Preliminary Research On The Swine Foot-And-Mouth Disease Subunit Vaccine Using FC Or LTB As Carriers

Foot and mouth disease is an acute, febrile and highly contagious anthropozoonosis which has been identified by OIE as type A. FMD can cause tremendous losses to many countries due to its quickly spread, economically and politically. Now, we control this disease by inactivated vaccine which has a complete immunogenicity and a good protection, but it do exist some vulnerabilities like incomplete inactivation and even virus escape. As the gene technology develops, many novel vaccines are attractive by its safety, reliability and easily quality control. Among these, recombinant protein vaccine is the best direction attribute to the advantages of its mature manufacture and low cost.Protein VP1-the main antigen of FMDV-has a G-H circle which concludes B cell epitopes:141-160aa and 200-213aa. These epitopes can induce antibody and thereby induce protective immunity. Thus vp1 is the perfect antigen to FMD vaccine research.1.Preparation and identification of the FMDV VP1 fusion protein using swine IgG Fc as carrier.We select the sequence of the 141-160aa and 200-213aa fo VP1 in FMDV-O/MYA98, insert into expression plasmid pQZ with the vector IgG FC fragment, get the recombination plasmid of pQZ-Fc-loop. Then we express the fusion protein by soluble expression of Escherichia coli platform CVC1102. The result shows that the protein obtained is 42KDa, soluble in the supernatant. Western-blotting shows fusion proteins Fc-loop is positive when reacting with rabbit anti FMDV VP1.2.Preparation and identification of the FMDV VP1 fusion protein using escherichia coli heat-labile enterotoxin B-subunit as carrier.We select the sequence of the 141-160aa and 200-213aa fo VP1 in FMDV-O/MYA98, insert into expression plasmid pQZ with the vector LTB fragment, get the recombination plasmid of pQZ-LTB-loop. Then we express the fusion protein by soluble expression of Escherichia coli platform CVC1102. The result shows that the protein obtained is 23KDa, soluble in the supernatant. Western-blotting shows fusion proteins Fc-loop is positive when reacting with rabbit anti FMDV VP1.3.Immunogenicity of the fusion protein FMDV VP1We get the experimental vaccines by mix and emulsify the Fc-loop and LTB-loop with the adjuvant 206, protein quantifying by BCA 2mg/ml、1mg/m respectivelyl. We classify the mice into 5 groups, immunizing them by neck and back with Fc-loop, LTB-loop, fusion protein add to CVC1302 and PBS. Strengthening immunity after 14 days. We collect the blood through eye-orbit after 14,22,28,55days after first immunization and detect the antibody level by ELISA. The result shows that both Fc-loop and LTB-loop can stimulate the production of specific antibodies in mice. The antibody level of Fc-loop is higher than that of LTB-loop. Also, immunopotentiator CVC1302 can raise the level of antibodies.4. Preliminary purification of recombinant proteins.In order to improve the immune protection effect of recombinant subunit vaccine without purification, we need to purificate recombinant proteins, the experiment tried several different purification methods in this chapter to gain in the previous chapter of Fc-loop and LTB-loop, for subsequent subunit vaccine research. We tried to use staphylococcus protein A, Ni2+-NT A, saturated ammonium sulfate precipitation in turn.But have not achieve the desired purpose of purified protein, may be associated with soluble expression change its space conformation, in future studies need to try other purification methods.