| Infectious bursal disease(IBD)is a highly contagious immunosuppressive disease that seriously affects the healthy development of the world’s chicken industry.Infectious bursal disease virus(IBDV)is the pathogen of IBD.It mainly infects the developing B lymphocytes in the bursa of fabricius of chickens,causing the bursa of fabricius of the infected chickens to shrink and reduce the immune function,eventually leading to immunosuppression and increasing susceptibility to other pathogens.Currently,IBD is mainly controlled through vaccination.As traditional vaccines,live vaccines and inactivated vaccines have the defect of strong virulence or low antigen content.IBDV VP2 is an important structural protein that can induce the body to produce neutralizing antibodies.In this paper,Saccharomyces cerevisiae was used as an expression system to construct a recombinant Saccharomyces cerevisiae strain expressing IBDV VP2 protein and used as an oral preparation,in order to bring possibilities for the prevention and control of IBDV infection.The main research results of this subject are as follows:(1)Construction of recombinant Saccharomyces cerevisiae strains expressed VP2 proteins: the Yeast Fab Assembly method was used to construct five kinds of recombinant Saccharomyces cerevisiae strains expressed VP2 protein with yeast strains ST1814 G and JDY52 as the chassis: surface display recombinant yeast strain ST1814G/VP2,secretory expression recombinant yeast strain JDY52/VP2,surface display-secretion tandem expression recombinant yeast strain ST1814G/(aga2-VP2)-(xyn2s-VP2),surface display-secretion dual-chromosome expression recombinant yeast strain ST1814G/(xyn2s-VP2)IV/(aga2-VP2)Ⅺ and VP2 truncated body surface display recombinant yeast strain(ST1814G/d VP2).(2)Study on expression characteristics of the recombinant S.cerevisiae strains expressed VP2 proteins: Western blot and immunofluorescence were used to detect the expression characteristics of the five kinds of recombinant yeasts.The results showed that ST1814G/VP2 strain can express efficiently and display VP2 protein on the cell surface,and the best expression time is 24h;The JDY52/VP2 strain can express and secrete the target protein to the outside of the cell;ST1814G/(aga2-VP2)-(xyn2s-VP2)strain achieved dual expression of surface display and secretion of the target protein on the same chromosome;ST1814G/(xyn2s-VP2)Ⅳ/(aga2-VP2)Ⅺ strain realized the dual expression of VP2 protein surface display and secretion on double chromosomes;ST1814G/d VP2 strain can express and display the target protein on the cell surface.Analysis of fermentation kinetics showed that the expression of VP2 protein would affect the growth of recombinant yeast.(3)Evaluation of the immune effect of oral recombinant S.cerevisiae strains expressed VP2 proteins: After preparing the ST1814G/VP2 strain into an oral preparation to immunize mice,the viscera index of the spleen and thymus of mice were increased.ELISA detected the kinetic changes of antibodies in the serum and feces of immunized mice.The results showed that the specific Ig A and Ig G of the experimental group were significantly increased.The changes in the transcription levels of a variety of immune-related molecules in the spleen and thymus after immunization were measured by fluorescence quantitative PCR.The results showed that recombinant yeast can cause changes in the transcription levels of a variety of immune-related molecules,and significantly up-regulate the transcription levels of immune molecules such as IL-2,TBX21,Foxp3 and IDO.It showed that oral administration of S.cerevisiae strains expressed VP2 proteins can stimulate the body’s immunity to a certain extent.In addition,16 S r DNA sequencing results of the intestinal flora of immunized mice showed that oral recombinant yeast immune preparations can enrich the types of intestinal flora.This showed that it plays an important role in regulating the structure of the intestinal flora and maintaining healthy and steady state of the intestinal tract.In summary,this study successfully constructed five kinds of recombinant S.cerevisiae strains expressed VP2 proteins,preliminarily characterized its expression characteristics,verified the immune efficacy of recombinant S.cerevisiae oral preparations through mouse immunization experiments,and provided references for future vaccine prevention and control of IBDV. |
