Construction Of Virus-like Particles Of AF/72 Strain Of Foot-and-mouth Disease Virus Type A And Their Immunogenicity

Foot-and-mouth disease(FMD), a highly contagious disease typically infects cloven-hoofed animals, is caused by foot-and-mouth disease virus(FMDV) which can lead to severe economic and political consequences. In this study, we investigated the construction of virus-like particles of FMDV Type A in insect cells using baculovirus expression system based on the AF/72 strain of FMDV, and with the hope to provide a new idea for FMDV vaccine.1.The P12 A and 3C genes of the AF/72 strain of foot-and-mouth disease virus type A were amplified, and then were cloned into the p MD19-T and sequenced. Sequence analysis showed that the whole length of structural protein P1 coding region was 2211 base pairs(bp) and encoded 737 amino acids(aa). The nucleotide sequences and deduced amino acid sequences of four structural proteins, VP4(255bp), VP2(654bp), VP3(663bp), and VP1(639bp) were 85 aa, 218 aa, 221 aa, 213 aa in length, respectively. 2A and 3C were 48 bp and 639 bp in length and the deduced amino acid sequences were about 16 aa and 213 aa, respectively.2. Two different strategies were used to investigate the construction of virus-like particles of the AF/72 strain of FMDV Type A.(1) We cascaded the structural protein P12 A gene and proteinase 3C gene and inserted them into baculovirus transfer vector p Fast BacTMHTB by restriction enzyme cutting sites and stduied the expression of target genes.(2)The P12 A and 3C genes were cloned into the baculovirus expression vector p Fast BacTM Dual which simultaneously expressed the target genes by two individual promoters(Pp10 and PPH). The two baculovirus expression vectors,which carried the target genes, were transformed into E.coli DH10 Bac competent cells to make white and blue screening. After transfecting the sf9 cells,we obtained two recombinant baculovirus(Bacmid-HTB-P12A3 C and Bacmid-Dual-P12A3C). As shown by Western blotting, only recombinant baculovirus(Bacmid-Dual-P12A3C) appeared five protein bands that represented the P12 A protein, VP0 and VP3 protein,VP3 and VP1 protein,VP0 protein, and VP1 or VP3 protein, respectively. This means thecapsid proteins could be processed by 3C proteinase. However, the recombinant baculovirus Bacmid-HTB-P12A3 C appeared only one bands represented the P12 A protein. Using electron microscopy, VLPs with a diameter of about 30 nm were observed in Bacmid-Dual-P12A3 C.3.Finally, using the sucrose density gradient centrifugation,the crude extracts from Sf9 cells, which were infected with recombinant baculovirus Bacmid-HTB-P12A3 C and Bacmid-Dual-P12A3 C, were mixed with ISA206 to evaluate its immunogenicity in guinea pigs.The humoral immunity showed that all two experimental groups and inactivated vaccine group were produce high level of FMDV sepcific antibodies and neutralizing antibodies.The cellular immunity showed,for instance, the IL-4 level in all two experimental groups had a significant increase similar to the inactivated vaccine group.