人细胞色素P450 1A1与谷胱甘肽S-转移酶的融合蛋白及其抗体的制备

采用融合蛋白技术原核表达 CYP1 A1 (第 2 4 1- 381个氨基酸 )与谷胱甘肽 S-转移酶 (GST)的融合蛋白作为抗原 ,用于制备 CYP1 A1多克隆抗体 .根据正反重组质粒 p GEX/ 1 A1表达的融合蛋白大小不同的原理 ,直接表达筛选得到正向重组质粒p GEX/ 1 A1 .通过优化表达条件 ,提高了目的蛋白的表达水平 .包涵体蛋白经制备型聚丙烯酰胺凝胶电泳 (PAGE)分离 ,获含纯化融合蛋白 GST- 1 A1的 PAGE凝胶 .直接用含 GST- 1 A1的凝胶悬液免疫 BALB/ c小鼠 ,自腹水中获取 CYP1 A1多克隆抗体 (1 A1 p Ab) . 1 A1 p Ab用切胶纯化的融合蛋白GST- 2 B6交叉吸收 ,蛋白 A- Sepharose亲和层析柱来纯化 .用切胶纯化的融合蛋白 GST- 1 A1及 GST-2 B6的免疫印迹反应初步鉴定 1 A1 p Ab的特异性 .纯化的 1 A1 p Ab对融合蛋白 GST- 1 A1反应特异性较强 ,但仍对 GST- 2 B6有弱交叉反应 .在实际应用中可根据反应强度来加以区分

Expression of glutathione S-transferase fusion proteins of human CYP1A1 and preparation of anti CYP1A1 polyclonal antibody

Our aim is to get high amounts of pure antigens to raise specific antibodies and to perform quantifications. Fusion proteins constructed between glutathione S transferase(GST) and CYP1A1 was expressed in Escherichia coli DH5α. We identified recombinants pGEX/1A1 by direct expression screening. Insoluble proteins were isolated from the bacteria and the fusion proteins were purified from a preparative (2 mm) SDS PAGE. The polyacrylamide gel containing the fusion proteins GST 1A1 was used to immunize BALB/c mice from which polyclonal ascites fluid was prepared. Anti CYP1A1 polyclonal antibodies(1A1pAb) was purified by cross absorption with GST 2B6(GST fusion protein of CYP2B6 204-353 amino acid expressed in this laboratory, purified by preparative SDS PAGE)and then by passing through protein A Sepharose affinity column. The purified GST 2B6 and GST 1A1 were used to test the specificity of 1A1pAb. 1A1pAb is specific to GST 1A1 and is still weakly crossreactive with GST 2B6. But CYP1A1 can be discriminated from other CYP subtypes.