Objective: To construct and express the CD80 extracellular region-anti-human CD33 single chain fragment of variable region (ExCD80-CD33scFv) fusion gene, and detect the biological activity of the fusion protein.
Methods: Extracellular region of CD80 (ExCD80) was amplified and then linked with anti-CD33scFv using 403Aa-427Aa (hydrophilic fragments) in domain III of human serum albumin (HAS) as interlinker. The recombinant fusion gene was subcloned into the prokaryotic expression vector PET22b(+) and expressed in E.coli Rosetta (DE3) after induction by IPTG. 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. The biological activity of the fusion protein was detected with indirect immunofluorescence technique.
Results: The ExCD80 (633bp) was amplified by PCR and 403aa-427aa from domain III of HAS as interlinker was synthesized correctly. SDS-PAGE and Western blotting demonstrated that ExCD80-CD33scFv fusion gene expression vector was successfully constructed and expressed in E.coli Rosetta (DE3). The relative molecular mass (Mr;) of the fusion protein was 55 000, which has human CD33-binding specificity after renaturation as shown by indirect immunofluorescence technique.
Conclusion: Recombinant ExCD80-CD33scFv fusion gene was successfully constructed and expressed in E.coli Rosetta (DE3), which could provide a foundation for the future target therapy to the myeloid leukemia.