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CYP1A1（第241-381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CYP1A1多克隆抗体. 根据正反重组质粒pGEX/1A1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pGEX/1A1. 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-1A1的PAGE凝胶. 直接用含GST-1A1的凝胶悬液免疫BALB/c小鼠,自腹水中获取CYP1A1多克隆抗体（1A1pAb）. 1A1pAb用切胶纯化的融合蛋白GST-2B6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-1A1及GST-2B6的免疫印迹反应初步鉴定1A1pAb的特异性. 纯化的1A1pAb对融合蛋白GST-1A1反应特异性较强，但仍对GST-2B6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.