Preparation And Immunological Study Of H5N1 And H7N9 Avian Influenza Subunit Vaccines

H5N1 and H7N9 highly pathogenic avian influenza virus not only seriously affect the healthy development of poultry breeding,but also pose a serious threat to human public health.At present,the prevention and control of avian influenza virus mainly depends on the whole virus inactivated vaccine,most inactivated avian influenza vaccines are produced by the chicken embryo,which has a large biosafety threat in the production process.Therefore,it is urgent to develop a novel candidate avian influenza vaccine.In this study,the avian influenza genetic engineering subunit vaccine was prepared using the hemagglutinin（HA）protein of the avian influenza virus expressed by the insect baculovirus expression system,and its immune effect was initially evaluated.In this study,the HA genes of H5N1（D889）and H7N9（E157）strains were inserted into the transfer plasmid p ACEBac1,Recombinant baculovirus（BV-HA5 and BV-HA7）were subsequently generated according to the manufacturer’s manual in the Bac-to-Bac^?system.The expressed HA protein was identified by SDS-PAGE,Western Blot and hemagglutination test,and the results of SDS-PAGE and Western Blot showed that the HA protein size of H7N9 was about 63 k Da,and the HA protein size of H5N1 was about 70 k Da;the results of hemagglutination test showed that the hemagglutination titer of HA protein of H7N9 AIV was 2¹⁶,and the hemagglutination titer of HA protein of H5N1 AIV was 2¹⁵.The immunogenicity of HA protein was evaluated by SPF chicken experiment.H7N9 and H5N1animal experiment were randomly divided into three groups,namely high dose group,low dose group and PBS control group,wherein the hemagglutination titer of the HA protein in the high-dose group was 2¹⁴,and the hemagglutination titer of the HA protein in the low-dose group was 2¹⁰,and vaccinated subcutaneously with 0.5 m L vaccine.Moreover all chickens in each group were intranasally challenged with 10⁶ EID₅₀/200μL of homologous strain after three weeks following a first immunization.Oropharyngeal and cloacal swabs were taken for detected virus at 3,5,7,9,11 day post infection（dpi）.The results showed that the average HI（Hemagglutination inhibition,HI）titer of H7N9 high dose group was6.4log2 and the low dose group was 6.1log2,HI titer between two groups was not significant（P>0.05）;the average HI titer of H5N1 high dose group was 4.6log2 and the low dose group was 3.8log2,HI titer between two groups was not significant（P>0.05）;the survival rate of SPF chickens in H7N9 high dose group and low dose group was 100%after viral challenge,and in H5N1 high dose group and low dose group was 89%after viral challenge.In the high dose group of H7N9,viral shedding was detectable in one chickens at 3 and 5 dpi,respectively;In the low dose group of H7N9,viral shedding was detectable in three chickens at 3 dpi;In the low dose group of H5N1,viral shedding was detectable in five chickens at 3dpi and two chickens at 5 dpi;In the high dose group of H5N1,viral shedding was detectable in seven chickens at 3 dpi and four chickens at 5 dpi.In this study,the HA protein of avian influenza virus was expressed by the insect baculovirus expression system,and the avian influenza genetic engineering vaccine was prepared using HA protein,which provided a new idea and technology platform for the prevention and control of highly pathogenic avian influenza.