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医学论文范文:聚乙二醇修饰海葵神经毒素rhk2a的分离与纯化
【摘要】 目的 对聚乙二醇化海葵神经毒素(mPEGrhk2a)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SP650S强阳离子交换层析柱和CM650S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、SDSPAGE电泳检测，确定分离纯化mPEGrhk2a的色谱条件。结果 在保证mPEGrhk2a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mPEGrhk2a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SP650S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,mPEGrhk2a能被洗脱下来，而且盐离子浓度梯度变化越缓，mPEGrhk2a修饰产物各洗脱峰的分离度越高;用CM650S弱阳离子交换层析柱进行分离纯化时，修饰产物mPEGrhk2a主要在穿透峰中出现。结论 使用SP650S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mPEGrhk2a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
Isolation and purification of pegylated sea anemone neurotoxin rhk2aHUANG Hui,PAN Yufang,GUO Xiaojuan,SHU Yajun,YANG Fan
(School of Pharmacy,Guangdong Pharmaceutical College,Guangzhou,Guangdong 510006,China) Abstract:Objective To isolate and purify mPEGrhk2a.Methods The mixture obtained from pegylated reactions was separated by Toyopearl SP650 column for strong ion exchange and Toyopearl CM650 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 freezedried,detected by SDSPAGE electrophoresis to determine chromatographic condition of isolate and purify mPEGrhk2a. Results With the preconditions of keeping mPEGrhk2a stable and reduced pH of buffer solutions,the binding capacity of both cation exchange columns and mPEGrhk2a increased. However,the capacity of the strong cation exchange was more than the week one. MPEGrhk2a was eluted by Toyopearl SP650 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 CM650 column for weak cation exchange,the mPEGrhk2a 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 using Toyopearl SP650 column for strong cation exchage. The products including pegylated and unmodified rhk2a were successfully isolated,which has laid a foundation for our further research on the properties of purified mPEGrhk2a医.学全.在.线网站www.med126.com.
Key words:mPEGrhk2a; cation exchange column chromatography; isolation and purification
PEG修饰蛋白质技术由于能够在保证药用蛋白活性的同时降低其在体内的免疫原性和毒副作用，延长血浆半衰期，增强其化学稳定性，增大溶解性，提高药用蛋白在体内的生物利用度，增加患者的顺应性，已成功用于改善多种蛋白质药物的性质。2000年中山大学生命科学院利用基因工程技术获得一种重组海葵神经毒素rhk2a，其对心肌组织产生很强的正性肌力作用，在治疗充血性心力衰竭方面有极好的应用前景[1]。为解决海葵肽类毒素rhk2a临床应用时存在稳定性差、毒性较大等问题，本课题组将PEG修饰技术应用于rhk2a，通过对各种反应条件的优化，得到了最佳工艺条件下的甲基聚乙二醇(mPEG)修饰的海葵神经毒素产物mPEGrhk2a[2]。但在mPEG修饰rhk2a过程中，由于大部分mPEG与rhk2a的偶联反应都是随机性亲核反应，所以偶联修饰产物往往都是由多种不同的偶联蛋白异构体组成的混合物。本研究旨在对mPEGrhk2a修饰产物进行分离与纯化。
