Study On Immunodominant B-cell Epitopes Of Hn Protein And Anti-V Protein Monoclonal Antibody In Newcastle Disease Vaccine Strain LaSota

Newcastle disease（ND）is a highly infectious disease of birds leading to poultry infection and death.It is caused by Newcastle disease virus（NDV）.The hemagglutinin-neuraminidase（HN）and V protein are virulence factors of NDV and closely related to the viral pathogenicity.As an important host protective antigen,the HN can induce neutralizing antibodies in animals.It is also a target protein for clinical vaccine design and immunological detection.The epitopes of HN have not been fully elucidated.Therefore,the identifying epitopes of HN would be insight into the understanding for antigenic characteristics of the HN and development of vaccine.The V protein plays very important roles on virus replication,pathogenicity and antagonism of interferon.However,it is very difficulty for further studies without anti-V protein monoclonal antibody,including little known about the epitopes of V protein.The preparation of V protein-specific monoclonal antibodies can provide a powerful tool for biological characteristics of the V protein.In this study,we identified the dominant epitopes of HN and prepared the monoclonal antibodies against V protein that improved the understanding of viral protein epitopes and provided more support for the research on the pathogenic mechanism and vaccine development of NDV.The main contents and results are as follows:1. Screening and identification of immunodominant B-cell epitopes of the HN（1）Screening of immunodominant epitopes（IDE）.Five B-cell IDE candidates of the HN of vaccine strain La Sota were screened by using a pepscan approach with the NDV-specific chicken hyperimmune antisera.The five IDE candidates were fused with RFP genes for eukaryotic expression and five peptides were synthesized.Using IFA and Dot blot analysis,the five IDEs were verified.（2）Immunogenicity of the IDEs.The mice were immunized with the synthetic peptides of five IDEs.The antisera were prepared for serological analysis.The Dot blot,IFA,ELISA,and Western Blot analysis showed that the five IDEs stimulated mice to produce the high levels of antibodies,which specifically bound the HN,indicating that they had good immunogenicity.The antisera of IDE1,IDE2,IDE3 and IDE4 could recognize Calss II and Class I strains and their epitope amino acid sequences are highly conserved,while IDE5antisera could only recognize Class II strains that could be used to identify viruses strains between Class II and I.（3）Identification of the virus-neutralizing epitope.The virus-neutralizing assay in vitro showed that the IDE4 mouse antisera partially blocked the infection of avirulent La Sota and slightly inhibited the infection of virulent viruses JS17 and F48E9,while other IDE antisera had no neutralizing activity.This results indicated that the IDE4（242-256aa）is a potential neutralizing epitope of the HN.2. Study on monoclonal antibody against V protein（1）Preparation of monoclonal antibody（m Ab）.The C-terminal domain（CTD）of V protein of vaccine strain La Sota was expressed in prokaryotic system,and then the BALB/c mice were immunized for preparing the m Ab agianst V protein.The m Abs were screened by ELISA,IFA and Western Blot method.Two positive hybridoma cells 5C5 and 6D4 were obtained,and they could specifically recognize the V protein.（2）The characteristics of 5C5 m Ab.The 5C5 m Ab could only specifically recognize La Sota（Class II genotype II）,but do not respond to Class I strains（Pigeon/QH-1/Ch/14）and Class II virulent strains SX10（genotype VI）,JS17（genotype VII）,F48E9（Genetic type IX）.The results indicated that the 5C5 m Ab could be used to differentiate between the La Sota and other strains.Using the truncated V proteins in eukaryotic expression system,a linear epitope ¹⁴⁶TSDSTAGEST¹⁵⁵ recognized by the 5C5 m Ab was identified.This antigenic epitope was located in the variable region of V-CTD and their amino acid sequence greatly varied among strains.In conclusion,our studies identified five linear IDEs and a potential neutralizing epitope IDE4（242-256aa）of the HN using pepscan technology.Two mAbs 5C5 and 6D4were successfully obtained against V-CTD.The 5C5 m Ab and recognized a linear epitope¹⁴⁶ TSDSTAGEST ¹⁵⁵,which was La Sota-specific.These results provided basic support for the HN epitopes,development of vaccines and the investigation of V protein function.