Optimization Of Oral Recombinant Yeast-Based Immunization System And Application Research Of Yeast Vaccine In The Carp

Since Steinman et al.discovered dendritic cells in 1973,the development of immunotherapy targeting the dendritic cell has achieved remarkable progress.However,the immunotherapy by using this discovery has been often tried and developed using the patients' own dendritic cells,and the cost is high and it is not easy to promote.To target the dendritic cells for inducing the specific immune response,the various microbial-mediated immune vaccine delivery systems have gradually gained people's attention because of their advantages such as convenience and low cost.However,there are still bottlenecks such as biosafety and inconvenient inoculation methods,which greatly limits the further application of these immunization systems.To solve the problems in the development of this type of immunization system,our laboratory has been committed to the development and optimization of oral recombinant yeast immunization system since 2011.At present,the development of this system mainly focuses on two fields: the immune regulation of endogenous genes through the oral recombinant yeast immunization system and oral recombinant yeast DNA vaccines.The experimental subjects involve a variety of animals,which provide important reference and more choices for the development of convenient,practical,safe and low cost new oral recombinant yeast vaccine technology.Based on our previous research results,this research first performed targeted knockout of genes that inhibits glucan synthesis in yeast,and uses the Cre-loxP system to delete drug resistance-gene to avoid biosafety problems;afterward,oral feeding experiments were performed on the living animals using an oral recombinant yeast immunization system prepared from the gene-knockout strains without drug resistance-gene,and the better strain' screening was performed compared to the wild type yeast strain.And subsequently based on the selected gene-knockout strain,the DNA expression plasmid in the oral recombinant yeast immunization system is optimized for providing a useful reference to subsequent construction of a more efficient oral recombinant yeast immunization system.At the same time,combined with bioinformatics,two oral recombinant yeast-mediated DNA vaccines were screened and constructed.After a series of verification experiments,oral yeast DNA vaccine was found to have an obvious preventive effect on the motile aeromonad septicemia.The main findings of this research are as follows:1.A more efficient oral recombinant yeast immunization system was screened.The four genes that inhibits glucan synthesis in S.cerevisiae genome were knocked out by traditional genome editing technology,combining with the Cre-loxP technology.Then the oral recombinant yeast immunization system prepared by above 15 strains was validated by the mouse model,and the weight data was combined with Western blotting and enzyme-linked immunosorbent assay,and used to verify the immune effects of different experimental groups,and the strains(GUP1? EXG1? and PBS2?MNN9? gene-knockout strain)with the better immune effect were selected.2.The DNA expression plasmid in the oral recombinant yeast immunization system was optimized.Based on the selected gene-knockout strains,three optimized DNA expression vectors were constructed by fusion of the chicken OVA gene and co-expression of shRNA of IL-21 gene.The immune responses of different experimental groups were verified by Western blotting and enzyme-linked immunosorbent assay,and the DNA expression plasmid with better immune response was screened to further optimize the oral recombinant yeast immunization system.3.Oral yeast DNA vaccines for the carp that can be used to prevent the motile aeromonad septicemia caused by Aeromonas hydrophila has been constructed.Bioinformatics means were first used to optimize and obtain two antigens with higher immunogenicity,thus ensuring the high antigenicity of the immunizing antigen;then the oral yeast feeding method was optimized for the fish,by mixing the recombinant yeast with the feed to increase the amount of yeast in the intestine of the fish;to visually confirm the position of the oral vaccine into the intestine,the GFP reporter vectors were constructed: when the antigenic protein was expressed in the intestinal specific cells,the expression of the GFP gene was simultaneously transcribed,and further green fluorescence can be observed;in addition,the presence of antibodies against specific antigens was confirmed by Western blotting and the better vaccine was further confirmed by enzyme-linked immunosorbent assay.The ova-ompG group can induce better immune effects,laying the foundation for subsequent challenge experiments.Finally,the oral yeast DNA vaccine(ova-ompG group)was fed orally to verify the vaccine effect of preventing the motile aeromonad septicemia by using a challenge experiment.In summary,this study used homologous repair mechanism to generate 15 strains of knockout yeast and screened the better strains that has better immune response: GUP1?EXG1? and PBS2?MNN9? gene knockout strain,which can be used for constructing oral recombinant yeast immunization system.Three optimized DNA expression vectors were then constructed by fusion of chicken OVA gene.By co-expression of IL-21 gene interfering RNA,the DNA expression plasmid with better immune response was determined.Meanwhile,the feasibility of an oral recombinant yeast DNA vaccine was tested in the carp.This research lays the foundation for the application and further investigation of oral yeast vaccine in the future.