我国不同地区东亚钳蝎(Buthus martensii Karsch)毒在三种类型电泳中的比较研究

用SDS—不连续聚丙烯酰胺凝胶板电泳、pH4.5—不连续聚丙烯酰胺凝胶板电泳和聚丙烯酰胺凝胶圆盘等电聚焦电泳,分离我国不同地区东亚钳蝎毒。其图谱表明:不同产地的东亚钳蝎毒蛋白的种类及含量存在一定的差异,地区相近,差异较小,反之亦然。

Effect of scorpion venom analgesic active peptide extracted from Buthus martensii Karsch on evoked potential in the thalamic posterior nucleus group in rats

BACKGROUND： Buthus martensii Karsch is a rare medicinal animal, and dried integral Buthus rnartensii Karsch is an important drug in traditional Chinese medicine. OBJECTIVE： To investigate the effects of scorpion venom analgesic active peptide （SAP） extracted from Buthus martensii Karsch on evoked unit discharge of the common peroneal nerve in the posterior nucleus group of the thalamus using a stereotaxic electrophysiological extracellular microelectrode recording. DESIGN, TIME AND SETTING： One-way designed study, performed in the Physiological Laboratory of Shenyang Medical College on September 15, 2006. MATERIALS： Fifty 3-4 months old Wistar rats （25 males and 25 females） were used. SAP was provided by Shenyang Pharmaceutical University. Morphine solution was made by the First Drug Manufactory, Northeastern Drug Manufacture Group （batch number： H20013351）. Naloxone solution was made by Hunan Pharmaceutical Co., Ltd. （batch number： H43021669）. Type ATAC-350 medical data processing equipment was made by the Photoelectricity Company, Japan. METHODS： Fifty rats were randomly divided into the SAP group （n=20）, saline group （n=10）, morphine group （n=10）, or naloxone group （n=10）. In the SAP group, the common peroneal nerve was separated and stimulated with a single square wave （17-19 V intensity; 0.2 ms width; 20 ms retardation time）. Subsequently, SAP （0.01%, 2 μL） was injected into the posterior nucleus group of the thalamus. Rats in the naloxone group were injected with naloxone （1.0 mg/kg i.v.） before SAP injection. Rats in the saline group and the morphine group were injected with saline （2 μL） or morphine （0.01%, 2μL）, respectively, before SAP injection. Other procedures were the same as those in the SAP group. MAIN OUTCOME MEASURES： Evoked discharge in the posterior nucleus group of the thalamus and effects of SAP alone and SAP in combination with saline, morphine, or naloxone on discharges in the posterior nucleus group of the thalamus as measured by TQ-19 medical data processing equipment. RESULTS： SAP group： At 1-3 minutes after SAP injection, evoked discharges in the posterior nucleus group of the thalamus were inhibited, and the inhibitory time lasted for （45.0±0.7） minutes. Saline group： Evoked discharges in the posterior nucleus group of the thalamus did not change after saline injection. Morphine group： At 1-3 minutes after morphine injection, evoked discharges in the posterior nucleus group of the thalamus were inhibited, and the inhibitory time lasted for （35.0±7.8） minutes. Naloxone group： SAP had no effects on evoked potentials in the posterior nucleus group of the thalamus. CONCLUSION： The inhibitory effect of SAP on evoked potentials was superior to that of morphine at the same concentration （2 μL of 0.01% solution）. Naloxone resupination demonstrated that the inhibitory effects of SAP on evoked discharges were influenced by the opioid receptor.

Cloning and sequencing of two depressant insect selective neurotoxin cDNAs from Buthus martensii Karsch

During evolution, scorpions have developed the ability to produce a series of toxins. Scorpion toxins, which are reserved in terminal segments of scorpion, serve as the arms for predation and self-defence. They have also been used as medicine for some human sickness. As far as the targets that they act on are concerned, these toxins can be

Molecular characterization of a K+ channel blocker in the scorpion Buthus martensii Karsch

K+ channel blockers of scorpion venoms are of important value in studyingpharmacology and physiology of specific K+ channel of celis. Based on the amino acid sequences of BmP01 previously characterized as a small-conductance Ca2+-activated K+ channel blocker, two 'back to back' degenarate primers have been designed and synthesized for inverse PCR strategy, its full-length cDNA has been cloned from the venom gland of the Chinese scorpion Buthus martensii. The cDNA is composed of 3 parts: 5’ UTR, oRF and 3’ UTR. The flanking sequence of translation initiation codon ATG is AAAKTGA, which is highly conserved in scorpion Na+ channel toxin and protozoan genes, suggesting that these genes may have followed a common mechanism for translation initiation. The 3’ UTR contains poly(A) signal AATAAA. The open reading frame encodes a precursor of 57 residues with a signal peptide of 28 residues and a mature peptide of 29 residues. The signal peptide is rich in hydrophobic amino acid residues and its length is

Purification,Characterization and ~1H NMR Resonance Assignment of an α-Like Neurotoxin BmK 16 from the Venom of Chinese Scorpion Buthus martensii Karsch

A natural scorpion toxin BmK 16 was purified for the first time from the venom of the Chinese scorpion Buthus martensii Karsch (BmK) by using combined gel filtration, ion exchange and reversed phase chromatography. The sequence of the N terminal 8 amino acid residues was determined by Edman degradation. Using the N terminal sequence as a tag, the database searching revealed a hit in the scorpion cDNA Bank. The sequence for N terminal 8 amino acid residues, molecular weight and amino acid compositions of BmK 16 were identical with the calculated values according to the first 64 residues' sequence of the precursor peptide alpha neurotoxin TX16 derived from the sequence of the cDNA AF156597 (EMBL). The sequence specific resonance assignment of BmK 16 was achieved and the intact sequence of BmK 16 was determined as followings: VRDAY IAKPH NCVYE CARNE YCNDL CTKNG AKSGY CQWVG KYGNG CWCKE LPDNV PIRVP GKCH. Furthermore, the results from the sequence homology analysis and the toxicity assays indicated that BmK 16 was an α like scorpion neurotoxin.

Crystal structure determination of an acidic neurotoxin (BmK M8) from scorpion Buthm martensii Karsch at 0.25nm resolution

The crystal structure of an acidic neurotoxin, BmK M8, from Chinese scorpion Buthus martensii Karsch was determined at 0.25 nm resolution. The X-ray diffraction data of BmK M8 crystals at 0.25nm resolution were collected on a Siemens area detector. Using molecular replacement method with a basic scorpion toxin AaH II in a search model, the cross-rotation function, PC-refinement and translation function were calculated by X-PLOR program package. The correct orientation and position of BmK M8 molecule in crystal were determined in a resolution range of 1.5 - 0.35nm, The oystallographic refinement was further performed by stereo-chemical restrict least-square technique, followed by simulated annealing, slow-cooling protocols. The final crystallographic R-factor at 0.8-0.25 nm is 0.171. The standard deviations of bond length and bond angle from ideality are 0.001 7nm and 2.24° , respectively. The final model of BmK M8 structure is composed of a dense core of secondary structure elements by a stretch of α-helix with two and a half turns (residues 19-28) and a three-stranded antiparallel β-sheet (residues 2-4, 32 - 37, 45- 51). In addition, three loops protruded from the structural core. The general folding properties of BmK M8 molecule were described; a common structure motif which may appear in all scorpion neurotoxins was identified. The conserved aromatic residues and charged residues were found to be distributed on two roughly opposite surfaces of the molecule. The relationship between these two faces and receptor-binding sites are also discussed.

Two non-coding transcripts of toxins from the scorpion But-hus martensii Karsch: Evidence for the existence of NMD in scorpion venom gland cells

Two non-coding transcripts of toxins were iso-lated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch, named BmαTX15-SP and BmTXKβ-SP, respectively. Analysis of nucleotide sequences shows that their 5’ sequences are identical to the 5 ’UTR and upstream open reading frame (ORF) sequences of the cDNAs encoding BmαTX15 and BmTXKb, two toxins from Buthus martensii Karsch. But no detectable homology exists in other regions. Two polyadenylated non-coding transcripts encode multiple short ORFs with no more than 34 amino acid resi-dues. The tailing signal (AATAAA) is situated at 14 or 18 bp downstream from the poly(A). The data of genomic se-quences provides support for Bmα TX15-SP and BmTXK β-SP not deriving from splicing errors of pre- mRNAs. Our finding provides evidence for the existence of non-sense-mediated mRNA decay (NMD) pathway in scorpion venom gland cells.

基于高通量测序的东亚钳蝎不同部位转录组分析

目的:探究东亚钳蝎蝎身和蝎尾中活性多肽成分及相关基因的表达差异。方法:通过Illumina Hiseq测序平台分别对东亚钳蝎蝎身和蝎尾进行转录组学测序和生物信息学分析。结果:转录组测序共获得70160条unigenes,对其中5859个差异表达基因进行生物信息学基因本体(GO)功能注释。东亚钳蝎转录组测序数据中存在大量与人类疾病治疗相关的活性多肽/转录本。BmKIT1、BmKITc、BmKAS、BmK-YA、agp-sypu1、BmKAGP-SYPU2、Martentoxin、BmKCT、Charybdotoxin均在蝎尾中特异性表达,其中BmKIT1、BmKITc、BmKAS、BmK-YA、agp-sypu1、BmKAGP-SYPU2与镇痛有关,Martentoxin与治疗一氧化氮(NO)过度生成引发的心脑血管疾病有关,BmKCT、Charybdotoxi与抑制神经胶质瘤细胞的增殖侵袭和抑制黑色素瘤细胞转移有关。结论:蝎尾中活性多肽成分多于蝎身,与镇痛和抗肿瘤相关的多肽主要存在于蝎尾中。

东亚钳蝎昆虫毒素BmKIT的cDNA序列分析

利用ＲＴ－ＰＣＲ方法从东亚钳蝎（ＢｕｔｈｕｓｍａｒｔｅｎｓｉＫａｒｓｃｈ，ＢｍＫ）的尾腺总ＲＮＡ中扩增出挛缩型昆虫毒素ＢｍＫＩＴ的ｃＤＮＡ，并对其核苷酸序列进行了分析。将含该基因的重组分泌型表达质粒ｐＥｘＳｅｃＩ－ＢｍＫＩＴ转化大肠杆菌ＢＬ２１（ＤＥ３），表达出分泌型的融合蛋白。

全蝎化学成分和药理作用的研究进展

全蝎为蛛形纲蝎目钳蝎科昆虫,为息风平肝的要药,味辛、平,有毒。其药用历史悠久,具息风镇痉、通络止痛、攻毒散结之功效,主要用于治疗风湿、癫痫、中风、慢性疼痛等疾病。其主要活性成分为蝎毒、甾体衍生物、生物碱等。全蝎具镇痛抗炎、抗癌、抗癫痫、抗惊厥、抗凝、抗菌、促生长、提高免疫力等药理作用。本研究对全蝎化学成分、药理作用进行综述,以期为全蝎的深入研究和开发利用提供参考。

全蝎蛋白抗肿瘤活性研究

目的比较全蝎蛋白超滤分段后各分子量段对肺腺癌细胞A549的抑制作用,筛选有效部分。方法采用蛋白酶酶解法提取全蝎总蛋白,利用80%乙醇沉淀并利用改良Lowry法测定蛋白质含量,再经超滤进行分段、冷冻干燥后通过MTT法研究其对肺腺癌细胞(A549)的抑制作用。结果 80%乙醇沉淀全蝎蛋白具有较高的选择性,其<3 kDa分子量段对A549细胞无抑制作用,3~10 kDa分子量段对A549细胞的抑制作用不明显,>10 kDa分子量段对A549细胞具有显著的抑制作用,IC_(50)=204.7μg·mL^(-1)。结论 80%乙醇沉淀物中蛋白质含量为89.4%;>10 kDa分子量段全蝎蛋白对A549细胞具有显著的抑制作用。

东亚钳蝎神经毒素在大肠杆菌中的表达

以山西风陵渡东亚钳蝎（ＢｕｔｈｕｓｍａｒｔｅｎｓｉＫａｒｓｃｈ）的尾腺总ＲＮＡ为模板，根据已知的蝎神经毒素保守氨基酸序列设计引物，利用ＰＣＲ技术，扩增并克隆了两个蝎神经毒素基因．序列分析表明，由两个基因导出的氨基酸序列（ＢｍＫＭｍ１和ＢｍＫＭｍ２）与已知的蝎神经毒素ＢｍＫⅠ、ＢｍＫⅡ、ＢｍＫⅢ、ＢｍＫＭ１、ＢｍＫＭ９有很高的同源性．将ＢｍＫＭｍ２基因重组到大肠杆菌分泌型表达载体ｐＥｘＳｅｃ１中进行表达．ＳＤＳ－ＰＡＧＥ证明表达产物被分泌到细胞周间质及培养液中．

蝎及蝎毒抗肿瘤作用研究进展

目的介绍蝎、蝎毒及其中活性组分抗肿瘤作用研究进展。方法根据近年来国内外公开发表的有关文献,从蝎及蝎毒的抗肿瘤作用、蝎毒抗肿瘤成分及其生物学机制、全蝎及蝎毒抗肿瘤的临床应用等方面综述。结果与结论全蝎粗提物可使体外培养的人体子宫颈癌细胞全部死亡脱壁,全蝎的醇制剂在体外能显著抑制人肝癌细胞呼吸,并对结肠和人肝癌细胞的生长均有明显的抑制作用,蝎尾提取物对肿瘤兼有预防和治疗的双重作用;蝎毒粗毒对肿瘤有确切的抑制作用,且优于化疗药物;蝎毒中具有抗肿瘤活性的单组分活性肽为Chlorotoxin和东亚钳蝎镇痛抗肿瘤活性肽;Chlorotoxin特异性抑制神经胶质瘤细胞的迁移与浸润,而不影响正常脑细胞;东亚钳蝎镇痛抗肿瘤活性肽是Na+离子通道抑制剂,其抗肿瘤作用机理可能与Chlorotoxin不同。

Intein介导的重组昆虫毒素BmKIT在大肠杆菌中的可溶性表达、纯化及活性分析

为获得重组蝎昆虫毒素BmKIT,通过PCR方法在BmKIT基因的3′端融合了编码6个组氨酸残基的核苷酸序列,将其插入原核表达载体pTWIN1的内含肽Ssp DnaB Intein基因下游的多克隆位点(MCS)。将获得的表达质粒转化大肠杆菌BL21(DE3)中,用IPTG诱导融合蛋白表达。用Ni-NTA亲和层析柱从菌体裂解液中纯化了CBD-Intein-BmK IThis6融合蛋白,并在柱上诱导Intein自剪切,成功去除融合子CBD-Intein。通过Superdex75凝胶过滤层析获得了纯度达95%以上的BmK IThis6蛋白,该蛋白不仅具有正确的二级结构而且有生物活性。

不同方法加工全蝎的氨基酸含量及药理作用对比研究

目的:研究不同加工方法对全蝎药材质量的影响,为确定合理的加工方法提供理论依据。方法:分别用清水煮制、盐水煮制两种《中国药典》规定的加工方法对全蝎进行加工,观察比较其外观性状、醇浸出物含量、氨基酸含量及镇痛药效差异等指标,依据上述指标对两种加工方法进行综合评价。结果:两法制得全蝎外观性状稍有差异;盐水煮制全蝎醇浸出物含量高,清水煮制全蝎醇浸出物含量低;清水煮制全蝎总氨基酸含量高;清水煮制全蝎的镇痛药效指标在数值上优于盐水煮制全蝎。结论:以总氨基酸含量和镇痛药效为评价指标时,进行全蝎加工宜采取清水煮制。

蝎毒对白血病HL-60细胞生长的抑制和诱导凋亡作用研究

目的 :研究蝎毒对HL 60细胞的生长抑制和诱导凋亡作用 ,并探讨其作用机制。方法 :应用MTT法观察蝎毒对细胞的抑制作用 ,光镜、荧光显微镜和透射电镜观察细胞结构的改变 ,原位末端标记检测DNA断裂 ,流式细胞术分析凋亡细胞和bcl 2蛋白表达。结果 :蝎毒可明显地抑制HL 60细胞生长和诱导细胞凋亡 ,作用在一定范围内呈对浓度和时间的依赖性 ,蝎毒并能下调HL 60细胞bcl 2蛋白表达。结论 :蝎毒能抑制HL 60细胞生长和诱导细胞凋亡 ,其作用机制可能与bcl 2蛋白表达的下降有关。

两步PCR快速扩增东亚钳蝎BmKIT3 3'cDNA末端序列

对3′RACE方法进行了修改,采用一条特异性引物和一条通用OligodT引物成功地克隆了东亚钳蝎BmKIT33′cDNA末端,与常规3′RACE方法相比,两步PCR方法具有节约成本、节省时间、增加反应特异性等优点,尤其适用于生物活性肽基因的快速克隆和鉴定.

一个东亚钳蝎蝎毒素BmTXKβ基因的克隆及其基因表达调控

从东亚钳蝎 (ButhusmartensiiKarsch ,BmK)毒腺组织cDNA文库中分离的长链钾通道毒素BmTXKβcDNA序列 ,克隆了BmTXKβ基因组序列 .BmTXKβ基因含有一个长度为 886bp的内含子 ,定位于BmTXKβ成熟肽中 ,与其它蝎毒素基因内含子定位于信号肽的基因结构不同 .并且 ,BmTXKβ基因的内含子特征也与其它蝎毒素基因不同 .研究结果从基因水平上证实了BmTXKβ是一个新的蝎毒素样肽 .以BmTXKβcDNA序列为探针与蝎基因组DNASouthern杂交出现 2条特异性杂交带 .杂交结果为蝎毒素基因可能通过DNA重排、多拷贝或多基因家族来调控基因表达提供了证据 .

东亚钳蝎对小菜蛾的捕食效应

在实验室条件下，研究东亚钳蝎对小菜蛾的捕食效应。结果表明，东亚钳蝎对小菜蛾3~4龄幼虫及成虫的捕食功能反应属于Holling 功能反应模型Ⅱ型，对幼虫和成虫的瞬间攻击力分别为0.661和0.687，最大捕食量则分别为36.36头和125头。东亚钳蝎有较强的种内干扰作用，随着捕食者密度的增大，捕食作用率相应降低。猎物和天敌相互干扰会降低东亚钳蝎对3~4龄小菜蛾幼虫的寻找效应，但对其捕食量影响不大。在小菜蛾幼虫和成虫的混合种群中东亚钳蝎偏嗜小菜蛾成虫。