Screening And Identification Of A B-cell Epitope On HN Protein Of Gene Ⅶ Newcastle Disease Virus

Newcastle disease(ND)is an acute,febrile and septic infectious disease of poultry that caused by Newcastle disease virus(NDV)infection,and has caused huge economic losses to poultry breeding industry all over the world.Newcastle disease virus(NDV)is a single-stranded unsegmented negative strand RNA virus belonging to paramyxoviridae and avian mumps virus genus,and is the viral factor causing Newcastle disease.The length of its genome is about 15 kb.ND is classified by the Office International Des Epizooties(OIE)as a Class I disease.It must be reported immediately to OIE once discovered.NDV encoded nucleocapsid protein(NP),the phosphoprotein(P),matrix protein(M),fusion protein(F),hemagglutinin-neuraminidase(HN)and Large RNA-dependent RNA polymerase protein(L).Studies have shown that the pathogenicity of NDV is directly related to F and HN proteins.F protein plays an important role in virus particle penetration into and fusion with cell membrane;HN protein is one of the important protective antigens of NDV,with three active functions of hemagglutination,neuraminidase activity and fusion promoting.It plays a role in identifying receptors,mediating virus to infecting cells,and cooperating with F protein in the process of virus infecting cells and promoting the fusion of virus with cell membrane.At present,there are about 12types of NDV genotypes that have been identified.The epidemiological investigation shows that the main viral strain prevalent in poultry in China is genotypeⅦ,which has a very high fatality rate after poultry infection.Therefore,it is important to studying the epitopes on HN protein ofⅦtype NDV for the development of new vaccines in the future.In this study,the HN gene of genetype VII NDV was randomly cut into small fragments between 60 bp-150 bp using DNaseⅠenzyme,and then these small fragments were inserted into the yeast expression vector PCT-XcmⅠ,the newly constructed recombinant ligation product was transformed into yeast competent cells,build genotypeⅦNDV HN protein yeast surface display random library.After calculation,the library capacity obtained is 5×10~5CFU.Eighty-two clones were randomly selected and sent to the sequencing company to determine the randomness and quality of the library.The results showed that forward and reverse insertions of HN fragments are 44 and 38 clones,accounting for 53.7%and 46.3%of the selected clones.The proportion between forward and reverse insertion was close to 1:1,and the distribution was uniform.These results indicated that the library is able to meet the needs of this research.The constructed yeast display library was transformed into yeast competent cells to induct expression,then stained with anti-genetypeⅦNDV positive serum,and sorted by flow cytometry.the obtained No.150 polypeptide reacted strongly with the positive serum.In order to verify whether the 150 peptide is the minimum antigen epitope sequences,the truncation from the N-terminal and C-terminal of the polypeptide were carried out,and the truncated peptides were displayed in the yeast cell surface and analysis using FACS after being stained with genotypeⅦchicken NDV positive serum.The results shown that the protein sequence VHDPDYIGGI is the minimum amino residues of B-cell epitope on No.150 polypeptide.In conclusion,this study successfully constructed the yeast random display library of HN protein from genotypeⅦNDV,and obtained 150G(VHDPDYIGGI)B-cell antigen epitope after screening the library by flow cytometry,which provides a theoretical foundation for the future development of novel Enzyme Linked Immunosorbent Assay(ELISA)kit or epitope vaccine based on this epitope.