Production, Characterization And Epitope Mapping Of Monoclonal Antibodies Against P/V Proteins Of NDV

Newcastle disease?ND?is one of the most serious infectious diseases of birds causing heavy economic losses to the poultry industry.Strick control measures are administrated in our country,including culling and mandatory immunization against NDV,which suppressed the ND epidemicand out break.However,NDV is still a threat to China poultry industry.The prevention and control of NDV are confronted with a series of problems.The use of live vaccines and the transmission of natural avirulent strains have made mixed infection very common,which seriously affected the rapid diagnosis of virulent NDV.Domestic routine determination of NDV virulence through ICPI testing and F protein gene sequencing failed to achieve rapid and timely diagnosis of NDV.It is very necessary and urgent to strengthen comprehensive researches on the etiology,epidemiology,diagnostic methods and prevention measures of NDV.In our previous work,a monoclonal antibody named McAb4 against P/V/W proteins was generated,which displayed high reactivity in IFA assay,WB test and ELISA with all genotypes of NDV.The V protein-specific CTD region was cloned into pET-28a.The His-tagged form of the ZJ1 V protein CTD region was highly expressed in E.coli by the recombinant plasmid pET-28a-ZJ1/VCD,and purified through Ni-chelating affinity chromatography under denaturing conditions.Six-week-old female BALB/c mice were subcutaneously immunized with His-VCD protein per mouse,emulsified with an equal amount of Freund's complete adjuvant.Five days after last injection,spleen cells were collected from immunized miceand mixed and fused with SP2/0 myeloma cells.After cell fusion,the supernatantof hybridoma cells in each well were detected in ELISA assay,coating with prepared His-VCD proteins.At last,a hybridoma cell line against V proteins was screened out and detected by ELISA,WB and IFA,which reacted with the genotype VII NDV strain.We found that the V protein-specific McAb3D7 could also be applied to determine the location of V protein during NDV replication in infected cells.Dynamic expression of V protein in NDV-infected cells was seen in IFA and WB.We established a series of recombinant plasmids expressing consecutively truncated His-tagged V protein and synthesized a panelof peptides for fine mapping of the epitope using WB and ELISA.These experiments further defined the McAb3D7 recognized epitope to be amino acid ¹⁵²RGPAELWK¹⁵⁹ and McAb4 recognized epitope tobe amino acid ⁴⁶ALSLAWEKHG⁵⁵.Sequence alignment showed that the sequence recognized by McAb3D7 was a region in the NDV V protein that variable among genotypes but was conserved in sequence and structure among NDV strains in the same genotype,while the sequence recognized by McAb4 was conserved in sequence and structure among all NDV strains.It suggests they could be used to differentiate NDVs genotypes or even subgenotypes.In this research,monoclonal antibodies specific for NDVP and V protein were established and identified for specificity.The epitopes targeted by those monoclonal antibodies were precisely mapped.These results would lay the foundation for the development of rapid diagnosis of NDV and novel epitope-based genotype differentiation of NDV genotypes and extend our understanding of the antigenic structure of V protein.