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     

眼镜王蛇神经毒素的镇痛作用研究

神经毒素(neurotoxin，NTX）是眼镜王蛇毒(Ophiophagus hannah Venom)的主要活性成分，我们应用离子交换层析、凝胶过滤及疏水层析等色谱方法从眼镜王蛇毒中分离纯化得到一种突触后神经毒素，暂定名为Neurotoxin-K(NTX-K)。经SDS-PAGE电泳鉴定为均一蛋白，分子量约为7776道尔顿，其小鼠静脉注射的LD50     

眼镜蛇毒L-氨基酸氧化酶分离纯化及对血管内皮细胞的抑制作用

目的从眼镜蛇毒中分离纯化L-氨基酸氧化酶(L-a-mino acid oxidase of Naja atra venom,NAV-LAAO),观察其对血管内皮细胞是否有抑制作用。方法应用阳离子交换层析和亲和层析方法分离纯化NAV-LAAO;采用MTT法、Western blot测定caspase及细胞小管形成实验观察NAV-LAAO对人脐静脉内皮细胞(HUVEC)生长和血管生成的影响。结果眼镜蛇毒粗毒经两步层析法得到的LAAO,分子量约为58 ku。NAV-LAAO抑制HUVEC细胞的生长和细胞小管形成,其抑制细胞生长的IC50为21.42 mg.L-1。与对照组相比,LAAO组caspase-3、caspase-8升高。结论应用两步层析法从眼镜蛇毒中分离出的NAV-LAAO具有剂量依赖性的抑制HUVEC细胞的生长和影响细胞小管形成的作用。     

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。     

中华眼镜蛇毒核糖核酸酶的分离纯化及其特殊生物学活性

目的从中华眼镜蛇毒中分离纯化核糖核酸酶,并研究其生物学活性。方法以中华眼镜蛇毒为原料,采用SP-Trisacryl阳离子交换色谱、Sephadex G-75凝胶色谱、C8反相色谱等纯化方法,分离纯化具有核糖核酸酶活性的蛋白质,表征其酶学性质,检测其抑菌性、抗肿瘤细胞作用及抗氧化作用。结果 SDS-PAGE电泳显示纯化的中华眼镜蛇毒核糖核酸酶(Na-RNase)为相对分子质量为13 000的单一成分。该酶最适反应温度为40℃,最适pH值为6.5,米氏常数为3.67μmol/L,最大反应速率为3.52 pmol/s。在体外抑菌实验中,在8μmol/L的浓度下对大肠艾希菌和金黄葡萄球菌均未显示出显著抑制作用;在体外细胞毒性实验中,对于Hela肿瘤细胞和正常的人成纤维细胞在8μmol/L的浓度下无明显抑制作用;在抗氧化实验中,浓度达到71.5μmol/mL时,对小鼠肝微粒体脂质过氧化反应的抑制率为41.30%。结论中华眼镜蛇毒中的核糖核酸酶具有抗氧化作用,可能作为氧化还原的调节剂,在有关的疾病治疗方面具有应用的潜能。     

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。     

发酵液中重组人血清白蛋白的纯化

采用摇瓶培养重组毕赤氏酵母（Pichia pastoris）表达并分泌重组人血清白蛋白（recombinant human se-rum alburmin，rHSA）至胞外，发酵液经（NH4）2SO4沉淀、离子交换层析、疏水层析和凝胶过滤分离纯化rHSA，纯化样品经SDS-PAGE检测为单一条带。     

中华眼镜蛇蛇毒神经生长因子生物活性和理化性质测定

目的：控制中华眼镜蛇蛇毒神经生长因子产品质量,研究其理化性质及生物学活性的定性和定量。方法：通过离子交换色谱、凝胶过滤及FPLC色谱分高纯化得到中华眼镜蛇蛇毒神经生长因子,按国家新药审批有关要求对其进行了SDS－PAGE电泳,N端蛋白质序列规定,HPLC色谱分析,UV光谱图谱扫描,并利用PC12细胞培养法和鸡胚背根神经节培养法检测其生物活性。结果：电泳为一条带,亚基分子量为13500,N端蛋白质序列测定后确证为神经生长因子（NGF）,HPLC为单峰,相对百分含量为95％以上,279．6nm处呈现出蛋白质样特征吸收峰。生物活性测定为,PC12细胞培养法灵敏度可达1ng／ml,鸡胚背根神经节培养法需30ng／ml的浓度梯度才能在神经节上有所反应。结论：此实验样品为具有较高生物活性的高纯度NGF多肽。     

大鼠再生肝中肝细胞生长因子的纯化和生物活性的研究

用大鼠的再生肝经过匀浆、加热、高速离心和酒精沉淀获得粗制的肝细胞生长因子（RHGF－1），可检测出肝细胞生长因子的活性。RHGF－1再经DEAE离子交换层析，Sephacryls-300凝胶柱层析及FPLCMonoQ色谱，纯化为单体（RHGF－4），经SDS－PAGE证实为单一成份，分子量为18KD。此纯化的RHGF－4相对活性比RHGF－1提高884倍。     

HPLC多柱系统在重组人肿瘤坏死因子衍生物鉴别分析中的应用

采用反相高效液相色谱（ＲＰ－ＨＰＬＣ）、高效凝胶过滤色谱（ＨＰＳＥＣ）、高效疏水相互作用色谱（ＨＰＨＩＣ）和高效离子交换色谱（ＨＰＩＥＣ）及肽图谱测定等技术对国内３个厂家的重组人肿瘤坏死因子样品进行鉴别和同一性检定，对样品中存在的杂质峰给予了解释．为同类基因工程产品的质量控制提供了切实可行的方法和实验依据．     

L-amino acid oxidase from Vipera lebetina venom: isolation, characterization, effects on platelets and bacteria.

The L-amino acid oxidase from Vipera lebetina venom was purified to homogeneity using combination of size exclusion, ion exchange and hydrophobic chromatography. The monomeric molecular mass of the homodimeric enzyme is 60.9kDa. The N-terminal and the tryptic peptides share high homology with other snake venom L-amino acid oxidases. The enzyme displays high specificity towards hydrophobic L-amino acids, the best substrates are L-Met, L-Trp, L-Leu followed by L-His, L-Phe, L-Arg and L-Ile. Six substrates-Gly, L-Ser, L-Thr, L-Pro, L-Cys, L-Asp--were not oxidized. The enzyme has antimicrobial activity inhibiting the growth of both Gram-negative and Gram-positive bacteria. V. lebetina LAAO dose-dependently inhibited platelet aggregation induced by ADP or collagen. In case of ADP-induced aggregation the inhibitory effect was more pronounced on the second wave of aggregation.     

Cytotoxicity and L-amino acid oxidase activity of animal venoms

The cytotoxicity of animal venoms (snakes, insects and marine animals) was measured against SNU-1 (stomach cancer cells) by dye uptake assay (MTT method). And also L-amino acid oxidase (AAO) activity of the venoms was compared. Among them, the venom fromOphiophagus hannah (king cobra) showed a strong AAO activity as well as a high potent cytotoxicity. Cytotoxic protein having a AAO was then partially purified by HPLC-GPC and two fractions (Fr. I and Fr. II) were collected. The IC₅₀ values of Fr. I and Fr. II were 0.19 μg/ml and 1.36 μg/ml, respectively. The results suggested that the cytotoxicity of king cobra venom may be due to its AAO activity.     

眼镜蛇毒L-氨基酸氧化酶对人膀胱癌ECV304细胞增殖和细胞周期的影响及机制

目的研究眼镜蛇毒L-氨基酸氧化酶(Naja atra L-a-mino acid oxidase,NA-LAAO)对人膀胱癌细胞株ECV304的生长抑制作用,探讨其作用机制。方法 CCK-8法测定NA-LAAO的细胞毒性及对细胞增殖抑制能力;流式细胞术分析NA-LAAO对ECV304细胞周期的影响;实时定量PCR和Western blot分析细胞周期相关基因mRNA和蛋白质表达。结果 NA-LAAO对ECV304细胞有强烈抑制作用,且呈剂量-效应和时间-效应关系。NA-LAAO处理6、12、24 h对ECV304的IC50分别为(1.54±0.23)、(1.09±0.15)和(0.48±0.14)mg.L-1;细胞生长曲线显示,0.156 mg.L-1的NA-LAAO作用4 d可明显抑制ECV304细胞增殖;0.313 mg.L-1从d 2起就有明显效果;0.625 mg.L-1处理后细胞多数死亡;流式细胞术分析提示NA-LAAO可使ECV304细胞阻滞在G1期,并呈量效关系;实时定量PCR结果显示,NA-LAAO可下调Cyclin A2、B1、D1、E1、CDK2、CDK6、Survivin、Skp2,上调p21、p16、p27的表达,Western blot结果显示NA-LAAO可下调Cyclin A、CDK2、CDK6,上调p21,与实时PCR结果一致。结论 NA-LAAO可通过调节细胞周期相关分子,使ECV304细胞阻滞在G1期,抑制细胞增殖。     

Antitumoural effect of an L-amino acid oxidase isolated from Bothrops jararaca snake venom

An L-amino acid oxidase (BjarLAAO-I) from Bothrops jararaca snake venom was highly purified using a stepwise sequential chromatography on Sephadex G-75, Benzamidine Sepharose and Phenyl Sepharose. Purified BjarLAAO-I showed a molecular weight around 60,000 under reducing conditions and about 125,000 in the native form, when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. BjarLAAO-I is a homodimeric acidic glycoprotein, pI approximately 5.0, and N-terminal sequence showing close structural homology with other snake venom LAAOs. The purified enzyme catalysed the oxidative deamination of L-amino acids, the most specific substrate being L-Phe. Five amino acids, L-Ser, L-Pro, L-Gly, L-Thr and L-Cys were not oxidized, clearly indicating a significant specificity. BjarLAAO-I significantly inhibited Ehrlich ascites tumour growth and induced an influx of polymorphonuclear cells, as well as spontaneous liberation of H(2)O(2) from peritoneal macrophages. Later, BjarLAAO-I induced mononuclear influx and peritoneal macrophage spreading. Animals treated with BjarLAAO-I showed higher survival time.     

Molecular characterization of L-amino acid oxidase from king cobra venom.

An L-amino acid oxidase from Ophiophagus hannah snake venom (Oh-LAAO) was purified by successive gel filtration, ion-exchange and heparin chromatography. Oh-LAAO did not induce platelet aggregation; however, it had potent inhibitory activity on platelet aggregation induced by ADP and U46619, but showed no effect on platelet aggregation induced by thrombin, mucetin, ristocetin and stejnulxin. By RT-PCR and 5'-RACE methods, the complete Oh-LAAO cDNA was cloned from the venom gland total RNA preparations. The cDNA sequence contains an open-reading frame (ORF) of 1476-bp, which encodes a protein of 491 amino acids comprising a signal peptide of 25 amino acids and 466-residue mature protein. The predicted protein sequence of Oh-LAAO was confirmed by N-terminal and trypsin-digested internal peptides sequencing together with peptide mass fingerprinting. cDNAs encoding for ORF of LAAOs from Bungarus fasciatus and B. multicinctus were cloned and reported in this study. In addition, partial cDNA encoding for Naja atra LAAO was also reported. Oh-LAAO shared approximately 50% protein sequence identity with other known snake venom LAAOs. Phylogenetic analysis indicated that Oh-LAAO is evolutionary distant to other snake venom LAAOs.     

眼镜蛇毒L-氨基酸氧化酶诱导人胃癌细胞MGC-803细胞凋亡的实验研究

目的:研究眼镜蛇毒L-氨基酸氧化酶(NA-LAAO)对人胃癌细胞MGC-803的细胞毒性、诱导凋亡作用及可能机制。方法:采用CCK-8法测定细胞毒性;采用DNA倍体分析和An-nexin V/碘化丙啶染色测定细胞凋亡;采用发色底物法测定Caspase-3酶活性;采用Western-blot方法测定聚二磷酸腺苷核糖多聚酸-1(PARP-1)切割。结果:NA-LAAO可抑制MGC-803细胞增殖,6、12、24h的IC50分别为(2.48±0.41)、(1.74±0.27)和(0.83±0.19)mg/L;NA-LAAO可使细胞DNA分布出现亚二倍体凋亡峰,并可促使细胞膜磷脂酰丝氨酸外翻;NA-LAAO可激活Caspase-3并可促使其底物PARP-1降解。结论:NA-LAAO可能通过激活Caspase-3这一分子机制而诱导细胞凋亡,从而抑制肿瘤细胞增殖。     

Enzymatic activities of Malayan cobra (Naja naja sputatrix) venoms

The enzymatic activities of four samples of Malayan cobra venom were investigated. There was significant variation in the contents of L-amino acid oxidase, alkaline phosphomonoesterase, acetylcholinesterase, phospholipase A, 5'-nucleotidase and hyaluronidase. The phosphodiesterase content was, however, constant. Storage of the lyophilized venom powder at 25 degrees C for 1 month did not affect the enzymatic activities. The venom enzymatic activities were generally also stable at 4 degrees C in 0.85% saline solution. After incubation at 37 degrees C for 39 days in 0.85% saline solution, the venom still retained considerable amounts of enzymatic activities. SP-Sephadex C-25 ion-exchange chromatography of the venom showed that the phospholipase A, L-amino acid oxidase, 5'-nucleotidase, phosphodiesterase and alkaline phosphomonoesterase exist in multiple forms.     

L-amino acid oxidase from Trimeresurus jerdonii snake venom: purification,characterization, platelet aggregation-inducing and antibacterial effects

An L-amino acid oxidase (LAO), designated as TJ-LAO, was purified to homogeneity from the venom of Trimeresurus jerdonii by Sephadex G-100 and Q Sepharose HP chromatography. The molecular weight of this enzyme was 110 kD as estimated by analytical gel filtration and was 55 kD by SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is composed of two subunits. The enzyme has an absorption spectrum characteristic of flavoproteins, containing 2 moles of FMN per mole of enzyme. The N-terminal sequence of TJ-LAO shares high homology with other viperid snake venom LAOs. Homology with elapid venom LAO is lower. TJ-LAO inhibited the growth of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus megaterium. The antibacterial effect associated with LAO activity was elminated with the addition of catalase. Platelets in platelet-rich plasma aggregated upon the addition of TJ-LAO. The enzyme-induced aggregation was inhibited by catalase, suggesting formation of H2O2 was essential for TJ-LAO to induce platelet aggregation. These results showed H2O2 formation is important for the biological effects of LAO.     

Purification and pharmacological characterization of a cardiotoxin-like protein from Formosan banded krait (Bungarus multicinctus) venom

By ion exchange chromatography followed by gel filtration, a polypeptide toxin was purified from Formosan banded krait (Bungarus multicinctus) venom. The toxin had a molecular weight of 7000 ± 300 and showed an amino acid composition characteristic of cardiotoxin from cobra venom. The i.p. ld₅₀ value of the toxin was 2.5 (1.9–3.2) mg per kg mouse. Pharmacological studies showed that the toxin (10 μg/ml) induced contracture in chick and mouse skeletal muscles, depolarized the cell membrane of the mouse diaphragm, arrested the contraction of spontaneously beating atria and the electrically driven ventricle strip of the rat, and caused direct hemolysis of guinea-pig erythrocytes. From these chemical and pharmacological characterizations it was concluded that this toxin has characteristics similar to those of cobra venom cardiotoxins.