Study On Expressing Linear Epitopes Of FMDV In Canine Adenovirus Type 2

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals. It is also infectious to human beings and follows with extensive interest as a zoonosis. Once FMD occurs, all the infected and exposed animals would be subjected to slaughter (OIE Manual, 1996). An economic devastation might result from the massive animal death and the economic sanction from other countries on account of the FMD. Therefore, prevention of FMD is the only way to solve the problem. Although the conventional vaccines made by chemically inactivated whole viral particle have been efficiently controlled FMD in many countries, it was found to cause outbreaks of FMD by release of incompletely inactivated virus from the vaccine. Attempts to produce safer vaccines for FMD have been made on the synthesized peptides or bacterially expressed proteins contain viral epitopes. Although antibodies induced by these peptides or protein are able to efficiently neutralize the FMD virus in experimental animals, protection of livestock from virus challenge are not successful.It was reported that FMD's VP1 structual protein was it's main antigen.There are many linear epotipes on the VP1 protein. For example, B cell pi-position is VP1 141-160AA,200-213AA,and T cell pi-position is 20-34AA.Human adenovirus type-5 was also used as a vector for FMDV recombinant vaccine and has been reported to induce immune response in swine. Other viruses, such as vaccinia virus, pseudorabies virus , and adenovirus were also used as potential vaccine vectors for FMDV. Similar to these vector viruses, canine adenovirus type-2 also possesses the following properties: it replicates effectively in pigs after inoculation; the HI antibodies it induces in pigs can reach 1: 210; CAV-2 has been proved in our previous experiment to be safe and has no side-effect in pigs (data not shown); the antibody it induces persists for ~6 months with only a one-time vaccination, which is enough for the production period of pork breeding; unlike vaccinia virus, other poxvirus, and human adenovirus, CAV-2 does not naturally cause infection in human beings and it reduces the public concern about its transmission to human beings; CAV-2 infects a wide variety of cell types in both dividing and nondividing cells. These characteristics, together with its relative ease of gene manipulation, virus production, and the high viral titer, make it valuable as a live vector for pig vaccine development.In this study, the purpose gene is three linearity epi-position which lie on FMDV's proteinVP1.The purpose gene encode B cell pi-psition that is VP1 20-30AA, 141-160AA, 200-213AA are T cell pi-position .These purpose genes were chemically synthesized and were linked together via pMD18-T Vectorand and pEGFP Vector, then we obtained the co-expression protein gene expession box. After that, the plasmid pEGFP-I-II-III was digested by Ase I and Mlu I. Then the expression box was cloned into pPoly II CAV-2. At last,we digested the plasmid with Asc I and Pme I and co-transfected the two gene fragment in MDCK cell by Lipofectamine?. Typical cytopathogenic effect was observed 5 passages after transfection. Using the restriction endonuclease digestion and PCR, we confirmed that the recombinant canine adenovirus type 2 expressing FMDV linear epitopes (CAV-2-F-I-II-III) had been obtained.We studied the biological feature of the CAV-2-F- I-II-III, which is a recombinant canine adenovirus type 2 expressing FMDV linear epitopes, using general virus and serology method, using the RT-PCR and Western blotting to examine the transcription and expression of the epitope gene., using PCR to analyse the hereditary feature of the CAV-2- F- I-II-III. It was concluded that FMDV linear epitopes and green fluorescence protein gene could transcript and express and in vitro the hereditary feature of the CAV-2- F- I-II-III is stable.On immunization experiment, using the Western blotting, ELISA, HI and Micro-neutralization test of serum samples to examine the titer of the sera, we found that the recombinant virus could stimulate specific immune responses to the protein VP1 of foot and mouth disease virus. There was no neutralizing antibody in the CAV-2 immunized animals. In contrast, protective level neutralizing antibody was detected in the CAV-2- F- I-II-III immunized animals. Using FMD virus inhibition test and micro-neutralization test of serum samples, we demonstrated that no neutralizing antibody was detected until 3 weeks after vaccination with the recombinant virus CAV-2- F- I-II-III. The neutralizing antibody level against FMDV reached peak at 4–5 weeks after the vaccination and was between 1:22 and 1:24. All the vaccinated pigs with CAV-2- F- I-II-III produced neutralizing antibodies and the antibodies lasted for 25 weeks with slight decline. In conclusion, the CAV-2- F- I-II-III would benefit the development of foot and mouth disease vaccine.