Preliminary Study On The Bacteria-like Particle Carrier Vaccine Against Swine Type O Foot-and-Mouth Disease Virus

Foot-and-mouse disease is an acute and severe infectious disease of cloven-hoofed animals caused by Foot-and-mouse disease virus?FMDV?,which seriously harm the animal husbandry.Preveting and controlling of swine type O Foot-and-mouth disease was achieved by immunizating with inactivated vaccine.However,the inactive vaccine still has some deficiencies,such as short immunization period,higher costs of production and can not stimulate cellular immune response.Thus,it is a meaningful to develop a new vaccine for swine type O FMDV.Previous studies have shown that surface display system of gram positive enhancer matrix?GEM?has displayed soluble exogenous protein fused with anchor protein PA3 and improved the immunogenicity of exogenous protein.It also has been proved by the reseach that VP1_141-160?VP1_200-21300-213 and 3A_21-351-35 from type O FMDV were B-cell epitope,Th2-cell epitope and Th1-cell epitope,respectively.Therefore,three multi-epitope peptides were designed with those epitopes and displayed on the surface of GEM particles to prepare the bacterial-like particle vaccine?BLP?,and to study its immunogenicity.The main study content and results as follows:First,Constructed prokaryotic expression recombinant plasmid carrying antigen epitopes of FMDV.Three multi-epitope peptides which contains Epi_141-160,Epi_200-213andEpi_21-35aswellasGGlinker,namelyBTTB?140¹60aa-GG-200²13aa-GG-21³5aa-GG-140¹60aa?TB?140¹60aa-GG-200²13aa??andVP1*C?VP1Complete genome sequences,substitute P for C in 158?,respectly,and clonedinto the pasmid pUC57 through commissioned biological company.Three multi-epitope peptide genes and the expression vector pQZ-PA3 were digested and then linked with enzyme to construct recombinant plasmids pQZ-TB-PA3,pQZ-BTTB-PA3 and pQZ-VP1*C-PA3.Then,recombinant plasmids were transformed into BL21.The three soluble fusion proteins were obtained by IPTG induction,and they possessed better immunoreactivity identified by western-blotting.Second,prepared three BLP carrier vaccines of FMDV and studied its immunogenicity.Three soluble fusion proteins purified and dispayed with GEM particles.There were protein layers around the surface of GEM particles observed by transmission electron microscope.The concentration of soluble fusion proteins were85?g/mL?rTB-PA3?,213?g/mL?rBTTB-PA3?and 870?g/mL?rVP1*C-PA3?,respectly.The three soluble fusion proteins were emulsified with oil adjuvant,and immunized five-week-old female ICR mice.Blood samples were collected at 21,35,49,and 63 days post vaccination,respectly,for detecting VP1-specific antibody and FMDV-specific liquid blocking antibody.The level of lymphocyte proliferation and the mRNA expression levels of IL-4,IL-10,IFN-?and TNF-?in the spleen were detected on the 35th day after immunization.The results showed that the vaccine BLP/rBTTB-PA3 has induced higher levels of VP1-specific antibody and FMDV-specific liquid blocking antibody?LPB?.The BLP/rBTTB-PA3-immunized group had induced stronger proliferative response than other groups.The mRNA expression levels of IFN-?and IL-4 in the BLP/rBTTB-PA3-immunized group were raised significantly than other BLP immune groups,but there was no significant difference with synthetic peptide vaccine group.The mRNA expression levels of IL-10 and TNF-?have no significant difference among all experimental groups.The results indicated that BLP/rBTTB-PA3 has a good immunogenicity and induced humoral immune response and Th1 type cell immune response.Finally,evaluated the adjuvanticity of CVC1302 for BLP/rBTTB-PA3 vaccine.ICR mice and 45-day-old FMDV negative piglets were immunized with BLP/rBTTB-PA3 vaccine plus CVC1302 as adjuvant,then the levels of VP1-specific antibody and FMDV-specific liquid blocking antibody in serum and the level of spleen lymph proliferation in peripheral blood were detected.The results showed that the level of VP1-specific antibody and LPB antibody in BLP/rBTTB-PA3+CVC1302 immunized mice and piglets groups were significantly higher than those of BLP/rBTTB group.In addition,the level of peripheral blood lymphocytes proliferation in BLP/rBTTB-PA3+CVC1302 immunized mice and piglets groups were markedly higher than that of BLP/rBTTB groups,which indicated CVC1302could further improve the immunogenicity of BLP/rBTTB-PA3.In summary,the BLP/rBTTB-PA3 with better immunogenicity was successfully prepared in our study.Moreover,CVC1302 could further improve the immunogenicity of BLP/rBTTB-PA3.