Construction Of Fowl Adenovirus Serotype 4 Nucleic Acid Vaccines And Preliminary Evaluation Of Its Immune Effect

Avian adenovirus was a most harmful pathogens to poultry.Hydropericardium hepatitis syndrome(HHS)was an infectious disease caused by fowl adenovirus serotype 4(FAV-4).The disease had a rapid onset and high mortality.At present,inactivated vaccines and live attenuated vaccines were the main ways to prevent the disease.However,there were many shortcomings in these two vaccines,so it was very helpful to develop a safe and efficient new nucleic acid vaccine for the prevention and control of the disease.In this study,its structural proteins Hexon and Penton were expressed in tandem in prokaryotic and eukaryotic respectively.Hexon-Penton tandem proteins were obtained and their immune effects were preliminarily studied.The results were as follows:1.Prokaryotic Expression of Fowl Adenovirus Serotype 4 Hexon-Penton Tandem Protein and Preparation of Its Polyclonal AntibodiesIn this study,we designed the specific primers according to the published gene sequences of Hexon and Penton in NCBI GenBank,and the Hexon gene and the Penton gene were amplified by PCR.The Hexon gene and Penton gene were linked by overlapping PCR to obtain the Hexon-Penton(HP)tandem gene,and the HP gene was inserted into prokaryotic expression vector pET32a(+).The recombinant plasmid pET32a-HP was obtained and transfromd into BL21 induced with IPTG for expression.The SDS-PAGE analysis showed that the HP prokaryotic protein had a molecular weight of 108 kDa.After purification,the HP prokaryotic protein was used as immunogen to immunize the BALB/c mouse.The result showed that HP prokaryotic protein had good immunogenicity,which would establish the foundation for the further research on the serological detection method of FAdV-4.2.Expression and Analysis of fowl adenovirus serotype 4 Hexon-Penton tandem proteinIn order to obtain a Hexon-Penton tandem protein of fowl adenovirus serotype 4(FAdV-4)expressed by 293 T cells,the HP gene was inserted into the eukaryotic expression vector pcDNA3.1(+).The recombinant plasmid pcDNA3.1-HP was obtained and transfected into 293 T cells,the expression of HP eukaryotic protein in 293 T cells was detected by indirect immunofluorescence assay(IFA)and Western blot.The analysis results of Western blot and IFA of the HP eukaryotic protein showed that the HP eukaryotic protein which was well expressed in 293 T cells could be specifically recognized by the polyclonal anti-HP antibody,and the molecular weight was 90 kDa.In this study,the eukaryotic expression plasmid pcDNA3.1-HP was successfully constructed and expressed in 293 T cells,which would establish the foundation for the further research on FAdV-4 nucleic acid vaccine.3.Preliminary study on immune effect of nucleic acid vaccineIn this study,FAdV-4 HP tandem gene nucleic acid vaccine was prepared,and chickens were immunized with different doses.The immunogenicity and titer of the antibody produced by chickens were tested.The results showed that the specific antibody was transmitted to chickens after immunization with the vaccine.The antibody level reached the highest level 21 days after immunization,and the immune dose of 100?g was better.