Expression And Immunogenicity Of The Multi-antigenic Epitopes Recombinant Protein Against Swine O-type Foot-and-mouth Disease Virus

Food-and-mouth disease(FMD)is a hoofed animal infectious disease caused by food-and-mouth disease virus(FMDV),which seriously threatens the development of the global animal husbandry.At present,inoculation of FMDV inactivated vaccine is an important measure to effectively prevent and purify FMD,but inactivated vaccines have problems such as poor thermal stability and short duration of immunization.Therefore,the development of a safe and effective new FMDV vaccine is the current trend.The genetic engineering technology and the immunogenicity of synthetic peptides provide a new direction for the development of new FMDV vaccines.The study intends to use the insect cell/baculovirus eukaryotic expression system to study the multi-epitope vaccine against the current porcine O-type FMDV in China.The FMDV VP1 protein has good immunogenicity and contains multiple dominant epitopes.In this study,the VP1 dominant T and B cell epitope sequences in the currently domestic porcine O-type FMDV were repeatedly tandemly to obtain the recombinant gene RBT,and the recombinant gene RBT was ligated in tandem with the porcine immunoglobulin Ig G Fc to obtain the recombinant gene RBTFc.In this study,the target genes RBT and RBTFc were cloned into the transfer vector p Fast Bac Dual,respectively.After PCR identification,double enzyme digestion and gene sequencing,the recombinant transfer plasmids p Fast Bac Dual-2RBT and p Fast Bac Dual-2RBTFc were obtained.The recombinant transfer plasmids p Fast Bac Dual-2RBT and p Fast Bac Dual-2RBTFc were transformed into competent cells DH10Bac carrying the baculovirus plasmid Bacmid,respectively.After screening with blue and white spots,PCR was used to detect the recombination of the target gene and Bacmid.The results showed that the target genes RBT and RBTFc were specifically recombined with Bacmid,and the recombinant baculovirus plasmids r Bac-RBT and r Bac-RBTFc were obtained.In this study,recombinant baculovirus plasmids r Bac-RBT and r Bac-RBTFc were transfected into Sf9 cells,respectively,to obtain recombinant baculovirus Ac-RBT and Ac-RBTFc.Recombinant baculovirus Ac-RBT and Ac-RBTFc were infected into Sf9 cells,respectively.The expression of RBT and RBTFc in the cells was detected by Western blot and Indirect immunofluorescence(IFA).Western blot analysis showed that there were specific bands around 45 k Da and 65 k Da,which were consistent with the size of the target proteins RBT and RBTFc.IFA results showed that Sf9 cells infected with recombinant baculovirus Ac-RBT and Ac-RBTFc were under fluorescence microscope.Green fluorescence can be observed.The results showed that the target proteins RBT and RBTFc were successfully expressed in Sf9 cells and had good reactogenicity.In this study,the absolute titer of the third generation recombinant baculovirus Ac-RBT and Ac-RBTFc was determined by absolute fluorescence quantification.The results showed that the viral titers of the 3rd generation recombinant baculovirus Ac-RBT and Ac-RBTFc were 4.9×10~9pfu/m L and 3.07×10~9 pfu/m L,respectively.In this study,the expression conditions of the target proteins RBT and RBTFc were optimized,and the optimal expression conditions were used to express the RBT and RBTFc proteins.After sonication and lysis of the cells,the solubility of RBT and RBTFc proteins was detected by Western blot.The results showed that the ultrasonic supernatant samples RBT and RBTFc had specific bands around 45 k Da and 65 k Da,respectively,which were consistent with the RBT and RBTFc protein sizes,indicating that the RBT and RBTFc proteins were soluble.We prepared vaccines with RBT and RBTFc proteins in combination with ISA 201VG adjuvant.The vaccine was immunized with SPF grade BALB/c mice to initially assess the immunogenicity of the vaccine.The experimental groups were RBT group,RBT+Nucleic acid adjuvant(NAA)group,RBTFc group,RBTFc+NAA group,FMDV inactivated vaccine group and PBS control group.The first dose was given to the second dose 14 d after the first immunization.The animals in each group were collected at 7 d,14 d,21 d and28 d after the first immunization,and the specific antibody levels in the serum were detected.The results showed that,except for the PBS control group,FMDV-specific antibodies were produced in each experimental group,and there was no significant difference in antibody levels between the groups.The results showed that the multi-epitope vaccine prepared in this study can effectively induce FMDV-specific antibodies in mice.To further assess the immunogenicity of the vaccine to pigs,the vaccine was immunized to pigs of 2 months of age,and the experimental groups were RBT group,RBT+NAA group,RBTFc+NAA group,FMDV inactivated vaccine group and PBS control group.Two doses were given at the same dose 28 d after the first immunization.Blood samples were taken at14 d,21 d,28 d,35 d,and 42 d after the first immunization,and the specific antibody levels in the serum of pigs were detected.The results showed that,except for the PBS control group,each group produced higher levels of FMDV-specific antibodies;after the first exemption,the level of specific antibodies in the RBT+NAA group was significantly higher than that in the RBT group(p<0.05);The serum specific antibody level of pigs in the RBTFc+NAA group was significantly higher than that in the RBT+NAA group(p<0.05).The results showed that the multi-epitope vaccine prepared in this study can effectively induce FMDV-specific antibodies in pigs,and the NAA and Ig G Fc domains have obvious immunopotentiating effects on multi-epitope vaccines.In summary,the multi-antigenic epitope of porcine O-type foot-and-mouth disease virus expressed in this study has good immunogenicity and provides theoretical basis and technical support for the development of FMDV multi-epitope vaccine.