醫(yī)學(xué)論文范文:聚乙二醇修飾海葵神經(jīng)毒素rhk2a的分離與純化

醫(yī)學(xué)論文范文:聚乙二醇修飾�？�神經(jīng)毒素rhk2a的分離與純化

【摘要】 目的 對(duì)聚乙二醇化�？�神經(jīng)毒素(mPEG�瞨hk2a)修飾產(chǎn)物進(jìn)行分離與純化。方法 將聚乙二醇(PEG)化反應(yīng)所得混合產(chǎn)物超濾除鹽后，采用SP��650S強(qiáng)陽(yáng)離子交換層析柱和CM��650S弱陽(yáng)離子交換層析柱進(jìn)行分離純化，收集不同NaCl離子濃度下的梯度洗脫液，經(jīng)超濾除鹽、冷凍干燥、SDS�睵AGE電泳檢測(cè)，確定分離純化mPEG�瞨hk2a的色譜條件。結(jié)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(yáng)離子交換柱與mPEG�瞨hk2a的結(jié)合量均增加;在同一pH值下，強(qiáng)陽(yáng)離子交換柱的結(jié)合情況更好。用SP��650S強(qiáng)陽(yáng)離子交換層析柱進(jìn)行分離純化時(shí)，洗脫液中溶液電導(dǎo)率在5～7 ms/cm時(shí),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(chǎn)物各洗脫峰的分離度越高;用CM��650S弱陽(yáng)離子交換層析柱進(jìn)行分離純化時(shí)，修飾產(chǎn)物mPEG�瞨hk2a主要在穿透峰中出現(xiàn)。結(jié)論 使用SP��650S強(qiáng)陽(yáng)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(jìn)行梯度洗脫，修飾產(chǎn)物mPEG�瞨hk2a與未修飾的rhk2a得到了很好的分離。

【關(guān)鍵詞】 聚乙二醇化�？�神經(jīng)毒素;陽(yáng)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(xué)全.在.線網(wǎng)站52667788.cn.

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

　 PEG修飾蛋白質(zhì)技術(shù)由于能夠在保證藥用蛋白活性的同時(shí)降低其在體內(nèi)的免疫原性和毒副作用，延長(zhǎng)血漿半衰期，增強(qiáng)其化學(xué)穩(wěn)定性，增大溶解性，提高藥用蛋白在體內(nèi)的生物利用度，增加患者的順應(yīng)性，已成功用于改善多種蛋白質(zhì)藥物的性質(zhì)。2000年中山大學(xué)生命科學(xué)院利用基因工程技術(shù)獲得一種重組�？�神經(jīng)毒素rhk2a，其對(duì)心肌組織產(chǎn)生很強(qiáng)的正性肌力作用，在治療充血性心力衰竭方面有極好的應(yīng)用前景[1]。為解決�？�肽類毒素rhk2a臨床應(yīng)用時(shí)存在穩(wěn)定性差、毒性較大等問題，本課題組將PEG修飾技術(shù)應(yīng)用于rhk2a，通過對(duì)各種反應(yīng)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�？�神經(jīng)毒素產(chǎn)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(yīng)都是隨機(jī)性親核反應(yīng)，所以偶聯(lián)修飾產(chǎn)物往往都是由多種不同的偶聯(lián)蛋白異構(gòu)體組成的混合物。本研究旨在對(duì)mPEG�瞨hk2a修飾產(chǎn)物進(jìn)行分離與純化。

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