抗p185单抗5E12Fab段基因的克隆及表达

Cloning and expression of Fab gene of anti-p185 monoclonal antibody

目的克隆抗p185单抗5E12的Fab段基因并在原核细胞进行表达。方法用逆转录-聚合酶链反应技术(RT-PCR),以可变区第一骨架区的通用引物从分泌抗p185单抗的杂交瘤细胞系5E12中克隆Fd段和κ链的基因,重组到Fab表达载体中,在大肠杆菌中表达噬菌体抗体和可溶性Fab;根据前导肽序列设计引物,通过PCR介导的定点突变将V区基因氨基端序列恢复为5E12的原始序列;以NIH3T3/erbB-2细胞ELISA法、免疫组化法等进行特异性鉴定。结果以第一骨架区的通用引物从小鼠杂交瘤细胞5E12中克隆到Fd段和κ链的基因,在大肠杆菌中表达出Fab段抗体但无特异性抗原结合活性,分别将Fd段和κ链V区基因的氨基端序列矫正为亲本单抗的原始序列后,恢复了Fab段的抗原结合活性。单独恢复Fd段可变区氨基端序列可恢复抗原结合活性。但同时恢复κ链后活性却有所下降。结论成功构建了抗p185小分子抗体Fab并进行功能性表达,为进一步构建抗p185鼠单抗其他小分子抗体及其人源化改造打下基础;进一步证实抗体氨基端序列对抗体活性的重要性,为今后以PCR方法构建小分子抗体的工作提供了有益的借鉴。

Objective：To clone Fab genes of anti-p185 monoclonal antibody 5E12 and express it in E. coli.Methods： Fd and k genes were cloned by RT-PCR, inserted into Fab expression vector and expressed in E. coli. The N-terminal sequences of V regions was resumed by PCR mediated mutagenesis. The antigen-binding activity of the Fab were tested by ELISA and immunohistochemistry.Results：Fd and k genes were cloned and expressed in E. coli. But the bacterially expressed Fab fragments showed no antigen binding activity. After the N-terminal sequences of V regions was corrected to original sequences, the Fab expressed in bacterial was able to target HER2/neu-expressing cells （NIH3T3/erbB-2 cells）. Correction of Fd N-terminal sequences could partially resume the antigen binding activity. But correction of k chain N terminal sequences was shown no expected result. Condusion：Successful in constructing and expressing anti-p185 Fab, which will benefit the construction of other engineering antibody and humanization of murine anti-p185 McAb. We also found that the V region N terminal changes introduced with PCR primers may affect antigen binding activity seriously, to which more attention should be paid during antibody engineering.