Construction Of Recombinant Adenovirus Vector Containing The Capsid And 3C Or 3ABC Protease Coding Regions Of Foot-and-Mouth Disease Virus

Foot-and-mouth disease (FMD) is a highly contagious, acute disease affecting cattle, pigs, sheep and other cloven-hoofed animals. FMD is caused by a positive-strand RNA virus belonging to the Aphthovirus genus of the Picornaviridae. Currently, in developing countries, FMD was routinely controlled and eradicated by systematic vaccination of inactivated whole-virus preparation. Such conventional vaccines, although are generally effective, do have some disadvantages. These include thermal instability, the short-term immunity and extra cost due to the high-security containment necessary for their preparation. Thus, the potentially safe alternatives were been explored. In this study, we successfully constructed two recombinant adenovirus vectors expressing the capsid proteins P1 and non-structural proteins 3C/3ABC of serotype A FMD virus (FMDV), which focused on the development of a potential vector vaccine against FMD.First, the interest genes corresponding to P1-2A,3C,3ABC were amplified from two recombinant plasmids containing FMDV P1-2A and 3ABC coding regions. Three fragments were sub-cloned into pGEM-T Easy vector. The three recombinant plasmids were then digested with enzymes of Xbaâ… /BamHâ… and BamHâ… /Kpnâ… , respectively. P1-2A, 3C and 3ABC fragments were ligated into Xbaâ… /Kpnâ… digested pshuttle2 vector. Two recombinant pshuttle plasmids named pSh-P12A3C and pSh- P12A3ABC were successfully constructed. Sequence analysis proved that the coding regions of two target gene cassettes were highly homologous with it's parent strain (99.7%, 99.6%).Two target gene cassettes contain P12A3C and P12A3ABC were obtained by double digestion of pSh-P12A3C and pSh-P12A3ABC plasmids with I-Ceuâ… and PI-Sceâ… enzymes, and were ligated with BD Adeno-X Virus DNA in vitro. The ligated products were digested with Swaâ… , and then transformed into DH5αcompetent cell. Two recombinant adenovirus plasmids containing P12A3C and P12A3ABC coding region of FMDV were confirmed by PCR, enzyme digestion and sequencing, respectively, which were given the name of A-P12A3C and A-P12A3ABC.The recombinant adenovirus plasmids were linearized with Pacâ… and then transfected HEK293 cells mediated with liposome. The cell pathogenic effect (CPE) could be observed within one week after transfection, and complete CPE appeared within 24⁴8h after two or four round passages, respectively. The concentration of recombinant adenoviruses were 2.4×10^-8 TCID50/0.2mL and 1×10^-8 TCID50/0.2mL in virus culture supernatant of 10^th round passages respectively. The expression of target genes in HEK293 cells were demonstrated with immunofluorescence and sandwich-ELISA tests. Two recombinant adenoviruses were proved to be stable during cell culture. The virion of recombinant adenovirus were observed by electron microscope. These results indicated the recombinant adenoviruses expressing capsid protein were successfully constructed. These preliminary results made a good foundation for subsequent study of live vector vaccine against A serotype FMD.