蕲蛇蛇毒抗凝血因子的分离纯化与特性

目的 寻找蕲蛇蛇毒中的抗凝血因子。方法 利用硫酸铵沉降、阴离子交换层析、阳离子交换层析及高效液相色谱层析,从蕲蛇蛇毒中分离纯化到一个抗凝血因子。结果 纯化的这一组份在PAGE、SDS—PAGE上均呈单一区带,分子量约为25．4kD,由两条分子量分别为15．0kD和16．0kD的肽链通过二硫键连接在一起。这一组份在体外显著地延长血浆复钙时间和凝血酶原时间,但不延长牛凝血酶时间,也不具有磷脂酶A2活性、纤溶活性和出血活性。结论 蕲蛇蛇毒中舍右一种新的抗凝血因子。     

枯草芽孢杆菌BS-26菌株纤溶酶的性质分析及活性组分的分离纯化

[目的]溶栓疗法是血栓性疾病安全且有效的治疗手段,从微生物中寻找溶栓药物是一种理想有效的途径,枯草芽孢杆菌(Bacillus subtilis)BS-26菌株发酵液具有很强的体外纤溶活性,本文分析了发酵液中纤溶酶的性质并对活性组分进行了分离纯化.[方法]利用纤维蛋白平板法检测纤溶酶活性,利用硫酸铵分级盐析、DEAE-Sepharose Fast Flow阴离子交换层析和聚丙烯酰胺制备电泳等方法,进行分离纯化.[结果]此菌株产生的纤溶酶在50℃以下和pH5.0～11.0范围内具有较好的稳定性,最适作用温度为42℃;最适pH值为9.0;Mg2 、Ca2 对此酶有明显的激活作用,而Cu2 能完全抑制酶的活性;174.2μg/mL的苯甲基磺酰氟、1000μg/mL的鸡卵类粘蛋白和1000μg/mL大豆胰蛋白酶抑制剂能完全抑制酶活性,初步说明此酶属于丝氨酸蛋白酶类;体外溶纤作用表明,该酶溶解纤维蛋白的方式是直接溶解,而不是通过激活纤溶酶原.从该菌株的发酵液中获得了一种纤溶酶组分,比活力达8750 U/mg,回收率为3.2%,所获得样品纯度相对于发酵液提高了41倍,该酶在SDS-PAGE中是单肽链蛋白,分子量为32 kDa.[结论]获得了一种纤溶酶的单一组分,为纤溶酶发酵产品的大规模纯化及进一步研制和开发新的溶栓药物提供重要理论依据.     

尖吻蝮蛇毒内一种新抗凝血因子ACF II 的纯化及特性

利用阴离子交换层析、凝胶过滤及阳离子交换层析三步方法, 从皖南尖吻蝮蛇毒中分离纯化到一个新的抗凝血因子ＡＣＦＩＩ（ａｎｔｉｃｏａｇｕｌａｔｉｏｎ ｆａｃｔｏｒＩＩ） 。纯化的ＡＣＦＩＩ在ＰＡＧＥ、ＳＤＳＰＡＧＥ和ＩＥＦＰＡＧＥ图谱上均呈单一区带。ＡＣＦＩＩ由两条分子量为１４．６ ｋＤ 的肽链通过二硫键连接在一起, 其等电点为７．０。ＡＣＦＩＩ具有显著的抗凝血活性, 在体外延长ＰＰＴ 时间的最终浓度为０ ．４ ｍｇ／Ｌ。ＡＣＦＩＩ不具有类凝血酶活性、磷脂酶Ａ２ 活性和纤溶活性,也没有出血活性和毒性, 是一种潜在的高效抗凝药物。     

白灵侧耳纤溶酶的纯化及酶学性质分析

白灵侧耳子实体浸提液经过硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换层析、凝胶过滤层析和羟基磷灰石色谱柱层析后,纯化得到一种纤溶酶。该酶在SDS-PAGE中显单条带,其分子量约为30kDa。该酶在45℃以下,pH6.5-10.0的范围内稳定,最适pH为8.0,最适温度为25℃。金属离子K+对该酶有明显的激活作用,Zn2+、Mg2+、Cu2+对酶有部分抑制作用。金属离子鳌合剂EDTA和丝氨酸蛋白酶抑制剂PMSF不抑制该酶活性,初步说明此酶既不是金属酶,也不是丝氨酸类蛋白酶。该酶既具有纤溶酶作用,又具有激活纤溶酶原的作用。     

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…     

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。     

木霉β-1,3-葡聚糖酶的分离纯化

目的:对木霉菌株LE02所产β-1,3-葡聚糖酶的分离纯化方法进行研究。方法:粗酶液分别用硫酸铵、乙醇和丙酮进行沉淀,再用DEAE-Sepharose CL-6B离子交换层析进一步分离纯化,并用SDS-PAGE法测其分子量。结果:硫酸铵分段盐析法沉淀酶蛋白的效果优于乙醇和丙酮沉淀;盐析得到的酶蛋白经透析浓缩后,再经DEAE-Sepharose CL-6B层析分离,可得到单一酶蛋白,总酶活回收率达78.71%,比酶活达到689.9U/mg,提高了53.74倍,经SDS-PAGE法测得该β-1,3-葡聚糖酶的分子量为80.137kDa。结论:采用硫酸铵分段盐析和离子交换层析法可获得电泳纯的β-1,3-葡聚糖酶,且酶活回收率高。     

尖吻蝮蛇毒去纤酶及其临床应用研究

Ottyang,C.等(1967)从尖吻蝮(Agkistrodon acutus)蛇毒中分离纯化得到凝血酶样成分,分子量33500,由17种263个氨基酸组成,具有体内去纤作用,但未见用于临床的报道。杉原久义等(1978)也从尖吻蝮蛇毒中分离纯化得到了一个促凝组分,分子量52000,具有精氨酸酯酶和凝血酶活性,未见体内血液学效应及临床应用的报道。我们从1974年起,对尖吻蝮蛇毒凝血酶样酶进行7较为系统的研究,并研制成“去纤酶注射液”,于1978年首先用于临床,1981年鉴定通过。这是我国最先研制并用于临床的蛇毒酶制剂。它由四个凝血酶样同工酶组成,其分子量分别是33500,68000,19500,30000;它们均由17种氨基酸组成,但组成     

五带虻溶纤活性蛋白的纯化和性质

五带虻Tabanus qutnquectnctus Rlcardo腹部匀浆液经硫酸铵沉淀、Sephadex G-75凝胶层析、Fibrin-Sepharose 4B亲和层析和电泳制备等方法纯化后,获得在SDS—PAGE图谱上呈现单一区带的溶纤活性蛋白。该蛋白质既具有纤溶酶作用,又具有激活纤溶酶原的作用,其分子量为40kD,等电点为4.5,最适作用pH为9.0,最适作用温度为28℃,37℃处理2h活性完全丧失,Ca²⁺、Mn²⁺、Cu²⁺、Zn²⁺、Hg²⁺和PMSF能抑制其活性。     

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…     

五步蛇蛇毒类凝血酶N端的部分氨基酸序列

从五步蛇蛇毒中纯化得到的类凝血酶,在SDS-PAGE及IEF均为一条带,且分子质量约38 ku,等电点约为4.0。测定该酶N端15个氨基酸的序列是VIGGVECDINEHRFL,与其他的蛇毒类凝血酶有高度同源性。     

蛇毒类凝血酶calobin在毕赤酵母中的表达

蛇毒类凝血酶是临床上防治血栓栓塞性疾病的有效药物。参照朝鲜蝮蛇(Agkistrodon caliginosus,Korean Viper)类凝血酶calobin基因序列(GenBank AccessionNo.U32937.1),将人工合成的calobin基因克隆到酵母表达载体pPICZαA,于毕赤酵母中表达,得到了分子量约为32kD的重组calobin蛋白,经甲醇诱导培养,表达产物可获得3.5g/L的高表达量。重组蛋白经过阴离子交换柱Q-Sepharose Fast Flow和分子筛Sephacryl-S-100凝胶过滤层析等纯化步骤进行了初步纯化。纯化后的重组calobin可以在纤维蛋白原平板上形成水解圈,经SDS-PAGE实验显示,重组蛋白能水解纤维蛋白原的Aα链,产生一条约40kD左右的降解带。在实验中未能发现重组calobin对纤维蛋白原的凝固作用。     

cDNA cloning and expression of acutin

Acutin, a thrombin-like enzyme was purified from Agkistrodon acutus venom in three steps by DEAE-Sepharose CL-6B, Superose 12 column on FPLC and Mono-Q column chromatographies. Its first 15 N-terminal amino acid residues sequence was then determined and the acutin cDNA was isolated from venom gland total RNA using RT-PCR. Determination of its nucleotide sequence allowed elucidation of the amino acid sequence of mature peptide for the first time. The mature acutin has 233 amino acids and its amino acid sequence exhibits significant homology with those of thrombin-like enzymes from crotaline snakes venoms. Based on the homology, the catalytic residues and disulfide bridges of acutin were deduced to be as follows: catalytic residues, His41, Asp84 and Ser179; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys231, Cys118-Cys185, Cys150-Cys164, Cys175-Cys200. The recombinant acutin has been expressed in E. coli and purified by affinity column. The renatured recombinant acutin is reported for the first time to have the activity of clotting fibrinogen and arginine-esterase.     

Purification and characterization of the fibrinolytic principle of Agkistrodon acutus venom.

By means of DEAE-Sephadex A-50 column chromatography, Agkistrodon acutus venom was separated into twelve fractions. The fibrinolytic activity was concentrated in Fraction 9. This fraction was rechromatographed on Sephadex G-75 three times and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single, symmetrical boundary with a value of 2.44 S was obtained by ultracentrifugation, the molecular weight of which was estimated to be 24 100, and the isoelectric point 3.8. The specific activity was four times higher than that of crude venom. The optimal pH value on fibrinolysis was 7.4. In addition to fibrinolytic activity, the purified principle also had fibrinogenolytic and caseinolytic activities. The purified fibrinolytic principle had a specific action on the a(A) chain subunit of fibrinogen, leaving the beta(B) chain and the gamma chain unaffected.     

制备式等电聚焦鉴定湖南产五步蛇蛇毒磷脂酶A2的等电点及纯度

制备式等电聚焦测得湖南产五步蛇蛇毒磷脂酶Ａ２为单一的吸收峰（ＯＤ２８０）,等电点为５．３２,证明了作者前报道的纯化的磷脂酶Ａ２为纯品     

Isolation, purification, and separation of the complex preparation of extracellular proteinases with fibrinolytic and anticoagulant properties from Aspergillus ochraceus 513]

The extracellular proteinase complex of the microscopic fungus Aspergillus ochraceus 513 was isolated, purified, and separated by affinity chromatography on bacillichin-silochrom and subsequent column chromatography on DEAE-Toyopearl 650 M. The extracellular enzyme of the protein C activator type had a molecular mass of 36.5 kDa and activity close to that of the Agkistrodon snake venom protein C activator. The fibrinolytic and anticoagulant activities of the enzyme were investigated.     

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.     

HPLC-based two-step purification of fibrinolytic enzymes from the venom of Agkistrodon contortrix contortrix and Agkistrodon piscivorus conanti

In investigations aimed at characterizing snake venom blood clot-dissolving enzymes, we have developed a rapid two-step high-performance chromatography method for the isolation of these fibrinolytic enzymes from the venoms of Agkistrodon contortrix contortrix and Agkistrodon piscivorus conanti. The first step consisted of hydrophobic interaction chromatography on a propyl-aspartamide column. Fractions containing the fibrinolytic activity were then concentrated and applied to a hydroxylapatite column. The resulting preparation, assessed for purity by reverse-phase chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was homogeneous. The molecular weight of both venom fibrinolytic enzymes was approximately 23,000 and amino acid analysis, immunological cross-reaction, cyanogen bromide, and tryptic digestion indicate a significant degree of structural similarity. However, the general proteolytic activity of the A. p. conanti venom enzyme was significantly lower than the corresponding activity of the A. c. contortrix venom, whereas their fibrinolytic activities were quite similar.     

五步蛇毒降纤酶的快速分离与纯化

目的：建立快速分离纯化降纤酶工艺方法适合于规模化生产。方法以五步蛇毒为原料。采用ＥＤＴＡ络合－硫氰酸钾沉淀初步分离与快速离子交换和凝胶层析化相结合。分离纯化出符合新部颁标准的降纤酶成分。结果经初步分离五步蛇粗毒可使出血毒去除９２％,非类凝血酶（ＴＬＥ）成分去除６５．１７％,ＴＬＥ活性回收保持９２．６８％以上。层析组份中至少含有４种ＴＬＥ,其中１种为降酶成分。初步化学分离,。离子交换柱层析和凝胶过滤