柴燕涛 姜棋予 谢国明 胡 燕 邬顺全 杨锐创 貌盼勇 侯 俊解放军第三〇二医：临床研究管理中心，北京100039

    包涵体蛋白3种纯化方法的比较

    柴燕涛*姜棋予*谢国明胡燕邬顺全杨锐创貌盼勇侯俊▲

    解放军第三〇二医：临床研究管理中心，北京100039

    目的比较3种纯化方法对表达的肠道病毒71型(EV71)VP1区包涵体蛋白的纯化效果。方法分别用差速离心法、固定化金属离子亲和层析(IMAC)及不同浓度饱和硫酸氨沉淀法3种包涵体蛋白纯化法对原核表达EV71 VP1包涵体蛋白进行纯化，蛋白垂直(SDS-PAGE)电泳检测蛋白纯化效果。结果差速离心法可以进行大批量表达蛋白纯化，纯化效果较好，但存在蛋白损失较多，且纯化蛋白不能反复冻融的问题。IMAC柱纯化获得的蛋白纯度较高，且蛋白性状稳定，但仍存在纯化蛋白量较少的问题。饱和硫酸氨沉淀纯化法无法获得纯度较高的包涵体蛋白。结论差速离心法可用于大批量蛋白纯化，纯化蛋白应避免反复冻融，可小量分装冻存后备用。IMAC柱纯化可获得纯度较高且性状相对稳定的纯化蛋白，其在相应抗体检测方法学的建立及疫苗制备方面均有广泛用途。饱和硫酸氨法只适用于表达蛋白的初步浓缩纯化。

    包涵体蛋白曰蛋白纯化曰蛋白垂直电泳

    [Abstract]Objective To compare the expressed inc1usion body protein of EV71 VP1 with three purification methods. Methods The expressed EV71 VP1 inc1usion body protein was purified by differentia1 centrifugation,IMAC co1umn purification and ammonium su1fate precipitation.SDS-PAGE experiments determinate the purity effects.Results Expressed inc1usion body purification in 1arge quantities cou1d been undertaken by differentia1 centrifugation and the purification effect was better but with a 1ot of protein 1oss and the purified protein cou1d not be repeated1y frozen and thawed.The purification effect of IMAC was good and stab1e,but 1ess purified protein was purified.High purity of inc1usion body protein wasn′t obtained by ammonium su1fate precipitation.Conclusion Differentia1 centrifugation is app1icab1e in 1arge quantities of protein purification,but avoid repeated1y freezing and thawing.The protein purification of IMAC can be used to estab1ish corresponding antibody detection methods.The ammonium su1fate precipitation is on1y app1icab1e for the pre1iminary purification of the expressed proteins. ......