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

Purification of cytotoxin from Naja naja atra venom and its antitumor activity in vitro

PurposeTo isolate and purifiy cytotoxin from Naja naja atra venom.MethodsSeparation of whole venom by gel filtration chromatography on Sephadex G 50 .Separation the fraction Ⅳ which contain cytotoxin by cation exchange chromatography on SP Sephadex C 50. Purification of the cytotoxic fraction by gel filtration chromatography on Superdex 75 and hydrophobic interaction chromatography on Phenyl Sepharose HP respectively. The inhibition of cytotoxin on the cultured tumour cells were tested by Sulforhodamine B(SRB).ResultsGel filtration of cobra venom on Sephadex G 50 yield 4 fractions, of which fraction Ⅳ had cytotoxic activity. Chromatography of fraction Ⅳ on SP Sephadex C 50 produced A,B,C,D,E,F and G fractions, of which E,F and G had cytotoxic activity. Fraction F was purified by Superdex 75 and Phenyl Sepharose HP. The product, named CTX F exhibited no detectable phospholipase A 2(PLA 2) activity, migrated as a single band on SDS polyacryamide gel electrophoresis (SDS PAGE). CTX F showed a potent antitumor activity on the cultured tumor cells.ConclusionCTX isoforms containing no PLA 2 activity can be obtained by gel filtration, cation exchange chromatography and hydrophobic interaction chromatography.