蝮蛇(Agkistrodon halys Pallas)蛇毒类凝血酶的研究——Ⅰ.分离提纯及理化、酶学性质的鉴定

(1) 浙江产蝮蛇蛇毒中含有三种具有精氨酸酯酶活力的组分。它们分别是激肽释放酶、类凝血酶及类溶血纤维酶。其中类凝血酶的精氨酸酯酶活力最高,约占总酯酶活力的60%。(2) 提纯后的类凝血酶在聚丙烯酰胺凝胶电泳上呈一条区带。经凝胶过滤及SDS聚丙烯酰胺凝胶电泳测定,其分子量约43,000。氨基酸组成分析表明含有较多的酸性氨基酸及脯氨酸,此外还含有约6%的中性糖,9%的己糖胺及3.3%的唾液酸。(3) 类凝血酶能直接使血纤维蛋白原凝聚,水解BAEE的活力约是胰蛋白酶的2.7倍,K_m为3.4×10~(-4)M,高于其它已知的蝮亚科蛇毒类凝血酶活力。它不作用于BANA、BAPA及其它蛋白底物。蛇毒类凝血酶与人凝血酶一样,对专一萤光底物Boc-Val-Pro-Arg-MCA都有明显活力,但其凝结血纤维蛋白原的活力却远低于人凝血酶。(4) 类凝血酶能被DFP及PMSF所抑制,但抑制速度缓慢,不能被胰蛋白酶的专一抑制剂TLCK所抑制,因而是专一性很强的丝氨酸蛋白酶。     

蝮蛇(Agkistrodon halys Pallas)蛇毒类凝血酶的研究——Ⅰ.分离提纯及理化、酶学性质的鉴定

(1)浙江产蝮蛇蛇毒中含有三种具有精氨酸酯酶活力的组分。它们分别是激肽释放酶、类凝血酶及类溶血纤维酶。其中类凝血酶的精氨酸酯酶活力最高,约占总酯酶活力的60%。(2)提纯后的类凝血酶在聚丙烯酰胺凝胶电泳上呈一条区带。经凝胶过滤及SDS 聚丙烯酰胺凝胶电泳测定,其分子量约43,000。氨基酸组成分析表明含有较多的酸性氨基酸及脯氨酸,此外还含有约6%的中性糖,9%的己糖胺及3.3%的唾液酸。(3)类凝血酶能直接使血纤维蛋白原凝聚,水解BAEE 的活力约是胰蛋白酶的2.7倍,K_(?)为3.4×10~(-4)M,高于其它已知的蝮亚科蛇毒类凝血酶活力。它不作用于BANA、BAPA 及其它蛋白底物。蛇毒类凝血酶与人凝血酶一样,对专一萤光底物Boc-Val-Pro-Arg-MCA 都有明显活力,但其凝结血纤维蛋白原的活力却远低于人凝血酶。(4)类凝血酶能被DFP 及PMSF 所抑制,但抑制速度缓慢,不能被胰蛋白酶的专一抑制剂TLCK 所抑制,因而是专一性很强的丝氨酸蛋白酶。     

蛇毒类凝血酶的研究概况

自 1 936年 Klobusitzki和 Konig首次从美洲矛头蝮蛇 ( Bothrops jararaca)毒中获得部分纯化的类凝血酶以来 ,迄今已发现 30余种蛇毒中含有类凝血酶组份 ,并有 2 0余种先后得到分离和纯化。尤其是近几年来 ,有关蛇毒类凝血酶分子结构及酶学性质的研究取得了很大进展 ,部分已作为治疗药物而广泛应用于临床。兹就近几年来蛇毒类凝血酶的研究进展作一简要综述。1　蛇毒类凝血酶的分布　　以前认为蛇毒类凝血酶仅在于蝮亚科蛇毒中 ,蝰亚科中只有一种沙蝰 ( Vipera ammodytes)具有凝血酶样活性。随后不但从另一种蝰蛇加蓬咝蝰( Bitis gabonica)…     

蛇毒类凝血酶的基因工程研究进展

自 1 936年 Klobusitzki和 Konig首次从美洲矛头蝮蛇 ( Bothrops jararaca)毒中获得部分纯化的类凝血酶以来 ,迄今已发现 30多种蛇毒中含有类凝血酶组份 ,并有 2 0多种先后得到分离和纯化。尤其近年来基于有关蛇毒类凝血酶分子结构及酶学性质的研究成果 ,部分蛇毒类凝血酶已经作为治疗药物而广泛应用于临床 ,如国外的 Ancrod和Batroxobin蛇毒抗凝剂 ,国内的五步蛇毒去纤酶、东北白眉蝮蛇抗栓酶 (清栓酶 )、江浙蝮蛇抗栓酶等已广泛用于临床治疗脑血栓形成、脉管炎、冠心病、心肌梗死 ,也有用于治疗癌痛综合症 [1]。但目前有关蛇毒类凝血酶…     

长白山白眉蝮蛇乌苏里亚种类凝血酶（TLE）基因克隆及分析

目的为了获得长白山白眉蝮蛇乌苏里亚种类凝血酶基因。方法根据GeneBank自眉类凝血酶eDNA5．和3保守序列设计了引物,通过RT-PCR从白眉蝮蛇乌苏里亚种毒腺TotalRNA中扩增得到1条长714bp的特异cDNA片段,将该cDNA片段重组到SimpleTvector,转化进E．coliJM109competentcell,阳性克隆委托生物公司测序,利用生物信息学方法对测序结果进行分析。结果该特异性片段与蛇毒类凝血酶同源性为95％,它为一个开发阅读框架,其编码的蛋白质序列与其他蛇毒类凝血酶序列同源性为94％,与其他蛇毒类凝血亲缘关系非常近。结论本实验获得了一种新型白眉蝮蛇乌苏里亚种类凝血酶基因。     

东北白眉蝮蛇毒类凝血酶的分离纯化

我国多年来因技术原因 ,一直使用多组份混合成分的蛇毒制剂 ,临床上虽有一定疗效 ,但存在严重的副作用。作者利用细胞融合技术成功地建立了一株高表达类凝血酶抗体的细胞系 ,该抗体经纯化后 ,可高效亲和白眉蝮蛇类凝血酶。该技术使纯化蛇毒类凝血酶工艺有了很大的飞跃 ,也使蛇毒制品质量达到世界先进水平。方法 :采用天然蛇毒经 DEAE-琼脂糖凝胶层析 ,收集活性组份 ,再经葡聚糖凝胶 G- 75柱层析后 ,收集活性组份 ,最后经类凝血酶亲和柱层析 ,测定 2 80 nm的光吸收度 ,收集蛋白峰 ,测定活性 ,获得了单一组份的类凝血酶 ,即为纯化的类凝血酶…     

五步蛇蛇毒的分离纯化及综合利用

五步蛇蛇毒冻干粉经过SephadexG-75分子筛层析,使纤溶酶和类凝血酶初步分离;DEAE阴离子交换层析对2种酶进一步分离纯化,分别得到了纤溶酶和类凝血酶。2种酶在HPLC图谱上均呈单一峰,在SDS-PAGE图谱上均为单一条带,纤溶酶分子量大约为24．1kDa,类凝血酶分子量大约为14．4kDa,与以往报道相符。酶的总活力回收率大大提高,纤溶酶的活力回收率达23．9％,类凝血酶的活力回收率达34．5％。实现了对蛇毒的综合利用,为进一步开发利用蛇毒探索了一条有效的途径。     

蛇毒类凝血酶的分子生物学研究进展及其应用

蛇毒类凝血酶在体外可以作用于纤维蛋白原使其凝固,具有类似凝血酶的功能。但在体内却表现出抗凝、降纤的功能。本概述了蛇毒类凝血酶对纤维蛋白原的识别和作用、序列同源性特点、cDNA克隆的表达以及在临床中的应用。     

五步蛇毒中低分子量蛇毒类凝血酶的分离纯化

目的 寻找五步蛇毒新的蛇毒类凝血酶组份。方法 用ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ－Ｆａｓｔ Ｆｌｏｗ（－ＦＦ）,ｃｍ－Ｓｅｐｈａｒｏｓｅ－ＦＦ纯化经常规化学提纯的五步蛇毒;以血凝活性和精氨酸酯酶活性（ＢＡＥＥ）检测酶活力;以ＳＤＳ－ＰＡＧＥ电泳法测定分子量。结果 得到分子量为１４０００左右的电泳纯蛇毒类凝血酶组份。结论 五步蛇毒中含有低分子量蛇毒类凝血酶。     

蛇毒抗栓酶制剂质量标准探讨

从蛇毒中提取的类凝血酶（简称ＴＬＥ）成分,对治疗血栓性疾病有较好的药用价值。但是,它的质量标准,十几年来一直比较混乱。虽然,近几年经过整顿有一定好转,但仍然不尽人意。且有些标准不结合实际,没有实际应用价值。我们认为对以类凝血酶成分为主的蛇毒抗栓酶的质...     

High-level expression of a soluble snake venom enzyme,gloshedobin, in E. coli in the presence of metal ions

The mature gene of gloshedobin, a snake venom thrombin-like enzyme from the snake, Gloydius shedaoensis, was cloned and expressed in strain E. coli BL21(DE3). Having been induced by IPTG, the recombinant gloshedobin was in both soluble and insoluble forms. To avoid inclusion body formation, expression was optimized at 25 °C. Furthermore, a 50% increase in solubilization of the target protein was obtained by adding 0.1 mM Mg²⁺ to the medium. The purified recombinant gloshedobin gave a 44 kDa band on SDS-PAGE gel.     

Molecular characterization of BjussuSP-I, a new thrombin-like enzyme with procoagulant and kallikrein-like activity isolated from Bothrops jararacussu snake venom

A thrombin-like enzyme, named BjussuSP-I, isolated from Bothrops jararacussu snake venom, is an acidic single-chain glycoprotein with M(r)=61,000, pI approximately 3.8 and 6% sugar. BjussuSP-I shows high proteolytic activity upon synthetic substrates, such as S-2238 and S-2288. It also shows procoagulant and kallikrein-like activity, but is unable to act on platelets and plasmin. These activities are inhibited by specific inhibitors of this class of enzymes. The complete cDNA sequence of BjussuSP-I with 696bp encodes open reading frames of 232 amino acid residues, which conserve the common domains of thrombin-like serine proteases. BjussuSP-I shows a high structural homology with other thrombin-like enzymes from snake venoms where common amino acid residues are identified as those corresponding to the catalytic site and subsites S1, S2 and S3 already reported. In this study, we also demonstrated the importance of N-linked glycans to improve thrombin-like activity of BjussuSP-I toxin.     

长白山白眉蝮蛇毒类凝血酶Gussurobin基因的克隆、表达与纯化

根据同源性 ,在高度保守的上游信号肽区域设计引物 ,通过RT PCR反应 ,从长白山白眉蝮蛇 (Gloydiusussurensis)毒腺总RNA中克隆得到类凝血酶 gussurobincDNA ,双向测序得到 gussurobin基因的全序列并由此推测出相应的氨基酸序列。与其他已知的类凝血酶不同 ,gussurobin只含有一个可能的糖基化位点 ,即Asn12 4 Ser12 5 Thr12 6。将gussurobin基因克隆到表达载体 pPIC9K中 ,电极转化至毕氏酵母菌株GS115中 ,经G418抗性筛选和营养缺陷型筛选获得重组子。经摇瓶培养 ,获得表达。经过柱层析分离 ,获得SDS PAGE电泳纯的重组gussurobin。     

BjussuSP-I: a new thrombin-like enzyme isolated from Bothrops jararacussu snake venom

A thrombin-like enzyme named BjussuSP-I, isolated from B. jararacussu snake venom, is an acidic single chain glycoprotein with approximately 6% sugar, Mr=61,000 under reducing conditions and pI approximately 3.8, representing 1.09% of the chromatographic A(280) recovery. BjussuSP-I is a glycosylated serine protease containing both N-linked carbohydrates and sialic acid in its structure. BjussuSP-I showed a high clotting activity upon human plasma, which was inhibited by PMSF, leupeptin, heparin and 1,10-phenantroline. This enzyme showed high stability regarding coagulant activity when analyzed at different temperatures (-70 to 37 degrees C), pHs (4.5 to 8.0), and presence of two divalent metal ions (Ca(2+) and Mg(2+)). It also displayed TAME esterase and proteolytic activities toward natural (fibrinogen and fibrin) and synthetic (BAPNA) substrates, respectively, being also inhibited by PMSF and leupeptin. BjussuSP-I can induce production of polyclonal antibodies able to inhibit its clotting activity, but unable to inhibit its proteolytic activity on fibrinogen. The enzyme also showed crossed immunoreactivity against 11 venom samples of Bothrops, 1 of Crotalus, and 1 of Calloselasma snakes, in addition of LAAO isolated from B. moojeni venom. It displayed neither hemorrhagic, myotoxic, edema-inducing profiles nor proteolytic activity on casein. BjussuSP-I showed an N-terminal sequence (VLGGDECDINEHPFLA FLYS) similar to other thrombin-like enzymes from snake venoms. Based on its biochemical, enzymatic and pharmacological characteristics, BjussuSP-I was identified as a new thrombin-like enzyme isoform from Bothrops jararacussu snake venom.     

Cloning and Functional Expression of the Mucrosobin Protein, a β-Fibrinogenase of Trimeresurus mucrosquamatus (Taiwan Habu)

A venom-specific cDNA encoding for a thrombin-like enzyme designated as mucrosobin has been cloned and sequenced from the cDNA library of the venomous gland of Trimeresurus mucrosquamatus. The full-length cDNA of mucrosobin was assembled by oligonucleotide screening and 5′-rapid amplification of cDNA ends. The amino acid sequence deduced from the cDNA consists of 257 amino acid residues with a putative signal peptide of 24 residues. It is highly homologous to the other thrombin-like enzymes (batroxobin, mucofirase, and calobin), suggesting that it is a serine proteinase with a conserved catalytic triad of His⁴¹, Asp⁸⁴ and Ser¹⁷⁹ in the deduced form of mucrosobin protein. Northern blot analysis revealed that the mucrosobin gene encodes an mRNA of 1.5 kb and suggested a tissue-specific expression in the venomous gland. In an effort to study the biological property of mocrosobin, we have expressed the 28-kDa protein as inclusion bodies in Escherichia coli. For analyzing enzymatic activity, the inclusion bodies were solubilized and the recombinant protein was refolded with a two-step dialysis protocol. The refolded recombinant protein exhibited a specific β-fibrinogenolytic activity. This study offers a possibility of using genetic engineering to acquirie a functional snake venom protein with therapeutic potential.     

Characterization of cerastobin, a thrombin-like enzyme from the venom of Cerastes vipera (Sahara sand viper)

Cerastobin, a thrombin-like enzyme, was isolated from the venom of Cerastes vipera (Sahara sand viper) in homogeneous form. Cerastobin had a molecular weight of 38,000 with 348 amino acid residues. It had an isoelectric point of 7.7 (a pH optimum of 7.9 and a temperature optimum of 45 degrees C). Cerastobin hydrolyzed arginine-containing synthetic substrates such as TAME, BAME, and BAEE, but BAPNA was not hydrolyzed. Cerastobin had thrombin-like activity, producing fibrin from fibrinogen and also hydrolyzing chromogenic substrates for thrombin such as 2AcOH.H-D-CHG-But-Arg-pNA (CBS 34.47) and H-D-Phe-Pip-Arg-pNA (S-2238). It showed kallikrein-like activity and hydrolyzed kallikrein substrates 2AcOH.H-D-Phe-Gly-Arg-pNA (CBS 33.27) and H-D-Pro-Phe-Arg-pNA (S-2302). It produced bradykinin from bradykininogen, as uterus contraction was observed. A serine inhibitor, DFP, exerted a pronounced inhibitory effect, suggesting that cerastobin is a serine-type protease. The sequence of 37 residues from the amino-terminal end was investigated. The amino-terminal amino acid was valine as it is in most other thrombin-like enzymes. The amino acid sequence of cerastobin was similar to that of thrombin in some residues and had some homology with that of kallikrein. However, cerastobin showed a high degree of homology to thrombin-like enzymes isolated from various snake venoms. Factor X was partially degraded by cerastobin. It was also found that antithrombin III was degraded by the enzyme. The alpha and beta chains of fibrin monomer were preferentially hydrolyzed by cerastobin, but the gamma chain was quite resistant.     

Isolation and characterization of the thrombin-like enzyme from Cryptelytrops albolabris (white-lipped tree viper) venom

A thrombin-like enzyme (termed albolabrase) was isolated in purified form from the venom of Cryptelytrops albolabris (white-lipped tree viper) using high performance anion ion exchange and gel filtration chromatography. The molecular mass of albolabrase was 33.7 kDa as determined by SDS-PAGE and 35.8 kDa as determined by Superose gel filtration chromatography. The N-terminal sequence was determined to be VVGGDECNINE which is homologous to many snake venom thrombin-like enzymes. Albolabrase exhibits both arginine ester hydrolase and arginine amidase activities and the enzyme is fastidious towards tripeptide chromogenic anilide substrates. The fibrinogen clotting activity was optimum at 3 mg/mL bovine fibrinogen, and showed distinct species differences in the following decreasing order: bovine fibrinogen > dog fibrinogen ≈ human fibrinogen > goat fibrinogen. The enzyme failed to clot both rabbit and cat fibrinogens. Reversed-phase HPLC analysis on the breakdown products of fibrinogenolytic action of albolabrase indicated that the enzyme belongs to the AB class of snake venom thrombin-like enzyme. In the indirect ELISA, IgG anti-albolabrase reacted extensively with most crotalid venoms, except with Tropidolaemus wagleri and Calloselasma rhodostoma venoms. The double sandwich ELISA, however, showed that anti-albolabrase reacted strongly only with venoms from the Trimeresurus complex, and that the results support the proposed new taxonomy changes concerning the Trimeresurus complex.     

Cloning, sequence analysis and expression in E. coli of the cDNA of the thrombin-like enzyme (pallabin) from the venom of Agkistrodon halys pallas

The cDNA of the thrombin-like enzyme (pallabin) from the venom of Agkistrodon halys pallas was cloned and sequenced. The length of the cDNA is 923bp which includes 120bp of noncoding region and 780bp of coding region. Pallabin was synthesized as a prozymogen with 260 amino acids, which includes a signal peptide of 18 amino acids, a proposed propeptide of 6 amino acids and a matured peptide of 236 amino acids. Pallabin exhibits a strong amino acid similarity to the serine proteases isolated from other snake venoms. It contains 12 cysteins which form 6 disulfide bridges. Like other serine proteases, it also has three conserved catalytically active sites: His41, Asp86 and Ser182. To our knowledge, this study is the first report concerning the cDNA of a thrombin-like enzyme from Agkistrodon halys pallas. The cDNA was cloned into the expression plasmid pT7ZZa and expressed in E.coli. The recombinant pallabin immunologically reacted with its specific antibody.     

Soluble expression, purification, and characterization of Gloydius shedaoensis venom gloshedobin in Escherichia coli by using fusion partners

Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, is usually produced as inclusion bodies in Escherichia coli cell. In this work, gloshedobin was separately fused with three fusion partners NusA, GST, and TrxA at its N terminus and then was expressed as fusion proteins in E. coli. The results showed that the NusA was the most efficient fusion partner to improve the solubility of recombinant gloshedobin. The purified NusA-fused gloshedobin with an overall yield of 64.6% was resolved as one band in the SDS-PAGE gel with molecular mass of about 90 kDa. Both fibrinogen clotting and fibrinogenolytic activities were found for the recombinant product. The purified NusA-fused gloshedobin exhibited amidolytic activity of 506 U/mg under optimal conditions of pH of 8.0 and 40°C. The inhibition study of NusA-fused gloshedobin by various inhibitors showed that serine protease inhibitors, phenylmethylsulphonyl fluoride, and N-tosyl-l-phenylalanine chloromethyl ketone, strongly inhibited its admidolytic activity, whereas ethylenediaminetetraacetic acid as well as heparin and hirudin did not, suggesting that NusA-fused gloshedobin exhibited the same characteristics as the native form of gloshedobin. The strategy of this work may contribute to improve the soluble expression level of other thrombin-like enzymes from snake venom in E. coli.