Preparation and Activity Analysis of Canine Derived Chimeric Single Chain Antibody scFv-Fc against Canine Distemper Virus
To reduce and improve the heterogeneity and efficacy of mouse-derived monoclonal an-tibodies in clinical treatment of dogs,the variable region of heavy chain(VH)and variable region of light chain(VL)were amplified from mouse-derived hybridomas secreting monoclonal antibody against canine distemper virus(CDV)using RT-PCR,the single chain antibody(scFv)gene was obtained by linking VH and VL through Linker,and the canine-derived chimeric single chain anti-body(scFv-Fc)gene was obtained by selecting the constant region(Fc)of canine IgG B to con-nect to the C-terminus of scFv;The prokaryotic expression vectors for expressing mouse-derived scFv and canine-derived scFv-Fc were respectively constructed,which were expressed in Esche-richia coli for purification,and the specificity and neutralizing activity of the expressed antibodies were detected by ELISA,IPMA,cell fusion inhibition,and neutralization assay.The detection limit of ELISA for CDV mouse-derived scFv and canine-derived scFv-Fc are 14.38 and 102.75 μg·mL-1,respectively;Both of scFv and scFv-Fc specifically react with the expressed CDV H protein and CDV;The cell membrane fusion mediated by CDV H and F proteins can be inhibited by scFv and scFv-Fc,and the minimum concentrations of them for complete protection of cells from CPE formation are 0.72 and 0.64 μg·mL-1,respectively.The mouse-derived scFv and canine-derived chimeric antibody scFv-Fc are successfully developed,providing candidate an-tibody drugs for the clinical treatment of canine distemper.
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