Expression And Immunogenicity Of Newcastle Disease Virus F Protein In Different Systems

Newcastle Disease(ND)is a contagious infectious disease that affects poultry,whichcreated by Newcastle Disease virus(NDV).It causes serious harm to the poultry breeding industry.Fusion(F)protein mainly mediates the fusion of NDV with the cell membrane of the host cell that plays a major virulence role.Therefore,the extracellular domain of the NDV-F protein of the virulent strain of Newcastle disease virus F48E9was expressed in prokaryotic and eukaryotic cells respectively.Furthermore,the antibody titer and immunogenicity produced by FIPV-N were identified.First,the recombinant plasmid is constructed according to the screening of theantigen epitope.The antigenicity and hydrophobicity of NDV-F protein were predicted by biological software,and the N-terminal 267-500 aa(234 aa)of F protein was selected as the main antigenic domain.The 267?500 aa fragments encoded by the F gene were amplified by PCR and ligated to the p Cold-Trx A prokaryotic expression vector.The p Cold-Trx A-F_{267⁵00 aa} recombinant plasmid was obtained by bacterial liquid PCR detection,double enzyme digestion and sequencing.The expression of recombinant protein was induced by E.coli(T7 shuffle),and the recombinant protein Trx A/F_267⁵00aa with a molecular weight of about 40 k Da was obtained.Secondly,the eukaryotic recombinant plasmid of NDV-F was constructed and expressed.The extracellular region of NDV virus F protein(NDV-F_32⁵00aa)was amplified by PCR and ligated to p Fuse vector.The p Fuse-F_32⁵00aa recombinant plasmid was obtained through bacterial liquid PCR detection,double enzyme digestion technology and sequencing.The recombinant plasmid was transfected into 293 F cells for protein expression,and the protein His/F_32⁵00aawith a molecular weight of about 70k Da was obtained.The 10 SPF chickens were divided into two groups,5 in each group.The first groupwas named the PBS group,and chickens in this group were immunized with 200?L PBS.The second group was the Trx A/F_267⁵00aa protein group.Chickens in this group were immunized with 200?g of purified recombinant protein Trx A/F_267⁵00aa.Blood was collected before immunization as a negative control,and blood was collected 2weeks after the first immunization,1 week after the second and third immunizations as the experimental group.The ELISA plates were coated with PBS and recombinant protein Trx A/F_267⁵00aa respectively.The serum collected from each group was used as the primary antibody,and the commercial HRP-labeled goat anti-chicken antibody was used as the secondary antibody.The antibody titer of PBS group and specific Trx A/F_267⁵00aa protein group was detected by ELISA.The results showed that there was almost no immune titer in the immune serum of the PBS group.The results showed that there was almost no immune titer in the serum of the PBS group.However,the antibody titers in the serum after Trx A/F_267⁵00aa immunization were higher than those in the negative serum.The antibody titer of 2 and 3 immunity is higher than that of 1immunity,and the titers of 2 and 3 immunity antibodies are similar(1:40500).The antibody titer of the second and third immunization serum was higher than that of the first immunization,and the antibody titers of the second and third immunizations were similar(1:40500).Afterwards,the activity of the recombinant protein Trx A/F_267⁵00aawas further verified by Western Blotting.The result showed that a band appeared at40Kda.These results indicated that the Trx A/F_267⁵00aa protein expressed in prokaryotic cells was immunogenic.Finally,the ELISA plate was coated with His/F_32⁵00aa protein.Serum collected from SPF chickens immunized with Trx A/F_267⁵00aa protein group was used as the primary antibody,and commercial HRP-labeled goat anti-chicken antibody was used as the secondary antibody.The results showed His/F_32⁵00aa protein activity was similar to that of recombinant protein Trx A/F_267⁵00aa.These results indicated that Trx A/F_267⁵00aaprotein with immunogenicity was successfully obtained.