醫(yī)學論文范文:聚乙二醇修飾�？�神經毒素rhk2a的分離與純化

醫(yī)學論文范文:聚乙二醇修飾�？�神經毒素rhk2a的分離與純化

【摘要】 目的 對聚乙二醇化海葵神經毒素(mPEG�瞨hk2a)修飾產物進行分離與純化。方法 將聚乙二醇(PEG)化反應所得混合產物超濾除鹽后，采用SP��650S強陽離子交換層析柱和CM��650S弱陽離子交換層析柱進行分離純化，收集不同NaCl離子濃度下的梯度洗脫液，經超濾除鹽、冷凍干燥、SDS�睵AGE電泳檢測，確定分離純化mPEG�瞨hk2a的色譜條件。結果 在保證mPEG�瞨hk2a穩(wěn)定性的前提下，隨著緩沖液pH值的降低，2種陽離子交換柱與mPEG�瞨hk2a的結合量均增加;在同一pH值下，強陽離子交換柱的結合情況更好。用SP��650S強陽離子交換層析柱進行分離純化時，洗脫液中溶液電導率在5～7 ms/cm時,mPEG�瞨hk2a能被洗脫下來，而且鹽離子濃度梯度變化越緩，mPEG�瞨hk2a修飾產物各洗脫峰的分離度越高;用CM��650S弱陽離子交換層析柱進行分離純化時，修飾產物mPEG�瞨hk2a主要在穿透峰中出現(xiàn)。結論 使用SP��650S強陽離子交換層析柱，用pH 4.0的0.02 mol/L醋酸鹽緩沖液與含0.5 mol/L NaCl 的pH 4.0的0.02 mol/L醋酸鹽緩沖液(后者比例變化為0%→50%)進行梯度洗脫，修飾產物mPEG�瞨hk2a與未修飾的rhk2a得到了很好的分離。

【關鍵詞】 聚乙二醇化�？�神經毒素;陽離子交換層析;分離;純化

Isolation and purification of pegylated sea anemone neurotoxin rhk2aHUANG Hui,PAN Yu�瞗ang,GUO Xiao�瞛uan,SHU Ya�瞛un,YANG Fan

(School of Pharmacy,Guangdong Pharmaceutical College,Guangzhou,Guangdong 510006,China) Abstract:Objective To isolate and purify mPEG�瞨hk2a.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�瞕ried,detected by SDS�睵AGE electrophoresis to determine chromatographic condition of isolate and purify mPEG�瞨hk2a. Results With the preconditions of keeping mPEG�瞨hk2a stable and reduced pH of buffer solutions,the binding capacity of both cation exchange columns and mPEG�瞨hk2a increased. However,the capacity of the strong cation exchange was more than the week one. MPEG�瞨hk2a 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�瞨hk2a 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 SP��650 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 mPEG�瞨hk2a醫(yī).學全.在.線網站gydjdsj.org.cn.

Key words:mPEG�瞨hk2a; cation exchange column chromatography; isolation and purification

　 PEG修飾蛋白質技術由于能夠在保證藥用蛋白活性的同時降低其在體內的免疫原性和毒副作用，延長血漿半衰期，增強其化學穩(wěn)定性，增大溶解性，提高藥用蛋白在體內的生物利用度，增加患者的順應性，已成功用于改善多種蛋白質藥物的性質。2000年中山大學生命科學院利用基因工程技術獲得一種重組�？�神經毒素rhk2a，其對心肌組織產生很強的正性肌力作用，在治療充血性心力衰竭方面有極好的應用前景[1]。為解決海葵肽類毒素rhk2a臨床應用時存在穩(wěn)定性差、毒性較大等問題，本課題組將PEG修飾技術應用于rhk2a，通過對各種反應條件的優(yōu)化，得到了最佳工藝條件下的甲基聚乙二醇(mPEG)修飾的海葵神經毒素產物mPEG�瞨hk2a[2]。但在mPEG修飾rhk2a過程中，由于大部分mPEG與rhk2a的偶聯(lián)反應都是隨機性親核反應，所以偶聯(lián)修飾產物往往都是由多種不同的偶聯(lián)蛋白異構體組成的混合物。本研究旨在對mPEG�瞨hk2a修飾產物進行分離與純化。

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