| In this study,the receptor binding domain(RBD)of porcine epidemic diarrhea virus(PEDV)and transmissible gastroenteritis virus(TGEV)based on aminopeptidase N were selected as the research objects.Through evolutionary analysis with RBD gene of other typical strains in China,the epidemic strain RBD was screened,the vaccine based on RBD was constructed,and its immunogenicity to mice and piglets was analyzed.In this study,PEDV and TGEV RBD genes were synthesized,the recombinant protein was expressed by mammalian expression system,and mouse polyclonal antibody was prepared.An indirect ELISA method for antibodies against PEDV and TGEV was established.The recombinant protein was obtained by baculovirus expression system,and the immunogenicity of mice and piglets was analyzed.The following results were obtained:1.Using mammalian expression system,pc DNA3.4-PER and pc DNA3.4-TGR recombinant plasmids were successfully constructed and recombinant protein were obtained,and mouse polyclonal antibody was successfully prepared.The screened RBD gene was codon optimized and added strep-Ⅱtag,and successfully constructed into pc DNA3.4 vector,the recombinant plasmid was transfected into 293F suspension cells for expression,and the recombinant protein was obtained.The polyclonal antibody was successfully obtained by immunizing BALB/c mice with 50μg protein and Freund adjuvant.The titer of PEDV polyclonal antibody was 1:204 800 and that of TGEV antibody was 1:12 800.2.The indirect ELISA method of PEDV antibody was successfully established,and the antigen coating concentration was 5μg/ml,the blocking solution was 5%skimmed milk-TBST,the blocking conditions were 37℃,1 h,the dilution of serum to be tested was 1:400,the reaction conditions were 37℃,30 min,the dilution of enzyme labeled secondary antibody was 1:8 000,the reaction conditions were 37℃,30 min,the substrate reaction time was 37℃,15 min,and the critical value was determined to be 0.053;The indirect ELISA method of TGEV antibody was successfully established,and the antigen coating concentration was 10μg/ml,the blocking solution is 5%skimmed milk-TBST,the blocking condition is 37℃,1 h,the dilution of serum to be tested is 1:400,the reaction condition is 37℃,40 min,the dilution of enzyme labeled secondary antibody is 1:5 000,the reaction condition is 37℃,40 min,the substrate reaction time is 37℃,20 min,and the critical value of yin and yang is determined to be0.115.3.Using baculovirus expression system,p FBD-PER and p FBD-TGR recombinant plasmids were successfully constructed,and the recombinant protein and recombinant baculovirus were obtained.The concentrated recombinant baculovirus was used to immunize mice and piglets,and its immunogenicity was analyzed.The selected RBD gene was codon optimized,strep-Ⅱtag was added,and was successfully constructed in p Fast Bac?Dual vector,the recombinant plasmid was transformed into DH10Bac?In the competent state of E.coli,the recombinant bacmid was screened by blue and white spots.The recombinant bacmid was transfected into SF9 adherent cells to save the recombinant baculovirus.After 5-7 days,the supernatant of cell culture was obtained as P1 generation recombinant baculovirus.The P1 generation recombinant baculovirus infected SF9 adherent cells to P3 generation,and the titer of the recombinant baculovirus was determined by indirect immunofluorescence.The protein expression was identified by Western blot and indirect immunofluorescence,and the infection number and harvest time of suspension SF9 cells infected with P3 generation recombinant baculovirus were optimized.After the protein was expressed according to the best expression conditions,the target protein was purified by strep-Ⅱlabeling purification system.At the same time,the recombinant protein was expressed according to the best expression conditions.The supernatant of cell culture was collected by low-speed centrifugation and concentrated and purified by ultracentrifugation.After protein quantification,mice and piglets were immunized:BALB/c mice were immunized with the recombinant protein prepared by baculovirus expression system,and the empty vector control and PBS control were set.The experimental results showed that the specific antibody and neutralizing antibody of the immunized group were significantly higher than those of the control group.The spleen lymphocytes of mice were isolated after the 8th week of the first immunization.After staining,the results were analyzed by flow cytometry.The results showed that the CD3~+CD4~+double positive T lymphocytes in PER vaccine group were slightly higher than those in empty vector control group and significantly higher than those in PBS group;The CD3~+CD4~+double positive T lymphocytes in TGR vaccine group were significantly higher than those in PBS group and empty vector control group,indicating that PER protein and TGR protein can effectively stimulate humoral and cellular immunity in mice.The piglets were immunized with PER protein and set up empty vector control group and adjuvant control group.The experimental results showed that the specific antibody and neutralizing antibody of piglets in the immunization group were significantly higher than those in the control group.The peripheral blood lymphocytes of piglets were isolated on the 32nd day after the first immunization.After staining,the results were analyzed by flow cytometry.The results showed that the level of CD3~+CD4~+double positive T lymphocytes in PER vaccine group was slightly higher than that in empty vector control group and significantly higher than that in adjuvant control group.The results showed that PER protein could effectively stimulate the humoral and cellular immunity of piglets. |
