中国生物工程杂志

Objective: Single chain fragment variable(ScFv) is the smallest functional structural unit with the antigen-binding specificity of the parent antibody. Due to its affinity and low immunogenicity, ScFv has broad application prospects in medical treatment and diagnosis. To reduce the host immune rejection during clinical treatment with heterologous murine monoclonal antibody (McAb) in canine, ScFv were prepared against canine parvovirus (CPV) using a prokaryotic expression system. Methods: Total RNA was extracted from hybridoma cell lines specific for CPV,amplification of the antibody heavy chain variable region (VH) gene and the light chain variable region (VL) gene from the reverse transcription cDNA into the expression vector pOPE101. The recombinant plasmid was transformed into E. coli for expression, and the expressed protein was identified by Western blot. The activity of the ScFv was detected by ELISA, and the ScFv purified by affinity chromatography was identified by virus neutralization test. Results: The recombinant plasmid pOPE101-ScFv was successfully constructed. The correct expression of single-chain antibody in E. coli was determined by western blot. The ability of the fusion protein to specifically bind to the virus was verified by ELISA and virus neutralization test. The potency was 1∶40 (0.014 μg/ml). Conclusion: A ScFv with neutralizing activity was obtained using an E. coli expression system, which provides a basis for clinical immunotherapy for CPV disease.