孕蝎饲养繁殖器具

马氏钳蝎(Buthsu martensi)是我国分布范围最广、种群数量最多的一种传统的药用野生动物。属节肢动物门、蛛形纲。近年来有些地方在人工养蝎方面取得了一定的成就。由于孕蝎产仔期间性喜安静场所,一般都需要单独饲养,避免惊扰。在其产仔之后还要及时补充水分和食物,否则母蝎会残食仔蝎。因此,解决母蝎孕期的饲养繁殖器具是人工养蝎一项急待解决的课题。本文介绍了我室在对马氏钳蝎开发利用的研究过程中,设计并制造的一种行之有效、简单方便的孕蝎饲养繁殖器具,以供参考。     

东亚钳蝎K+通道阻断肽cDNA的克隆与表达

目的：克隆东亚钳蝎毒素基因,以进一步研究其生物学和药理学功能。方法：利用已知蝎神经毒素基因序列,设计引物,用RT-PCR方法克隆从蝎毒腺组织蝎毒素cDNA。结果：成功地克隆了一个新的东亚钳蝎毒素基因,该基因开放阅读框架编码59个氨基酸残基,其中前22个为信号肽,成熟肽为37个氨基酸残基,经PCR扩增除去信号肽序列,克隆到pTreHisA质粒中,在E．coli中表达了分子质量为7ku左右融合蛋白,表达产物占菌体总蛋白的21％左右。结论：其结构中含有三对二硫链,6个Cys残基组成蝎K^ 通道毒素共同特征序列-CXXXC-、-GXC-、-CXC-,推断其为K^ 通道阻断肽,命名为KChTX1。已被Gene-bank收录,收录号为AY129234。     

我国东亚钳蝎的研究概况

东亚钳蝎又称马氏钳蝎(Buthus martensi),是我国分布最广,数量最多的蝎种。中药全蝎为名贵药材之一。全蝎的养殖和综合利用已列入国家星火计划。蝎子养殖专业户,正从综合利用方面谋求新路。本文作者根据文献资料和实验研究报告,简单介绍我国东亚钳蝎的研究概况。 (一)全蝎的加工与蝎毒的提取 中药全蝎是活蝎制成干品,以干蝎配伍人药,因此,全蝎的质量很大程度取决于加工方法和技术。要使干品得到符合《药典》的要求,首先除去蝎体上的泥沙,以每0.5公斤活蝎用盐量75克为宜,     

钳蝎系统分类研究概况

传统观点认为蝎类是蛛形纲中最原始的类群,但是最近的形态分析和分子系统发生研究表明蝎类与单胫类(Haplocnemata=Solifugae Pseudoscorpiones)构成姐妹群,并与盲蛛类一起在蛛形纲形成一个独立的分支.钳蝎科是蝎目中种类最多分布最广的一个科,目前已知82属600余种.钳蝎一直被认为是蝎目中最原始的类群,但是随着Pseudochactidae的发现和建立,其原始的地位也被取代.钳蝎科中亚科的划分一直存在很大的争议,至今尚没有一个亚科划分意见被广泛接受.同样,钳蝎科中属种的界定也存在问题,一些建立较早的属亟待修订.本文对钳蝎的系统发生和分类进行了综述,并对国内外文献中已记载的中国钳蝎种类进行了汇总和记述,即东亚钳蝎Mesobuthus martensii(Karsch,1879),条斑钳蝎蒙古亚种M.eupeus mongolicus(Birula,1911)、凶恶亚种M.e.thersites(C.L.Koch,1839),高加索钳蝎间型亚种M.caucasicus intermedius(Birula,1897)、普氏亚种M.c.przewaklskii(Birula,1897),细尖狼蝎Lychas mucronatus(Fabricius,1798),斑等蝎Isometrus maculates(DeGeer,1778),凹直钳蝎Orthochirus scrobiculosus(Grube,1873).     

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 .     

蝎毒的采集与加工

蝎毒是由蝎尾部毒腺分泌的一种成份复杂的有毒蛋白质。近几年来,国内外对蝎毒进行大量研究结果表明,蝎毒中具有生物活性很高的蝎毒素,不但在生物学研究中有广泛的应用,而且具有许多重要的生理功能。我国中医历来认为,全蝎是治疗神经系统疾病必不可少的良药,而其中起主要作用的就是蝎毒。可见,蝎毒是一种有着广阔应用前景的动物毒素。我国蝎资源极为丰富,仅东亚钳蝎一个种类就分布在长江以北到辽宁铁岭以南的宽广区域。提取蝎毒,能更有效地利用这一宝贵的生物资源,必将成为我国广大产蝎区农民的新的经济来源。提取蝎毒主要有三种方…     

东亚钳蝎蝎毒素BmKBT基因组序列的克隆及其分析

东亚钳蝎 (ButhusmartensiiKarsch ,BmK)蝎毒素BmKBT(又名BmKabT)是一个在初级结构上相似于β类哺乳动物毒素和功能接近于α类哺乳动物毒素的Na+ 通道毒素 .基于从毒腺cDNA文库中筛选得到的全长BmKBT前体核苷酸序列设计引物 ,以蝎基因组总DNA为模板进行聚合酶链式反应 (PCR) ,将PCR产物克隆至T载体、测序 .序列分析表明 :在BmKBT信号肽编码区的 3′端的- 4位Gly密码子的第 1位与第 2位碱基中有 1个长 2 2 5nt的内含子 ,插入位点距离该基因的起始密码子 4 6nt ,AT含量为 78 7% ,其内含子可能的剪接分枝位点距离 3′剪接受体位点 4 7nt.内含子的大小及其基因组织结构分析表明 :BmKBT具有与α类哺乳动物毒素类似的基因组织结构 ,进一步说明BmKBT是一个介于α类和β类Na+ 通道毒素之间的中间型蝎毒素 ,可以作为研究蝎毒素分子进化的合适材料     

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 .     

具有中等毒性的马氏钳蝎神经毒素的纯化和初步晶体学…

运用柱层析技术对产自淅川和常德的马氏钳蝎毒素进行分离纯化,得到８种哺乳动物神经毒素。运用制备型等电聚焦电泳技术,对常德样品中具有中等毒性的蝎神经毒素进一步纯化,获得了高纯度样品。两个产地的蝎毒素ＢｍＫ５均已经成功地获得了大晶体。空间群均为Ｐ２１２１２１,晶胞参数分别为：ａ＝３８．４６埃,ｂ＝３７．２８埃,ｃ＝３６．９７埃;ａ＝３８．４４埃,ｂ＝３７．５５埃,ｃ＝３６．８３埃。对两个产地的晶体分别收     

海南岛的蝎子地理分布研究

蝎目是蛛形动物中较早登陆且又比较原始的类群,在蛛形动物进化历程中占重要地位,是已知最古老的陆生节肢动物之一,具有较高的药用价值和食用价值.本文对海南岛蝎子的生态地理分布进行了研究,从理论上丰富了热带地区蝎目动物的地理分布和物种丰富度等方面的内容;在实践上,拓宽了应用的领域,有利于对不同蝎目动物的资源开发和利用,同时也为保护这类古老的动物提供了依据.经调查,共发现蝎目动物2科3属3种,均为海南已有记录种类,其中钳蝎科2种,分别为等蝎属的斑等蝎和狼蝎属的细尖狼蝎;瘦尾蝎科1种,系链尾蝎属的澳链尾蝎.     

Cloning and characterization of a novel calcium channel toxin-like gene BmCa1 from Chinese scorpion Mesobuthus martensii Karsch

Many studies have been carried on peptides and genes encoding scorpion toxins from the venom of Mesobuthus martensii Karsch (synonym: Buthus martensii Karsch, BmK), such as Na+, K+ and Cl- channel modulators. In this study, a novel calcium channel toxin-like gene BmCa1 was isolated and characterized from the venom of Mesobuthus martensii Karsch. First, a partial cDNA sequence of the Ca2+ channel toxin-like gene was identified by random sequencing method from a venomous gland cDNA library of Mesobuthus martensii Karsch. The full-length sequence of BmCa1 was then obtained by 5'RACE technique. The peptide deduced from BmCa1 precursor nucleotide sequence contains a 27-residue signal peptide and a 37-residue mature peptide. Although BmCa1 and other scorpion toxins are different at the gene and protein primary structure levels, BmCa1 has the same precursor nucleotide organization and cysteine arrangement as that of the first subfamily members of calcium channel scorpion toxins. Genomic DNA sequence of BmCa1 was also cloned by PCR. Sequence analysis showed that BmCa1 gene consists of three exons separated by two introns of 72 bp and 1076 bp in length, respectively. BmCa1 is the first calcium channel toxin-like gene cloned from the venom of Mesobuthus martensii Karsch and potentially represents a novel class of calcium channel toxins in scorpion venoms.     

Molecular characterization of a possible progenitor sodium channel toxin from the Old World scorpion Mesobuthus martensii

Toxins affecting sodium channels widely exist in the venoms of scorpions throughout the world. These molecules comprise an evolutionarily related peptide family with three shared features including conserved three-dimensional structure and gene organization, and similar function. Based on different pharmacological profiles and binding properties, scorpion sodium channel toxins are divided into alpha- and beta-groups. However, their evolutionary relationship is not yet established. Here, we report a gene isolated from the venom gland of scorpion Mesobuthus martensii which encodes a novel sodium channel toxin-like peptide of 64 amino acids, named Mesotoxin. The Mesotoxin gene is organized into three exons and two introns with the second intron location conserved across the family. This peptide is unusual in that it has only three disulfides and a long cysteine-free tail with loop size and structural characteristics close to beta-toxins. Evolutionary analysis favors its basal position in the origin of scorpion sodium channel toxins as a progenitor. The discovery of Mesotoxin will assist investigations into the key issue regarding the origin and evolution of scorpion toxins.     

蝎毒液肽基因内含子剪接信号分析

从中国蝎Buthus martensii Karsch基因组DNA中分离到两个毒液基因的内含子。在此基础上,通过编辑和分析目前已出版的蝎毒液肽基因内含子序列,确定了这类基因内含子剪接信号的共有序列,并将其与其它其它物种进行了比较。本文的结果对于研究蝎毒液肽前体mRNA的剪接机制以及比较不同物种之间内含子进化和功能的关系具有参考价值。     

Molecular cloning and sequencing of two 'short chain' and two 'long chain' K(+) channel-blocking peptides from the Chinese scorpion Buthus martensii Karsch.

Five full-length cDNAs encoding the precursors of two 'short chain' scorpion non-toxic peptides active on Ca(2+)-activated K(+) channels (BmP02 and BmP03) and two novel putative long chain K(+) channel-blocking peptides (named BmTXKbeta and BmTXKbeta2) were first isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch (BmK). BmTXKbeta2 showed a high similarity with AaTXKbeta, while BmTXKbeta was completely different in the deduced primary structure from the long chain and short chain scorpion toxins already characterized. Thus, BmTXKbeta expands the scorpion long chain K(+) channel-blocking peptide family. Although little sequence similarity exists between the above two short and two long peptides, they are similar at the positions of six cysteines, suggesting that they should all share a similar scaffold composed of an alpha-helix and a three-stranded beta-sheet.     

BmK-YA, an enkephalin-like peptide in scorpion venom

By screening extracts of venom from the Asian scorpion Buthus martensii Karsch (BmK) for their abilities to activate opioid receptors, we have identified BmK-YA, an amidated peptide containing an enkephalin-like sequence. BmK-YA is encoded by a precursor that displays a signal sequence and contains four copies of BmK-YA sequences and four of His(4)-BmK-YA, all flanked by single amino acid residues. BmK-YA and His(4)-BmK-YA are amidated and thus fulfill the characteristics expected of bioactive peptides. BmK-YA can activate mammalian opioid receptors with selectivity for the δ subtype while His(4)-BmK-YA is inactive at opioid receptors. The discovery of BmK-YA suggests that scorpion venom may represent a novel source of bioactive molecules targeting G protein-coupled receptors (GPCRs) and reveal additional insights on the evolution of the opioid precursors.     

马氏蝎毒毒素的分离纯化及其对红细胞膜作用的初步研究

本文用山东产马氏蝎(Buthus martensii kashi)粗毒为材料,经SephadexG-50和Sp-Sephadex C-25二次柱层析,分离纯化获得三个毒峰部分,毒性比粗毒分别提高40—100倍。 纯度鉴定表明三个毒峰的聚丙烯酰胺凝胶电泳和等电聚焦电泳均为一条带,等电点分别为8.7,9.1,9.1,分子量用SDS-不连续聚丙烯酰胺凝胶电泳测定分别为6,600,5,000和8,500。对纯化蝎毒毒素的氨基酸组分也作了分析。 蝎毒毒素对人红细胞膜作用的初步探索结果表明:它使人红细胞膜的Na.K-ATP酶活性和膜脂流动性有所降低。     

Molecular dissection of venom from Chinese scorpion Mesobuthus martensii: identification and characterization of four novel disulfide-bridged venom peptides

Scorpion venom is composed of a large repertoire of biologically active polypeptides. However, most of these peptides remain to be identified and characterized. In this paper, we report the identification and characterization of four novel disulfide-bridged venom peptides (named BmKBTx, BmKITx, BmKKx1 and BmKKx2, respectively) from the Chinese scorpion, Mesobuthus martensii (also named Buthus martensii Karsch). BmKBTx is composed of 58 amino acid residues and cross-linked by three disulfide bridges. The sequence of BmKBTx shows some similarities to that of the toxin, birtoxin, and its analogs. It is likely that BmKBTx is a beta-toxin active on Na+ channels, which is toxic to either insects or mammals. BmKITx is composed of 71 amino acid residues with four disulfide bridges. It is the longest venom peptide identified from M. martensii so far. BmKITx shows little sequence identity with scorpion alpha-toxins toxic to insects. It is likely that BmKITx is a new type of Na+ -channel specific toxin active on both insects and mammals. BmKKx1 contains 38 amino acid residues cross-linked by three disulfide bridges and shows 84% sequence identity with BmTx3, an inhibitor of A-type K+ channel and HERG currents. BmKKx1 has been classified as alpha-KTx-15.8. BmKKx2 is composed of 36 residues and stabilized by three disulfide bridges. BmKKx2 is a new member of the gamma-K+ -channel toxin subfamily (classified as gamma-KTx 2.2). The venoms of scorpions thus continue to provide novel toxins with potential novel actions on targets.     

The Mesobuthus martensii genome reveals the molecular diversity of scorpion toxins

Recent complete sequencing of the genome of the Asian scorpion, Mesobuthus martensii, highlights the molecular diversity of its venom neurotoxin/defensin genes and interesting features of their evolution.     

具有中等毒性的马氏钳蝎神经毒素的纯化和初步晶体学研究

运用柱层析技术对产自淅川和常德的马氏钳蝎毒素进行分离纯化,得到８种哺乳动物神经毒素。运用制备型等电聚焦电泳技术,对常德样品中具有中等毒性的蝎神经毒素（ＢｍＫ５）进一步纯化,获得了高纯度样品。两个产地的蝎毒素ＢｍＫ５均已经成功地获得了大晶体。空间群均为Ｐ２１２１２１,晶胞参数分别为：ａ＝３８．４６埃,ｂ＝３７．２８埃,ｃ＝３６．９７埃（淅川）;ａ＝３８．４４埃,ｂ＝３７．５５埃,ｃ＝３６．８３埃（常德）。对两个产地的晶体分别收集了２．１埃（淅川）和１．６２埃（常德）分辨率的衍射数据。