舟山眼镜蛇毒神经毒素的分离纯化及镇痛作用研究

Purification,characterization and analgesic activity of the neurotoxins from Naja atra venom

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．SephadexC-50离子交换色谱法分离眼镜蛇毒粗毒，用SephadexG-50凝胶过滤色谱、CM—SephadexC-25离子交换色谱纯化神经毒素；SDS．PAGE（Tfis-Tfieine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—SephadexC-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Ⅻ经SephadexG-50、CM．SephadexC-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LDso分别为1．9875、2．2217mg·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。

OBJECTIVE To isolate and purify neurotoxin from Naja atra venom and further appraise its properties and analgesic activity. dex C-50. The fraction whi adex C-25. The purity METHODS Separation of whole venom by ion-exchange chromatography on SP-Sephach contain neurotoxin was purified by successive separation on Sephadex G-50, CM-Sephneurotoxin of was appraised by SDS-PAGE（ Tris-Tricine system）. The LD50 of neurotoxin in mice were determined by Meier and Theakston method. The analgesic activity of neurotoxin was observed by hot-plate test. RESULTS Ion-exchange chromatography of Naja atra venom yielded 14 fractions（ Ⅰ - ⅩⅣ）. The neurotoxin was purified from fraction Ⅺ which has most neurotoxic activities by gel filtration, ion-exchange, named NTXI. NTXI migrated as a single band on SDS-PAGE. The molecular weight of NTXI was 12. 3kDa. The LDso of NTXI in mice （i. v. and i. p. ） were determined as 1. 9875,2. 2217 mg · kg^-1 respectively. The hot-plate test showed that NTXI had analgesic activity. An i. p. dose of 0. 2 mg · kg-lincreased latency time to 84. 35 percent. In the hot-plate test, the analgesic effect of NTXI effected at 2hr and peaked at 4 hr. CONCLUSIONS NTXI obtained by ion exchange, gel filtration chromatography from Naja atra venom had analgesic activity.