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

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.