Study On Genetically Engineered Antibodies Of Canine Parvovirus

Canine parvovirus disease is an infectious disease caused by the infection of canine parvovirus?CPV?,which is harmful to dog's health.It is a disease with high incidence rate,high infectivity and high mortality.Currently,no effective drugs against CPV are available.At present,the monoclonal antibody has a good therapeutic effect in the early stage of CPV infection,but the monoclonal antibody is murine origin antibody,which has high immunogenicity and cannot be injected continuously.In this study,the existing monoclonal antibodies were modified to produce anti CPV drugs with low immunogenicity.First of all,it was identified that the antibody subtype of the hybridoma cell line4H8 against CPV was IgG_2b,and the average value of the six Ka of binding affinity is 1.02×10¹11 L/mol,which only reacted with CPV VLPs.The sequence of the variable region of 4H8 antibody was extracted by RACE-PCR and analyzed by NCBI IgBLAST.The eukaryotic expression vectors pcDNA3.1?+?-L and pcDNA3.1?+?-H were constructed by assembling the variable region sequence of the4H8 antibody and the constant region sequence of the mouse antibody found on the ENA website.They were co-transfected into HEK-293 and HEK-293F cells for expression.The expression level of murine CPV genetically engineered antibody?CA?was detected by indirect ELISA,and the expression level of CA in HEK-293F cells was 5.97 mg/L.The biological activity of CA was detected by the hemagglutination inhibition and neutralization test.The hemagglutination inhibition and neutralization titers of CA were 1:2⁴ and 1:152 in the supernatant of HEK-293cells,1:2⁶and 1:1 290 in the supernatant of HEK-293F cells.After CA was purified,SDS-PAGE analysis showed that the heavy chain and light chain sizes of CA were 55and 25 Ku,respectively.And the results of indirect immunofluorescence detection showed that CA can specifically bind to CPV.After the transformation,CA was still kept bioactivity,and then the 4H8antibody was caninised.The eukaryotic expression vectors pcDNA3.1?+?-cL and pcDNA3.1?+?-cH were constructed by assembling the variable region sequence of the 4H8 antibody and the constant region sequence of the canine antibody found on the NCBI website.The expression and detection of caninised CPV genetically engineered antibody?CCA?was performed according to the method of CA.The hemagglutination inhibition and neutralization titers of CCA were 1:2⁵ and 1:203 in the supernatant of HEK-293 cells,1:2⁹ and 1:2 580 in the supernatant of HEK-293F cells.The purity of CCA after purification by Protein A affinity chromatography column reached over 95%,and the hemagglutination inhibition and neutralization titers were 1:2¹²and 1:32 768.The expression level of CCA in HEK-293F cells was89.65 mg/L by BCA.SDS-PAGE analysis showed that the heavy chain and light chain sizes of CCA were 55 and 25 Ku,respectively.The Western-blot results showed that the heavy chain could bind to rabbit anti-canine IgG-HRP.The results of indirect immunofluorescence detection indicate that CCA can neutralize CPV.The above results indicate that the caninised CPV genetically engineered antibody was successfully expressed in HEK-293F cells in this study,which has the characteristics of neutralizing activity,high purity and low immunogenicity.