Construction Of IBV S1 And Cps MOMP Gene Combined Epitope Vaccine And Immune Effect Evaluation

Infectious bronchitis(IB)is a respiratory disease that affects poultry and is caused by the infectious bronchitis virus(IBV).The disease is highly contagious and can make poultry more susceptible to secondary infections by bacteria or other pathogens,ultimately increasing mortality rates.The S1 protein is the major structural protein of IBV and plays an important role in the induction of specific neutralising antibodies,which contribute to the immune response against the virus.Avian chlamydiosis(AC)is a disease caused by the bacterial infection with Chlamydia psittaci(Cps).This infection can lead to stunted growth and increased mortality in infected chicks,and can also be a source of prolonged infection due to shedding of Chlamydia.IBV and Cps are common respiratory diseases in the poultry industry.Mixed infections are common and result in significant economic losses to the industry.Additionally,these infections pose a serious threat to human health.In this study,we employed multiple online biological software tools to identify the dominant antigenic epitopes of S1 and MOMP proteins.Subsequently,we designed the multi-epitope gene M and predicted and analysed its biological properties including physicochemical properties,hydrophilicity,transmembrane region,protein secondary and tertiary structures using online software such as Prot Param,Ex PASy,Vaxi Jen,TMHMM and SOPMA.The genes M,S1,and MOMP were combined and inserted into the eukaryotic expression vector p EGFP-N1.The size of the target gene was confirmed by PCR and double digestion techniques.The three recombinant plasmids p EGFP-M,p EGFP-S1-MOMP and p EGFP-M-S1-MOMP were transfected into DF-1 cells,and the safety of the recombinant plasmids was evaluated by CCK-8,Western blot assay to detect the expression of target proteins and IFA assay to verify the immunolocalisation of recombinant plasmids in cells.The study demonstrated that the screened multi-epitope gene M exhibited strong immunogenicity.The researchers constructed three sets of recombinant plasmids using double digestion,PCR,and sequencing techniques.The plasmids showed specific target bands at 837 bp,2952 bp,and 3789 bp,respectively,without any alterations in the nucleotide sequence.Furthermore,the expression of these plasmids in DF-1 cells did not cause any apparent damage to the cells.In this study,a total of fifty chicks were randomly assigned to the p EGFP-M group,p EGFP-S1-MOMP group,p EGFP-M-S1-MOMP group and control group(p EGFP-N1 group and PBS group).The chicks were immunised with recombinant plasmids with an interval of14 days between each immunisation,for a total of four immunisations.Serum samples were collected from the chicks at 28 and 56 days after the first immunisation and the levels of specific antibody Ig G,cytokines and T lymphocytes were measured.Compared with the control group,after four immunisations,the serum levels of p EGFP-M,p EGFP-S1-MOMP and p EGFP-M-S1-MOMP groups were highly significantly higher(P<0.01),and there was no significant difference between the three recombinant plasmids(P>0.05);the serum levels of cytokines IFN-γand T lymphocytes in chickens of p EGFP-M,p EGFP-S1-MOMP and p EGFP-M-S1-MOMP groups were significantly higher(P<0.01).The levels of cytokines IFN-γand IL-4 and T lymphocyte antigens CD3~+,CD4~+and CD8~+were significantly or highly significantly higher in the p EGFP-M,p EGFP-S1-MOMP and p EGFP-M-S1-MOMP groups compared to the control groups(p0.05).The results of organ ratio analysis showed that there was no significant difference between the three recombinant plasmid groups and the control group(P>0.05).In addition,no significant pathological changes were observed in the parenchymal organ tissue sections.In conclusion,the duplex epitope gene vaccines of epitope genes M and S1 and MOMP genes constructed in this study were able to induce humoral and cellular immune responses in chicks after immunisation of each group was immunized,and were able to produce specific immune responses,which laid the foundation for the development of a multi-epitope vaccine for the simultaneous prevention of infectious bronchitis and avian chlamydiosis in chickens.