Primary Structure of a Short Toxin from the Venom of a Vietnamese Scorpion (Buthus sp.)

The complete amino acid sequence of an important toxin (toxin 14) from the venom of a Vietnamese scorpion (Buthus occitanus sp.) has been determined, which includes 35 amino acid residues and three disulfide bridges (molecular weight, 3843 Da). The comparison of the sequence with known sequences of short scorpion toxins led to the conclusion that toxin 14 belongs to a novel group of toxins affecting the excitability of myelinated nerves.     

梁氏异蝎毒素促进KYSE-510细胞凋亡的作用机制研究

为了研究蝎毒抑制肿瘤细胞生长的机理,提取梁氏异蝎Heterometrus liangi蝎毒干预人食管癌细胞(KYSE-510细胞),运用流式细胞术、电镜技术、逆转录PCR和Western blot方法分别检测蝎毒对KYSE-510细胞凋亡的影响以及对caspase-3基因表达。结果表明:经蝎毒处理的实验组KYSE-510活细胞数量显著减少,其原因是蝎毒引起细胞大量凋亡和坏死。经不同浓度(50μg·m L~(-1),100μg·m L~(-1),200μg·m L~(-1))蝎毒处理的KYSE-510细胞与对照组的caspase-3基因在mRNA水平表达相近,而在蛋白表达水平上,实验组细胞与对照组细胞相比,蝎毒促进了caspase-3的剪切活化,且以200μg·m L~(-1)蝎毒处理的KYSE-510细胞caspase-3的剪切活化最强,由此证明蝎毒导致KYSE-510细胞凋亡的机理之一可能是其促进了caspase-3的剪切活化。     

蛇毒蛋白triflin功能区TFPR1在大肠杆菌中的表达及其功能初步研究

目的:获得具有生物学活性的蛇毒蛋白triflin的致病相关蛋白1(PR-1)功能区TFPR1。方法:分析triflin空间结构及其保守结构域,获得TFPR1序列,并进行密码子优化、全基因合成,构建原核表达质粒p ET-TFPR1,在大肠杆菌BL21中以IPTG诱导表达,表达产物经Western印迹鉴定后,镍柱纯化、复性,对复性后的产物进行特性分析;以复性的TFPR1肌肉途径接种BALB/c雌性小鼠,分析其生物学活性。结果:TFPR1以包涵体形式表达,复性后蛋白纯度大于95%,无需佐剂免疫小鼠即可诱导机体产生效价为20万的抗TFPR1抗体。结论:制备了具有生物学活性的重组蛋白TFPR1,为后续研究TFPR1的生物学功能奠定了物质基础。     

北非蝎昆虫毒素AaHIT1基因的原核表达和转基因分析

将北非蝎昆虫毒素 (AndroctonusaustralisHectorinsecttoxin ,AaHIT1)基因克隆入温度诱导表达载体pBV2 2 0 ,并在宿主菌E .coliDH5α中进行诱导表达 .表达定位分析发现 ,AaHIT1大部分存在于包涵体中 .蛋白质N 端测序证实了体外表达的AaHIT1蛋白的正确性 .将AaHIT1基因转化入烟草中并获得了转基因植株 ,基因组PCR、RT PCR及Northern印迹分别证实AaHIT1基因已正确地插入到烟草基因组中并已得到表达 .抗虫实验表明 ,该转基因烟草对白粉虱有明显的抗性 .该研究为蝎毒AaHIT1的生物学毒性实验以及获得抗虫转基因作物奠定了基础     

The Effects of Scorpion Venom Toxin on the Release of Amino Acid Neurotransmitters from Cerebral Cortex In Vivo and In Vitro

Tityustoxin, the active component of the venom of the Brazilian yellow scorpion Tityus serrulatus, caused specific release of the neurotransmitter amino acids glutamate, aspartate and GABA in vivo from the superfused sensori-motor cortex of conscious unanesthetised rats and in vitro from rat cortical synaptosomes. The effects on synaptosomes appear to be due to a depolarising action. Synaptosomal potassium levels were depleted by the toxin. The action was also blocked both in vivo and in vitro by tetrodotoxin and was Ca2+-dependent. The uptake of [U-14C]GABA was inhibited by tityustoxin but this action was prevented by tetrodotoxin (1 microM). Since the release of [U-14C]GABA from synaptosomes due to the tityustoxin was also prevented by tetrodotoxin under identical circumstances, it is concluded that the tityustoxin has a primary action on release of neurotransmitters rather than on uptake.     

湖南尖吻蝮蛇毒出血毒素的圆二色性和化学修饰

用远紫外ＣＤ谱研究了湖南产尖吻蝮蛇毒的两个出血毒素（ＤａＨＴ－１、ＤａＨＴ－２）的溶液构象,计算得ＤａＨＴ－１的α螺旋、β折叠、无规卷曲的含量分别为３６．９％、３５．５％、２７．６％;ＤａＨＴ－２的α螺旋、β折叠、无规卷曲分别为２３．４％、３１．３％、４５．３％。随ｐＨ的增大或减小,峰位蓝移,酸性条件下的变化比碱性条件下的变化大。构象单元含量计算表明：α螺旋减少,无规卷曲增多,β折叠基本未变。温度和ｐＨ对ＣＤ谱的影响相似,５０℃时峰位蓝移,α螺旋减少,无规卷曲增多．ＥＤＴＡ对ＣＤ谱影响显著,０．０２ｍｏｌ／ＬＥＤＴＡ便导致两个出血毒素呈极度的无序状态。ＥＤＴＡ完全抑制,半胱氨酸部分抑制,胰蛋白酶不影响它们的出血活性。     

Isolation,Stabilization, and Characterization of a Toxin from Timber Rattlesnake Venom

A rapid and convenient method for the purification of a toxin from timber rattlesnake, Crotalus horridus horridus, venom using carboxymethyl cellulose ion-exchange chromatography has been devised. The toxicity of this venom component is labile, but it is stabilized by the addition of 20% V/V glycerol to the buffer solution. This toxin has a molecular weight of 15,000 + 700 as determined by SDS gel electrophoresis. It is both heat and protease resistant. Treatment of this venom component with 2-mercaptoethanol followed by G-50 Sephadex chromatography causes no loss of toxicity although incubation of the toxin with 1% SDS and 1% 2-mercaptoethanol prior to electrophoresis does result in a faster migrating species. The toxin does not affect neuromuscular junctions but does appear to act on the nervous system. It causes no local responses in mice.     

一个新的东亚钳蝎毒素(BmKT_1)全长cDNA的克隆和分析

首先构建了东亚钳蝎毒腺组织 c DNA文库 ;根据已知的东亚钳蝎哺乳动物毒素氨基酸序列保守区设计引物 ,并用 PCR从 c DNA文库中扩增出一个 c DNA片段作为筛选 c DNA文库的探针 ;从 c DNA文库中筛选到二个编码同一个新的蝎毒素多肽的 c DNA,它们除 3′- UTR外 ,其余序列完全一致 .它们均含有 2 55bp长的开放阅读框 ,编码 85肽的前体毒素 ,包括 1 9个氨基酸残基的信号肽 ,66个残基的成熟毒素 (命名为 Bm KT1) ;Bm KT1氨基酸序列与已知的蝎毒素具有较大的同源性 ,与 Bm KM1,Lqq ,Lqhα IT和 Bm K M10 的同源性分别为 77%、67%、67%和 65% .Bm KT1的 C端不存在末端修饰步骤且具有一个与这些毒素不相同的特征结构 ,即在末端延伸了两个氨基酸残基 - P- S,推测 Bm KT1具有新的活性功能特征 .     

Identification and Structural Characterization of a New Three-Finger Toxin Hemachatoxin from Hemachatus haemachatus Venom

Snake venoms are rich sources of biologically active proteins and polypeptides. Three-finger toxins are non-enzymatic proteins present in elapid (cobras, kraits, mambas and sea snakes) and colubrid venoms. These proteins contain four conserved disulfide bonds in the core to maintain the three-finger folds. Although all three-finger toxins have similar fold, their biological activities are different. A new three-finger toxin (hemachatoxin) was isolated from Hemachatus haemachatus (Ringhals cobra) venom. Its amino acid sequence was elucidated, and crystal structure was determined at 2.43 Å resolution. The overall fold is similar to other three-finger toxins. The structure and sequence analysis revealed that the fold is maintained by four highly conserved disulfide bonds. It exhibited highest similarity to particularly P-type cardiotoxins that are known to associate and perturb the membrane surface with their lipid binding sites. Also, the increased B value of hemachotoxin loop II suggests that loop II is flexible and may remain flexible until its interaction with membrane phospholipids. Based on the analysis, we predict hemachatoxin to be cardiotoxic/cytotoxic and our future experiments will be directed to characterize the activity of hemachatoxin.     

SPEX, a System for the Expression of Recombinant Proteins from Gram-Positive Bacterial Vectors

Using a conserved pathway for surface protein extrusion, a system has been developed for the expression and secretion of proteins from gram-positive bacteria. As proof-of-concept, theStreptococcus gordoniiChallis strain has been engineered to express a series of recombinant proteins fused to the conserved region of the M6 protein ofStreptococcus pyogenes.In the prototype surface protein expression system, the recombinant M6 protein is anchored to the surface ofS. gordoniicells expressing it. In order to overexpress the protein and easily purify it away from the bacteria, the protein was modified to enable it to be secreted into the medium. To accomplish this, a stop codon was introduced into the gene just prior to the anchor region using site-directed mutagenesis. Using enzyme-linked immunosorbent assays, it was possible to quantitate the amount of protein expressed using this system. With little or no optimization, 3 mg of protein per liter of culture was expressed and secreted into the medium of a bacterial culture grown to an OD₆₀₀equal to 1.0. This system should be broadly applicable for the expression and secretion of a variety of proteins (antigens, hormones, and enzymes) directly into the medium.     

A Novel Insecticidal Toxin from Photorhabdus luminescens, Toxin Complex a (Tca), and Its Histopathological Effects on the Midgut of Manduca sexta

Photorhabdus luminescens is a bacterium which is mutualistic with entomophagous nematodes and which secretes high-molecular-weight toxin complexes following its release into the insect hemocoel upon nematode invasion. Thus, unlike other protein toxins from Bacillus thuringiensis (δ-endotoxins and Vip’s), P. luminescens toxin (Pht) normally acts from within the insect hemocoel. Unexpectedly, therefore, the toxin complex has both oral and injectable activities against a wide range of insects. We have recently fractionated the protein toxin and shown it to consist of several native complexes, the most abundant of which we have termed Toxin complex a (Tca). This complex is highly active against the lepidopteran Manduca sexta. In view of the difference in the normal mode of delivery of P. luminescens toxin and the apparent communality in the histopathological effects of other gut-active toxins from B. thuringiensis, as well as cholesterol oxidase, we were interested in investigating the effects of purified Tca protein on larvae of M. sexta. Here we report that the histopathology of the M. sexta midgut is similar to that for other novel midgut-active toxins. Following oral ingestion of Tca by M. sexta, we observed an acceleration in the blebbing of the midgut epithelium into the gut lumen and eventual lysis of the epithelium. The midgut shows a similar histopathology following injection of Tca into the insect hemocoel. These results not only show that Tca is a highly active oral insecticide but also confirm the similar histopathologies of a range of very different gut-active toxins, despite presumed differences in modes of action and/or delivery. The implications for the mode of action of Tca are discussed.     

蝎毒多肽提取物对卵巢癌SKOV3 细胞HPA 与VEGF 抑制作用 的实验研究

目的:研究蝎毒多肽提取物(polypeptide extract from scorpion venom,PESV)对卵巢癌SKOV3细胞HPA与VEGF表达的影响,进一步探讨其抗血管生成的分子机制.方法:卵巢癌SKOV3细胞分为对照组、PESV组,免疫组织化学方法检测各组HPA、VEGF的表达,western-blot方法检测各组HPA在蛋白水平的表达.结果:与对照组相比,PESV组HPA、VEGF表达明显降低(P＜0.05),进一步检测发现PESV组HPA在蛋白水平表达亦明显下调(P＜0.05).结论:PESV能够抑制卵巢癌SKOV3细胞的生长,其机制可能与抑制HPA、VEGF的表达有关.     

Production of Recombinant Allergens Phl p 1 and Amb a 1 for Detection of Class E Immunoglobulins

Russian Journal of Bioorganic Chemistry - Recombinant major allergens Phl p 1 from meadow timothy (Phleum pratense) and Amb a 1 from ragweed (Ambrosia artemisiifolia) were obtained in E. coli...     

二聚体蝎钠通道毒素的1.4(A)分辨率晶体结构:反常非脯氨酸顺式肽键及其内在结构因素

报道了以二聚体存在的dimo-BmK M1的1．4A分辨率晶体结构．蛋白质中的肽键是局部双键,不可旋转,因此具有顺式（cis）和反式（trans）两种构型,它们不能通过旋转操作相互转换．非脯氨酸顺式肽键是指形成该肽键的氨基是由脯氨酸以外的氨基酸提供的（Xaa-nonPro）,这类肽键的顺式构型的自由能远比反式高,因此极少出现在天然蛋白质结构中．事实上,在长时间中,多肽链的“反式肽键连接”被视为蛋白质结构的一条基本规则,把顺式肽键视为不可能．随着高分辨率精确蛋白质结构数量的增加,近年来有详细的统计分析揭示,非脯氨酸顺式肽键（Xaa-nPro）在蛋白质结构中出现的几率为0．03％～0．05％,而且大多存在于功能敏感的结构区域,可能具有重要意义．但由于所用的基本结构数据都来自晶体结构,对这种反常肽键是否由结晶环境影响而形成,存在疑问．此前曾在以单体形式存在的蝎神经毒素mono-BmK M1的高分辨率结构中发现其中肽键Pr09-His10是非脯氨酸顺式肽键,并详细分析了其结构．功能意义．以二聚体存在的dimo-BmK M1的1．4A分辨率晶体结构表明,它与mono．BmK M1有不同的空间群、不同的分子堆积方式,不同的晶体环境．结构模型被高度精化,Rcryst达到0．109．dimo-BmK M1结构显示,在不对称单位中的两个M1分子在同一位置（残基9．10之间）都清晰地存在顺式肽键．立体化学分析显示,这一肽键的几何参数和局部结构与mono．BmK M1中的（9．10）顺式肽键基本相同．这一结果表明,非脯氨酸顺式肽键9．10的存在与结晶环境无关,是BmK M1分子的固有结构特征．在此基础上,综合分析了与顺式、反式肽键相关的结构元素,发现与残基（8．19）序列模体-KPXNC-（X为任意氨基酸）所决定的特征回折结构可能是分子内在的主要结构因素,其中第8位残基是Lys或Asp对决定肽键是顺式还是反式有关键作用．近来的突变实验及其晶体结构测定已证实,Lys8／Asp8是（9-10）肽键顺式／反式异构的结构开关,它们对该类分子与不同种属钠通道作用的专一选择性具有重要作用．通过BLAST搜索,发现在其他18个蛋白质中也存在相同的序列模体．KPXNC-,推测在这些蛋白质的相应肽键位置也可能存在反常的脯氨酸顺式肽键。     

Production and Characterization of N- and C-terminally Truncated Mtx2: a Mosquitocidal Toxin from Bacillus sphaericus

Mosquitocidal toxin 2 (Mtx2) is a mosquito-larvicidal protein produced during vegetative stage of Bacillus sphaericus. The toxin consists of 292 amino acids and has a molecular weight of 31.8 kDa. To determine the active core region of the toxin, amino acids at N- and C-termini were sequentially removed. Deletion up to 23 amino acids from the N-terminus (Met1-Tyr23) did not significantly affect protein production and the toxin activity, whereas removal of 26 amino acids from the N-terminus (Met1-Lys26) completely abolished toxicity even though the protein production remained unchanged. Deletion of only 5 amino acids from the C-terminal end yielded the protein that could not be solubilized and rendered the toxin inactive. The results demonstrated that the C-terminal end of Mtx2 is required for proper folding and toxicity. Amino acids at the N-terminus up to Tyr23 did not play a significant role in protein production and toxicity whereas amino acids between Thr24 and Lys26 are required for full toxicity.     

Leftward Shift in the Voltage-Dependence for Ca2+ Currents Activation Induced by a New Toxin from Phoneutria reidyi (Aranae, Ctenidae) Venom

Summary Various neurotoxins have been described from the venom of the Brazilian spider Phoneutria nigriventer, but little is known about the venoms of the other species of this genus. In the present work, we describe the purification and some structural and pharmacological features of a new toxin (PRTx3-7) from Phoneutria reidyi that causes flaccid paralysis in mice. The observed molecular mass (4627.26 Da) was in accordance with the calculated mass for the amidated form of the amino acid sequence (4627.08 Da). The presence of an α-amidated C-terminus was confirmed by MS/MS analysis of the C-terminal peptide, isolated after enzymatic digestion of the native protein with Glu-C endoproteinase. The purified protein was injected (intracerebro-ventricular) into mice at dose levels of 5 μg/mouse causing immediate agitation and clockwise gyration, followed by the gradual development of general flaccid paralysis. PRTx3-7 at 1 μM inhibited by 20% the KCl-induced increase on [Ca²⁺]_i in rat brain synaptosomes. The HEK cells permanently expressing L, N, P/Q and R HVA Ca²⁺ channels were also used to better characterize the pharmacological features of PRTx3-7. To our surprise, PRTx3-7 shifted the voltage-dependence for activation towards hyperpolarized membrane potentials for L (−4 mV), P/Q (−8 mV) and R (−5 mV) type Ca²⁺ currents. In addition, the new toxin also affected the steady state of inactivation of L-, N- and P/Q-type Ca²⁺ currents. L. B. Vieira and A. M. C. Pimenta contributed equally to this work.     

大连蛇岛蝮蛇类凝血酶基因在毕赤酵母中的克隆表达及分离纯化

根据同源性分析设计引物,通过RT-PCR方法从大连蛇岛蝮蛇毒腺总RNA中合成扩增出类凝血酶基因,之后将该基因克隆到表达载体pPIC9K中,经电激转化后整合至毕赤酵母细胞基因组中.经筛选得到甲醇快速生长型转化子His+Mut+在500 ml摇瓶中培养,甲醇诱导分泌表达.上清液中重组类凝血酶是通过两步柱层析得到:Q Sepharose FF和Benzamidine-Sepharose 4BCL.与天然蛇毒类凝血酶一致,分泌表达的重组类凝血酶具有较强的酯酶活性,但精氨酸甲酯如TAME的水解活性较弱.此重组类凝血酶在37℃中性溶液中保存过夜将分解成小肽,但在0℃下很稳定.该酶的最适pH为8.0.     

Polyclonal Antibody Against a Recombinant Chlorotoxin-like Peptide from the Chinese Scorpion and Detection of its Putative Receptors in Human Glioma Cells

The nucleotide sequence of a type of chlorotoxin-like peptide, an inhibitor of small-conductance Cl⁻ channels, from the scorpion, Buthus martensii Karsch, was synthesized (named rBmK CTa) according to the sequence optimized for codon usage in E. coli. It was over-expressed using a pExSecI expression system and purified to homogeneity. Polycolonal antibodies to the purified protein were raised in rats. Overlay assay and pull-down assay showed that this toxin specially binds to two proteins in the glioma cells with corresponding molecular weights of about 80 and 35 kDa. They may serve as candidate receptors or alternative cellular component for interaction with rBmK CTa.