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

Abstract

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.