重组蛋白Fpr3的原核表达、纯化与多克隆抗体的制备  

Overexpression,purification and polyclonal antibody preparation of recombinant protein Fpr3

作　　者：伍翔[1] 祁超[1] 李伟国[2] 刘中来[1] 

机构地区：[1]华中师范大学生命科学学院,武汉430079 [2]华中师范大学化学学院农药与化学生物学教育部重点实验室,武汉430079

出　　处：《华中师范大学学报（自然科学版）》2010年第1期120-124,共5页Journal of Central China Normal University：Natural Sciences

基　　金：国家教育部科学技术研究重点项目(107082);湖北省自然科学基金资助项目(2007ABA141)

摘　　要：为了从分子水平上进一步研究PPIase与PP1的相互作用,将构建好的pGEX-5X-1重组表达质粒转化于受体菌BL21(DE3)感受态细胞中,用IPTG诱导后得到了可溶形式表达的融合蛋白.采用GST亲和层析法对重组蛋白进行了纯化,并用Factor Xa对融合蛋白进行柱上酶切,SDSPAGE检测表明可获得较高纯度的GST-Fpr3融合蛋白以及去除GST标签的目的蛋白.用其免疫日本雄性大耳白兔,成功制备了抗GST-Fpr3的多克隆抗体,为Fpr3的空间结构的解析和相应的分子识别过程奠定了基础.In order to get insights into the structural and molecular interaction mechanisms between PPIase and PP1, the recombinant plasmid pGEX-SX-1-Fpr3 was constructed and overexpressed in E. eoli as a soluble protein. The fusion protein was purified by GSTrap affinity chromatography and the GST tag was removed by the method of on-column cleavage with Factor Xa. The recombinant GST-Fpr3 and Fpr3 was assayed and identified by SDS-PAGE and Western blot, respectively. The recombinant GST- Fpr3 was used to immunize rabbits for preparing polyclonal antibody. The polyclonal antibody with high titer and high specificity against the GST-Fpr3 has been successfully prepared.

关 键 词：Fpr3 肽基脯氨酰顺反异构酶 重组检验点活性 表达 纯化 多克隆抗体 

分 类 号：Q786[生物学—分子生物学]