论文部分内容阅读
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k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k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 fragment of anti-bladder cancer monoclonal antibody BDI and express in Escherichia coli. Methods: The k chain and Fd gene were cloned from murine monoclonal antibody (McAb) secreting anti-human bladder cancer by reverse transcription polymerase chain reaction (RT-PCR) and cloned into Fab expression vector. Expressing phage antibody and soluble Fab; using PCR-mediated site-directed mutagenesis to transform V_H amino terminal sequence; using ELISA, immunohistochemistry and other specific identification. Results: The heavy chain Fd fragment and k chain gene were cloned from mouse monoclonal antibody secreting anti-bladder cancer monoclonal antibody, and the phage antibody and soluble Fab with antigen-binding activity were obtained in E. coli. However, After the amino acid sequence of V_H was corrected to the original sequence of parent monoclonal antibody, the activity of V_H was improved obviously. The specific binding of the obtained antibody fragment and its availability in antibody library technology were confirmed by ELISA, immunohistochemistry and screening of antibody library. CONCLUSION: The functional small antibody against bladder cancer was obtained and the importance of the amino terminal sequence of antibody against the activity of antibody was again demonstrated.