Expression And Immunogenicity Analysis Of Fiber2 Protein Of Different Serotypes Of Avian Adenovirus In Baculovirus

Fowl adenovirus(FAdV)includes 5 species(A-E species)and 12 serotypes(1-8a,8b-11).Several serotypes of FAdV are prevalent in chickens in China.In recent years,the prevalence of serum type 2 avian adenovirus(FAdV-2),serum type 4 avian adenovirus(FAdV-4)and serum type 8b avian adenovirus(FAdV-8b)in chickens has become more and more serious,which brings difficulties to the prevention and control of the disease.Fiber2,located on the surface of avian adenovirus,is an important structural protein of avian adenovirus.Fiber2 contains neutralizing epitopes related to the infectivity of the virus and can recognize host specific receptors.FAdV-2-Fiber2,FAdV-4-Fiber2 and FAdV-8b-Fiber2 proteins were expressed by insect baculovirus expression system and the immunogenicity of the recombinant protein was further studied.According to this study included on Gen Bank FAdV-2,FAdV-4,FAdV-8b gene sequence,design contains Xhol ? and Kpn l enzyme loci of upstream and downstream primers,amplification FAdV-2-Fiber2,FAd-4-Fiber2,FAdV-8b-Fiber2 coding sequence.The recombinant plasmid was cloned into eukaryotic expression vector p Fast Bacdaul to construct baculovirus donor plasmid containing FAdV-2-Fiber2,FAdV-4-Fiber2 and FAdV-8b-Fiber2.Recombinant baculovirus plasmid was obtained by transforming it into DH10 Bac competent cells.Recombinant baculovirus was obtained by transforming the recombinant plasmid into insect cells.The expression of recombinant FAdV-2-Fiber2,FAdV-4-Fiber2 and FAdV-8b-Fiber2 in insect cells infected with baculovirus was verified by indirect immunofluorescence,and the results showed that the recombinant baculovirus with stable expression of Fiber2 protein was successfully constructed.The recombinant proteins of FAdV-2-Fiber2,FAdV-4-Fiber2 and FAdV-8b-Fiber2 were extracted by ultrasonic crushing,and SPF chickens were immunized to prepare chicken polyclonal antibodies against recombinant Fiber2 protein.ELISA method was used to detect SPF chicken antiserum.The results showed that the titer of chicken serum antibody of the immune recombinant protein was 1:4096,1:16,384 and 1:4096,respectively,indicating that the obtained recombinant Fiber2 protein had good immunogenicity.The neutralization assay was used to detect the neutralization and FAdV activity of the recombinant protein antisera on LMH cells.The results showed that the neutralization titers of the three recombinant protein antisera were 1:158,1:133 and 1:2233,respectively,indicating that the antisera could neutralize the infection of FAdV on LMH cells.The recombinant proteins of FAdV-2-Fiber2,FAdV-4-Fiber2 and FAdV-8b-Fiber2 were extracted by ultrasonic crushing and immunized with Kunming white mice to prepare mouse polyclonal antibodies against the recombinant Fiber2 protein.Indirect immunofluorescence assay showed that the rat antiserum still had good activity and specificity when diluted at 1:200 times.In conclusion: Recombinant baculovirus with stable expression of Fiber2 protein was successfully constructed in this study,which further confirmed that the expressed Fiber2 protein has good immunogenicity.These results lay a solid foundation for the study of the biological function of Fiber2 protein of FAdV,and also provide scientific reference for the prevention and control of FAdV.