Classical Swin Fever Virus (CFSV) And Foot-and-mouth Disease Virus (FMDV) Antigen Gene Recombined And Expression In Plant

Both classical swine fever (CSF) and Foot- and- mouth disease (FMD) are highly contagious disease of livestock, which are classified by the Office International des Epizooties (OIE) as a list A disease. Classical swine fever virus (CSFV) and Foot-and-mouth disease virus (FMDV) are the causative agent of classical swine fever (CSF) and Foot- and- mouth disease (FMD) respectively. At present, Vaccine is effective method to prevent the disease. Conventional vaccines are based on the utilization of inactivated and attenuated virus which have considerable risk although they have proved to be effective for the prevention and controlling of the disease. The most widely used conventional vaccination of classical swine fever is The Chinese strain which was safe and effective but interferes with serodiagnosis. So it is necessary to develop safe and effective alternative vaccines.The structural protein VP1 of foot-and-mouth disease virus, which has been shown to contain critical epitopes responsible for the induction of neutralizing antibodies, has been expressed in different expression systems and plants. Production of complete or partial VP1 in a diversity of expression systems has been performed in the search for an effective and inexpensive alternative which would be highly immunogenesis. In this study, we developed a expression vector pBI121CTBVP1 by fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene with a flexible linker tetrapeptide (GPGP) at the c-terminal of CTB, and then cloned into intermediate vector pBI121-patatin containing specific-tuber patatin promoter of potato. Furthermore, we fused the ER targeting signal (SEKDEL) encoding sequence at the c-terminal of VP1 in order to improve expression of fused gene and distribution. The transgenic potatos were obtained by Agrobacterium-mediated transformation and selection on kanamycin resistance agent. The transgenic plantlets were identified by PCR, Southern-blot and the production of fused protein was confirmed and quantified by Western-blot and ELISA assays. The results demonstrated that the fused genes were expressed stablely under control of specific-tuber patatin promoter up to 0.1%-0.13% though it was fant in leaves, and thetransgenic potato could be as oral vaccine candidate defense of foot-and-mouth disease.The E2 protein of CSFV contains major antigenic determinants that is conserved and involved in neutralization by antibodies. The expression of E2 protein in insect and in E. coli has been reported. Animal immunization and the challenge against CSFV experiments have been conducted using the recombinant E2 protein. Here, we studied the expression of CSFV E2 gene of shimen strain in tobacco and C. reinhardti chloroplast by biolistic bombardment transformation. The transformants were identified by PCR, Southern blotting, Western blotting after selection on resistant medium. ELISA quantification assay showed that the expressed E2 protein accumulated up to 1.0-2% and 2% of the total soluble protein in tobacco and C. reinhardti chloroplast respectively. In order to confirm the neutralizing activity of the protected antibodies induced by E2 antigen, neutralization assay were carried out on mices. The results of the study showed that the protein E2 expressed in tobacco and C. reinhardti chloroplast could elicit animal bodies to produce antibodies. This indicated that the expressed E2 proteins have a certain of immunogenicity. Our finding provides a new way to develop plant-drived vaccine against CSFV.