BIG-3的原核表达、纯化及其多克隆抗体的制备与鉴定

Prokaryotic Expression and Purification of BIG-3 Fusion Protein and the Antibody’s Preparation and Identification

原核表达、纯化骨形态发生蛋白诱导基因(BIG-3,BMP-2-induced gene 3kb)所编码的融合蛋白并制备其多克隆抗体。将BIG-3基因插入原核表达载体PGEX-4T-2的多克隆位点获得pGEX-4T-2-BIG-3重组表达载体,转化大肠杆菌BL21(DE3)菌株,诱导表达,SDS-PAGE鉴定融合蛋白表达情况及Sepharose4B层析柱亲和层析法纯化的融合蛋白;用纯化的融合蛋白免疫新西兰大白兔制备其多克隆抗体,并以Western blot鉴定其特异性。结果在大肠杆菌中获得BIG-3-GST融合蛋白高水平的诱导表达,经Sepharose4B层析柱亲和层析,GST-BIG-3融合蛋白在电泳图片上显示较为清晰的单一条带,所制备的多克隆抗体具有较高的特异性。说明在大肠杆菌中成功表达且以亲和层析法纯化得到了BIG-3-GST融合蛋白,并制备了特异性较高的多克隆抗体。

Prokaryotic expression and purification of fusion protein coded by bone morphogenetic protein induced gene （ BIG-3 ） and preparation of its polyclonal antibody, BIG-3 gene was inserted into the expression vector pGEX-4T-2 to get recombinant plasmid pGEX-4T-2-BIG-3. The recombinant plasmid pGEX-4T-2-BIG-3 was transformed into E. coli BL 21 and fusion protein expression was induced by IPTG. New Zealand rabbits were immunized with GST-BIG-3 fusion proteins to prepare a polyclonal antibody of BIG-3. GST-BIG-3 antiserum was obtained and polyclonal antibody was characterized by western blot. It is resulted that GST-BIG-3 fusion protein was effectively expressed in E. coli. After purification, a single clear band of GST-BIG-3 fusion protein appeared in SDS-PAGE gel. Polyclonal antibody prepared from purified fusion protein has obvious characteristic. It is conclused that GST-BIG-3 fusion protein can be effectively expressed in E. coli, SDS-PAGE and Western blot analysis showed that purified GST-BIG-3 fusion protein and polyclonal antibody with characteristics was obtained.