Abstract: The aim of this study was to prepare a mouse genetically engineered antibody against canine parvovirus (CPV) using HEK-293 cell line and to detect its biological activity.The CPV monoclonal antibody subtype was detected by antibody subtype detection kit.The affinity and specificity of CPV monoclonal antibody were detected by indirect ELISA,the variable region of CPV monoclonal antibody was obtained by RACE-PCR,and linking the variable region sequence to the murine antibody constant region sequence.The eukaryotic expression vectors of pcDNA3.1(+)-L and pcDNA3.1(+)-H were constructed,respectively.The expression vectors was co-transfected into HEK-293 cells,and the biological activity of mouse CPV genetically engineered antibody was detected by hemagglutination inhibition techniques and neutralization testing.The mouse CPV genetically engineered antibody was expressed by using HEK-293F cells and the expression of mouse CPV genetically engineered antibody in HEK-293F cells was detected by indirect ELISA.The mouse CPV genetically engineered antibody was purified by Protein A affinity chromatography column and detected by SDS-PAGE.The activity of the purified mouse CPV genetically engineered antibody was detected by indirect immunofluorescence.The results showed that CPV monoclonal antibody was belong to IgG_2b,and the average of affinity constant of 6 Ka was 1.02×10¹¹ L/mol,which only reacted with CPV VLPs.The results of agarose gel electrophoresis showed that pcDNA3.1(+)-L and pcDNA3.1(+)-H were successfully constructed,and the hemagglutination inhibition titer of culture supernatant of HEK-293 and HEK-293F cells were 1:2⁴ and 1:2⁶,respectively;The neutralization experiments showed that the neutralization titer of culture supernatant of HEK-293 and HEK-293F cells were 1:152 and 1:1 290,respectively;The expression of mouse CPV genetically engineered antibody in HEK-293F cells was 5.97 mg/L.SDS-PAGE analysis showed bands at 55 and 25 ku.The results showed that the mouse CPV genetically engineered antibody was successfully expressed and purified in HEK-293F cells.Indirect immunofluorescence assay showed that the purified mouse CPV genetically engineered antibody had good biological activity.In this study,we successfully expressed mouse CPV genetically engineered antibody with high neutralizing activity and high purity in HEK-293F cells,it yet laid a foundation for study of CPV genetically engineered antibody drugs in the future.

Key words: canine parvovirus(CPV); genetically engineered antibody; co-transfected; HEK-293F cell