蝮蛇毒纤溶酶的分离纯化及性质研究

目的 :寻找一种分离纯化蝮蛇毒纤溶酶的工艺并研究其理化性质。方法 :采用DEAE SepharoseCL 6B和HeparinCL 6B层析方法 ,从蝮蛇毒中分离纯化纤溶酶。结果 :蝮蛇毒纤溶酶经HPLC为单一峰 ,等电聚焦电泳为一条带 ,其等电点为 4.5 5 ,经SDS 聚丙烯酰胺凝胶电泳测得分子量为 2 9.4kD。该酶对热不稳定 ,在 pH6～ 9时稳定 ,氨基酸组成分析表明含酸性氨基酸较多。结论 :用此工艺可制得高纯度的蝮蛇毒纤溶酶。     

黑眉蝮蛇蛇毒纤溶酶的纯化及其性质研究

目的从黑眉蝮蛇蛇毒中纯化出无出血毒的纤溶酶。方法用DEAE-Sepharose FF阴离子交换树脂、CM-sepharose FF阳离子交换树脂和Sephacryl S-100凝胶过滤树脂三步分离方法,从黑眉蝮蛇蛇毒中纯化出一种纤溶酶活性成分。结果经SDS-聚丙烯酰胺凝胶电泳检测为单一蛋白质,相对分子质量为31 800,小鼠皮下注射无出血反应。该酶与纤维蛋白原保温15 min能迅速水解Bβ(β)链,随后缓慢水解Aα(α)链,而对γ(γ-γ)链无影响。结论此工艺可以快速纯化黑眉蝮蛇蛇毒纤溶酶。     

双胸蚓纤溶酶的分离纯化及性质研究

通过选择性热变性、大豆胰蛋白酶抑制剂－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析，ＤＥＡＥ－纤维素离子交换层析和Ａｒｇ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析，从双胸蚓中分离纯化得到具有强烈纤溶活性的酶组分。     

白眉蝮蛇蛇毒纤溶酶的分离纯化

目的用白眉蝮蛇蛇毒分离纯化纤溶酶。方法利用离子交换层析、亲和层析、分子筛层析技术进行分离纯化纤溶酶。结果得到了电泳纯的纤溶酶,其相对分子质量为24000。结论该工艺可以快速地分离纯化白眉蝮蛇蛇毒纤溶酶：     

烙铁头蛇毒中一个具有抗补体活性金属蛋白酶的纯化和性质研究

目的从烙铁头蛇毒中筛选分离抗补体活性蛋白,并对其部分生物学活性开展研究,以了解其在蛇伤中的病理生理作用和潜在的应用价值。方法采用蛋白层析技术分离纯化抗补体活性蛋白,测定其分子量、等电点、抗补体作用、多种蛋白水解酶活性、水肿活性及出血活性。结果从烙铁头蛇毒中分离纯化出一个抗补体活性蛋白TMAC-1,表观分子量约为25ku,等电点为9.0。TMAC-1能够抑制补体经典途径和替代途径的溶血,预孵条件下,其IC50分别为62、29mg·L-1;不预孵条件下,其IC50为263、246mg·L-1。TMAC-1能够裂解C3、C5,但裂解产物不能诱导内皮细胞P-selectin的表达。TMAC-1能依次降解纤维蛋白原的Aα、Bβ、γ链,该活性能被EDTA、1,10-phenanthroline、EGTA完全抑制,不受PMSF、SBTI的抑制。TMAC-1具有水肿活性和微弱的偶氮酪蛋白水解活性,没有精氨酸酯酶水解活性和皮下出血活性。结论 TMAC-1是一个非出血性的金属蛋白酶,它可通过酶切补体特定成分抑制补体激活。     

蝮蛇毒纤溶酶的分离纯化及其制剂的临床应用

目的:采用凝胶电泳法分离纯化长白山白眉蝮蛇毒纤溶酶并将其制剂应用于临床。方法:采用DEAE- Sepharose CL-6B和Heparin CL-6B层析方法,从蝮蛇毒中分离纯化纤溶酶,通过临床应用分析成品制剂的治疗效果。结果:蝮蛇毒纤溶酶经HPLC为单一峰,等电聚焦电泳为一条带,其等电点为4．55,经SDS-聚丙烯酰胺凝胶电泳测得分子量为29．4kD,将其成品制剂应用于临床,结果表明有效减少了缺血性脑血管病血栓的形成,使缺血部位迅速恢复功能。     

尖吻蝮蛇毒纤溶酶的柱层析分离

应用Sephadex G100、DEAE-Sepharose CL6B、Sephadex G75柱层析分离方法,对尖吻蝮蛇毒进行分离。实验表明,采用上述3种柱层析分离得到的纤溶酶在聚丙烯酰胺凝胶电泳和免疫电泳上为一条区带的单一蛋白。     

长白山白眉蝮蛇蛇毒纤溶酶的纯化

从长白山白眉蝮蛇蛇毒中纯化具有溶栓功效的纤溶酶。方法：用 ＤＥＡＥ Ｓｅｐｈａｄｅｘ Ａ５０离子交换、Ｓｅｐｈａｄｅｘ Ｇ７５凝胶过滤层析和 ＤＥＡＥ Ｓｅｐｈａｄｅｘ Ａ ５０三步柱层析分离方法,对长白山白眉蝮蛇蛇毒进行分离纯化,得到了一种纤溶酶活性组分。结果：经ＳＤＳ－聚丙烯酰胺凝胶电泳和基质辅助激光解吸电离飞行时间质谱表征该酶为单一蛋白,其分子量为 ２３．３６７ ｋＤ。结论：为进一步研究其结构和功能提供了可靠依据。     

蚓激酶中纤溶酶谱鉴别方法的研究

摘 要 目的：建立蚓激酶的纤溶酶谱鉴别方法,同时考察5个企业产品及同一企业不同批次的一致性。方法: 纤溶酶谱法,纤维蛋白原终浓度为5×10^-4g·mL^-1,2.5%Triton-x-100溶液中复性30 min,在PBS缓冲液(pH=7.4)中37℃孵育30 min,供试品蛋白质浓度为5.0～37.5 μg。结果: 该方法灵敏度高,5家企业的活性蛋白条带均分布在15～40KD间,有6个相同特征活性蛋白条带;5个企业 产品之间酶谱图略有差异,同一企业不同批次产品批间无差异。结论:该方法专属性较强, 能够反映蚓激酶活性成分的分子量大小分布及种类,也能反映产品的批间一致性,生产工艺稳定性,且易于标准化,可作为蚓激酶的鉴别方法,同时为评价上市后产品质量一致性奠定了基础。     

蛇毒类抗血栓蛋白酶的研究进展

血栓栓塞性疾病严重威胁人类健康,研究具有优势的抗栓药物是当代医学的重点和热点之一.蛇毒中存在3类具有抗栓作用的蛋白酶,分别为蛇毒类凝血酶、纤维蛋白(原)溶解酶、纤溶酶原激活剂.对这3类抗血栓蛋白酶进行了综述,分别介绍了各类酶的分布、结构、生化性质以及作用机制,并结合其性质及机制分析在抗栓方面存在的优点和不足.另外,还介绍了蛇毒类抗血栓蛋白酶的研究现状,临床应用情况,以及临床常用的一些成熟药物.     

Comparison of Protein Variation in Protobothrops mucrosquamatus Venom between Northern and Southeast Taiwan and Association with Human Envenoming Effects

Reports of bite from Protobothrops mucrosquamatus (Pmu) are frequent in Taiwan, and its wide-spread distribution and diverse habitats drove us to investigate its envenoming effects and relevant venom variations. We used reversed-phase high-performance liquid chromatography and mass spectrometry to analyze 163 Pmu venom samples collected from northern and southeastern Taiwan. Twenty-two major protein fractions were separated and analyzed, and their contents were determined semi-quantitatively. The results showed that despite the trivial differences in the protein family, there is an existing variation in acidic phospholipases A₂s, serine proteinases, metalloproteinases, C-type lectin-like proteins, and other less abundant components in the Pmu venoms. Moreover, clinical manifestations of 209 Pmu envenomed patients hospitalized in northern or southeastern Taiwan revealed significant differences in local symptoms, such as ecchymosis and blistering. The mechanism of these local effects and possibly relevant venom components were examined. Further analysis showed that certain venom components with inter-population variation might work alone or synergistically with others to aggravate the local effects. Therefore, our findings of the venom variation may help one to improve antivenom production and better understand and manage Pmu bites.     

Fibrin(ogen)olytic activity of bumblebee venom serine protease

Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis.     

白唇竹叶青蛇(Trimeresurus albolabris)毒降纤酶的分离纯化以及性质

目的从白唇竹叶青蛇(T.albolabris)毒中分离纯化无出血作用的降纤活性组分,探讨其理化性质及部分生物功能。方法用DEAE-SephadexA-25,SephadexG-100和CM-SephadexC-50三步色谱法进行分离纯化。SDS-PAGE和HPLC鉴定其纯度和相对分子质量,平板法测定其降纤活性。结果从白唇竹叶青蛇毒中分离纯化获得单一的降纤组分,能迅速水解纤维蛋白原或纤维蛋白原Aα链,缓慢水解Bβ链,而对γ链无作用,SDS-PAGE鉴定其相对分子质量为56000。EDTA能抑制其纤维蛋白原水解活性,而PMSF、β-巯基乙醇对其活性无影响,提示该组分为单链α金属蛋白酶。结论从白唇竹叶青蛇毒中分离纯化得到1种无出血作用且降纤活性强的新蛇毒降纤酶。     

Expression and characterization of a recombinant fibrinogenolytic serine protease from green pit viper (Trimeresurus albolabris) venom

Viper venom serine proteases (SPs) display several effects on hemostatic system. Molecular cloning showed that Trimeresurus albolabris venom comprised a mixture of five SPs with thrombin-like (2), fibrinogenase (2) and plasminogen-activating (1) activities. Because only few fibrinogenolytic SP sequences were reported, we decided to express albofibrase, a novel fibrinogenase from T. albolabris using Pichia pastoris system. The recombinant active form of enzyme was 30 kDa including 2.2 kDa of glycosylation. Albofibrase showed an fibrinogenase activity. In addition, a plasminogen activating and clotting effect were detectable. Albofibrase prolonged APTT and PT in a time-dependent manner. The effect was neutralized by pre-incubation with equine antivenom to T. albolabris. Therefore, the protein is potentially useful as a new anticoagulant as the antidote is clinically available. Sequence analysis compared with other snake venom fibrinogenases and SPs could not find any unique residues responsible for their various effects. Structure–function relationship should be further studied using mutagenesis in order to explore the mechanisms of venom protease functional diversity.     

Purification and characterization of alpha-mucrofibrase,a novel serine protease with alpha-fibrinogenase activity from the venom of Chinese Habu (Trimeresurus mucrosquamatus)

A novel fibrinogenolytic protease, named alpha-mucrofibrase, was purified from the venom of Chinese Habu (Trimeresurus mucrosquamatus) by DEAE-Sephadex A-50 ion-exchange chromatography and Sephadex G-100 (super fine) gel filtration alpha-Mucrofibrase is a single-chain polypeptide of approximately 29 kDa. It is stable even at 95 degrees C, and the most susceptible hydrolysis substrate is S-2302. It cleaved primarily the Aalpha chain of fibrinogen followed by the Bbeta chain, while the gamma chain was partially affected. N-terminal sequence of this fibrinogenolytic enzyme has great homology with those of other snake venom serine proteases. The esterase activity of alpha-mucrofibrase is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by metal chelator (EDTA), suggesting this fibrinogenase belongs to the venom serine protease family.     

cDNA cloning, expression and fibrin(ogen)olytic activity of two low-molecular weight snake venom metalloproteinases

Two cDNA clones, AplVMP1 and AplVMP2, were isolated from a snake (Agkistrodon piscivorus leucostoma) venom gland cDNA library. The full-length cDNA sequence of AplVMP1 with a calculated molecular mass of 46.61 kDa is 1233 bp in length. AplVMP1 encodes PI class metalloproteinase with an open reading frame of 411 amino acid residues that includes signal peptide, pro-domain and metalloproteinase domains. The full-length cDNA of the AplVMP2 (1371 bp) has a calculated molecular mass of 51.16 kDa and encodes PII class metalloproteinase. The open reading frame of AplVMP2 with a 457 amino acid residues is composed of signal peptide, pro-domain, metalloproteinase and disintegrin domains. AplVMP1 and AplVMP2 showed 85% and 93% amino acid identical to PI class enzyme Agkistrodon contortrix laticinctus ACLPREF and PII class enzyme Agkistrodon piscivorus piscivorus piscivostatin, respectively. When expressed in Escherichia coli, most of recombinant proteins of AplVMP1 and AplVMP2 were in insoluble inclusion bodies, with soluble yields of 0.7 mg/l and 0.4 mg/l bacterial culture, respectively. Both affinity purified recombinant proteins show proteolytic activity on fibrinogen, although having an activity lower than that of crude A. p. leucostoma venom. Proteolytic activities of AplVMP1 and AplVMP2 were completely abolished after incubation with a final concentration of 100 μM of EDTA or 1,10-phenanthroline. Both AplVMP1 and AplVMP2 were active in a fibrin-agarose plate but devoid of hemorrhagic activity when injected (up to 50 μg) subcutaneously into mice, and had no capacity to inhibit platelet aggregation.     

蝮蛇毒小分子多肽的分离、纯化及其抗肿瘤作用研究

目的从长白山岩栖蝮蛇蛇毒中分离纯化1种小分子多肽Saxis,测定理化性质,研究其对肿瘤细胞的生长抑制作用。方法利用有机溶剂沉淀和Sephadex G-50S、ephadex G-25凝胶过滤色谱等方法分离纯化长白山栖岩蝮蛇蛇毒中的1种小分子多肽Saxis;采用Tricine-SDS-PAGE鉴定;利用Bradford蛋白质测定法测定蛋白质浓度;运用MTT比色法检测该小分子多肽对宫颈癌细胞Hela、肝癌细胞SMMC-7721和胃腺癌细胞SGC-7901的生长抑制作用。结果该小分子多肽Saxis的相对分子质量约为5 200,它能抑制Hela、SMMC-7721和SGC-7901细胞的增殖,并且对这3种肿瘤细胞的抗增殖作用具有药物剂量依赖关系,24 h的IC50分别为66.3,54.5,62.2μg/mL。结论利用有机溶剂沉淀和凝胶过滤色谱法可以从长白山岩栖蝮蛇蛇毒中分离纯化1种小分子多肽Saxis,其对多种肿瘤细胞的生长有抑制作用。     

Purification, cloning and biological characterization of a novel disintegrin from Trimeresurus jerdonii venom.

A novel disintegrin, jerdonin, was purified from the Trimeresurus jerdonii venom by means of gel filtration and reverse phase high pressure liquid chromatography. Its coding cDNA was also isolated from the venom gland. The jerdonin coding cDNA is part of a precursor composed of proprotein, metalloproteinase, and disintegrin domains. From the deduced amino acid sequence, jerdonin is composed of 71 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonin was determined to be 7483Da by matrix-assisted laser desorption ionization time of flight mass spectrometry. Jerdonin inhibited ADP- and collagen-induced human platelet aggregation with IC(50) of 220 and 240 nM, respectively. In vivo, jerdonin inhibited the growth of subcutaneously inoculated B16 solid tumor in C57BL/6 mice and improved the survival time of the tumor-bearing mice.