聚乙二醇修饰海葵神经毒素rhk2a分离及纯化.docVIP

聚乙二醇修饰海葵神经毒素rhk2a分离及纯化 　 作者：黄慧,潘育方,郭晓娟,舒雅俊,杨帆 【摘要】 目的 对聚乙二醇化海葵神经毒素(mPEGrhk2a)修饰产物进行分离与纯化。方法 将聚乙二醇(PEG)化反应所得混合产物超滤除盐后，采用SP650S强阳离子交换层析柱和CM650S弱阳离子交换层析柱进行分离纯化，收集不同NaCl离子浓度下的梯度洗脱液，经超滤除盐、冷冻干燥、SDSPAGE电泳检测，确定分离纯化mPEGrhk2a的色谱条件。结果 在保证mPEGrhk2a稳定性的前提下，随着缓冲液pH值的降低，2种阳离子交换柱与mPEGrhk2a的结合量均增加;在同一pH值下，强阳离子交换柱的结合情况更好。用SP650S强阳离子交换层析柱进行分离纯化时，洗脱液中溶液电导率在5～7 ms/cm时,mPEGrhk2a能被洗脱下来，而且盐离子浓度梯度变化越缓，mPEGrhk2a修饰产物各洗脱峰的分离度越高;用CM650S弱阳离子交换层析柱进行分离纯化时，修饰产物mPEGrhk2a主要在穿透峰中出现。结论 使用SP650S强阳离子交换层析柱，用pH 4.0的0.02 mol/L醋酸盐缓冲液与含0.5 mol/L NaCl 的pH 4.0的0.02 mol/L醋酸盐缓冲液(后者比例变化为0%→50%)进行梯度洗脱，修饰产物mPEGrhk2a与未修饰的rhk2a得到了很好的分离。 【关键词】 聚乙二醇化海葵神经毒素;阳离子交换层析;分离;纯化 Abstract:Objective To isolate and purify mPEGrhk2a.Methods The mixture obtained from pegylated reactions was separated by Toyopearl SP650 column for strong ion exchange and Toyopearl CM650 column for weak cation exchange after desalted by ultrafiltration and the effluent of gradient elution was collected at various concentrations of sodium chloride,ultrafiltrated and freezedried,detected by SDSPAGE electrophoresis to determine chromatographic condition of isolate and purify mPEGrhk2a. Results With the preconditions of keeping mPEGrhk2a stable and reduced pH of buffer solutions,the binding capacity of both cation exchange columns and mPEGrhk2a increased. However,the capacity of the strong cation exchange was more than the week one. MPEGrhk2a was eluted by Toyopearl SP650 column for strong cation exchange when the concentration of NaCl was in the range of 5%～7%. The more slowly the concentration gradient changed,the higher of the resolution of diverse eluting peaks amongst modified products was. When using the Toyopearl CM650 column for weak cation exchange,the mPEGrhk2a mainly presented in penetration peaks. Conclusion The gradient elution was finished under the condition of 0.02 mol/L acetate buffer solution with pH 4.0 and the same acetate buffer solution (with the proportion change from 0% to 50%) containing 0.5 mol/L NaCl by