包涵体蛋白3种纯化方法比较.docVIP

包涵体蛋白3种纯化方法比较 　　[摘要] 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）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。 　　[关键词] 包涵体蛋白；蛋白纯化；蛋白垂直电泳 　　[中图分类号] R392.33 [文献标识码] A [文章编号] 1673-7210（2016）04（a）-0004-03 　　[Abstract] Objective To compare the expressed inclusion body protein of EV71 VP1 with three purification methods. Methods The expressed EV71 VP1 inclusion body protein was purified by differential centrifugation， IMAC column purification and ammonium sulfate precipitation. SDSexperiments determinate the purity effects. Results Expressed inclusion body purification in large quantities could been undertaken by differential centrifugation and the purification effect was better but with a lot of protein loss and the purified protein could not be repeatedly frozen and thawed. The purification effect of IMAC was good and stable， but less purified protein was purified. High purity of inclusion body protein wasnt obtained by ammonium sulfate precipitation. Conclusion Differential centrifugation is applicable in large quantities of protein purification， but avoid repeatedly freezing and thawing. The protein purification of IMAC can be used to establish corresponding antibody detection methods. The ammonium sulfate precipitation is only applicable for the preliminary purification of the expressed proteins. 　　[Key words] Inclusion body protein； Protein purification； SDSelectrophoresis 　　包涵体是外源基因在细胞中表达量过高形成的一种膜包裹的高密度、不溶性蛋白颗粒。该蛋白不具有生物学活性，其分子的空间结构表现为非折叠态的聚集体，复性后可恢复其生物学活性[1]。研究发现外源基因低表达时很少形成包涵体，蛋白表达合成速度越快、量越高越容易形成包涵体[2-4]。包涵体纯化方法有色谱法、离子交换层析、亲和层析、疏水层析、双水相萃取技术、反胶团相转移技术、凝胶过滤层析等[5-13]，不同的方法间纯化效果有明显差异。为寻找一种简单、快速的纯化方法，本研究用3种实验室常用的包涵体蛋白纯化方法对肠道病毒71型（EV71）VP1包涵体蛋白进行纯化，并对其纯化效果进行比较，希望能为相关抗体检测试剂盒的研制提供参