亲和层析纯化重组中性粒细胞激活蛋白  

Purification of recombinant neutrophil-activating protein fusion expressed with maltose-binding protein by affinity chromatography

作　　者：丁亚骏[1] 何璐云[1] 王亚楠[1] 张亚娟[1] 康巧珍[1] 

机构地区：[1]郑州大学生物工程系,河南郑州450001

出　　处：《河南医学研究》2012年第3期271-273,共3页Henan Medical Research

基　　金：全国大学生创新创业训练计划项目(121045944);国家科技重大专项(2012ZX09103-301-022)

摘　　要：目的:直链淀粉树脂亲和层析法分离纯化重组麦芽糖结合蛋白-中性粒细胞激活蛋白(recombinantmaltose-binding protein-neutrophil-activating protein,rMBP-NAP)。用于支气管哮喘模型小鼠进行免疫调节实验。方法:IPTG诱导基因工程菌E.coli TB1(pMAL-c2X-nap)表达rMBP-NAP,离心收获细胞,过柱缓冲液重悬菌体并超声破碎,再次离心取上清和沉淀,SDS-PAGE分析rMBP-NAP可溶性;直链淀粉树脂亲和层析法分离纯化rMBP-NAP;SDS-PAGE凝胶扫描分析纯化后rMBP-NAP纯度;蛋白定量试剂盒测定rMBP-NAP浓度。结果:IPTG诱导工程菌表达的rMBP-NAP主要以可溶性形式存在于超声上清中,相对分子质量约为57 kD,与预期结果一致;亲和层析法纯化rMBP-NAP纯度可达96.5%,浓度可达0.625 g/L。结论:用直链淀粉树脂亲和层析法,可纯化得到高纯度、高浓度的融合蛋白rMBP-NAP。Objective： To purify recombinant neutrophil-activating protein of H.pylori fusion expressed with maltose-binding Protein（rMBP-NAP） in E.coli TB1（pMAL-c2X-nap） by affinity chromatography on Amylose Resin.Methods： To obtain rMBP-NAP,E.coli TB1 was induced by IPTG,harvested by centrifugation.The pellet was resuspended in Column Buffer and broken by sonicate followe centrifugation.The solubility of rMBP-NAP was analyzed by SDS-PAGE.rMBP-NAP was purified by affinity chromatography on Amylose Resin.The purity of rMBP-NAP was detected by SDS-PAGE gel scanning and concentration of purified fractions was tested by Bradford method.Results： The expressed fusion protein was soluble mainly in the supernatant.Conformed to prediction,molecular weight of rMBP-NAP expressed in TB1 cell was approximate 57 kD.The purity of rMBP-NAP was 96.5% after one step purification by affinity chromatography.The concentration of eluted rMBP-NAP was 0.625 g/L.Conclusion：Affinity chromatography can purify rMBP-NAP with high quantity and purity from E.coli TB1.

关 键 词：亲和层析 融合蛋白 麦芽糖结合蛋白 中性粒细胞激活蛋白 

分 类 号：R392[医药卫生—免疫学]