Eukaryotic Expression Of VP4 And VP7 Protein Of Donkey Rotavirus And Immunogenicity Of Subunit Vaccine

Rotavirus(RV)is one of the common pathogens causing acute diarrhea and dehydration of many young livestock,which can cause infection of piglets,calves,foals and other animals with high incidence and low mortality.However,it is often infected with other pathogenic factors,resulting in the mortality of young livestock increased to about 50%.RV is transmitted through direct contact and mainly infects and replicates within intestinal villous epithelial cells.Donkey Rotavirus(Do RV)was first discovered in Pakistan in 2017 and then introduced into China,causing great economic losses to the donkey industry in China.In view of this,we expressed the proteins VP4 and VP7 against Do RV in this study,and established a rapid and sensitive indirect ELISA method for the detection of Do RV antibodies.At the same time,subunit vaccines against VP4 and VP7 proteins were developed and immunogenicity was studied,respectively,which provided new ideas for the design of novel subunit vaccines for Do RV.The main research is as follows:1.Eukaryotic expression and purification of Dorv VP4 and DoRV VP7 proteinsThe extracellular domain and signal peptides of VP4 and VP7 genes of Do RV G3P12 strain were analyzed.After codon optimization,eukaryotic expression plasmids p CAGGS-Do RV-VP4 and p CAGGS-Do RV-VP7 were constructed,and human signal peptides,His tags,Strep tags and restriction sites were inserted.With HEK-293F suspension cell eukaryotic protein expression system,Do RV VP4 and VP7 proteins were expressed respectively.After affinity chromatography and molecular sieve purification,Do RV VP4 protein(1.0mg/ml)and Do RV VP7 protein(0.6mg/ml)were obtained.2.Establishment of indirect ELISA methodTaking DoRV VP4 and VP7 proteins as coated antigens respectively,the best coating concentration,coating time,color development time and critical value of the antigens were explored through experiments,and the indirect ELISA methods for Do RV VP4 and VP7 proteins were established by further specificity and repeatability experiments.The results showed that the method was specific,sensitive,accurate,rapid and convenient,which laid a foundation for the detection of Do RV antibody.3.Study on immunogenicity of DORV VP4 and VP7 subunit vaccinesThe purified DoRV VP4 and VP7 proteins were used as immunogens and emulsified with VG206 adjuvant to immunize five-week-old Balb/c mice.Serum samples of mice were collected at 0d,14d,28d and 42d,respectively,and antibody levels were determined.At 42 days,the proportion of lymphocyte subtypes in mice was detected by flow cytometry.MTT assay was used to detect the proliferation of mouse lymphocytes.At the same time,the cytokines of mice were measured.Then the neutralizing antibody titer of mouse serum was detected.The results showed that the level of Ig G antibody,the proportion of CD3~+CD4~+,CD3~+CD8~+lymphocytes and the amount of IFN-γand IL-4 expressed by lymphocytes in the experimental group increased in different degrees.Both proteins can induce humoral immunity and activate B cells in mice,and at the same time improve the immune function and cytokine expression ability of peripheral blood T lymphocytes.MTT assay was used to detect the proliferation of mouse lymphocytes.After Con A stimulation,the experimental group could stimulate T cells to transform into immune memory cells,among which VP4 group was better.Both subunit vaccines can produce immune effect in mice,and the subunit vaccine using Do RV VP4 protein as antigen is better than Do RV VP7 protein.Therefore,this study provides theoretical support for the prevention of DoRV and the preparation of subunit vaccine.