新型抗体结合蛋白CBD-SPG的设计与表达

Design and expression of new antibody binding protein CBD-SPG

设计并表达可用于纯化IgG的新型高载量抗体结合蛋白CBD-SPG。利用基因重组技术将纤维素结合结构域(Cellulose Binding Domain,CBD)基因插入到表达载体pET28a-SPG中,获得重组质粒pET28a-CBD-SPG,并转化大肠杆菌BL21(DE3)。IPTG诱导CBD-SPG融合蛋白表达,并用SDS-PAGE和Western blot对表达产物进行鉴定。重组表达质粒pET28a-CBD-SPG经双酶切及测序验证元误;表达产物经SDS-PAGE和Western Blot分析表明融合蛋白的表观分子量约为40kD;CBDSPG具有良好的结合纤维素和抗体的能力,晶体纤雏素Avicel ph101对CBD-SPG的载量可达11.6l mg/g(w/w)。成功构建并运用原核系统表达CBD-SPG;CBD-SPG在保持良好抗体结合能力的同时,更具有了结合纤维素的能力,有望成为一种新型的亲和材料。

In order to design and express a new CBD-SPG fusion protein with a high antibody binding capacity for purifying IgG, a cellulose binding domain was inserted into pET28a-SPG using the gene reeombination method. Then the recombinant plasmid pET28a-CBD-SPG was transformed into E. coli BL21 （ DE3 ） and induced to express CBD-SPG by IPTG. The fusion protein was identified by SDS-PAGE and Western Blot. The DNA fragment encoding the fusion protein showed correct size and correct sequence after digested with Nco I and Xho I ~ The expression of CBD-SPG was visual- ized by SDS-PAGE with a MW around 40 kDa. CBD-SPG was able to bind to both IgG and cellulose. The binding capacity of CBD-SPG to avicel phl01 was 11.61mg/g （w/w）. CBD-SPG gene was successfully cloned and expressed in E. coll. The fusion kept the ability of SPG part, at the same time, it could also bind to the cellulose. In the future it may be used as a new affinity matrix.