改良Blue Native PAGE方法的建立及对注射用A型肉毒毒素活性药用成分天然结构的分析

Establishment of Improved Blue Native PAGE and Analysis for Native Structure of Botulinum Toxin Type A for Injection

建立具有较高分辨率的温和的Blue native PAGE方法,用于分析国产注射用A型肉毒毒素(商品名为衡力■)复合物活性药用成分(Pharmaceutical active ingredient,API)天然结构。通过改进样品处理方法解决蛋白样品分解的问题。由7种电极缓冲液构成8个组合,从中筛选出最优组合并改进染色方法,电泳结果的分辨率明显提高。以基于改良Blue native PAGE进行二维SDS-PAGE,以基于二维电泳的Western blotting对各组分定性分析,以改良对Blue Native PAGE对A型肉毒毒素API天然结构相对分子质量进行测定。改良Blue Native PAGE方法能够分析注射用A型肉毒毒素API天然结构,明确了注射用A型肉毒毒素API天然结构的存在形态及组成,注射用A型肉毒毒素API相对分子质量为(918±18. 38) k。

Method of high resolution ratio and mild Blue native PAGE was established in order to analyze the native structure of Botulinum toxin type A for injection of pharmaceutical active ingredient( API). To resolve the difficulty of sample decomposing,the treatment method of sample was optimized. 7 kinds of electrode buffer were compounded to arrange 8 groupes combination and filtered the optimal group of electrode buffer. Finally,improving the methods of stain. The resolution ratio was improved obviously. Qualitative analysis to components were performed successively by means of two dimensional Native/SDS-PAGE based on improved Blue native PAGE and Western blotting based on two dimensional Native/SDS-PAGE. The relative molecular weight of native structure of Botulinum toxin type A API was measured by improved Blue native PAGE. The improved Blue native PAGE clarified the structure and composition of native structure of Botulinum toxin type A API for injection. The molecular weight of native structure of Botulinum toxin type A API is( 918 ± 18. 38) k.