[Construction, expression and functional characterization of single chain variable fragments (scFv) against human CD33 antigen].

AIM

To construct and express the single chain variable fragments (scFv) gene against human CD33 antigen, and characterize its bioactivity.

METHODS

The genes encoding the light and heavy chain variable regions were cloned by RT-PCR from a murine hybridoma cell line, which could produce monoclonal antibody(mAb) against human CD33 antigen. Then the light and heavy chain variable regions were fused together by a short peptide linker containing 15 amino acid (Gly(4)Ser)(3) using splice-overlap extensive PCR. The recombinant anti-CD33 scFv was subcloned into the expression vector pET28a(+) and expressed in E.coli Rosetta after induction by IPTG.

RESULTS

SDS-PAGE and Western blot analysis showed that the recombinant anti-CD33 scFv gene was expressed in the form of inclusion body in E.coli Rosetta, and the purified fusion protein was obtained after a series of purification steps including cell lysis, inclusion body solubilization, Ni(2+) metal affinity chromatography and protein refolding. Flow cytometry(FCM) analysis showed that the scFv could react with human CD33 antigen.

CONCLUSION

Recombinant anti-CD33 scFv gene has been successfully constructed and expressed in E.coli Rosetta, which could provide foundation for the future target therapy to the myeloid leukemia.