舟山眼镜蛇毒神经毒素的分离纯化及镇痛作用研究

目的从舟山眼镜蛇毒中分离纯化神经毒素,并测定其部分理化性质及镇痛活性。方法应用SP-Sephadex C-50离子交换色谱法分离眼镜蛇毒粗毒,用Sephadex G-50凝胶过滤色谱、CM-Sephadex C-25离子交换色谱纯化神经毒素;SDS-PAGE(Tris-Tricine系统)鉴定纯度;用Meier方法测定毒性;用热板法观察神经毒素的镇痛作用。结果应用SP-Sephadex C-50离子交换柱层析,从舟山眼镜蛇毒中分离得到14个蛋白峰,其中6个组分(NNAV-Ⅶ ~Ⅻ)经鉴定为含有神经毒素组分,将神经毒活性最强的组分XI经Sephadex G-50、CM-Sephadex C-25纯化得神经毒素纯品NTⅪ。该纯品分子量12.3kD,小白鼠静脉注射和腹腔注射的LD50分别为1.9875、2.2217 mg·kg-1。NTXI能显著提高小鼠对热板刺激的痛阈。腹腔注射0.2mg·kg-1NTXI可使小鼠的热板痛阈提高84.35%。NTXI的镇痛作用具有时效性,给药后2h起效,4h作用达峰值。结论舟山眼镜蛇毒中分离得到一个神经毒素纯品NTⅪ,具有镇痛作用。     

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。     

皖南尖吻蝮蛇蛇毒中抗肿瘤活性蛋白分离纯化及其活体外性研究

目的:分离纯化皖南尖吻蝮蛇蛇毒(Wannan Agkistrodon acutus venom)中抗肿瘤活性蛋白并研究其活性。方法:利用DEAE-sepharose Fast Flow和SP-sepharose Fast Flow阴阳离子交换层析以及Sephadex G-75凝胶过滤层析等分离方法从皖南尖吻蝮蛇蛇毒中分离纯化得一种抗肿瘤活性蛋白,用CCK-8法检测该蛋白对体外培养的白血病K562细胞、结肠癌细胞(SW480)、胃癌细胞(SGC7901)、肝癌细胞(HepG2)的增殖抑制作用。结果:分离纯化得一相对分子质量约23700的抗肿瘤活性组分(ATF1-c),CCK-8检测对体外培养的K562、SW480、SGC7901、HepG2细胞的增殖抑制作用,呈剂量-时间依赖关系。结论:ATF1-c对体外培养的人癌细胞有明显的抑制和杀伤作用。     

长白山白眉蝮蛇蛇毒中类凝血酶的分离纯化方法研究

目的:研究长白山白眉蝮蛇蛇毒中类凝血酶的简单分离纯化方法。方法:采用DEAE-Sephadex A-25及Sephadex G-25层析的方法,比较二者对长白山白眉蝮蛇蛇毒中类凝血酶简单分离纯化的效果。结果:从长白山白眉蝮蛇蛇毒中分离出类凝血酶,SDS-PAGE电泳显示为一条带,分子量大约为35.5kDa,达到电泳纯。理化性质研究表明,此类凝血酶具有体外凝血活性,体外凝血酶比活力为12.57IU.mg-1,用N-苯甲酰-L-精氨酸乙酯盐酸盐测得该酶的精氨酸酯酶比活力为137.65IU.mg-1。用蛋白酶抑制剂和乙二胺四乙酸对该酶进行抑制实验,结果表明该酶属于丝氨酸蛋白酶,而不是金属蛋白酶。结论:本方法可用于长白山白眉蝮蛇蛇毒中类凝血酶的分离纯化。     

蝰蛇(缅甸亚种)毒促凝—纤溶双相活性组分FⅥbb的分离纯化及生物活性

目的从蝰蛇(缅甸亚种)毒分离纯化一种促凝—纤溶双相活性组分FⅥbb,并研究其理化性质和生物活性。方法应用CM-Sephadex C-50阳离子交换层析Sephadex G-150(超细)凝胶过滤层析Chelating Sepharose Fast Flow金属离子螯合亲和层析分离纯化蛇毒,反相HPLC检测组分FⅥbb纯度,采用MALDI质谱测定法测定分子量,以发色底物法、纤维平板法和SDS-PAGE测定FⅥbb的酶学特征和生物活性。结果从蝰蛇(缅甸亚种)毒分离得到的促凝—纤溶双相活性组分FⅥbb为单体蛋白,相对分子质量为59 138,最适温度为40℃,最适pH为10.0,为金属蛋白酶。结论应用CM-Sephadex C-50阳离子交换层析和Chelating Sepha-rose Fast Flow金属离子螯合亲和层析等方法可以从蝰蛇(缅甸亚种)毒纯化出促凝—纤溶双相活性组分FⅥbb,具有促凝—纤溶双相活性。     

大麦根磷脂酶的分离纯化

考察了Sephadex G-100凝胶色谱法和CM-Sephadex C-50离子交换色谱法分离纯化大麦根磷脂酶的条件.结果显示,用Sephadex G-100凝胶色谱分离磷脂酶,得到2个洗脱峰,其中仅S1峰有酶活力,SDS-PAGE电泳检测S1峰有5条蛋白条带,纯度较浓缩粗酶液约提高了3.9倍,酶活力回收率为40.51%.用CM-Sephadex C-50离子交换色谱分离,得5个洗脱峰,其中仅C3峰有酶活力,SDS-PAGE电泳检测该活力峰仅1条蛋白条带,相对分子质量约5.28×104,纯度为浓缩酶液的8.8倍,酶活力回收率为16.3%.     

五步蛇毒纤溶组分FⅨcaⅠ的分离纯化和生物活性的测定

目的从五步蛇(安徽产)毒分离纯化一种具有纤溶活性的组分FⅨcaⅠ,并研究其理化性质和生物活性。方法应用DEAE-Sephadex A-50阴离子交换层析、Sephadex G-75凝胶层析、Chelating Sepharose Fast Flow金属离子螯合亲和层析和Sephadex G-50凝胶层析四步分离纯化目的组分FⅨcaⅠ;纤维蛋白平板法和SDS-PAGE测定FⅨcaⅠ的生物活性;通过小鼠皮下注射不同剂量的FⅨcaⅠ,测量皮下出血斑的面积并求出最小出血剂量。结果从五步蛇毒分离纯化的FⅨcaⅠ组分为单体蛋白,相对分子量23 ku,等电点为4.8。FⅨcaⅠ可降解纤维蛋白和纤维蛋白原,优先降解α链,呈时效、量效关系。蛇毒纤溶酶FⅨcaⅠ最小出血剂量为54.9μg,为非出血性蛇毒纤溶酶。结论从五步蛇(安徽产)毒分离纯化的FⅨcaⅠ是一种分子量较小,比较稳定,纤溶活性强,可直接降解纤维蛋白且非出血性的新蛇毒纤溶酶。     

用亲和层析法分离纯化降纤酶

目的用亲和层析法从尖吻蝮蛇蛇毒中分离纯化降纤酶。方法用离子交换色谱、单克隆抗体亲和色谱技术进行分离纯化。结果得到电泳纯的降纤酶，其分子量为36000。结论用单克隆抗体亲和色谱法纯化降纤酶是切实可行的。     

广东眼镜蛇毒低分子量多肽的分离纯化与性质鉴定

目的从广东眼镜蛇粗毒中分离纯化出色谱纯的低分子量多肽并对其部分性质进行鉴定。方法采用化学沉淀法对蛇粗毒进行预处理,经Sephadex G-50凝胶过滤和UND Sphere S阳离子交换层析得到高纯度低分子量多肽,用SDS-PAGE及HPLC对产物纯度进行鉴定,并用SDS-PAGE和质谱法测定分子量,采用蛙心灌流法考察产物是否具有心脏毒性。结果结果表明,经分离纯化后得到两种低分子量多肽GI2和GI3,均为单一组分,可达到色谱纯,SDS-PAGE法测得GI2分子量为14 300 Da,质谱法测得GI3分子量为6 725.8 Da,仅GI2具有心脏毒性,收率分别为9.5%和10.1%。结论通过化学沉淀预处理,并经2次柱色谱可从广东眼镜蛇毒中分离得到两种色谱纯的低分子量多肽,且收率较高。     

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。     

Isolation and some biochemical properties of a paralysing toxin from the venom of the wasp Microbracon hebetor (Say)

A toxin with paralysing activity was isolated from crude preparations of Microbracon hebetor (Say) venom by ion exchange chromatography on DEAE-Sephadex A-50, gel chromatography on Sephadex G-100, electrophoresis on polyacrylamide gel and gel chromatography on Sephadex G-75. The active component is very labile. A 28-fold purification was obtained with a recovery of about 2.5% of the original biological activity. The toxin shows one single protein band after disc electrophoresis. The molecular weight was assessed at 61,000 by gel chromatography, and at 62,700 by the sedimentation equilibrium method. The isoelectric point was at pH 6.8. The purified toxin was inactivated by a number of proteolytic enzymes.     

霍乱毒素B亚单位与胰岛素B链结构类似多肽融合蛋白的制备

为了在大肠杆菌中表达霍乱毒素B亚单位(Cholera toxin B subunit,CTB)与胰岛素B链(9-23)肽段结构类似多肽(Ins B(9-23))的融合蛋白,使用基因工程方法构建了CTB与Ins B(9-23)的融合基因CTB-Ins B-X若干,并将融合基因克隆到大肠杆菌表达载体pET28a(+)中,获得的重组质粒转化大肠杆菌BL21(DE3)。重组菌株经乳糖诱导后,其表达产物经过12%SDS-PAGE分析表明该菌株可以包含体形式表达融合蛋白,并制备得到纯度较高的重组蛋白。该重组蛋白的包含体经过变性、复性后,可以在体外自组装成五聚体结构。GM1-ELISA分析结果表明,重组蛋白在体外可以与神经节苷脂GM1(monosialoganglioside)特异性结合,表明该融合蛋白保持了CTB形成五聚体的生物活性。     

Partial purification of a toxin from tentacles of the sea anemone Anemonia sulcata

A toxin of low molecular weight was isolated from tentacles of the sea anemone Anemonia sulcata P. Partially purified toxin was obtained by precipitation with ammonium sulphate, ion exchange chromatography on DEAE cellulose and chromatography on Sephadex G-50. The molecular weight of a partially purified toxin, probably a basic protein was approximately 6000 as determined by gel filtration. Disc electrophoresis of the partially purified toxin revealed four proteins. Heating for 15 min at 90°C inactivated the toxin. The toxic activity remained unchanged over 12 months when stored in frozen solution at ?26°C. The ld₁₀₀ is 6 mg of the toxin per kg (rat) by i.v. injection. The toxin showed a cardiotropic action on the rat heart. When injected i.v. the toxin caused cardiac arrhythmia and fall of the arterial blood pressure.     

新疆葡萄干超氧化物歧化酶的分离提纯

本文首次对新疆葡萄干中超氧化物歧化酶进行了分离提纯.酶活力测定采用微量邻苯三酚自氧化法.结果表明:100g新疆葡萄干经粗抽提,丙酮两步沉淀,Sephadex G-100柱层析,DEAE-52柱层析,得2.73mg酶蛋白,比活为408.8u/mg pro,纯化倍数为92.9,达电泳纯.     

弹性蛋白水解物的制备

用碱性蛋白酶水解牛颈部韧带弹性蛋白,酶促反应条件是:pH10,温度不高于45℃,尿素浓度0.37M,反应时间10～11h。经葡聚糖凝胶 G-50层析制得的弹性蛋白水解物平均分子量1000～5000,主要成分是酸性多肽。     

螺旋藻多糖的化学研究

螺旋藻多糖的化学研究曾和平，郭宝江（华南师范大学化学系，广州５１０６３１）螺旋藻（ＳＰｉｒｕｌｉｎａｐｌａｔｅｎｓｉｓ）为蓝藻门、颤藻纲、螺旋藻属，含有极丰富营养成分和有抗辐射、抗肿瘤及调节免疫功能的耐卤藻类。近几年的研究表明，螺旋藻中抗肿瘤和免疫调...     

Isolation and characterization of a toxic protein from Canavalia ensiformis (jack bean) seeds,distinct from concanavalin A

A toxic protein present in the crude extract of Canavalia ensiformis seeds induces within 24 hr dyspnoea, ataxia, hypothermia, coma, tonic convulsions and death in mice injected i.p. with 100–200 mg of protein per kg. The toxin was separated from concanavalin A by affinity adsorption of the latter on Sephadex G-100, and further purified by sequential fractionation by (a) removal of polysaccharides with 30% v/v ethanol; (b) ammonium sulfate precipitation at 0.35–0.55 saturation and (c) DEAE-cellulose chromatography. The purified toxin, with an ld₅₀ of 2–5 mg of protein/kg mouse, is unstable. Gel-filtration of the purified toxin on Bio-Gel P-200 showed a single protein peak with a molecular weight corresponding to 88,000 daltons. Polyacrylamide gel electrophoresis of this material indicated the presence of two small contaminants. A central convulsive effect of the toxin can be proposed since no effects were found on isolated pharmacological preparations. The name CANATOXIN is suggested for this new toxic protein.     

An anticoagulant serine protease from the wasp venom of Vespa magnifica.

Wasp is an important venomous animal that can induce human fatalities. Coagulopathy is a clinical symptom after massive wasp stings, but the reason leading to the envenomation manifestation is still not known. In this paper, a toxin protein is purified and characterized by Sephadex G-75 gel filtration, CM-Sephadex C-25 cationic exchange and fast protein liquid chromatography (FPLC) from the venom of the wasp, Vespa magnifica (Smith). This protein, named magnvesin, contains serine protease-like activity and inhibits blood coagulation. The cDNA encoding magnvesin is cloned from the venom sac cDNA library of the wasp. The deduced protein from the cDNA is composed of 305 amino acid residues. Magnvesin shares 52% identity with allergen serine protease from the wasp Polistes dominulus. Magnvesin exerted its anti-coagulant function by hydrolyzing coagulant factors TF, VII, VIII, IX and X.     

多相脂质体139—2号注射液的包封率测定(SePhadex凝胶柱-荧光法)

本文研究了多相脂质体139-2号注射液中唐松草新碱(TD)的包封率测定法。建立了凝胶柱-荧光法。考察了Sephadex G-50凝胶柱对139-2号注射液中脂质体等微粒和药物分子的分离能力、凝胶柱对脂质体等微粒的回收率、包封率的测定等。操作简便,结果可靠,重现性较好。适用于139-2号注射液的包封率质量控制。