Acid-resistant Modification And Expression In Insect Cells Of O Type FMDV Capsid Antigen

Foot-and-mouth disease(Foot and mouth disease, FMD) is a animal disease caused by the foot-and-mouth disease virus(Foot and mouth disease virus, FMDV). It can infect cloven-hoofed animals such as cattle、sheep and pigs. FMD was listed as the A class infectious disease by the Office International Des Epizooties(Office International Des Epizooties, OIE)and was listed as the I class epidemic disease because its fast speed of spread and high efficiency of infection. At present, FMD vaccination is the most effective prevention and control measure to control FMD outbreak and FMD inactivated vaccine has played a very important role in the prevention and control. Although it is proved to be effective, there are many drawbacks of current inactivated vaccines, including the risk of inactivating the virus incompletely, and of virus escaping from the manufacturing facilities. According to reports, FMDV empty capsid protein 75 S has a very similar antigenic properties with the complete FMDV 146 S particles, it can cause the very similar immune response with complete FMDV virions, and the empty virus capsids contain no nucleic acid components, safety, it does not exist risk of virus escaping from the manufacturing facilities. Therefore, the development of FMDV empty capsids vaccine is extremely important.This study was aimed to select the acid stability sites of type O FMDV capsid. Using the bovine type O FMDV ON strain as research object, six acid resistant monoclonal strains were obtained after treatment in pH 6.0 acidic environment for serial passages. The P1 region which encoding their capsid proteins of the mutagenic viruses were sequenced and compared with the parental virus, then six mutation sites were found, of which the mutation sites A460 C and G462A(K154Q)on the VP3 and mutation sites A121G(K41E)、A74G(Q25R) and A253G、A254C(N85A) on the VP1.Then the capsid protein precursor P12 A and protease 3C coding P12A3 C gene of FMDV type O were amplified from the plasmid pMD19-P1A3 C, and the gene P12A3 C were inserted into the baculovirus transfer vector pFastBacDual to construct recombinant transfer vector pFastBacDual-P12A3 C, in which the P12A3 C gene were under the control of PH promoter and the six mutations above were mutanted. The recombinant baculovirus plasmids were transformed into Escherichia coli DH10 BacTM to construct the recombinant baculovirus bacmid Bacmid-P12A3 C, then transfected into Sf9 cells, the recombinant baculovirus was harvested when obvious cytopathic effect was observed. To amplied, the baculovirus were infected into the Sf9 cells. Then the expressed proteins were analyzed by the indirect immunofluorescent assay、ELISA and western-blot. The result indicated that the expressed protein were expressed in Sf9 cells, and displayed specificity to FMDV type O polyclonal antibody. Protein samples were detected by double-antibody sandwich ELISA after extracting by sucrose density gradient centrifugation, and higher amount of antigen expression was detected in 35% sucrose density position, consistent with the expected results, under the immune electron microscopy, a diameter hexagonal particles of about 27 nm was observed in protein sample rBac-P12A3 C, illustrating the FMDV empty capsids was successful assembled in Bac-to-Bac baculovirus expression system. From this study, the recombinant baculovirus which including the virus capsid P1 gene and 3C protease gene coding regions of FMDV type O were successfully obtained. This provides a basis for the empty capsid vaccine research.