Construction And Immune Potency Evaluation Of Recombinant S.cerevisiae Strains Expressed African Swine Fever Virus Proteins

African swine fever(ASF)is a highly contagious infectious disease caused by African swine fever virus(ASFV),leading to high mortality and chronic persistent infection.At present,the epidemic ASFV strain in China belongs to P72 gene type II,which shows the epidemic situation in multiple provinces and regions.The structure of ASFV particles and its pathogenic mechanism are peculiar,indicating the great challenge of vaccines development.In this paper,Saccharomyces cerevisiae(S.cerevisiae)was used as a vector to express various ASFV proteins,and the possibility of preventing and controlling ASFV infection by oral administration was explored.The main results of our research are as following:(1)Screening for genes encoding major immunogen proteins of ASFV:Bioinformatics analysis of the structure and function of major ASFV immunogen proteins was performed,and then we synthesized a series of genes that play an important role in the process of ASFV infection and proliferation,as candidate genes in recombinant yeasts design.(2)Design and construction of different ASFV genes expression vectors: Three types of S.cerevisiae expression vectors(Type 1: ASFV genes tandem expression,Type 2: ASFV protein-porcine Ig heavy chain fusion expression,Type 3: membrane anchorage and secretion expression)were designed and constructed.We constructed Aga2-ASFV protein-coding gene transcription unit(GPD-Aga2-ASFV genes-ADH1)through the application of Yeast Fab module assembly method.Sequential connection and assembly of large genome fragments in vitro was completed as well as homologous recombination module URR1,URR2,and auxotrophic screening module LEU2,through a single tube method based on the principle of complementary terminal pairing of large fragments.The single tube reaction fragments are transformed into S.cerevisiae strain ST1814G(or ST1814G/TEV,ST1814G/R3C)using lithium acetate(Li Ac).The goal of stably integrating the Aga2-ASFV protein expression element into ST1814G(or ST1814 G /TEV,ST1814G/R3C)and the HO locus(URR1-HIS3-URR2)of the chromosome IV was achieved based on the mechanism of repair and homologous recombination achieves,which is selected by SD-LEU Culture medium screening.Primary recombinants were identificated by PCR and recombinant S.cerevisiae strains expressed ASFV proteins were obtained.Furthermore,two types of recombinant S.cerevisiae strains expressed TEV/R3 C protease on golgi and 10 recombinant S.cerevisiae strains expressed 14 ASFV proteins(Type 1: P72-A104R-CP312R-P12,CD2v-EP153R-EP152R-P17,PP62-P22;Type 2: P30-?Fc,P54-?Fc;Type 3: P30-F2 A,CD2v-F2 A,E248R-F2 A,E120R-F2A)were constructed.(3)Exploration of expression characteristics of the three types of recombinant S.cerevisiae strains expressed ASFV proteins: Western blot,immunofluorescence,and flow cytometry were performed to analyze the target proteins expression and fermentation kinetic characteristics of S.cerevisiae strains expressed ASFV proteins.The fermentation kinetics indicated that the expression of ASFV proteins would affect the growth of recombinant S.cerevisiae strains to a certain extent.(4)Immune potency evaluation of recombinant S.cerevisiae strains expressed ASFV proteins-porcine Ig heavy chain: The effects of different Ig heavy chains and Fc receptors on yeast-cell adhesion and internalization of phagocytosis were evaluated through the phagocytosis experiment of IPEC-J2 and PAM cells 3D4/21 and recombinant S.cerevisiae strains expressed P30-?Fc,P54-?Fc fusion proteins,indicating that Fc-ASFV proteins fusion expression promote the phagocytosis progress of recombinant yeasts by porcine cells,facilitating the presentation and processing of antigens.ASFV prokaryotic recombinant proteins P30,P54 and K145 R were prepared by purification for detection of the fermentation kinetic changes of antibodies in serum of porcine oral immunized with recombinant S.cerevisiae strains by indirect ELISA method.The results demonstrated that the levels of ASFV proteins P30,P54-specific Ig G/Ig A antibodies in the experimental group were significantly higher than those in the unvaccinated group.In summary,our research has initially established a technical platform based on S.cerevisiae strains,completing the construction of a single yeast strain multi-modal expressed multiple proteins,the assembly of large fragment,the screening,expression and identification of recombinants Furthermore,the major immunogenic proteins of ASFV was expressed by recombinant S.cerevisiae strains of which the expression characteristics were analyzed.The immune potency of recombinant S.cerevisiae strains expressed ASFV protein-porcine Ig heavy chains was evaluated,providing a novel tool for the prevention and control of ASFV.