Construction And Immune Potency Analysis Of Oral Yeast Expression E Protein Of Duck Tembusu Virus

Duck Tembusu Virus(DTMUV)spreads fast in poultry and has high pathogenicity.It is difficult to prevent and control and has the possibility of cross-species transmission,which seriously affects the healthy development of the poultry breeding industry.Although there are currently DTMUV attenuated vaccines and inactivated vaccines,these vaccines have certain risks and limitations in practice.The development of safe,efficient,and convenient immune preparations has strong application value and practical significance.This study is based on the expression system of Saccharomyces cerevisiae,constructing secreted expression,surface display and secretion-surface display double expression recombinant Saccharomyces cerevisiae strain expressing DTMUV protective antigen E protein,and its biological characteristics,immune-related indicators after oral administration in mice were evaluated.The main research results were listed as follows:(1)Construction of DTMUV-E protein recombinant Saccharomyces cerevisiae strain: The optimized DTMUV E protein expression frame was selected and the complete transcription unit was constructed.Using S.cerevisiae strains ST1814 G and JDY52 as vectors,the surface display recombinant yeast strain ST1814G/Aga2-E and the secretory expression recombinant yeast strain JDY52/xyn2s-E,secretion-surface display double expression type recombinant yeast strain JDY52/xyn2s-E-Aga2-E were successfully constructed.(2)Analysis of the main biological characteristics of the DTMUV-E protein recombinant yeast strain: The expression characteristics of the three recombinant yeasts were detected by Western blot,immunofluorescence and flow cytometry.The results showed that ST1814G/Aga2-E strain can express well and efficiently display the target protein on the surface,the best expression time is 36 h;JDY52/xyn2s-E can express well and secrete the target protein outside the cell,the best expression time is 48 h;the JDY52/xyn2s-E-Aga2-E strain achieves dual expression of surface display and secreted E protein,and the optimal expression time is 48 h.Studies on fermentation kinetics of three recombinant yeast strains showed that the insertion of exogenously expressed genes only slowed down the initial growth of yeast strains and had no effect on the later growth.(3)Analysis of changes in related immune indicators after oral administration of DTMUV-E protein recombinant yeast in mice: Saccharomyces cerevisiae oral immune preparations can significantly induce the body’s specific humoral immunity,cellular immunity and mucosal immune response;Yeast oral immune preparations regulate the body’s immunity in both directions.On the one hand,it improves the body’s immune response capacity and is safe and effective.On the other hand,it negatively inhibits the inflammatory response and avoids damage to the body due to excessive immune response.In addition,oral Saccharomyces cerevisiae immune preparations can improve the diversity of mice intestinal microbes,adjust the structure of intestinal flora,and maintain the steady state of intestinal health.In summary,the recombinant Saccharomyces cerevisiae strains successfully constructed in this study to efficiently and stably express DTMUV E protein with three different expression methods can be used as potential candidate strains for the prevention and treatment of duck tambusu virus disease.