Aim: To construct and express a chimeric antibody against human CD40 molecule by genetic engineering antibody transformation.
Methods: Total RNA was extracted from the murine hybridoma cell 5C11 which secreted anti-CD40 monoclonal antibody (mAb). The genes encoding V(H) and V(L) of mAb 5C11 were amplified by RT-PCR. According to sequence analysis, the primer was designed to amplify signal peptide sequences relative to V(H) and V(L) genes. The V(H) and V(L) genes of mAb 5C11 and relative signal peptide sequences were spliced with C(H) and C(kappa) genes of human IgG1 to construct expression plasmid pIRES/h5C11 of human-mouse chimeric antibody gene and the plasmid was transfected into 293T cells under Lipofectamine mediation for transient expression. Expressed product was analyzed by flow cytometry.
Results: The result of NCBI gene data bank blast revealed that cloned gene sequence accorded with mice' V(H) and V(L) genes and feature of signal peptide sequence. The constructed plasmid pIRES/h5C11 was correct. The restriction endonuclease digestion analysis and PCR showed that the recombinant genes were subsequently cloned into vector pIRES. FACS analysis showed that human-mouse chimeric antibody against human CD40 maintained the binding activity and specificity to human CD40 molecule.
Conclusion: The gene encoding variable region of human-mouse chimeric antibody against CD40 is cloned successfully and the human-mouse chimeric antibody against CD40 is expressed transiently in 293T cells.