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目的:分离纯化东亚钳蝎原毒,提取活性多肽成分SVⅡ-A4,并观察其抑瘤活性。方法:(1)分离纯化,采用分子筛凝胶柱层析和离子交换柱层析分离纯化蝎毒;(2)纯度分析,用SDS-PAGE电泳和HPLC色谱法对SVⅡ-A4进行纯度分析;(3)活性观察,用MTT法(酶反应比色法)观察比较不同浓度(1、10、100 mg/L)的蝎毒及其成分与SVⅡ-A4对4种肿瘤细胞(A549、HL-60、K562/S及K562/ADR)的抑制作用。结果:经过两步的分离纯化获得5个蝎毒多肽成分,其中的蝎毒抗肿瘤有效成分SVⅡ-A4对4种肿瘤细胞的抑制作用与阳性对照组相当。结论:经过优化提纯方法,两步即可从东亚钳蝎原毒中获得色谱纯度达96.73%的抑瘤活性单体成分SVⅡ-A4,初步观察,SVⅡ-A4对4种肿瘤细胞均显示出明显的抑制作用及一定的剂量-效应关系,而且它对布耐药细胞株(K562/ADR)的抑制作用较阳性对照组强有进一步开发利用的价值。
OBJECTIVE: To isolate and purify virulent venom from Scorpion, and to extract SVⅡ-A4, an active polypeptide, and observe its antitumor activity. Methods: (1) Separation and purification, separation and purification of scorpion venom using molecular sieve gel column chromatography and ion exchange column chromatography; (2) Purity analysis, purity of SVⅡ-A4 was analyzed by SDS-PAGE and HPLC; 3) The activity was observed by MTT method (enzyme reaction colorimetric method) to observe the effects of different concentrations (1, 10, 100 mg / L) of scorpion venom and its components and SVⅡ-A4 on four kinds of tumor cells , K562 / S and K562 / ADR). Results: Five scorpion venom peptide fractions were obtained after two steps of purification. The inhibitory effect of the scorpion venom antitumor active ingredient SVⅡ-A4 on the four tumor cells was similar to that of the positive control group. CONCLUSION: SVⅡ-A4, an anti-tumor active monomer with a chromatographic purity of 96.73%, can be obtained in two steps from virulent venom of Asarum And its dose-effect relationship, and its inhibitory effect on the Bcl-2-resistant cell line (K562 / ADR) is more valuable than the positive control group for further development and utilization.