The Effects Of FliC Protein And PEI As Adjuvants For H9N2 Inactivated Avian Influenza Vaccine

Low-pathogenic avian influenza virus（LPAIV）infection in chickens usually results in economic losses to the farming industry,and poses a threat to human health.Poultry vaccination is an important strategy for controlling avian influenza infection and transmission,the currently used bird flu vaccine is still an inactivated whole virus vaccine,because inactivated whole virus vaccines mainly induce humoral immune responses,their antibody production is slower and cannot induce effective cellular immunity,studies have shown that effective vaccines need to select appropriate adjuvants in addition to antigens to activate the corresponding immune response,therefore,the use of adjuvants is essential to enhance the immune efficacy and cellular immune response of inactivated vaccines.In this study,two immune adjuvants,flagellin（FliC）and polyethyleneimine（PEI）,were selected as the research objects,the eukaryotic expression system and prokaryotic expression system were used to explore the expression and purification of the protein adjuvant FliC,and the prepared adjuvants were respectively subcutaneously immunized with the H9N2 avian influenza inactivated virus,and commercial H9N2 inactivated vaccines were set up as contrast,SPF chicken was used as an animal model to compare the immune enhancement effects of different adjuvants.The main research contents are as follows:1.Eukaryotic expression,prokaryotic expression and purification of FliC proteinNCBI gene library comparative analysis to determine the flagellin（FliC）sequence of Salmonella typhimurium,and the target protein was expressed by the eukaryotic expression system and the prokaryotic expression system respectively.The target gene FliC was successfully inserted into the eukaryotic expression vector p Fast Bac^TM 1 and the prokaryotic expression vector p ET-32a,and further constructed to express the target proteins FliC-B（FliC protein expressed by insect baculovirus expression system）and FliC-E（FliC protein expressed by E.coli expression system）.The expressed FliC protein was identified by SDS-PAGE and Western-Blot analysis.The results showed that the size of FliC-B protein was about 54 k Da,and the size of FliC-E protein was about62 k Da,and most of the protein is released in the supernatant,and can react with His-tag monoclonal antibody,showing good reactogenicity.After purification of FliC-B protein and FliC-E protein by nickel column,a large number of target proteins were obtained.2.Preparation of inactivated H9N2 avian influenza vaccineReferences set the final content of the adjuvant to 20μg/0.3 m L,and configure the H9N2avian influenza inactivated vaccine according to the virus content of the H9N2 commercial inactivated vaccine(Every 0.1 ml of virus before inactivation content≥5×10⁷ EID₅₀),as follows:The H9N2 avian influenza virus was inactivated with formalin.The inactivated H9N2avian influenza virus was mixed with FliC-B protein,FliC-E protein,and PEI adjuvant at a volume ratio of 13:2 as the aqueous phase.The vaccine aqueous phase and the SEPPIC MONTANIDETM ISA 201VG adjuvant were emulsified at a volume ratio of 1:1 to obtain an H9N2 inactivated avian influenza vaccine.3.Comparison of immune enhancement effects of FliC and PEI adjuvant on H9N2 avian influenza inactivated vaccineTwo-week-old SPF chickens were used as animal models,which were divided into the following five groups:PBS group,H9N2 commercial inactivated vaccine group,H9N2+FliC-B group,H9N2+FliC-E group,H9N2+PEI group.According to the immune dose of each chicken was 0.3 m L,the adjuvant activity was compared by detecting the humoral and cellular immune response levels of each group.By detecting the serum HI antibody titer within 3 weeks after immunization,the results showed that one week after immunization,the addition of different adjuvants could quickly cause the body’s humoral immune response.and the titers of HI antibodies were above 2⁴,which was extremely different from the H9N2 commercial inactivated vaccine group（P<0.01）;Compared with different adjuvant groups,the humoral immune activation ability of PEI was the strongest,and the FliC-E group was the weakest,and the HI antibody titer between the two groups was significantly different（P<0.05）.The expression of pre-inflammatory cytokines in the spleen of chickens was detected 24 hours after immunization,the expression levels of pre-inflammatory cytokines in the H9N2+FliC-B group were slightly higher than those in the other groups,but there was no statistical difference.Peripheral blood lymphocytes were isolated 3 weeks after immunization,and lymphocyte proliferation was measured,compared with the PBS group,the H9N2+FliC-E group can significantly increase the specific proliferation of chicken peripheral blood lymphocytes,which was 1.7 times that of the PBS group（P<0.05）,the H9N2+FliC-B group had a higher proliferation index,which was 3.0 times that of the PBS group（P<0.01）;IFN-γcytokine m RNA test results showed that the expression level of IFN-γin the H9N2+PEI group was significantly higher than that in the PBS group and the H9N2 commercial inactivated vaccine group,which were 3.0 times and 3.1 times,respectively,with significant differences（P<0.05）.In summary,H9N2 inactivated vaccines combined with different adjuvants showed good adjuvant activity in both humoral and cellular immunity.Among them,PEI showed greater potential than FliC-E in inducing humoral immunity.In this study,the FliC protein were successfully prepared using eukaryotic expression system and prokaryotic expression system,and the immune enhancement effect of different adjuvants combined with H9N2 avian influenza inactivated virus was evaluated,which not only provides a certain reference for the preparation method of protein adjuvants,it also provides new solutions for the use of immune adjuvants in the configuration of low-immunogenic vaccines other than inactivated vaccines.