Aim: To construct the eukaryotic expression vector of chimeric anti-human CD20 monoclonal antibody (mAb) and realize its expression.
Methods: The light- and heavy-chain genes were amplified from hybridoma cell line 1-28 secreting anti-human CD20 mAb by RT-PCR and were cloned to T vector and sequenced. Proteins of mAb 1-28 were separated by reducing SDS-PAGE. Light- and heavy-chain bands were excised from preparative gel, digested by trypsin, and subjected to peptide mass fingerprinting. Software Biolynx and pepeseq were used to evaluate the score of probability. Correctness of the light- and heavy-chain DNA sequences was verified by their protein sequences. Genes of V(H) and V(L) were amplified from T vector and cloned into chimeric antibody expression vector (pCMV-V(H) and pCMV-V(L)), generating the expression vectors of chimeric anti-human CD20 mAb (C1-28) including light chain expression vector C1-28L and heavy chain expression vector C1-28H. The two plasmids were co-transfected into 293T cells with Lipofectamine 2000. RT-PCR was used to detect the transcription at mRNA level. C1-28 expression was detected by Sandwich ELISA and Western blot methods.
Results: mAb 1-28's genes were successfully cloned and verified by peptide mass fingerprinting. Eukaryotic expression vectors of anti-human CD20 mAb were constructed and expressed in 293T cells with the expression amount reaching 257 mg/L and the molecular weight consistent with that of human IgG.
Conclusion: These experiments lay solid foundation for further study on the role of chimeric CD20 antibody in the treatment of non-Hodgkin's lymphoma.