| Infectious bronchitis virus(IBV)is a single-stranded positive-stranded RNA virus,which belongs to the coronavirus family gamma coronavirus.Infectious bronchitis(IB)caused by IBV has been widely spread worldwide since it was discovered in 1931,causing serious harm to farming enterprises.Although the use of vaccines has controlled IB to some extent,the disease is currently circulating in many places,the situation of prevention and control is still not optimistic.Vaccine immunization is currently the main means of preventing and controlling IBV,so it is particularly important to evaluate the effectiveness of vaccine use.At the same time,some commercial vaccines currently offer limited protection against different serotypes of IBV,so it is of great importance to develop new IBV vaccines that are safe and easy to apply.In this study,we analyzed the conserved sequences of IBV Spike protein(S)and nucleocapsid protein(N),established a peptide ELISA method to detect the antibodies against S protein and a N2-ELISA method to detect the antibodies against N protein,and generated a broad-spectrum monoclonal antibody against IBV N protein.The recombinant FAdV-4 viruses FAV4-EX1 and FAV4-EX2 expressing IBV multiple epitopes were constructed using CRISPR/cas9 technology,and the protection effect of the recombinant viruses against FAdV-4 and IBV was explored by attack protection assay,which provides new ideas for the vaccine development of IBV.1.Establishment of an ELISA method for detecting IBV antibodies based on conserved peptides of S2 proteinIn this study,the S protein of different IBV strains was analyzed by biological software,and a conserved antigenic peptide(P2)located on S2 was discovered and synthesized,which was used as the coating antigen,and the conditions of antigen coating concentration,serum dilution,antibody dilution,primary antibody incubation time and secondary antibody dilution concentration were explored,and finally a pELISA based on the conserved antigenic peptide(P2)for the detection of IBV.The pELISA method showed good specificity and sensitivity in the detection of IBV antibodies against different serotypes(QX,TW,GVI,4/91 and H120).The relationship between pELISA results(S/P values)and endpoint antibody titers of serum samples was initially established by semi-logarithmic regression equations(log10(titer)=(2.823-S/P)/7.045).pELISA-predicted antibody titers were totally consistent with those assessed by IFA.In addition,the accuracy between the pELIS A and the commercial IDEXX ELISA was 96.07%.All these data suggest that pELISA can be used as a rapid and reliable serological assay for monitoring IB V immunity or infection in chickens.2.Establishment of ELISA method based on conserved N protein sequences and preparation of N protein monoclonal antibodyIn this study,N genes of different genotypes of IBV strains were analyzed to select conserved and antigenic sequences.The antigenic regions with high conservation were cloned into pET-28a(+)vector,extensive expressed by prokaryotic expression system and purified.The purified fusion protein His-N2 were used to establish the ELISA method for detecting IBV antibody and to prepare monoclonal antibody.Finally,an ELISA method(N2-ELISA)for detecting IBV antibodies based on amino acids 120-265 of N protein was established,which can effectively detect antibodies in IBV infected and immunized chickens without reacting with antibodies to other pathogens.Meanwhile,the prokaryotic expression products of conserved N protein sequences were used as the immunogen for the preparation of mouse monoclonal antibodies,and finally,broad-spectrum monoclonal antibodies that cross-reacted with different serotypes of IBV were obtained,which laid the foundation for the establishment of a broad-spectrum and sensitive IBV antigen detection method.3.Construction of FAdV-4 recombinant virus expressing multiple epitopes of IBV and evaluation of its immune effectIn this study,the recombinant viruses FAV4-EX1 and FAV4-EX2 were successfully constructed and purified by replacing Fiber-2 with the expression cassette of tandem IBV multiple epitopes using CRISPR/Cas9 technology,using the EGFP and Fiber-2 fusion-expressed recombinant viruses as template viruses.Both FAV4-EX1 and FAV4-EX2 replicated stably in chicken hepatoma cell lines in LMH cells,but were slightly weaker than wild-type FAdV-4 due to the absence of Fiber-2.Both recombinant viruses produced neutralizing antibodies against FAdV-4 and IBV after inoculation with 14-day-old chicks,and were able to resist the strong virulent attack of FAdV-4 after 21 days.However,in the IBV infection and protection experiment,it was found that chickens inoculated with the recombinant virus were not able to resist the IBV QX virus attack and even showed higher mortality.Chicken serum infected with recombinant viruses for 21 days were mixed with IBV virus and infected CEK cells,it was found that the serum at this time could significantly promote IBV infection,which indicated that the antibodies induced by amino acids 19-69 epitope in S protein could cause ADE effect.Although the recombinant virus developed in this study can not provide effective protection against IBV,it provides a new idea for the construction of a recombinant multiplex vaccine between FAdV-4 and IBV. |
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