一种东亚钳蝎碱性神经毒素的纯化和初步晶体学研究

经ＳｅｐｈａｄｅｘＧ－５０和ＳＰ－ＳｅｐｈａｄｅｘＣ－２５两次柱层析,从河南淅川马氏钳蝎毒素中分得一种碱性神经毒素ＢｍＫＭＩ８。等电聚聚焦和ＳＤＳ电泳显示单一组分,其ＰＩ为９．１,Ｍｒ为７１００,毒性测试结果表明,该组分对小白鼠有较强的毒性,对昆虫也有一定的毒性。已获得该毒素的两种晶全型的大单晶,并测定其空间群为Ｐ２１２１２１,晶胞参数为：ＢｍＫＭＩ８－Ａ：ａ＝３６．７Ａ,ｂ＝２６．６Ａ,ｃ＝５２     

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     

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。     

一种强毒性中华马氏钳蝎哺乳动物神经毒素新晶型的初步晶体学研究

经ＳｅｐｈａｄｅｘＧ－５０和Ｓｐ－ＳｅｐｈａｄｅｘＣ－２５两次柱层析,从常德马氏钳蝎毒素中分离得到一种强毒性的哺乳动物神经毒（Ｂｍｋｌ）,等电聚焦及ＳＤＳ－电泳显示它为单一组份,其ｐＩ为９．４４,ＭＷ为７．１ＫＤ．毒性试验结果表明,该组份对小白鼠的最小致死刘量为０．５μｇ／ｇ小白鼠,对昆虫及甲壳动物也有毒性．已获得该毒素的一种新晶型的大晶体。其空间群为Ｐ２１２１２,晶胞参数为：α＝８３．４６Ａ,ｂ＝４０．３６Ａ,ｃ＝２４．００Ａ,单位晶胞体积为８０８４２．６９Ａ,一个不对称单位含１个分子,已收集了分辨率为１．７５Ａ的数据．     

两个东亚钳蝎抗哺乳动物神经毒素的cDNA序列

从我国山东东亚马氏钳蝎尾腺中分离纯化ｍＲＮＡ,经逆转录构建了ＢｍＫ蝎毒ｃＤＮＡ文库。利用ＰＣＲ扩增,筛选到两个抗哺乳动物毒素的ｃＤＮＡ基因,并测定了序列。这两个ｃＤＮＡ阅读框均为２５２ｂｐ组成,可翻译８４肽的毒素前体,包括Ｎ端１９个氨基酸组成的信号肽,６４个氨基酸残芭的成熟毒蛋白,以及Ｃ端一个额外的碱性氨基酸Ａｒｇ。其中由一ＣＤＮＡ所推导的氨基酸序列（ＢｍＫＭ１）一已知的天然毒素ＢｍＫ１蛋白序列完     

不同产地中华马氏钳蝎神经毒素的分离纯化和部分性质比较研究

经Ｓｅｐｈａｄｅｘ Ｇ－５０,Ｓｐ－Ｓｅｐｈａｄｅｘ Ｃ－２５两次柱层析,从没产地的东亚马氏钳歇粗毒中分别得到了一组碱性哺乳动物神经素和一种甲壳类神经毒素,对它们进行的等电点、分子量、动物毒性等部分性质的研究与比较结果表明,淅川、常德与益都产的蝎毒无论在柱层析行为上,还是在等电点、分子量、各组分的毒性大顺序及某些对应组分的结晶行为上有很大相似性,仅存在略微的差异。     

东亚钳蝎神经毒素基因BmK IT3的克隆及其在大肠杆菌中的表达

东亚钳蝎毒素基因ＢｍＫＩＴ3 编码是由 6 5个氨基酸残基组成的多肽物质。该类毒素为专一性作用于昆虫的抑制型神经毒素 ,它已被广泛用于研究离子通道作用机理[1 ] ;同时 ,它是研究蛋白质结构和功能的极好模型 ,是研究神经药理学的理想工具 ,将具有药理活性和昆虫毒性的基因导入细胞或动植物体内具有十分重要的应用价值。它对昆虫作用的专一性很高 ,对哺乳动物无害或毒性很小 ,可作为一种安全、有效的生物杀虫剂[2 ,3 ] 。我们的研究是对该基因密码子进行优化 ,采用化学合成的方法合成了适于在昆虫中表达的ＢｍＫＩＴ3 的两条长的引物 ,通过…     

东亚钳蝎中一个中性哺乳动物神经毒素全长cDNA的克隆和分析

构建了东亚钳蝎毒腺cDNA文库,根据东亚钳蝎中性哺乳动物神经毒素BmKM4的氨基酸序列设计并合成引物,用PCR方法从文库中筛选到BmKM4全长cDNA序列.它由5′UTR、可读框和3′UTR组成.与其他东亚钳蝎哺乳动物神经毒素cDNA的相应区域相比,BmKM4cDNA的5′UTR高度保守,而其3′UTR则变异较大.AUG的旁侧序列为AAAATGAA,与绝大多数蝎毒素基因一致.在BmKM4mRNApoly(A)尾上游17nt处,有一典型的腺苷化信号(AATAAA).可读框编码84个氨基酸的毒素前体,包括N端19个氨基酸残基组成的信号肽,中间64个氨基酸残基组成的成熟毒素,以及C末端的额外碱性氨基酸Arg.椐据一般规律,尾端Arg在毒素前体的成熟过程中会被切除。 Abstract:A full-length cDNA sequence encoding the precursor of a neutral mammalian neurotoxin,BmKM4,was first isolated from a cDNA library made from thc venom gland of Chinese scorpion Buthus martensii Karsch.ABmKM4-specific primer and a primer corresponding to the partial sequence of pSPORT1 vector were used as forward primer and reverse primer,respectively,to screen the cDNA library by PCR reaction.Sequence analysis of positive clones showed that the BmKM4 cDNA is composed of three parts:5'UTR,open reading frame and 3'UTR.Compared with the corresponding regions of other scorpion mammalian neurotoxin cDNAs,the 5'UTR of BmKM4 cDNA is highly conservative versus highly variable for 3'UTR.The lateral sequence of initiation codon (AUG) is AAATGAA which is in consistent with that of most scorpion toxin genes.On the 3'-end,a putative polyadenylation signal (AATAAA) was 1Tnt upstream of Poly (A) tail.The open reading frame encodes a precursor of 84 amino acid residues,including a signal peptide of 19 residues,a mature toxin(BmKM4) of 64 residues,and a basic residue (Arg) tailwhich would be removed in the processing step.     

东亚钳蝎昆虫毒素研究进展

述了我国东亚钳蝎（Buthus martensii Karsch)昆虫神经毒素的分离、,纯化、性质、基因、结构、结晶及晶体学研究的概况。     

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 .     

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     

东亚钳蝎毒素对Caco-2人结肠癌细胞的体外药理学研究

研究东亚钳蝎毒素对人结肠癌细胞Caco-2增殖的影响。以不同浓度的东亚钳蝎（Buthus martensii Karsch）毒素（10、20、40滋g/mL）干预体外培养的Caco-2细胞,分别于24 h、48 h后,用四甲基偶氮唑盐（MTT）比色法,观察毒素对Caco-2细胞的增殖抑制作用。运用淋巴细胞转化实验和乳酸脱氢酶（LDH）释放实验检测蝎毒素对Caco-2细胞的作用途径。结果表明：东亚钳蝎毒不仅能抑制Caco-2细胞的增殖而且能促进淋巴细胞转化,毒素对Caco-2细胞增殖的抑制作用与浓度和作用时间密切相关。     

Precursors of three unique cysteine-rich peptides from the scorpion Buthus martensii Karsch

Scorpion venoms contain a large number of small peptides with diverse primary structures and unique pharmacological functions. From a cDNA library prepared from venom glands of the Chinese scorpion Buthus martensii Karsch, clones encoding precursors of three unique cysteine-rich peptides named BmTXKS3, BmTXLP2 and BmAP1 have been isolated and sequenced. These precursors are composed of 54, 94 and 89 amino acids, respectively, containing a signal peptide in their N-termini. Sequence analysis shows that BmTXKS3 and BmTXLP2 are two novel members of a scorpion toxin family sharing cysteine-stabilized α-helical folds. BmAP1 possesses a distinctive cystine framework, which is similar to some serine protease inhibitors and the segments of several extracellular proteins.     

Molecular cloning, genomic organization and functional characterization of a new short-chain potassium channel toxin-like peptide BmTxKS4 from Buthus martensii Karsch(BmK)

Scorpion venom contains many small polypeptide toxins, which can modulate Na(+), K(+), Cl(-), and Ca(2+) ion-channel conductance in the cell membrane. A full-length cDNA sequence encoding a novel type of K(+)-channel toxin (named BmTxKS4) was first isolated and identified from a venom gland cDNA library of Buthus martensii Karsch (BmK). The encoded precursor contains 78 amino acid residues including a putative signal peptide of 21 residues, propeptide of 11 residues, and a mature peptide of 43 residues with three disulfide bridges. BmTxKS4 shares the identical organization of disulfide bridges with all the other short-chain K(+)-channel scorpion toxins. By PCR amplification of the genomic region encoding BmTxKS4, it was shown that BmTxKS4 composed of two exons is disrupted by an intron of 87 bp inserted between the first and the second codes of Phe (F) in the encoding signal peptide region, which is completely identical with that of the characterized scorpion K(+)-channel ligands in the size, position, consensus junctions, putative branch point, and A+T content. The GST-BmTxKS4 fusion protein was successfully expressed in BL21 (DE3) and purified with affinity chromatography. About 2.5 mg purified recombinant BmTxKS4 (rBmTxKS4) protein was obtained by treating GST-BmTxKS4 with enterokinase and sephadex chromatography from 1 L bacterial culture. The electrophysiological activity of 1.0 microM rBmTxKS4 was measured and compared by whole cell patch-clamp technique. The results indicated that rBmTxKS4 reversibly inhibited the transient outward K(+) current (I(to)), delayed inward rectifier K(+) current (I(k1)), and prolonged the action potential duration of ventricular myocyte, but it has no effect on the action potential amplitude. Taken together, BmTxKS4 is a novel subfamily member of short-strain K(+)-channel scorpion toxin.     

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-