The Construction And Biological Characteristics Of Recombinant Saccharomyces Cerevisiae Strains Displaying Avian Influenza Antigen

Avian influenza is an acute respiratory infectious disease caused by avian influenza virus(AIV),which is quite harmful to the poultry industry.Birds infected with AIV show different degrees of respiratory symptoms,decreased egg production rates and even systemic diseases,which has a high morbidity and mortality.Vaccination is currently the main strategy for preventing and controlling bird flu.Avian influenza virus has multiple serotypes,is susceptible to mutation,and has problems such as infectious infection after inactivated vaccines.A pan-cross-protective vaccine that can stimulate mucosal immunity has been developed.The HA head region,which contains large variability between different serotypes and provides the main protective antigenic epitope,and the HA stem region,which is relatively conserved among different influenza virus subtypes and can generate extensive cross-protection,is an effective strategy for designing genetically universal influenza vaccines.Saccharomyces cerevisiae surface display technology is an eukaryotic display system that immobilizes and expresses foreign proteins,which fuses exogenous gene sequence with a specific vector gene sequence and then introduces them into the yeast cells,and makes uses of protein transport to the membrane surface to anchor the target proteins to the cell surface for expression.In this study,based on the HA sequence,structural analysis of H5,H7 and H9 subtypes of avian influenza virus hemagglutinin,the anchored expression of HA truncated proteins containing three subtypes of avian influenza virus was constructed.The recombinant Saccharomyces cerevisiae strains were characterized for its biological characteristics and the immunopotency of the recombinant Saccharomyces cerevisiae strain as a candidate vaccine was preliminarily evaluated by oral administration of the strain to commercial laying hens.Main research methods and results are as follows:Immunogenicity analysis of HA protein of three subtypes of avian influenza virus H5,H7,and H9 and screening of expression domains: Conservative analysis of HA sequences of three subtypes AIV,prediction of B cell epitopes,and identification of HA structural simulation,from which highly conserved regions containing B-cell dominant epitopes were obtained.Head regions of HA proteins of H5 subtype(Genbank number: AAY56367.1)and H9 subtype(Genbank number: AAR98872.1)were selected,and part of the head and stem of HA proteins of H7 subtype(Genbank number: AGQ80952.1)were selected which contains the binding sites of currently known neutralizing antibodies.The truncated sequence encoding those regions were selected for use in constructing the recombinant Saccharomyces cerevisiae expression vectors;Construction and screening of recombinant Saccharomyces cerevisiae strains:The transformed Saccharomyces cerevisiae strain is used as an expression vector,and the transcription unit containing the target gene is introduced into the Saccharomyces cerevisiae host bacteria by means of lithium acetate transformation.The verification method screened and purified the recombinant Saccharomyces cerevisiae strain.The biological characteristics of the selected recombinant Saccharomyces cerevisiae strains were studied.The results of Western Blot,immunofluorescence and flow cytometry showed that AIV truncated proteins were successfully displayed on the surface of Saccharomyces cerevisiae;Immune characteristics of recombinant Saccharomyces cerevisiae: commercial laying hens take orally the vaccine prepared by recombinant Saccharomyces cerevisiae strain at the dose of 0.8 m L/feather.On the premise of containing mother antibody,the HI antibody titer of three subtypes of AIV(H5,H7 and H9)in serum after immunization of recombinant Saccharomyces cerevisiae strain is equivalent to that of commercial adjuvant vaccine,which can provide immune protection.In summary,we have obtained a recombinant Saccharomyces cerevisiae strain expressing three subtype AIV membrane protein HA truncated proteins.As an oral vaccine to immunize commercial laying hens in chicken farms,it has shown a certain immunogenicity,compared with traditional vaccines.It has the advantages of safety,convenience,low cost,and less stress response to poultry,and can effectively respond to influenza outbreaks.The selection of the HA head region and the design of the epitope of the stem region can provide a theoretical basis for the future development of a broad-spectrum avian influenza vaccine,and provide a reference for the prevention and treatment of influenza viruses.