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

Area of Science:

• Biotechnology
• Immunology
• Molecular Biology

Background:

• The CD33 antigen is a target for myeloid leukemia therapies.
• Monoclonal antibodies (mAbs) are crucial in targeted therapy.
• Developing recombinant antibody fragments like scFv offers therapeutic advantages.

Purpose of the Study:

• To construct and express a single chain variable fragment (scFv) gene targeting human CD33.
• To characterize the bioactivity of the recombinant anti-CD33 scFv.
• To lay the groundwork for novel targeted therapies against myeloid leukemia.

Main Methods:

• Cloning of variable light and heavy chain genes from a murine hybridoma using RT-PCR.
• Fusion of variable regions with a peptide linker using splice-overlap extensive PCR.
• Subcloning into pET28a(+) vector for expression in E. coli Rosetta and purification via Ni(2+) metal affinity chromatography.

Main Results:

• Successful construction and expression of recombinant anti-CD33 scFv in E. coli Rosetta as inclusion bodies.
• Purification of the fusion protein achieved through solubilization, chromatography, and refolding.
• Flow cytometry confirmed the scFv's ability to bind to the human CD33 antigen.

Conclusions:

• The recombinant anti-CD33 scFv gene was successfully constructed and expressed.
• The expressed scFv exhibits bioactivity, binding to the CD33 antigen.
• This work provides a foundation for developing targeted therapies for myeloid leukemia.