眼镜蛇毒细胞毒素CTX-d的分离纯化及抗肿瘤活性

目的 分离纯化眼镜蛇毒细胞毒素,测定其体内外抗癌作用.方法 应用柱层析及RP-HPLC,从眼镜蛇毒粗毒中分离纯化细胞素素(CTX).体内外抗癌作用利用噻唑兰(MTT)法及对荷瘤小鼠U14瘤的抑瘤作用.结果 分离纯化获得的CTX-d不混有PLA2,在体内外实验中显示明显的抗肿瘤作用.结论 结合阳离子交换层析和RP-HPLC可从眼镜蛇毒中高效分离获得不含PLA2的CTX.     

中华眼镜蛇毒诱导HL-60细胞凋亡及其机制探讨

目的　观察中华眼镜蛇毒体外对白血病细胞凋亡的影响 ,并探讨其可能的作用机制。方法　本研究采用血清药理学方法 ,以人白血病细胞HL 60细胞为靶细胞 ,通过流式细胞仪分析、DNA片段凝胶电泳、免疫组织化学 (S P法 )观察中华眼镜蛇毒兔血清体外对细胞凋亡的诱导作用及癌基因表达的影响。结果　经DNA电泳、流式细胞仪分析显示 :中华眼镜蛇毒兔血清与HL 60细胞共同培养 48h可诱导其细胞凋亡增加 ,bcl 2、c myc基因蛋白表达下调。结论　中华眼镜蛇毒兔血清体外抑制HL 60细胞增殖可能与其诱导细胞凋亡及使细胞bcl 2、c myc基因蛋白表达下调密切相关     

江西常见蛇血清中抗蛇毒sPLA_2蛋白的筛选与纯化

为研究特异性解蛇毒和抗炎症蛋白质药物,对6种江西常见蛇种血清sPLA2抑制蛋白进行筛选和分离纯化。利用实验室前期纯化的五步蛇毒sPLA2作为靶酶,以琼脂糖平板法和pH动态监测法联合筛选江西常见蛇种血清sPLA2抑制剂。基于初筛结果和资源量,确定以华游蛇血清为原料,分离纯化sPLA2抑制剂。分离过程包括Source 30Q、超滤、Uno Q柱精纯化等3个步骤,目的组分经SDS-PAGE分离,显示含有2个亚基,相对分子质量为25.4 ku和21.2 ku。结合文献初步推测该蛋白可能为-γPLI。     

中华眼镜蛇毒口服给药镇痛作用的研究

目的：研究中华眼镜蛇毒灌服给药对小鼠的镇痛作用。方法：将ICR和昆明种小鼠,采用3种致疼痛模型,即冰醋酸所致扭体反应、小鼠热板法致痛实验和甲醛致炎性疼痛反应;中华眼镜蛇毒经热变性复性处理,将其灌胃给药30-270μg／kg。结果：中华眼镜蛇毒粗毒灌服给药能减少腹腔注射醋酸引起的扭体次数,延长热引起的疼痛反应潜伏期和减少甲醛引起的舔足反应。结论：中华眼镜蛇毒灌服给药具有良好的镇痛作用,且给药途径方便、安全范围大,具有进一步开发成新型镇痛药的潜力。     

眼镜蛇毒蛋白酶natrahagin抑制兔颈动脉血栓形成的作用

眼镜蛇毒蛋白酶ｎａｔｒａｈａｇｉｎ是从中国眼镜蛇(Ｎａｊａｎａｊａａｔｒａ)毒中分离纯化出的蛋白酶,在体外可水解血小板膜糖蛋白Ⅰｂ(ＧＰⅠｂ)并具有抑制血小板聚集的作用[1]。研究表明,抑制ＧＰⅠｂ与其粘附蛋白配基之间相互作用的药物,能够抑制血小板的聚集与粘附反应,从而产生抗血栓的作用[2]。本文旨在观察ｎａｔｒａｈａｇｉｎ在体内对动脉血栓形成和血小板聚集的影响,为寻找新型抗血栓药物提供有价值的实验资料。1　材料和方法1.1　材料　ｎａｔｒａｈａｇｉｎ由第一军医大学药物研究所从中国眼镜蛇粗毒(由广州医学院蛇毒研究所…     

眼镜蛇神经毒素对豚鼠颈上神经节烟碱传递的易化作用(英文)

本研究用离休豚鼠颈上神经节细胞内生物电记录技术,对眼镜蛇毒神经毒素对交感神经节突触传递影响进行探讨。研究首次表明,眼镜蛇神经毒素对交感神经突触前ACh的释放有易化作用。     

眼镜蛇毒细胞毒素的分离、纯化及其抗癌活性

目的：研究眼镜蛇毒细胞毒素的抗癌活性。方法：应用Ｓｅｐｈａｄｅｘ　 Ｇ１００和 ＣＭ－Ｓｅｐｈａｒｏｓｅ ＦＦ柱层析,从眼镜蛇毒中分离、纯化细胞毒素组分,用ＭＴＴ法测定该组分对体外培养的人癌细胞的细胞毒性作用。结果：眼镜蛇毒细胞毒素对人癌细胞ＳＧＣ－７９０１、Ｂｅｌ－７４０２、Ｋ５６２和Ｕ９３７的抑制作用呈良好的量效关系,半数抑制浓度分别为 ４．１０、 ２． ０８、 ０． ２９和 ０．１７ μｇ／ｍｌ。结论：眼镜蛇毒细胞毒素对体外培养的人癌细胞有很强的杀伤作用。     

眼镜蛇毒因子的研究进展

目的:介绍眼镜蛇毒因子的性质,应用现状及发展前景。方法:参考国内外的文献,介绍眼镜蛇毒因子的理化性质、分子生物学特性、免疫学作用机制及其临床应用。结果与结论:眼镜蛇毒因子作为一种强效的补体抑制剂,具有突出的优点和特异的临床应用范围,有广阔的开发前景     

眼镜蛇毒的化学成分研究进展

眼镜蛇毒具有广泛的生物学活性。文章综述了近年来眼镜蛇毒中研究较为深入的一些组分的分离提纯、理化性质及其生物活性等方面的研究进展。     

舟山眼镜蛇毒细胞毒素的分离纯化及其体外抗肿瘤活性

目的从眼镜蛇毒中分离纯化细胞毒素 F(CTX F)并鉴定其活性。方法应用凝胶过滤、离子交换柱色谱及疏水柱色谱等方法从舟山眼镜蛇毒中分离纯化CTX F ,以SRB法观察CTX F对体外培养癌细胞的杀伤作用。结果眼镜蛇毒粗毒经凝胶过滤获得 4个蛋白峰 ,将CTX所在第Ⅳ峰用阳离子交换柱色谱获A、B、C、D、E、F和G等7个组分 ,其中E、F和G具CTX活性 ,将F组分再经凝胶过滤和疏水色谱进一步纯化得不含磷酯酶A2 (PLA2 )的CTX纯品 ,暂定名为CTX F ,它对多种癌细胞株有杀伤作用。结论应用凝胶过滤、离子交换和疏水色谱等方法可从眼镜蛇毒中获得不含PLA2 的CTX ,其组分F有抗肿瘤活性     

抗眼镜王蛇毒卵黄抗体的免疫活性及其对眼镜王蛇毒的中和作用

目的：探讨抗眼镜王蛇毒卵黄抗体的免疫活性及其在体内外对眼镜王蛇毒的中和作用。方法：采用嗜硫色谱法一步分离纯化抗眼镜王蛇毒卵黄抗体,通过间接ELISA法对制备的抗眼镜王蛇毒卵黄抗体进行免疫活性检测,采用小鼠体内外保护实验评估抗眼镜王蛇毒卵黄抗体对眼镜王蛇毒的中和作用。结果：嗜硫色谱法制备的卵黄抗体具有良好的免疫活性,并且对眼镜王蛇毒素显示较好的中和效应,在体外6mg/kg抗眼镜王蛇毒卵黄抗体可有效地中和约4LD50（约1.6mg/kg）的眼镜王蛇毒素,而在体内可有效地中和约3LD50（约1.2mg/kg）的眼镜王蛇毒素。结论：抗眼镜王蛇毒卵黄抗体对眼镜王蛇毒具有良好的中和作用,本项目为抗眼镜王蛇毒卵黄抗体的进一步研究开发提供实验依据。     

Biochemical characterization of a toxin from Indian cobra (Naja naja naja) venom

A major toxic component was isolated from the venom of Indian cobra (Naja naja naja) by ammonium sulfate fractional precipitation followed by carboxymethyl cellulose column chromatography and Sephadex gel filtration. This component constituted 2% of the venom and produced a block of neuromuscular transmission in nerve muscle preparations. Three other toxic fractions comprising 3% of the venom were also detected. The major toxic component was homogeneous on starch and polyacrylamide gel electrophoresis and on rechromatography on CM-cellulose. Its molecular weight was approximately 6300. This toxin contained 61 amino acid residues including 8 half-cystine residues whereas alanine, methionine and phenylalanine were totally absent. Its ld₅₀, as determined by i.p. injection in mice, was 0·2 mg per kg body weight. The fraction did not possess any enzymatic, hemolytic or hemagglutinin activities of crude venom but showed a close resemblance to the major neurotoxin of Formosan cobra venom.     

Proteome and immunome of the venom of the Thai cobra, Naja kaouthia.

The proteome of the Thai cobra, Naja kaouthia, venom, revealed by two-dimensional liquid chromatography/tandem mass spectrometry, was found to consist of peptides which could be matched with 61 proteins in the database. These proteins were classified into 12 groups according to the differences in their biological activities: cardiotoxins, cobra venom factors, a cysteine-rich toxin, cytotoxins, kaouthiagin, mocarhagin, muscarinic toxin-like proteins, neurotoxins, an oxoglutarate dehydrogenase, phospholipases, serum albumin, and a weak toxin. Horse derived- anti-N. kaouthia venom hyperimmune serum currently used for the treatment of cobra ophitoxaemia reacted only to the cobra venom factors and phospholipases in the cobra holovenom by two-dimensional gel electrophoresis based-immunoblotting. The venom proteomic insight of this study should pave the way for preparing a therapeutic anti-venom of improved quality, i.e. also containing antibodies to the newly revealed toxic, but poorly immunogenic, minor venom components. It is expected that such a preparation should have a higher effectiveness than the currently used anti-venom in resuscitating cobra-bite victims.     

Non-lethal polypeptide components in cobra venom

Snakes from several genera (mostly from Naja genus) belonging to the Elapidae family are usually named cobras. The effect of cobra bites is mainly neurotoxic. This is explained by the presence of highly potent alpha-neurotoxin in their venoms. The other two highly toxic components of cobra venoms are cytotoxins and phospholipases A(2). These three types of toxins constitute a major part of cobra venom. They have attracted the attention of researchers for many years and have been very well studied and thoroughly described. However cobra venoms contain also many other less abundant components which possess very low toxicity or even are not toxic at all. These components, mostly proteins, belong to different structural and functional types, and the reason for their presence in the venom is not always evident. Some of them are known for many years (e.g., nerve growth factor and cobra venom factor); others (e.g., cysteine rich secretory proteins, CRISPs) were discovered only recently. There are non-lethal proteins with unique biological activities that can be used as biochemical tools, while others may be regarded as potential leads for drug design. This review is the first attempt to systemize the available data on non-lethal components of cobra venom.     

Comparative characterization of two toxic phospholipases A2 from Indian cobra (Naja naja naja) venom.

Indian cobra venom contains many phospholipase A2 (PLA2) toxins. In the present study two toxic PLA2s have been purified from the Indian cobra (Naja naja naja) venom by column chromatography. The NN-XIa-and NN-XIb-PLA2s have mol. wts between 10,700 and 15,000. The NN-XIa-PLA2 induces myotoxic effects, oedema and neurotoxicity in mice and has an i.p. LD50 of 8.5 mg/kg body weight. The NN-XIa-PLA2 is also cytotoxic to Ehrlich ascites tumour cells. The other PLA2, NN-XIb, in contrast has an i.p. LD50 of 0.22 mg/kg body weight, and it induces acute neurotoxicity. The NN-XIb-PLA2 is devoid of the other biological activities which are exhibited by NN-XIa-PLA2.     

Extraction of α‐neurotoxins from equine plasma by receptor based affinity purification

Venoms were first identified as potential doping agents by the racing industry in 2007 when three vials of cobra venom were seized during an inspection of a stable at Keeneland Racecourse in the USA. Venoms are a complex mixture of proteins, peptides, and other substances with a wide range of biological effects, including inhibiting the transmission of nervous and muscular impulses. As an example of this, cobratoxin, an α‐neurotoxin found in cobra venom, is claimed to be an effective treatment for pain. Recent analysis of seized samples identified venom from two different species of snake. Proteomic analysis identified the first sample as cobra venom, while the second sample, in a vial labeled “Conotoxin”, was identified as venom from a many banded krait. Cobratoxin, conotoxins, and bungarotoxins (a component of krait venom) are all α‐neurotoxins, suggesting a common application for all three venom proteins as potential pain blocking medications. Using a peptide based on the nicotinic acetylcholine receptor, a one‐step affinity purification method was developed for the detection of α‐neurotoxins in plasma.     

Isolation of nerve growth factor (NGF) from human body fluids; saliva, serum and urine: comparison between cobra venom and cobra serum NGF

Several investigators have isolated nerve growth factor (NGF) from various tissues and organs of different animals. There is no published documentation about NGF from body fluids, such as blood serum, saliva, and urine. Contrary to the unsuccessful attempts to detect or isolate NGF in serum in the past, this investigation reports the isolation of NGF from human serum, saliva, and urine. It further reports the comparison of properties between NGFs derived from cobra venom and cobra serum. NGF from serum, saliva, and urine was isolated by high pressure liquid chromatography (HPLC) and was identified as described by Lipps (1998). The identified fractions of NGF were further purified to study the biological and immunological properties. The biological activities of NGFs from human body fluids and cobra serum on PC12 cells were miniscule in comparison to the cobra venom derived NGF. The molecular weights of NGFs from human serum, saliva, and urine were identical, 36.0 kDa. However, the molecular weights of cobra serum and cobra venom NGFs were different, 55.0 kDa and 13.5 kDa, respectively. NGF level is age dependent and varies under different conditions. Using anti-human NGF, diagnostic tests can be developed for neurological disorders. This investigation also emphasizes the replacement of invasive blood collection for serum by use of saliva and urine for clinical diagnostic use in general.     

Preparation of complement fragments C3b and C3a from purified rat complement component C3 by activated cobra venom factor

INTRODUCTION: Complement component C3 (C3) can be a target of pharmacological or toxicological agents. In the analysis of this, it is important to examine the involvement of fragments C3b and C3a since C3 function normally requires cleavage into these fragments. The present study describes a simple and efficient method for the preparation of rat complement C3b and C3a by using purified C3 and cobra venom factor (CVF) as a cleaving enzyme. METHODS: CVF was purified from lyophilized cobra venom (Naja naja kausia) by two-step chromatography and was activated by incubation with human factors B and D. C3 was cleaved by incubation with activated CVF (CVF,Bb), and C3b and C3a were isolated by anion- and cation-exchange chromatography, respectively. RESULTS: About 200 microg of CVF was purified from 100 mg of cobra venom. All the CVF was activated by incubation with factors B and D. The C3b and C3a obtained were pure as analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and no digestive by-products such as C3f were found. DISCUSSION: The advantage of the present method is that it is possible to prepare relatively large amounts of C3b by simple procedures without digestive by-products. C3a can be prepared from the flow through fraction of the C3b purification. C3b and C3a prepared by the present method would be useful for pharmacological or toxicological experiments involving receptor binding since their binding sites remain intact.     

Effects of phospholipase A2 from cobra and bee venom on the presynaptic membrane

L. G. Magazanik, I. M. Gotgilf, T. I. Slavnova, A. I. Miroshnikov and U. R. Apsalon. Effects of phospholipase A₂ from cobra and bee venom on the presynaptic membrane. Toxicon17, 477–488, 1979.—Phospholipases A₂ from bee venom and cobra venom have been isolated and studied. A parallelism was found between enzymatic activity and the ability to block spontaneous miniature end-plate potentials (m.e.p.p.'s) or end-plate potentials (e.p.p.'s) induced by nerve stimulation in the frog sartorius muscle. Different experimental procedures affected both enzymatic activity and blocking ability in qualitatively the same way. Thus, modification of the histidine residue in cobra venom phospholipase by bromophenacyl bromide or the removal of Ca-ions from the medium abolished both activities. Replacement of Ca²⁺ by Sr²⁺ inhibited both the enzymatic and presynaptic effects of cobra venom phospholipase, but did not inhibit the presynaptic action of bee venom phospholipase and decreased its enzymatic activity only 6-fold. Irreversible binding of cobra and bee venom phospholipase to the presynaptic membrane was found in Ca-free solution but Ca-ions were essential for the presynaptic blocking effect induced by these phospholipases. A reduction in the effect of high K⁺ on m.e.p.p. frequency was observed after cobra venom phospholipase treatment. The similar effects of hypertonic sucrose solution and the mitochondrial poison TTFB (4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole) were changed only slightly by bee and cobra venom phospholipase pretreatment. It is concluded that the mechanism of presynaptic blockade induced by bee venom and cobra venom phospholipase consists mainly of damage to sites of release at the presynaptic membrane. There are also some signs of disturbances of depolarization-secretion coupling and of the process of formation of new quanta. The possible functional role of enzymatic activity in the presynaptic effect is discussed.     

Naja melanoleuca cobra venom contains two forms of complement-depleting factor (CVF)

Two forms of complement-depleting cobra venom factor (CVFm1 and CVFm2), possessing molecular masses of 142.6 kDa (CVFm1) and 143.1 kDa (CVFm2), according to MALDI mass-spectrometry, were isolated from the Naja melanoleuca cobra venom. As shown by polyacrylamide gel electrophoresis in the presence of SDS, both forms similarly to factor from the Naja kaouthia cobra venom (CVFk) consist of three polypeptide chains with molecular masses of about 70, 50, and 30 kDa, the two large subunits being glycosylated. As determined by MALDI mass-spectrometry, 30 kDa subunits of CVFm1 and CVFm2 have considerably different finger-prints of tryptic digests that suggests differences in their amino acid sequences. A study of activity in vivo has shown no significant differences in C3 consumption by CVFm1, CVFm2 and CVFk in mouse blood. However, as shown by an immunoassay method, they differ in their ability to activate the complement system via C3 conversion, the ratio of these activities for CVFm1:CVFm2:CVFk being 2.5:1.6:1. Kinetic studies using a hemolytic test showed that complement depletion by CVFm1 is faster than that by CVFm2. Thus, for the first time the presence in a single venom of two forms of CVF differing by both amino acid sequence and biological activity has been shown.