理，综述了抗菌肽在生物学功能、作用机制等方面的研究进展。     

抗菌肽及其应用前景概述

抗菌肽是生物体产生的一种具有抗菌活性的多肽类物质,它对细菌、真菌和病毒等都具有明显的杀伤作用.抗菌肽有望开发成为防治人和动物疾病的药物,应用前景广阔.文章对抗菌肽的结构特征、作用机制及应用前景进行了综述,以期为抗菌肽的更深层次的研究与应用提供参考.     

重组家蝇抗菌肽Des-HF在酵母中的表达及对金黄色葡萄球菌的抗菌活性

参考家蝇防御素抗菌肽基因序列(AY260152) 设计4条引物,用降落PCR方法获得家蝇防御素抗菌肽成熟肽基因.将基因克隆人酵母表达载体pPICZα-A,构建分泌型重组酵母表达载体pPIC-Des-HF,转化Pichia pastoris受体菌SMD1168,在醇氧化酶(AOX) 启动子调控下,相对分子质量约5 000的重组抗菌肽Des-HF获得表达.抗菌试验结果表明,该表达产物对金黄色葡萄球菌有较好的抑菌活性.以小鼠为试验模型研究重组抗菌肽对金黄色葡萄球菌的体内抗菌活性,试验结果表明,240μg重组Des-HF可分别保护小鼠免受10倍最小致死浓度的金黄色葡萄球菌(ATCC26003) 和Cowan I的攻击.     

林蛙皮多肽凝胶制剂对兔皮肤损伤的修复作用

林蛙皮多肽是从林蛙皮中提取出的活性多肽,含有多种氨基酸和细胞因子,对皮肤修复具有重要作用,但其水溶液不利于临床使用。试验拟将提纯的林蛙皮多肽与其他皮肤调理剂制备成凝胶制剂并通过建立兔背部皮肤创伤模型来探究林蛙皮多肽凝胶制剂在兔皮肤创伤愈合中的作用。通过观察伤口红肿、出血、渗出和结痂,计算创面愈合,以及通过病理组织学变化评估林蛙皮多肽凝胶制剂的作用。结果表明:林蛙皮多肽凝胶制剂减少炎症反应,促进兔皮肤创伤愈合,提高肉芽组织中新生血管密度和成纤维细胞数量。     

Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase

The structure of a 20-amino acid peptide inhibitor bound to the catalytic subunit of cyclic AMP-dependent protein kinase, and its interactions with the enzyme, are described. The x-ray crystal structure of the complex is the basis of the analysis. The peptide inhibitor, derived from a naturally occurring heat-stable protein kinase inhibitor, contains an amphipathic helix that is followed by a turn and an extended conformation. The extended region occupies the cleft between the two lobes of the enzyme and contains a five-residue consensus recognition sequence common to all substrates and peptide inhibitors of the catalytic subunit. The helical portion of the peptide binds to a hydrophobic groove and conveys high affinity binding. Loops from both domains converge at the active site and contribute to a network of conserved residues at the sites of magnesium adenosine triphosphate binding and catalysis. Amino acids associated with peptide recognition, nonconserved, extend over a large surface area.     

The M43 domain-containing metalloprotease RcMEP1 in Rhizoctonia cerealis is a pathogenicity factor during the fungus infection to wheat

Wheat(Triticum aestivum L.) is an important staple crop for global human. The necrotrophic fungus Rhizoctonia cerealis is the causal pathogen of sharp eyespot, a devastating disease of wheat. Herein, we identified RcMEP1, a zinc metalloproteaseencoding gene from R. cerealis genomic sequences, and characterized its pathogenesis function. RcMEP1 expressed at markedly-high levels during R. cerealis infection process to wheat. The predicted protein RcMEP1 comprises of 287 amino acid residues and contains a signal peptide and a M43 metalloprotease domain harboring the active site motif(HEVGHWLGLYH). The assays of Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana leaves indicated that RcMEP1 is an apoplastic elicitor of cell death, and that the predicted signal peptide functions and is required for secretion and cell death-induction. The purified RcMEP1 protein and its M43 domain peptide were individually able to induce plant cell death and H2 O2 accumulation, and to inhibit expression of host chitinases when infiltrated into wheat and N. benthamiana leaves, while the M43 domain-deleting peptide and negative control lacked the capacity. Moreover, compared with the control pretreatment, the purified RcMEP1 protein or its M43-domain peptide resulted in enhanced pathogenesis in the inoculated wheat, whereas the M43 domain-deleting peptide failed. These results suggest that RcMEP1 acted as an important pathogenicity factor during R. cerealis infection to wheat and that its signal peptide and M43 domain are required for the secretion and pathogenesis of RcMEP1. This study provides insights into pathogenesis role of M43 domain-containing metalloproteases during R. cerealis infection to wheat.     

A chemically synthesized Antennapedia homeo domain binds to a specific DNA sequence

A peptide 60 residues in length that corresponds to the homeo domain of Antennapedia (Antp), a protein governing development in Drosophila, was synthesized by segment condensation with protected peptide segments prepared on an oxime resin. A footprinting assay showed that the homeo domain binds specifically to a TAA repeat DNA sequence in the Antp gene. Thus the Antp homeo domain has a sequence-specific DNA binding property. The circular dichroism spectra of the homeo domain peptide showed the presence of a significant amount of alpha-helical structure in aqueous solution and in 50 percent trifluoroethanol. The alpha helicity measured in water appears to depend on the peptide concentration, which suggests that the peptide aggregates. These results support the hypothesis that the homeo domain binds to DNA through a helix-turn-helix motif.     

法氏囊活性肽对传染性法氏囊病毒弱毒疫苗的免疫增强效果

将7日龄黄羽肉鸡300只均分为5组,即对照组、常规疫苗剂量配合活性肽组、常规疫苗剂量减半配合活性肽组、生理盐水组和活性肽组。通过间接ELISA测定血清抗体水平及计算法氏囊指数等试验方法,研究活性肽和传染性法氏囊弱毒疫苗联合应用的免疫效果。结果表明:常规免疫剂量配合活性肽组和疫苗剂量减半配合活性肽组的法氏囊指数显著高于对照组,常规免疫剂量配合活性肽组的血清抗体滴度显著高于对照组,而常规剂量减半组与对照组在血清抗体滴度上差异不显著。说明活性肽对传染性法氏囊弱毒疫苗有免疫增强作用,疫苗剂量减半配合法氏囊活性肽应用能达到常规疫苗免疫效果。     

水稻半胱氨酸蛋白酶OsCP2的特征分析及其原核表达与纯化

半胱氨酸蛋白酶是植物体中重要的蛋白水解酶,广泛参与种子萌发、幼苗发育、胁迫响应和组织分化衰老等生理过程.我们对水稻中半胱氨酸蛋白酶基因OsCF2 (Oryza sativa cysteine protease 2)序列进行分析.该基因cDNA全长2 279 bp,由2个外显子和1个内含子组成;包含1个1 089 bp的开放读码框,编码1个含362个氨基酸残基的蛋白,在蛋白氨基端有-36个氨基酸组成的信号肽.生物信息学分析表明,OsCP2与大麦半胱氨酸蛋白酶HvCP同源性最高,同属木瓜蛋白酶亚家族.另外,我们构建了pET32a/CP2原核.表达载体,通过转化大肠杆菌BL21DE3,成功表达了OsCP2重组蛋白.分子量约为58kD;通过纯化包涵体获得了纯度高达90%以上的重组OsCP2蛋白,这为进一步研究该基因的功能提供了基础.     

Construction of synthetic immunogen: use of new T-helper epitope on malaria circumsporozoite protein

The circumsporozoite (CS) protein of Plasmodium falciparum is the focus of intense efforts to develop an antisporozoite malaria vaccine. Localization of sites for T-cell recognition on this molecule is critical for vaccine design. By using an algorithm designed to predict T-cell sites and a large panel of H-2 congenic mice, a major nonrepetitive T-cell site was located. When a synthetic peptide corresponding to this site was covalently linked to the major B-cell site on the molecule, an immunogen capable of eliciting a high-titer antibody response was formed. This peptide sequence could prime helper T cells for a secondary response to the intact CS protein. The new helper T-cell site is located outside the repetitive region of the CS protein and appears to be the immunodominant T site on the molecule. This approach should be useful in the rational design and construction of vaccines.     

In vivo function and membrane binding properties are correlated for Escherichia coli lamB signal peptides

Wild-type and pseudorevertant signal peptides of the lamB gene product of Escherichia coli interact with lipid systems whereas a nonfunctional deletion mutant signal peptide does not. This conclusion is based on interaction of synthetic signal peptides with a lipid monolayer-water surface, conformational changes induced by presence of lipid vesicles in an aqueous solution of signal peptide, and capacities of the peptides to promote vesicle aggregation. Analysis of the signal sequences and previous conformational studies suggest that these lipid interaction properties may be attributable to the tendency of the functional signal peptides to adopt alpha-helical conformations. Although the possibility of direct interaction between the signal peptide and membrane lipids during protein secretion is controversial, the results suggest that conformationally related amphiphilicity and consequent membrane affinity of signal sequences are important for function in vivo.     

An antigenic peptide produced by peptide splicing in the proteasome

CD8 T lymphocytes recognize peptides of 8 to 10 amino acids presented by class I molecules of the major histocompatibility complex. Here, CD8 T lymphocytes were found to recognize a nonameric peptide on melanoma cells that comprises two noncontiguous segments of melanocytic glycoprotein gp100(PMEL17). The production of this peptide involves the excision of four amino acids and splicing of the fragments. This process was reproduced in vitro by incubating a precursor peptide of 13 amino acids with highly purified proteasomes. Splicing appears to occur by transpeptidation involving an acyl-enzyme intermediate. Our results reveal an unanticipated aspect of the proteasome function of producing antigenic peptides.     

Crystal structures of an antibody to a peptide and its complex with peptide antigen at 2.8 A

The three-dimensional structures of an antibody to a peptide and its complex with the peptide antigen have been determined at 2.8 A resolution. The antigen is a synthetic 19-amino acid peptide homolog of the C helix of myohemerythrin (Mhr). The unliganded Fab' crystals are orthorhombic with two molecules per asymmetric unit, whereas the complex crystals are hexagonal with one molecule per asymmetric unit. The Fab' and the Fab'-peptide complex structures have been solved independently by molecular replacement methods and have crystallographic R factors of 0.197 and 0.215, respectively, with no water molecules included. The amino-terminal portion of the peptide sequence (NH2-Glu-Val-Val-Pro-His-Lys-Lys) is clearly interpretable in the electron density map of the Fab'-peptide complex and adopts a well-defined type II beta-turn in the concave antigen binding pocket. This same peptide amino acid sequence in native Mhr is alpha-helical. The peptide conformation when bound to the Fab' is inconsistent with binding of the Fab' to native Mhr, and suggests that binding of the Fab' to conformationally altered forms of the native Mhr or to apo-Mhr. Immunological mapping previously identified this sequence as the peptide epitope, and its fine specificity correlates well with the structural analysis. The binding pocket includes a large percentage of hydrophobic residues. The buried surfaces of the peptide and the antibody are complementary in shape and cover 460 A2 and 540 A2, respectively. These two structures now enable a comparison of a specific monoclonal Fab' both in its free and antigen complexed state. While no major changes in the antibody were observed when peptide was bound, there were some small but significant side chain and main chain rearrangements.