大疣蛛毒素-Ⅵ的分离纯化及部分生物学活性鉴定

从雷氏大疣蛛(Macrothele raveni)粗毒中,结合阳离子交换和反相高效液相色谱分离到一种多肽神经毒素,命名为大疣蛛毒素-VI(Raventoxin-VI).经MALDI-TOF质谱分析,它的分子量为(5 371.6±0.5) Da.利用Edman降解气相蛋白质测序仪测得其氨基酸序列是NH2-NIIKGRVVKLCGGCAQKCCDREPRCDPCRTCVENVGT-GGGYLSSNKKCNGS-COOH,其中8个Cys形成4对二硫键.Raventoxin-Ⅵ能阻断小鼠膈神经膈肌标本神经肌肉接头传递,脑室注射能使小鼠瘫软.从一级结构上没有发现与该毒素有较高相似性的其他蜘蛛毒素.     

大疣蛛毒素-VI的分离纯化及部分生物学活性鉴定

从雷氏大疣蛛(Macrotheleraveni)粗毒中,结合阳离子交换和反相高效液相色谱分离到一种多肽神经毒素,命名为大疣蛛毒素 VI(Raventoxin VI).经MALDI TOF质谱分析,它的分子量为(5371.6±0.5)Da.利用Edman降解气相蛋白质测序仪测得其氨基酸序列是NH2 NIIKGRVVKLCGGCAQKCCDREPRCDPCRTCVENVGT GGGYLSSNKKCNGS COOH,其中8个Cys形成4对二硫键.Raventoxin Ⅵ能阻断小鼠膈神经膈肌标本神经肌肉接头传递,脑室注射能使小鼠瘫软.从一级结构上没有发现与该毒素有较高相似性的其他蜘蛛毒素.     

新型抗虫活性肽雷氏大疣蛛毒素4596的纯化与鉴定

雷氏大疣蛛是我国新近鉴定的蜘蛛新种,虽然动物学家们已经对其进行过分类地位与生态学特性研究,然而有关抗昆虫活性成分研究的报道较少.通过比较解剖蜘蛛毒囊、乳胶软管诱导与电脉冲刺激3种蜘蛛毒液采集技术,发现雷氏大疣蛛毒液的最佳采集方法是电脉冲刺激采毒法.该蜘蛛粗毒对蜚蠊50%致死剂量LD50值是1.486 mg/g.结合阳离子交换层析和反相高效液相色谱技术从该蜘蛛粗毒中分离纯化到一种新型多肽毒素,根据质谱分析确定其相对分子质量为4 596.38,该毒素被命名为雷氏大疣蜘蛛毒素-4596(raventoxin-4596,RVTX-4596).通过初步毒性实验证实RVTX-4596是一种新型抗昆虫毒素.     

大胡蜂蜂毒中多肽和蛋白质结构和功能的多样性

为证明大胡蜂Vespa magnifica(Smith)蜂毒具有较大的药用开发价值,本研究采用超高效液相色谱-质谱和电泳技术对其多肽和蛋白质的分布进行分析,发现其蛋白质的相对分子质量主要分布在17~45kDa范围内。蜂毒多肽类物质的相对分子质量呈"单峰"式分布,61%在500~3000Da范围内,为大胡蜂蜂毒中多肽含量最为丰富的部分。通过牛津杯法对蜂毒的抑菌活性进行研究,且以HepG2人肝癌细胞及B16黑色素瘤细胞为研究对象,用MTT法检测蜂毒的细胞毒性活性,证明其具有良好的抑菌作用和细胞毒活性,其结果与已报道的其他蜂类既有相似性又存在具体差异,展示了大胡蜂蜂毒的分子多样性,为后续该毒素的物质基础研究及药用价值开发提供参考。     

雷氏大疣蛛毒素—Ⅱ的纯化与初步毒性研究

雷氏大疣蛛是我国最近鉴定的蜘蛛新种。雷氏大疣蛛毒素－Ⅱ就是以其粗毒为材料,利用阴、阳离子交换层析和反相HPLC分离得到并命名的一种新型神经毒素肽,根据质谱分析得知它的相对分子质量为3021．56;通过初步毒性研究,证明它是一个神经毒素。     

雌性和雄性虎纹捕鸟蛛粗毒蛋白质含量比较分析

自然界虎纹捕鸟蛛雌蛛数量远多于雄蛛数量,为了探究雌、雄蛛粗毒的差异,用不同方法比较了单个虎纹捕鸟蛛雌、雄蛛粗毒的特征.雌蛛单次螫毒量为(26.5±2.47)μL,冻干后粗毒质量为(5.01±0.78)mg,明显高于雄蛛单次螫毒量(10.83±1.35)μL,冻干后粗毒质量(2.05±0.17)mg;用Lowry法和Bradford法测定雌蛛和雄蛛粗毒的蛋白质含量,两种方法均表明雄蛛粗毒中蛋白含量高于雌蛛.用反相高效液相色谱分离雌蛛和雄蛛粗毒蛋白,并215 nm检测色谱图,发现大部分洗脱峰重叠,而雄蛛色谱图中多两个主峰.Tricine SDS-PAGE电泳分析表明雌蛛粗毒相对分子质量小于10 kD的蛋白质含量较高;而Tris SDS-PAGE电泳分析表明雌蛛粗毒相对分子质量大于10 kD的蛋白质含量较低,基于雌蛛和雄蛛粗毒蛋白含量的差异,有必要对虎纹捕鸟蛛雌、雄蛛粗毒进行独立研究.     

敬钊缨毛蛛毒素-Ⅷ的分离纯化与鉴定

从敬钊缨毛蛛（Chilobrachys jingzhao）粗毒中,通过阳离子交换色谱和反相高效液相色谱分离得到一种新的多肽神经毒素,命名为敬钊缨毛蛛毒素-Ⅷ（jingzhaotoxin-Ⅷ,JZTX-Ⅷ）.经MALDI-TOF分析表明该多肽分子质量为4 329.37 Da,结合氨基酸序列分析和RACE的cDNA快速扩增法得到了敬钊缨毛蛛毒素-Ⅷ的氨基酸序列和全长cDNA,序列为LFECSFSCDIKKNGKPCKGSGEKKCSGGWRCKMNFCVKV-COOH,其中6个半胱氨酸形成3对二硫键.敬钊缨毛蛛毒素-Ⅷ能阻断小鼠膈神经-膈肌标本的神经肌肉接头传递,经初步膜片钳实验分析,表明该多肽是一种钙离子通道抑制剂.     

海南捕鸟蛛毒素-Ⅵ的分离纯化及鉴定

从分布于我国海南省的海南捕鸟蛛（Selenocosmia hainana)粗毒中,应用阳离子交换色谱和反相高效液相色谱分离得到1种多肽神经毒素,命名为海南捕鸟蛛毒素－Ⅵ（Hainantoxin-Ⅵ,HNTX-Ⅵ)。MALDI－TOF南谱分析及氨基酸序列分析表明该多肽毒素分子量为3.998 49kDa,序列为NH2－ECKYLWGTCEKDE－HCCEHLGCNKKHGWCGWDGTF－COOH,其中的6个Cys形成3对二硫键。HNTX－Ⅵ能阻断小鼠膈神经膈肌标本神经肌肉接头传递,脑室及硬膜外注射毒能使小鼠瘫软。同源性搜索表明该毒素与蜘蛛Grammostola spatulata毒素HaTx和蜘蛛Scodra griseipes中的SGTx1毒素有很高的同源性,而后两种毒素均为K^ 通道阻断剂。     

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。     

颜氏大疣蛛毒腺转录组学研究

颜氏大疣蛛Macrothele yani个体大、毒性强、易取毒,是农林生态系统中害虫的重要天敌,也是天然药物研究工作者进行新药挖掘和毒液开发利用的重要选择对象。本研究利用高通量测序平台Illumina Hiseq 2000对颜氏大疣蛛雌雄成体的毒腺进行转录组测序和生物信息学分析,共获得转录组样本数据37.8 G,总计301 024条unigenes,总长度170 512 372 bp,最短201 bp,最长36 105 bp,平均长度566 bp,GC含量平均值为38.17%,N50长度为705 bp。将unigenes序列与NR（非冗余蛋白序列数据库）、String（蛋白质相互作用数据库）、Swiss-prot（蛋白质序列数据库）、Pfam（蛋白质家族数据库）、GO（基因本体论）、KOG（真核生物蛋白质直系同源数据库）、KEGG（京都基因与基因组百科全书）数据库进行比对（e≤10^-10）,分别获得31 409、6 549、11 817、8 216、11 480、7 804条unigenes注释。通过生物信息学对雌雄颜氏大疣蛛unigenes进行表达量分析,筛选高表达量且高可信度的差异表达基因,并进行对这些差异基因进行GO和KEGG功能富集分析;与雄蛛相比,雌成体有205条unigenes表达量上调,113条unigenes表达量下调。最后在所有的转录本数据中共比对到68条毒素相关序列。本研究获得的颜氏大疣蛛转录组信息,为颜氏大疣蛛的功能基因挖掘提供了重要的信息资源。     

Characterization of rat adipose tissue lipoprotein lipase using a monospecific antibody

An antibody to a highly pure enzyme preparation was developed to facilitate detailed studies of rat adipose tissue lipoprotein lipase regulation. Lipoprotein lipase was purified by heparin-Sepharose affinity chromatography followed by preparative isoelectric focusing. The enzyme migrated as a single broad band on SDS disc gel and two-dimensional gel electrophoresis with an apparent molecular mass of 67 000 and 62 000 Da, respectively. The amino acid composition of the purified rat enzyme was virtually identical to that of bovine milk. A major protein component with no lipase activity co-eluted with the enzyme from the affinity column, but was separated by the isoelectric focusing step. The molecular mass was slightly lower (58 000 Da) but the amino acid composition of this protein was similar to that of the enzyme. An antibody raised against the purified rat enzyme was highly potent and was effective in inhibiting rat heart lipoprotein lipase, but not the salt-resistant hepatic lipase. Analysis of crude acetone-ether adipose tissue preparation on SDS slab polyacrylamide gel coupled to Western blotting revealed five protein bands = (62 000, 56 000, 41 700, 22 500, 20 000 Da). Similarly, following affinity purification by immunoadsorption, the purified antibody reacted with five equivalent protein bands. Fluorescent concanavalin A binding data indicated that the 56 kDa band is a glycosylated form of lipoprotein lipase. Pretreatment of adipose tissue with proteinase inhibitors revealed that the lower molecular mass proteins (41 700 and 20 000 Da) were degradation products of lipoprotein lipase, and the 22 500 Da band could be accounted for by non-specific binding.     

Proteomic and peptidomic analysis of the venom from Chinese tarantula Chilobrachys jingzhao

Chinese tarantula, Chilobrachys jingzhao is one of the most venomous spiders in southern China and its venom is a mixture of various compounds with diversified biological activities. The proteome of C. jingzhao venom was analyzed by proteomic techniques. Proteins with molecular weight of over 10 kDa, indicated by gel-filtration and SDS-PAGE, were analyzed using 2-DE and MALDI-TOF/TOF and LC/ESI-Q-TOF MS. More than 90 proteins were detected, with 47 confirmed by sequence similarity search using mass spectrum driven basic local alignment search tool (MS BLAST). On the other hand, peptides with MW lower than 10 kDa were separated by HPLC and identified by MALDI-TOF MS and Edman degradation sequencing. About 120 peptides were detected, 60 of which were fully or partially sequenced. Our results indicate that peptides with MW lower than 10 kDa are the major components in the crude venom of C. jingzhao. Like those of other tarantulas, these peptides are very likely to act on various ion channels. These results pave a way for further detailed structure-function correlation analysis of the individual toxins present in the venom of C. jingzhao.     

Induction, purification and characterization of an antibacterial peptide scolopendrin I from the venom of centipede Scolopendra subspinipes mutilans

The crude venom of the centipede Scolopendra subspinipes mutilans, injected with Escherichia coli K12D31 for 3-4 days showed broad-spectrum antimicrobial activity against Gram-positive. Gram-negative bacteria and fungi. It showed good antibacterial activity against E. coli K12D31 at different temperatures, pH, and ionic strengths. The crude venom was heated at 100 degrees C for 30 min, centrifuged at 10,000 rpm for 30 min at 4 degrees C and the supernatants were obtained, from which an antibacterial fraction having a molecular mass of 3000-5000 Da, was further separated by ultrafiltration. A homogeneous antibacterial peptide named scolopendrin I, having a molecular mass of 4,498 Da, was isolated using cation-exchange chromatography and two steps of reverse-phase high performance liquid chromatography (RP-HPLC). Scolopendrin I did not show any hemolytic and agglutination activities at the concentration below 30 microM.     

Tarantula (Eurypelma californicum) venom, a multicomponent system

The venom of the tarantula Eurypelma californicum was analysed biochemically, the components were isolated and characterized. The pH value of the crude venom is 5.3 +/- 0.3. After dilution with distilled water, UV-absorption spectra showed a single maximum at 258 nm (pH ca. 7.0). A second maximum at 328 nm emerged above pH 8.0. Protein concentration of the venom is ca. 65 mg/ml. After Coomassie staining SDS-PAGE patterns show three major bands with apparent molecular masses around 40 kDa, 4.3 kDa and 1.3 kDa besides some weak high molecular protein bands. The following low-molecular mass constituents were determined in the crude venom: ATP, ADP, AMP, glutamic acid, aspartic acid, gamma-aminobutyric acid, glucose and the ions potassium, sodium, calcium, magnesium and chloride; the osmolality was 361 micro0smol/ml. The LD50 value for female cockroaches was 0.15 microliters venom per g body weight and for male cockroaches 0.4 microliters venom per g body weight. Separation of the crude venom by gel chromatography yielded four elution peaks. Peak I contains the enzyme hyaluronidase. The activity is 200-900 U/microliters. Peak II contains a mixture of toxic peptides. Peak III contains the 1.3-kDa components of SDS-PAGE and peak IV mainly contains ATP. Venom proteins including the enzyme hyaluronidase were precipitated by 5% trichloroacetic acid. The supernatant was separated by HPLC into 13 fractions. Fraction 1 contains glutamic acid, aspartic acid, gamma-aminobutyric acid and ATP; fraction 2 contains ATP, ADP and AMP as well as a component 2' visible in SDS-PAGE as 1.3-kDa band and consisting of spermine and tryptophan; fraction 3 contains ATP and an unknown component 3'; fractions 4-6 also show a 1.3-kDa band in SDS-PAGE, fraction 4 being tyrosylspermine and fractions 5 and 6 containing compounds of spermine and aromatic molecules; fraction 7 contains a peptide which lacks aromatic amino acids, it was sequenced from the N-terminus; fractions 8-13 contain very similar toxic peptides. The peptides in fractions 11 and 12, labeled ESTX for Eurypelma spider toxin, were cleaved with different enzymes and sequenced. They differ in one amino acid in position 26. Homologies with scorpion toxins and with a toxin of the spider Segestria florentina were found.     

Snake venomics of Central American pitvipers: clues for rationalizing the distinct envenomation profiles of Atropoides nummifer and Atropoides picadoi

We report the proteomic characterization of the Central American pitvipers Atropoides nummifer and Atropoides picadoi. The crude venoms were fractionated by reverse-phase high-performance liquid chromatography (HPLC), followed by analysis of each chromatographic fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequencing, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass fingerprinting, and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) of tryptic peptides. Each venom contained a number of bradykinin-potentiating peptides and around 25-27 proteins of molecular masses in the range of 7-112 kDa, belonging to only nine different toxin families (disintegrin, DC fragment, snake venom vascular endothelial growth factor, phospholipases A2, serine protease, cysteine-rich secretory proteins, C-type lectins, L-amino acid oxidase, and Zn2+-dependent metalloproteases), albeit distinctly distributed among the two Atropoides species. In addition, A. nummifer expresses low amounts of a three-finger toxin not detected in the venom of A. picadoi. The major toxins of A. nummifer belong to the PLA2 (relative abundance, 36.5%) and the serine proteinase (22%) families, whereas the most abundant A. picadoi toxins are Zn2+-dependent metalloproteinases (66.4%). We estimate that the similarity of venom proteins between the two Atropoides taxa may be around 14-16%. The high degree of differentiation in the venom proteome among congeneric taxa emphasizes unique aspects of venom composition of related species of Atropoides snakes and points to a strong role for adaptive diversification via natural selection as a cause of this distinctiveness. On the other hand, their distinct venom toxin compositions provide clues for rationalizing the low hemorrhagic, coagulant, and defibrinating activities and the high myotoxic and proteolytic effects evoked by A. nummifer snakebite in comparison to other crotaline snake venoms and the high hemorrhagic activity of A. picadoi.     

Characterization of PPD protein antigens in whole cell lysates of Mycobacterium bovis BCG

The low molecular mass protein antigens in PPD from M. bovis BCG were chemically oligomerized using sulfosuccinimidyl-4-(p-maleimidophenyl)-butyrate (S-SMPB) as a crosslinking agent. Protein oligomers with molecular mass over 90 kDa were obtained and used for the preparation of hyperimmune polyclonal rabbit antiserum. Using this antiserum four protein bands with molecular mass 120, 90, 75 and 65 kDA were detected in immunoblotting analysis of sonic extract from M. bovis BCG separated in SDS-polyacrylamide gel. We suggest that these immunoreactive proteins in the sonic extract represent the native forms of the heat stable low molecular mass protein antigens in PPD.     

A novel dimer of a C-type lectin-like heterodimer from the venom of Calloselasma rhodostoma (Malayan pit viper).

We have isolated a potent platelet aggregation inducer from the crude venom of Calloselasma rhodostoma (Malayan pit viper), termed rhodoaggretin, with a novel oligomeric structure consisting of a dimer of C-type lectin-like heterodimers. On the basis of its native molecular mass of 66 kDa, and a M(r) of 30 kDa for its disulfide-linked alphabeta-heterodimer, we propose that rhodoaggretin exists as a (alphabeta)2 complex in the native state. We postulate that the di-dimer is stabilized by non-covalent interactions as well as by an intersubunit disulfide bridge between the two alphabeta-heterodimers. This conclusion is based on the following observations: (a) sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the non-reduced rhodoaggretin gave a major 28 and a minor 52 kDa band. (b) Prior treatment of rhodoaggretin with a limited amount of 2-mercaptoethanol (2-ME; 0.1%) resulted in the complete abolishment of the 52 kDa band in SDS-PAGE. (c) Two-dimensional SDS-PAGE in the presence of 3% 2-ME showed that both the 28 and 52 kDa bands gave two bands each with M(r)s of 18 (alpha-subunit) and 15 (beta-subunit) kDa. (d) Mass spectrometric analyses showed that purified rhodoaggretin had a M(r) of 30155.39+/-3.25 Da while its s-pyridylethylated alpha- and beta-subunits had M(r)s of 16535.62+/-2.98 and 15209.89+/-1.61 Da respectively. These molecular weight data suggested the presence of 15 cysteinyl residues in rhodoaggretin as compared to the 14 that are reported for the heterodimeric C-type lectin-like proteins. This extra cysteinyl residue is a candidate for the formation of the intersubunit disulfide bond in the (alphabeta)2 complex. (e) Homology structural modeling studies showed that the extra cysteinyl residue can indeed form a disulfide bond that covalently links the two alphabeta-heterodimers as proposed above.     

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.     

蝶蛹金小蜂毒液蛋白不同提取分离方法的比较

为了建立蝶蛹金小蜂Pteromalus puparum毒液抑制寄主血细胞免疫活性组分合适的分离纯化方法,就等电点沉淀法、乙醇沉淀法、75%硫酸铵沉淀法、75%硫酸铵沉淀法+40℃加热处理法,以及75%硫酸铵沉淀法分别与3种不同滤膜的分子大小截留法的组合等7种方法对毒液蛋白分离效果及活性的影响进行了比较。结果表明：等电点沉淀法获得的组分抑制寄主菜粉蝶Pieris rapae离体血细胞延展和包囊的活性最强,乙醇沉淀法次之,75%硫酸铵沉淀法最弱。从蛋白组分的SDS-PAGE图谱来看,等电点沉淀法获得毒液组分相对最纯,仅有3条主要谱带,分子量大小在45～116.2 kDa范围内;乙醇沉淀法次之,有5条主要谱带,分子量大小在24～116.2 kDa范围内;硫酸铵沉淀法的谱带组成与毒液蛋白粗提液相似。3种分子大小截留法获得的毒液组分的活性分析表明,强活性组分分子量大小可能都大于100 kDa。综合认为,7种方法中以等电点沉淀法提取分离蝶蛹金小蜂毒液蛋白相对为最适。