Study On Suicidal DNA Vaccine Of Capsid Gene From Swine Vesicular Disease Virus

Swine vesicular disease (SVD) was a highly contagious viral pig disease characterized by the appearance of vesicles on the coronary bands, heels of the feet, and less commonly on the snout and tongue. SVD was classified as a List A disease by the Office International des Epizooties (OIE) because the lesions produced by SVD virus (SVDV) were indistinguishable from those caused by foot-and-mouth disease virus. SVD was first identified in Italy in 1966 and several more outbreaks have been reported subsequently in Europe and Eastern Asia, however, in most recent, infection with SVDV has been reported only in Portugal and Italy. There was not an effective vaccine available for SVD that could help in the eradication of this disease. Once introduced, SVD could be a difficult disease to eradicate and improved methods of control would be highly beneficial. Therefore, it was urgent to develop safer and more effective vaccines to protect and control this disease.Suicidal DNA vaccines based on the alpha virus replicon, have been developed recently. The suicidal DNA vaccines were considered having more advantages of biosafety and enhanced immunogenicity than conventional DNA vaccines. It has been demonstrated the suicidal DNA vaccine could break immunological tolerance by activating innate antiviral pathways. All these advantages indicated that suicidal DNA vaccine was an attractive vaccine delivery vehicle.The suicidal DNA vaccine of capsid gene from SVDV was constructed and its immunogenicity and protective effectivity was investgated. The details were showed as below.The capsid gene, 1BCD, of SVDV HK'70 strain were amplified by RT-PCR and cloned into pMD18-T vector, followed sequencing. The nucleotide (nt) sequence and the deduced amino acid (aa) were megaligned and analyzed with biosoftware. The phylogenetic tree of SVDV was constructed by DNAStar software according to VP1 sequence. The result demonstrated that there were 2346nt of 1 BCD gene encoding 782aa and the potential lytic site between VP1, VP2 and VP3 were all Gln/Gly. As showed in phylogenetic tree, SVDV HK'70, J/'73, H3'76 and UKG/27/72 were all clustered into genotype Group II.The secondary structure of capsid protein was predicted by the methods of Chou-Fasman, Garnier-Robson and Karplus-Schultz based on the sequence of capsid protein gene of SVDV and hydrophilicity, surface probability plot and antigenic index for capsid protein were obtained by the methods of Kyte-Doolittle, Emini and Jameson-wolf respectively, Combining the results according to these methods, the B cell epitopes for capsid protein of SVDV were predicted. The results showed that there were much flexible region such as coil region and turn region in capsid protein of SVDV and there were more predominant B cell epitopes in VP1 than in VP2 and VP3. This study would be helpful for identification of B cell epitopes for capsid protein using experimental methods and research of reverse vaccine of SVDV.The capsid protein gene, 1BCD, of SVDV was inserted into semliki forest virus (SFV) replicon-based plasmid, namely suicidal plasmid, pSCAl. The recombinant plasmid was confirmed by restriction enzyme analysis and nucleic acid sequencing and named pSCA/lBCD. Then the recombinant plasmid was transfected into BHK-21 cells by Lipofectamine Plus? Reagent. The capsid proteins of SVDV expressed in BHK-21 cells were confirmed by RT-PCR and indirect immunofiuorescence assay (IFA) .The result showed that the 1BCD gene of SVDV was cloned into pSCAl correctly, and the recombinant plasmid pSCA/lBCD could express immunocompetence protein of SVDV in BHK-21 cells.To evaluate the immunogenicity of Semliki forest virus replicon-based recombinant plasmid, the guinea pigs were inoculated 3 times at 3-week interval with suicidal DNA vaccine encoding capsid protein of SVDV HK'70 by intramuscle of quadriceps and the immune responses induced by suicidal DNA vaccine were detected by MTT for peripheral blood lymphocytes (PBL) proliferation, Enzyme-linked immunosorbent assay (ELISA) and Virus neutralization test (VNT). The results showed that the suicidal DNA vaccine, pSCA/lBCD, could induce SVDV specific antibody and PBL proliferation in guinea pigs.The immune response induced by suicidal DNA vaccine of capsid gene from SVDV was studied in pigs. 2 groups animal were immunized by intramuscle injection with recombinant plasmid DNA on each of 3 times with a 2-week dose interval. Group 1 received non-coding plasmid DNA only as control;group 2 received the capsid protein coding plasmid DNA. All pigs were challenged by inoculation with homologous virus 3 weeks after the final immunization. Neutralizing antibodies were detectable in 4 of 6 vaccinated pigs on the day of challenge. Furthermore, PBL proliferation following in vitro re-stimulation with SVDV antigen was elevated in vaccinated group, the proliferation was not significant compared to control group (P>0.5). In summary, the administration of a suicidal DNA vaccine of SVDV induced a SVDV specific immune response and protected half of the vaccinated animals from clinical sign.The results of this work showed that the suicidal DNA vaccine could be used as candidate vaccine for SVDV, however, the effectivity must be further improved.