[Cloning of the variable region genes from hybridoma against bFGF and expression of single chain antibody fragments in E.coli HB2151]

Abstract

Aim: To construct and express the single chain antibody (scFv) in E.coli HB2151 by cloning the variable region genes from hybridoma against bFGF.

Methods: Total RNA was extracted from hybridoma cell line B2F3 secreting mAbs against bFGF and the cDNA was amplified by retropolymerase chain reaction (RT-PCR). V(L) and V(H) were fused by a short peptide linker containing 15 amino acids (Gly(4)Ser)(3) using splice-overlap extension PCR to construct the scFv gene. The sequences of the scFv were analyzed by Shanghai Sangon Biological Engineering Technology and Services Co. Ltd and Ig Blast data base in GenBank. The scFv gene was inserted into pCANTAB-5E vector and expressed in E.coli HB2151.

Results: The V(H) gene contained 375 base pairs and encoded 125 amine acid residues. The V(L) gene contained 399 base pairs and encoded 133 amine acid residues. There were four FRs, three CDRs and two characteristic cysteine residues in the V(H) gene and the V(L) gene, respectively. The scFv gene contained 789 base pairs and encoded 263 amine acid residues with the structure of V(H)-linker-V(L). Restriction endonuclease digestion and DNA sequencing proved that the expression vector of pCANTAB-5E-scFv was constructed correctly. SDS-PAGE and ELISA analysis showed that scFv was successfully expressed in E.coli HB2151 and the expression protein had specific antigen binding activity.

Conclusion: The variable region genes of anti-bFGF mAbs have been cloned successfully and single chain antibody fragments have been constructed and expressed, which will be a great help to the study of humanized antibodies against bFGF.