颜氏大疣蛛蛛毒中多肽和蛋白质的多样性分析

对颜氏大疣蛛Macrothele yani蛛毒所富含的多肽与蛋白质多样性进行探索,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和超高效液相色谱-电喷雾-四极杆-飞行时间质谱技术分离和鉴定颜氏大疣蛛蛛毒中的蛋白质和多肽,并对其相对分子质量分布多样性进行分析。结果显示:粗毒中所含蛋白质的相对分子质量主要分布在35kDa以上。在17~135kDa分离度较佳的共有11条电泳条带,主要集中于40~120kDa附近;在75kDa附近弥散着高丰度的蛋白条带,75kDa以上的蛋白质最丰富。粗毒经色谱分离后得到超过50个色谱峰,经质谱鉴定得到121个物质成分,其中,多肽类物质的相对分子质量呈双峰式分布,21%分布在500~2000 Da,76%分布在3000~5000Da,为粗毒中多肽含量最丰富的部分,且集中于35~60min的保留时间内被洗脱。研究结果表明,颜氏大疣蛛蛛毒中含有较为丰富的多肽和蛋白类物质,这些物质的相对分子质量分布特征与已报道的其他蜘蛛既有相似性又存在具体差异。本文展示了颜氏大疣蛛蛛毒的分子多样性,为后续该毒素的物质基础研究及药用价值开发提供参考。     

黄唇蜾蠃蜂蜂毒中两个过敏原全长基因的克隆、序列分析和表达

为了进一步研究透明质酸酶的过敏活性,利用昆虫杆状病毒成功地表达了黄唇蜾蠃蜂Rhynchium brunneum蜂毒的透明质酸酶。根据已报道的胡蜂科透明质酸酶和抗原5基因的氨基酸保守序列,设计合成简并引物,利用反转录多聚酶链式反应（RT-PCR）技术扩增了黄唇蜾蠃蜂蜂毒透明质酸酶和抗原5的基因片段,利用RACE技术进一步获得了它们的全长基因（GenBank登录号分别为EU624135和EU624136）。按照过敏原的命名法则,分别命名为Rhy b 2和Rhy b 5。序列比对分析发现,这两个基因与胡蜂科的相应序列高度相似,说明对胡蜂蜂毒过敏的人群也可能对蜾蠃蜂蜂毒有交叉过敏反应。但进一步分析发现,黄唇蜾蠃蜂蜂毒透明质酸酶的B细胞决定表位显著不同,9个保守性氨基酸在蜾蠃蜂中仅保留了3个,而且缺乏两个关键的精氨酸;黄唇蜾蠃蜂蜂毒抗原5序列N端的二级结构和具有过敏活性的常见黄胡蜂Vespula vulgaris蜂毒的抗原5显著不同,有可能因此而缺失依赖其N端二级结构的B细胞决定表位。据此认为,蜾蠃蜂蜂毒透明质酸酶和抗原5很可能是天然弱化的过敏原,在过敏原特异性免疫治疗上具有潜在的应用价值。     

黄唇蜾赢蜂蜂毒中两个过敏原全长基因的克隆、序列分析和表达

为了进一步研究透明质酸酶的过敏活性,利用昆虫杆状病毒成功地表达了黄唇蜾赢蜂Rhynehium brunneum蜂毒的透明质酸酶。根据已报道的胡蜂科透明质酸酶和抗原5基因的氨基酸保守序列,设计合成简并引物,利用反转录多聚酶链式反应（RT—PCR）技术扩增了黄唇蜾赢蜂蜂毒透明质酸酶和抗原5的基因片段,利用RACE技术进一步获得了它们的全长基因（GenBank登录号分别为EU624135和EU624136）。按照过敏原的命名法则,分别命名为Rhyb2和Rhyb5。序列比对分析发现,这两个基因与胡蜂科的相应序列高度相似,说明对胡蜂蜂毒过敏的人群也可能对蜾赢蜂蜂毒有交叉过敏反应。但进一步分析发现,黄唇蜾赢蜂蜂毒透明质酸酶的B细胞决定表位显著不同,9个保守性氨基酸在蜾赢蜂中仅保留了3个,而且缺乏两个关键的精氨酸;黄唇蜾赢蜂蜂毒抗原5序列N端的二级结构和具有过敏活性的常见黄胡蜂Vespula vulgaris蜂毒的抗原5显著不同,有可能因此而缺失依赖其N端二级结构的B细胞决定表位。据此认为,蜾赢蜂蜂毒透明质酸酶和抗原5很可能是天然弱化的过敏原,在过敏原特异性免疫治疗上具有潜在的应用价值。     

2种蜜蜂和4种胡蜂蜂毒明肽基因比较

从中华蜜蜂、意大利蜜蜂和大胡蜂、墨胸胡蜂、额斑黄胡蜂、亚非马蜂6种蜂的雌成蜂毒腺中快速抽提总RNA,用RT—PCR方法分别扩增各得到大小约为150bp的cDNA片段,进一步将这6个片段克隆人pGEM^5—T easy载体,进行测序和序列分析。结果表明,所扩增得到的6个片段长度均为141bp,均为编码蜂毒apamin前体的cDNA。序列比较发现,中华蜜蜂、大胡蜂、墨胸胡蜂和亚非马蜂序列完全一样,都与已发表意大利蜜蜂precursor of apamin核苷酸序列具有95％同源性,氨基酸序列具有95％的同源性。额斑黄胡蜂和意大利蜜蜂precursor of apamin核苷酸序列完全一样。本研究首次从中华蜜蜂与几种胡蜂中克隆到蜂毒precursor of apamin基因,并发现胡蜂科与蜜蜂科来源的蜂毒明肽序列是完全保守的。     

4种胡蜂前溶血肽原基因的克隆与序列比较

从雌性亚非马蜂、额斑黄胡蜂、墨胸胡蜂、大胡蜂毒腺中抽提总RNA,通过RT-PCR方法扩增得到4种胡蜂蜂毒前溶血肽原的cDNA,再将扩增产物克隆到pGEM⑥-Teasy vector上。测序结果表明：扩增得到的片段长度均为213bp,系蜂毒前溶血肽原编码区的cDNA。经序列比较,4种胡蜂蜂毒前溶血肽原之间的氨基酸序列同源性都超过95％。亚非马蜂、额斑黄胡蜂、墨胸胡蜂、大胡蜂各自与意大利蜜蜂蜂毒前溶血肽原氨基酸序列的同源性分别为95．8％、100％、97．2％、97．2％。结果表明：蜂毒前溶血肽原一级结构序列具有很高的保守性,尽管胡蜂和蜜蜂属于膜翅目不同的总科,但它们的前溶血肽原基因却非常相似。     

MMP-2结合肽-蜂毒素杂合基因的表达纯化及活性测定

为开发抗癌导向多肽药物,用从噬菌体十二肽库中筛选的与MMP-2具有高度亲和性的十二肽与蜂毒素连接,采用基因工程方法在大肠杆菌中进行了高效表达;并分别通过亲和层析、肠激酶酶切、凝胶层析获得高纯度的多肽(相对分子质量45000),经体外抑瘤作用测定该融合蛋白具有明显抑制肿瘤细胞的生长。该研究对肿瘤的导向治疗和临床应用提供了一定的帮助。     

蜂蛹多肽对巨噬细胞RAW264.7免疫活性的影响

蜂蛹多肽因具有丰富的营养价值,以及增强免疫、抗肿瘤及抗氧化等生物学活性,而受到了广泛关注,但目前关于蜂蛹多肽纯化组分的体外免疫调节活性的研究尚未见报道。为了探究蜂蛹多肽对巨噬细胞RAW264.7免疫活性的影响,以蜂蛹多肽纯化组分BPP-21为研究对象,研究其在不同浓度（12.5、25、50、100和200 μg·mL-1）下对RAW264.7巨噬细胞的细胞活力、吞噬能力、细胞因子分泌能力、NO分泌能力和氧化应激指标的影响。结果显示,在浓度12.5~200 μg·mL-1范围内,BPP-21对RAW264.7巨噬细胞无明显的细胞毒性作用,可显著提高干扰素-γ（interferon-gamma,IFN-γ）与NO的分泌水平（P<0.05）;在浓度25~200 μg·mL-1范围内,显著增加细胞吞噬能力以及白细胞介素-2（interleukin-2,IL-2）、肿瘤坏死因子α（tumor necrosis factor-alpha,TNF-α）的分泌量（P<0.05）;在浓度50~200 μg·mL-1范围内,显著提高细胞内超氧化物歧化酶（superoxide dismutase,SOD）活力（P<0.05）。研究表明,蜂蛹多肽纯化组分BPP-21可增强RAW264.7巨噬细胞的免疫活性,为蜂蛹多肽免疫调节剂的研究与开发提供了理论依据。     

天然活性多肽的发掘策略和生产技术

活性多肽可以参与生命机体的多种生理活动,对促进人体健康发挥着重要的作用,如降血压、降血糖、降血脂和抗癌等,其创制技术也逐渐成为重要的研究和应用转化方向。本综述旨在总结天然活性多肽的发掘策略和生产技术的研究进展。目前,天然活性多肽的发掘与生产技术主要包括自上而下和自下而上两种方法,其中自上而下方法在多肽发掘方面主要为直接提取鉴定法,在生产技术方面主要包括直接提取法、酶解法和微生物发酵法;自下而上方法在多肽发掘方面包括天然活性多肽改造和数据库发掘方法,在生产技术方面主要方法包括化学合成法、酶合成法、基因重组表达法和无细胞合成法。自上而下的天然多肽制备与功能验证方法存在步骤烦琐、耗费时间长、功能不确定性大、实验与生产成本高以及质量控制难度大等问题;而自下而上的活性多肽合成与功能验证方法适合多肽药物的开发,而难以用于功能食品。随着测序和质谱技术的发展,人们更容易从分子水平获取物种蛋白组信息。以此蛋白组信息为根据,将自上而下和自下而上两种方法结合,可以克服单独使用这两种方法存在的问题,从而为快速开发和生产天然活性多肽提供新的策略。     

蜜蜂毒溶血活性肽(melittin)的研究进展

蜜蜂毒(下称蜂毒)是很早以来人们就十分关注的毒性物质。在动物毒液中,它是研究得较早的。但对它的组成和作用模式的研究仅有二、三十年。就其组成成分的药理作用和治疗价值,无论在理论上还是在实践上都还很有限。蜂毒中含多种蛋白质和多肽,其多肽成分具较强的药理学和生物学活性,有抗炎镇痛等作用,临床上用于风湿、类风湿和牛皮癣等胶原性疾病的治疗,也可用于高血压及动脉粥样硬化、神经痛等的治疗,总有效率达80％。据报道在蜂毒中至少含有14个生物活性肽:melittin,melittin F,apamine,secapin,     

一种羊肚菌源多肽MIP-16的分离纯化及其神经保护活性

分离纯化人工栽培的梯棱羊肚菌子实体多肽(MIP-16),对其结构和神经保护活性进行研究。采用磷酸盐缓冲液提取,分子排阻色谱分离,反相高效液相色谱纯化获得梯棱羊肚菌子实体多肽,通过液相色谱-质谱连用技术完成氨基酸序列鉴定。构建6-羟基多巴胺处理后引起凋亡的PC12细胞模型,验证MIP-16对PC12细胞的保护作用。结果显示:MIP-16由16个氨基酸组成,相对分子质量为1 762 Da,氨基酸组成序列为Thr-Ile-Thr-Leu-Glu-Val-Glu-Ser-Ser-Asn-Ile-Thr-Asn-Asp-Val-Lys。细胞实验发现,MIP-16能够抑制PC12细胞丙二醛(MDA)产生,降低活性氧(ROS)水平,调节Bcl-2和Bax的比例,并降低Caspase蛋白的表达。MIP-16通过细胞线粒体途径抑制PC12细胞凋亡,具有神经保护活性,可作为一种辅助治疗帕金森综合症的药物开发。     

蜘蛛抗菌肽研究进展

蜘蛛活性多肽研究主要集中于蜘蛛毒液中作用于离子通道的神经毒素多肽。但近年来,一些蜘蛛抗菌肽不断被分离纯化,其结构和抗菌活性也被广泛深入研究,这将成为蜘蛛活性多肽研究领域的一个新热点。在蜘蛛毒液和血液中,存在不同种类的抗菌肽,其多肽长度、结构、抗菌作用各不相同。而且,有些抗菌肽甚至具有抗肿瘤作用。概述了蜘蛛抗菌肽在结构和功能方面的研究进展。     

Oxineur,a novel peptide from Caspian cobra Naja naja oxiana against HT-29 colon cancer

Colon cancer ranks fourth in mortality. This cancer is still an important clinical challenge worldwide due to its high prevalence and poor prognosis. Proteomic studies revealed that snake venom is a diverse and variable mixture of enzymatic and non-enzymatic proteins and peptides. Despite the toxic effects of these molecules, several proteins and peptides have been isolated that have practical applications and appear to induce apoptosis and prevent cell metastasis. In this study, we worked on cytotoxic effects and anticancer activity of Naja naja oxiana (Iranian Caspian cobra) snake venom components on HT-29 cell line colon cancer. Separated Fraction-5 by FPLC indicated the high cytotoxicity on HT-29 cell line colon cancer by MTT test. Further isolation of F5 by HPLC showed that the purified peak 2, nominated as Oxineur that contains a cytotoxic effect on HT-29 cells and reduces cell viability at 8 μg/ml to 4% in 24 h. Oxineur has the least cytotoxic effect on HEK-293 normal cells. Further studies on Oxineur peptide confirmed the apoptotic effects with high expression of CASP9 gene and DNA fragmentation in cancerous cells. The partial sequence of Oxineur revealed 71% homology with the neurotoxin II from Naja naja oxiana. Since our target molecule is a peptide in the molecular weight range of 7 kDa, it has potentially a therapeutic value.     

The structure and antimicrobial potential of wasp and hornet (Vespidae) mastoparans: A review

Wasp venom is a complex mixture of biologically active components, including high molecular weight proteins, small peptides, bioactive amines, and amino acids. Peptides comprise up to 70% of dried venom. In social wasp venoms, three of the major peptide types are mastoparans, which cause mast cell degranulation, chemotactic peptides, which promote chemotaxis of polymorphonucleated leukocytes, and kinin‐related peptides, which are known to produce pain and increase vascular permeability. Among these, the bioactive tridecapeptide mastoparan is the most common and may even have antimicrobial activity. Herein we summarize the results of studies on vespid mastoparans, focusing on hornets (Vespa spp.) identified following a systematic literature search for mastoparans of hornets in the genus Vespa, the most active mastoparan research taxon. The common features of hornet mastoparans are C‐terminal amidation, amphipathic helical structure, and multiple functions such as mast cell degranulation and hemolysis, as well as membrane permeabilization. Most interestingly, all tested hornet mastoparans have strong antimicrobial activities, suggesting that they can provide useful insights into and opportunities for development of novel antibacterial peptides.     

Characterization and biochemical analyses of venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

During parasitism, the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) induces a developmental arrest in host pupae that is sustained until the fly is either consumed by developing larvae or the onset of death. Bioassays using fluids collected from the female reproductive system (calyx, alkaline gland, acid gland, and venom reservoir) indicated that the venom gland and venom reservoir are the sources of the arrestant and inducer(s) of death. Infrared spectroscopic analyses revealed that crude venom is acidic and composed of amines, peptides, and proteins, which apparently are not glycosylated. Reversed phase high performance liquid chromatography (HPLC) and sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the proteinaceous nature of venom and that it is composed mostly of mid to high molecular weight proteins in the range of 13 to 200.5 kilodaltons (kDa). Ammonium sulfate precipitation and centrifugal size exclusion membranes were used to isolate venom proteins. SDS-PAGE protein profiles of the isolated venom fractions displaying biological activity suggest that multiple proteins contribute to arresting host development and eliciting death. Additionally, HPLC fractionation coupled with use of several internal standards implied that two of the low molecular weight proteins were apamin and histamine. However, in vitro assays using BTI-TN-5B1-4 cells contradict the presence of these agents.     

间斑寇蛛粗毒液的生理生化分析及两种采毒方法的比较

利用两种不同方法采集间斑寇蛛（Latrodectus tredecimguttatus）毒液并对其进行理化和生物学性质的比较分析。结果显示,粗毒液中的蛋白质成分主要是相对分子质量较大（>104）的酸性蛋白质。与毒囊粗毒液相比,电刺激粗毒液中相对分子质量在105左右的蛋白质含量明显高于毒囊粗毒液中的含量,而两者中相对分子质量较小（<104）的蛋白质与多肽的组成非常相似。电刺激粗毒冻干粉和毒囊粗毒冻干粉对小白鼠的LD50值分别为(0.16±0.03) mg/kg和(0.39±0.05)mg/kg体重;对美洲蜚蠊（Periplaneta Americana）的LD50值分别为1.87 μg/g和2.32 μg/g。在浓度为3.2×10-6g/mL时,电刺激粗毒冻干粉能在（25.0±2.2） min内完全阻断小鼠膈神经-膈肌标本的神经肌肉接头传递;毒囊粗毒冻干粉在浓度为6×10-6g/mL时的完全阻断时间为（23.3±2.2） min;粗毒液中的低相对分子质量（<104）部分在10-4g/mL浓度下对标本的神经肌肉接头传递无明显影响。上述结果表明,间斑寇蛛毒液是一种富含大分子量蛋白质的混合物;哺乳动物神经毒性主要基于其中的大的相对分子质量酸性蛋白质成分,而不是低的相对分子质量的多肽;电刺激粗毒液中的活性成分与毒囊粗毒液中的相似,但含量高于毒囊粗毒液。     

Proteomic and peptidomic characterization of the venom from the Chinese bird spider, Ornithoctonus huwena Wang

The bird spider Ornithoctonus huwena Wang is a very venomous spider in China. Several compounds with different types of biological activities have been identified previously from the venom of this spider. In this study, we have performed a proteomic and peptidomic analysis of the venom. The venom was preseparated into two parts: the venom proteins with molecular weight (MW) higher than 10,000 and the venom peptides with MW lower than 10 000. Using one-dimensional gel electrophoresis (1-DE), two-dimensional gel electrophoresis (2-DE), and mass spectrometry, 90 proteins were identified, including some important enzymes, binding proteins, and some proteins with significant biological functions. For venom peptides, a combination of cation-exchange and reversed-phase chromatography was employed. More than 100 components were detected by mass spectrometry, and 47 peptides were sequenced by Edman degradation. The peptides display structural and pharmacological diversity and share little sequence similarity with peptides from other animal venoms, which indicates the venom of O. huwena Wang is unique. The venom peptides can be classified into several superfamilies. Also it is revealed that gene duplication and focal hypermutation have taken place during the evolution of the spider toxins.     

Characterisation of neurotoxic polypeptides from <Emphasis Type="Italic">Cyanea capillata</Emphasis> medusae (Scyphozoa)

Cnidarian venoms include neurotoxins, which are able to paralyse prey organisms immediately. Important targets for neurotoxins are voltage-gated ion channels in membranes of excitable cells. By blocking specific receptor sites, neurotoxic components disturb the physiological ion channel functions. Here, we describe the isolation and characterisation of potential neurotoxic polypeptides from the crude tentacle venom of the boreal scyphomedusan Cyanea capillata. Partially purified venom fractions were obtained by size-exclusion and subsequent reversed-phase chromatography. To assess the blocking activity of the venom on voltage-gated sodium channels, we modified a mouse neuroblastoma (MNB) cell assay. Venom fractions containing channel-blocking activity were analysed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The resulting mass spectra revealed a cluster of singly charged peptides within a mass range from 3,900 to 7,000 Da. A group of three potentially neurotoxic peptides with molecular masses of 3983.4, 5795.4 and 6962.1 Da could be tracked throughout the purification process. This investigation of the crude venom is part of a multidimensional assay-guided approach for the isolation and structural characterisation of toxic polypeptides in northern Scyphozoa.     

Sulfated sialic acid-polymers inhibit the cytotoxic action of bee and snake venom

Colominic acid is an 2,8-linked sialic acid polymer produced by Escherichia coli. We found that synthetic sulfated-colominic acids (SC) remarkably inhibited the cytotoxicity of bee and snake venom toward mouse fibroblast cells, but colominic acids showed no inhibition themselves, indicating the important role of sulfate groups in the inhibitory activity of SC. Other sulfated carbohydrates such as chondroitin sulfates, heparin and heparan sulfate showed no inhibition. SC also exhibited potent inhibition of melittin, a highly basic peptide, which is a major cytotoxic component of bee venom. SC did not inhibit phospholipase A₂ activity in bee venom. This suggests that the inhibition of bee and snake venom by SC is due to inhibition of melittin and cardiotoxin, which is a cytolytic peptide in snake venom, respectively. SC with a higher sulfur content and a larger molecular mass showed more potent activity. The interaction between SC and melittin basically seems an ionic one, however, the conformation of SC is also likely important. For the binding of SC to melittin leading loss of its cytotoxic activity, the sulfate groups of SC must be properly arranged to interact with lysine and arginine residues of melittin molecules, which play an important role in the cytolytic activity. A higher molecular mass of SC substituted with more sulfate groups is required for more obvious inhibition of the cytotoxic activity.