Construction Of The Bivalent Chimeric Virus-like Particles Of H9N2 And H3N2 Subtypes Of Avian Influenza And Evaluation Of Its Immune Efficency

In this study,we replaced M1 protein of avian influenza virus with Gag protein of mouse leukemia virus,the HA and NA proteins of H9N2 subtype avian influenza virus and the HA protein of H3N2 subtype avian influenza virus were chimerized with Gag protein by baculovirus insect cell expression system to construct bivalent chimeric avian influenza virus like particles,the purpose of this study is to obtain a vaccine that can induce specific immunity of H9N2 and H3N2 subtypes AIV at the same time.The specific construction process was as follows: firstly,three recombinant shuttle plasmids pfastbac1-H9,pfastbac1-H3 and pfastbac1-Mgag N2 were constructed,and then they were transformed into E.coli DH10 Bac competent cells,and three recombinant bacmids,namely r Bacmid-H9,r Bacmid-H3 and r Bacmid-Mgag N2,were obtained through Bluewhite screening.Then,Sf9 adherent cells were infected with three kinds of recombinant bacmids.After 96 hours,P1 generation of baculovirus was collected.After three generations of blind passage,P4 generation of baculovirus was obtained,namely r BVH9,r BV-H3 and r BV-Bgag N2.After determining the titer of P4 generation of baculovirus,Sf9 suspension cells were co infected with three baculoviruses at MOI= 3.After 96 hours,the supernatant was purified by gradient centrifugation with 20%-30%-60% sucrose.The hemagglutination titer of the virus like particles was 28;western blot analysis showed that the target protein could be expressed correctly;Under the transmission electron microscope,the diameter of the virus like particles was about 180 nm,and the surface of the particles was embedded with fibrils;.These results indicated that the four proteins were successfully assembled into bivalent chimeric virus like particles(bc VLPs).In order to verify the immune efficency of bc VLPs,HI antibody titer and splenic lymphocyte proliferation of immunized chickens were determined.The results of HI antibody titer test showed that the HI antibody titers of bc VLPs immunized groups and vaccine immunized groups increased within three weeks after immunization.Even on the 35 th day after immunization,the HI antibody titers of bc VLPs immunized groups and vaccine immunized groups were still higher than 2log2,and the comparison between high-dose bc VLPs immunized group and vaccine immunized group showed that,there was no significant difference between high-dose bc VLPs immunized group and vaccine immunized group(P>0.05).These results indicate that bc VLPs have strong immunogenicity and can induce the body to produce specific antibodies at the same level as inactivated vaccine.The results of spleen lymphocyte proliferation showed that the Si of bc VLPs immunized group and vaccine immunized group increased with time,and reached the peak at the third week after immunization.The comparison between high-dose bc VLPs immunized group and vaccine immunized group showed that there was no significant difference between high-dose bc VLPs immunized group and vaccine immunized group at each time point(P > 0.05).These results indicate that bc VLPs can induce cellular immunity at the same level as inactivated vaccine.In order to evaluate the protective efficency of bivalent chimeric virus like particles bc VLPs constructed in this study,H9N2 and H3N2 subtypes of AIV were intranasally challenged until three weeks after immunization.The results of in vitro and in vivo detoxification showed that the time of in vitro detoxification and in vivo detoxification in high-dose and medium-dose bc VLPs immunized groups were shorter than that in vaccine immunized group,while the time of in vitro detoxification and in vivo detoxification in low-dose bc VLPs immunized group was shorter than that in vaccine immunized group or equivalent to that in vaccine immunized group.In addition,the results of IHC antigen test showed that the virus could not be detected in the lungs and trachea of the chickens in the immunized and vaccine immunized groups on the 7th day after challenge,while a large number of viruses still existed in the lungs and trachea of the chickens in the control group.Therefore,bc VLPs vaccine can better prevent virus replication and effectively protect chickens from virus attack.In conclusion,the bc VLPs constructed in this study can induce humoral and cellular immune responses of chickens at the same level as the vaccine group,and can effectively protect chickens from the attack of homologous viruses,which provides a new vaccine constructed strategy for the development of novel bivalent AIV vaccine.