C3d On Immunogenicity Of Fusion Protein In CSFV E2-ASFV P72 Epitope Region

African swine fever(ASF)is caused by the African swine fever virus(ASFV).The disease is characterized by high toxicity,blood fever,contact and infection.The infected pigs developed rapidly and had low survival rate.ASFV has brought a heavy blow to the development of aquaculture in the world,so it is urgent to protect pigs from virus infection with effective vaccines.However,for African swine fever,there is no effective vaccine for clinical use at home and abroad.Traditional inactivated virus,attenuated vaccine or gene-deficient vaccine strains have been shown to be ineffective in protecting pigs from ASFV infection.Therefore,the development of other types of vaccines,such as subunit vaccines,DNA vaccines and live carrier vaccines,has become an important direction in exploring ASFV and some progress has been made.Structurally,ASFV is a double-stranded DNA virus with regular icosahedral symmetry.The genome size is about 170-190 kb,and its viral particles contain nine proteins that play an important role in the induction of neutralizing antibodies: P72,P49,P54,P12,P14,P17,P22,PE248 R and CD2 v.P72 anchored to the capsid as an important antigenic protein.And some areas are exposed outside the capsid.It has become the focus of attention in the research of swine fever virus vaccine in Africa.In recent years,Chinese scientists have successfully analyzed the structure of P72 proteins.And recently,by analyzing P72 structure,found that each monomer in the P72 trimer structure forms four exposed regions on the surface of the virus particles,ER1,ER2,ER3 and ER4,Their length totals about 140 amino acids.The discovery of these four exposed areas,it provides an important research direction for the development of African swine fever virus vaccine.Classical swine fever(CSF)is highly infectious to pigs,and the probability of death after infection is 90-100%.And ASFV also become a major infectious disease threatening the pig industry.Owing to the serious economic crisis caused by CSF to countries,it is OIE listed as one of the animal diseases that must be reported.Its pathogenic mechanism and effective vaccine research have become the focus of various countries.The CSFV is different from the ASFV structure and is a linear single strand RNA virus.CSFV gene leader 12.3 kbp,can encode 12 viral peptides.The structural protein E2 anchored on the capsid and found that the first 200 amino acids at the E2 N end contain four important antigen binding regions(A,B,Cand D),which play an important role in the process of virus infection.As the main target protein,E2 has been used many times in the preparation of antibodies to achieve the immune effect of swine fever virus.As a result,this study focused on the preparation of CSFV E2-ASFV P72 bivalent high-efficiency vaccine and antibodies,and obtained the following results:(1)The E2s-P72 s bivalent polyclonal antibody was obtained by exogenous fusion protein E2s-P72 s immunity.The E2 s and P72 s containing epitope region were connected to the p CMV-HA-C simultaneously to construct eukaryotic expression vector p CMV-HA-E2s-P72 s.The recombinant vector was transfected into BHK-21 cell lines and the anti-serum of E2s-P72 s mice was prepared by extracting and purifying the fusion protein expressed in the cells.However,it is found that the efficiency of preparing serum by this method is low.The E2s-P72 s antibody prepared in this experiment can provide a material basis for the study of the influence mechanism of E2 protein and P72 protein on virus.(2)E2’ polyclonal antibody and C3 d polyclonal antibody were successfully prepared.To optimize the synthesis of 621 bp bases at CSFV E2 N end and C3 d bases of rat-derived adjuvant and construct p ET28a-E2’ and p ET28a-C3 d of prokaryotic expression vector.The E2’ and C3 d proteins were purified and immunized mice to obtain E2’ and C3 d anti-blood serum.The two kinds of serum immunity and specificity were identified by Western Blot and ELISA detection.The two antibodies provide a theoretical basis for the study of CSFV infection mechanism and vaccine and the subsequent detection of DNA vaccine with C3 d as adjuvant.(3)Next,we studied the effect of mouse molecular adjuvant C3 d on the immunogenicity E2s-P72 s fusion protein to obtain efficient vaccine and serum.The eukaryotic expression vector was constructed by homologous recombination with mouse C3 d molecular adjuvant and Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element(WPRE)of groundhog hepatitis virus p CMV-E2s-P72s-C3d-WPRE,p CMV-E2s-P72s-C3d-C3d-WPRE and p CMV-E2s-P72s-C3d-C3d-C3d-WPRE and immunized mice.The effect of different molecular adjuvant C3 d copies on the immunogenicity of fusion protein E2s-P72 s was studied by Western Blot identification,ELISA titer detection and comparison of serum IFN-γ produced by different DNA vaccines.It was found that the plasmid immune titer of three C3 d copies could induce the highest number of molecular adjuvant C3 d in mice to produce high titer serum,and the DNA vaccine which could induce mice to produce high efficient bivalent serum was successfully optimized.(4)The same homologous recombination method was used to connect the E2s-P72 s with the pig source C3d(PC3d)molecular adjuvant and the original transcription regulation WPRE to the p CMV-HA-C,and the eukaryotic expression vector p CMV-E2s-P72s-PC3d-WPRE and p CMV-E2s-P72s-PC3d-PC3d-PC3d-WPRE was constructed.The candidate DNA vaccine which may be used for pig immunization and efficient preparation of bivalent antibody was screened.To sum up,through exploring the effect of molecular adjuvant C3 d on the immunogenicity E2s-P72 s fusion protein,this experiment successfully screened and prepared CSFV E2 and ASFV P72 bivalent high-efficiency candidate vaccine and antibodies.It provides material basis and technical support for the study of high-efficiency immune strains and virus pathogenesis containing two viruses.