Establishment And Application Of Immunological Evaluation Method For Giant Panda-derived CDV/CPV Virus-like Particle Vaccine

The giant panda is a rare and vulnerable species in the world.The artificial captive breeding method leads to its high density,which is easy to cause infectious diseases.Among them,canine distemper virus（CDV）and canine parvovirus（Canine Parvovirus,CPV）are harmful to giant pandas.Panda population safety is the most serious threat,and its prevention and control is of great significance.It has been confirmed that the traditional canine-derived CDV/CPV dual vaccine has poor immune effect on giant pandas;the canary pox vector vaccine only contains the CDV antigen gene,although it is used clinically,it cannot protect giant pandas from CDV/CPV at the same time.In this study,using giant panda-derived CDV/CPV as the basic material,an ELISA detection method was established by expressing proteins related to giant panda-derived CDV/CPV virus-like particles（CDV/CPV Virus-Like Particles,CDV/CPV VLPs）,and used in large Immunological evaluation of panda-derived CDV/CPV VLPs vaccine.This study is divided into the following three parts:1.Prokaryotic expression of giant panda-derived CDV FH fusion gene and CPV VP₂geneThe giant panda-derived CDV F and H genes were amplified,sequenced,antigenic epitope analysis,screening,and FH fusion fragments were synthesized.The giant panda-derived CPV VP₂gene was amplified and sequenced using homologous arm primers,and then the FH fusion fragments and The VP₂gene was connected to the expression vectors p ET-32a and p ET-28a respectively,and transformed into the recipient bacteria BL21（DE3）for expression.The conditions were optimized（the optimum concentration of IPTG was 0.8mmol/L,the optimum induction time was 24h,the optimum After the best induction temperature is 37℃）,FH fusion protein（about 35k Da）and VP₂protein（about 70k Da）were successfully expressed,both proteins were expressed in the form of inclusion bodies,and the expression levels of FH fusion protein and VP₂protein were 0.884mg/m L and 0.925mg/m L.The FH fusion protein and VP₂protein were used as antigens,and the canine polyclonal antibody against CDV FH fusion protein and CPV VP₂protein was used as an antibody to carry out agar diffusion test.When the polyclonal antibody serum of CDV FH protein was diluted to 1:8,there was a white color visible to the naked eye Precipitation line;when the polyclonal antibody serum of CPV VP₂protein is diluted to 1:16,there is a white precipitation line visible to the naked eye,indicating that the expressed protein is immunogenic.The two proteins were verified by Western blotting,and the target protein could specifically react with the corresponding positive serum,indicating that the two recombinant proteins had good reactogenicity.2.Establishment of an indirect ELISA detection method for giant panda-derived CDV and CPV antibodiesThe FH fusion protein and VP₂protein were respectively coated on the enzyme-labeled plate as antigens,and the positive and negative judgment standard values were determined after optimizing the conditions such as antigen coating concentration,serum dilution,and enzyme-labeled secondary antibody dilution.The results show that when FH fusion protein is used as the coating antigen,the optimal coating concentration is 8μg/m L,the optimal dilution of serum is 1:800,the optimal dilution of enzyme-labeled secondary antibody is 1:2000,and the judgment standard is OD₄₅₀value≥0.281 is positive,otherwise it is negative.When VP₂protein is used as the coating antigen,the optimal coating concentration is 4μg/m L,the optimal dilution of serum is1:400,and the optimal dilution of enzyme-labeled secondary antibody is 1:2000,and the judgment standard is that the OD₄₅₀value≥0.297 is positive,otherwise negative.The ELISA detection method established by the two proteins as the coating antigen respectively,the positive serum dilution can reach 1:25600,and the sensitivity is high;the repeatability within the same batch and the coefficient of variation between different batches are both less than 10%,and the repeatability is relatively high.Good;and the two envelope proteins do not cross-react with other virus-positive sera,and the specificity is good.Compared with commercial detection kits,the ELISA detection method established in this study is more sensitive.3.Evaluation of immune effect of giant panda-derived CDV/CPV virus-like particle vaccineThe giant panda-derived CDV/CPV VLPs vaccine was used to immunize the experimental dogs,and the safety test showed that the body temperature of the experimental dogs was within the normal range（37.5°C-39°C）,and there were no abnormalities in the immune site,behavior,organs,and intestinal tract,indicating that the vaccine was safe.Obvious side effects occurred.Two established ELISA detection methods were used to detect specific antibodies in serum.The results showed that after the completion of VLPs vaccine immunization,the body could quickly produce a higher level of specific antibodies and lasted for up to 3 months,and the level of specific antibodies was in the top 3 Months is always higher than that of canine CDV/CPV dual vaccine,and the immune effect is better.At the same time,it is confirmed that the established ELISA detection method can be used to evaluate the specific antibody level of VLPs.In addition,after the VLPs vaccine stimulated the body,the Ig G antibody typing showed that the immunoglobulins Ig A,Ig G1 and Ig G2a were always significantly different from the non-immunized group in the first 3 months.The measurement of m RNA transcription levels of cytokines showed that within 3 weeks after the completion of immunization,the relative expression levels of IL-2,IL-4,IL-10,IL-13,IFN-α,and IFN-βcould be promoted;lymphocyte proliferation indicated,under the stimulation of Con A,there was a very significant difference in the proliferation of lymphocytes between the VLPs group and the canine dual vaccine group,and the VLPs group could effectively stimulate the body to produce cellular immunity and improve the level of the body’s cellular immunity.The results generally show that VLPs vaccine can stimulate humoral immunity and cellular immunity,the immune effect is good and safe,and it has a good clinical application prospect.In summary,this study successfully expressed the giant panda-derived CDV FH fusion protein and CPV VP₂protein,and successfully established two ELISA detection methods for the detection of giant panda-derived CDV and CPV antibodies in canine serum using the expressed proteins as antigens.The established ELISA method was used to detect the serum antibody levels of the dogs immunized with the giant panda CDV/CPV VLPs vaccine,and it was confirmed that the immunized dogs could only rapidly produce a higher level of specific antibodies and lasted for as long as 3 months,showing that the immune effect of the vaccine was relatively high.we have the conditions for a vaccine candidate.