新型重组免疫抑制融合毒素蛋白B7-2-PE40KDEL的分离纯化_医学论文.docVIP

新型重组免疫抑制融合毒素蛋白B7-2-PE40KDEL的分离纯化_医学论文 新型重组免疫抑制融合毒素蛋白B7-2-PE40KDEL的分离纯化_医学论文 作者：关海容，孙玉英，袁志宏，张惠丽，梁飞，刘楠，郭斯启，习彩霞，奚永志 【摘要】 为了建立高效快捷能分离大量重组B7-2-PE40KDEL融合蛋白的下游纯化路线，比较了不同组合的纯化策略和条件，包括反相层析、金属螯合层析、阴离子交换层析、蓝染料亲和层析和凝胶过滤层析等，并采用MTT法检测该融合蛋白的靶向杀伤活性。结果表明，在反相层析分离中疏水相分别采用了常规的甲醇和乙腈，结果为所分离的该重组融合蛋白均发生了变性，从色谱柱中分离出来就生成了絮状沉淀。在蓝染料亲和层析一步分离中，由于全菌体蛋白中与亲和树脂发生非特异结合的成分较多，极难区分目的蛋白和杂蛋白。但是，PRSETA-B7-2-PE40KDEL表达载体上带有翻译增强序列T7-g10，可在表达目的蛋白N端融合6个组氨酸，这十分有利于通过Ni-NTA金属鏊和层析法快速高纯度地纯化表达产物。根据这一特性，经反复筛选实验设计最终确定了金属螯合层析、阴离子交换层析和凝胶过滤层析三步法的纯化路线，用于纯化分离 B7-2-PE40KDEL融合蛋白。所得的目的蛋白的纯度可达95%以上，总回收率为8%。Western -blot实验显示，所得蛋白可与B7-2及PEA抗体特异性结合；MTT生物活性测定表明，该融合蛋白对表达CD28受体的人T细胞系Jurkat产生特异性杀伤作用，而对不表达CD28的淋巴细胞系Raji无任何杀伤作用。结论：建立了高效快速纯化大量重组B7-2-PE40KDEL融合蛋白的技术路线，所纯化的重组B7-2-PE40KDEL融合蛋白能特异性靶向杀伤表达CD28受体的人T细胞。 【关键词】 重组外毒素融合蛋白　　Establishment of Purification Procedure for Recombinant Fusion Protein B7-2-PE40KDEL 　　Abstract This study was aimed to establish downstream purification procedure by which the protein of interest can be purified to higher purity rapidly and efficiently. The different combinations of various purification strategies，methods and conditions were compared，including reversed phase chromatography，metal chelating chromatography，anion exchange chromatography，blue dye affinity chromatography，filtration chromatography and so on. The results showed that in reversed phase chromatography，isolated protein of interest was denatured and precipitated immediately after chromatography because methanol or acetonitrile were adopted as the organic phase. In blue dye affinity chromatography expecting to purify the protein of interest in one step，protein of interest was difficultly differentiated from mixed protein as much proteins bound to the chromatography media by non-specific affinity. While there is a translation-enhancing sequence T7-g10 in the PRSETA-B7-2-PE40KDEL expression vector，so it adds 6 histidines to the N terminus of the protein of interest，this allows to purify the protein of interest by metal chelating chromatograp