目的: 克隆抗膀胱癌单抗BDI的Fab段基因并在大肠杆菌中表达。方法: 用逆转录 聚合酶链反应技术（RT-PCR），从分泌抗人膀胱癌的鼠单抗杂交瘤细胞系中克隆κ链和Fd段基因，克隆到Fab表达载体中，在大肠杆菌表达噬菌体抗体和可溶Fab；运用PCR介导的定位点突变改造VH氨基端序列；用ELISA、免疫组化法等进行特异性鉴定。结果:从分泌抗膀胱癌的鼠单抗杂交瘤细胞系中克隆了重链Fd段和κ链基因，在大肠杆菌中获得有抗原结合活性的噬菌体抗体和可溶性Fab的表达，但活性很弱，将VH氨基端序列矫正为亲本单抗原始序列后，明显改善了其活性，通过ELISA、免疫组化及模拟抗体库筛选证实了所获抗体片段的特异性结合及在抗体库技术中的可用性。结论:获得了功能性抗膀胱癌小分子抗体，并再次提示抗体氨基端序列对抗体活性的影响的重要性。

Objective:To clone the Fab gene of a monoclonal antibody (mAb) BDI against human bladder cancer and its expression in E.coli. Methods: Fd and κ genes of mAb BDI were cloned by RT-PCRand inserted into an Fab expression vector. Phage displaying Fab and soluble Fab were expressed in E.coli. The N-terminal sequence of VH region was corrected by PCR mediated mutagenesis. The antigen-binding characteristics of the Fab were tested by ELISA and immunohistochemistry. Results: Fd and κ genes were cloned into the expressing vector p3MH and the phage displaying antibody and soluble Fab were expressed in E.coli, which showed weak binding activity to bladder cancer cells. Correction of the N-terminal sequence of the VHimproved the biding activity dramatically. The feasibility of the application of the Fab in phage antibody library screening was confirmed by a simulated panning procedure. Conclusion:The Fab gene of an anti-human bladder cancer mAb was expressed in E.coli. The importance of the N-terminal sequence on antibody binding activity was suggested.

国家自然科学基金项目（30070706）资助