Immunogenicity Analysis of Multi-epitopes Antigen of Senecavirus A VP2 Protein
TAO Shi-Yu1,2,3, RU Yi2, CHEN Jiao2, HAO Rong-Zeng2, LI Ya-Jun2, LU Bing-Zhou2, SHI Zheng-Wang2, LIU Xue-Rong3, ZHENG Hai-Xue2, WEI Yan-Ming1*
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China; 2 State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China; 3 China Agricultural Vet. Bio. Science and Technology Co., Ltd., Lanzhou 730046, China
Abstract：Senecavirus A (SVA), a new RNA virus belonging to the Picornaviridae family, which seriously endangers the development of China's pig industry. At present, there is no effective SVA vaccine. In order to study the new SVA subunit vaccine, this study was based on the viral structural protein 2 (VP2) gene sequences of SVA, and the B cell epitope sequences predicted by bioinformatic methods and the T cell epitope genes in the reported Foot-and-mouth disease virus (FMDV) 3A protein. The recombinant expression plasmids pET28a-rSVP2-B and pET28a-rSVP2-BT were constructed respectively after optimization codon and connecting in series with the flexible linkers. The recombinant tandem epitope proteins rSVP2-B and rSVP2- BT, which were expressed in prokaryotic system and purified by Ni+ ion chromatographic method, were identified by SDS-PAGE and Western blot, and then immunized BALB/c mice (Mus musculus) to evaluate their immunogenicity. The results showed that the target proteins were expressed correctly after the recombined plasmid pET28a-rSVP2-B and pET28a-rSVP2-BT were transformed into Escherichia coli BL21 (DE3) and induced by 1 mmol/L isopropy- β -D-thiogalactoside (IPTG). SDS-PAGE analysis showed that the relative molecular weights were 34 and 38 kD, respectively. Western blot results showed that rSVP2-B and rSVP2-BT had good reactivity with SVA positive sera. There was no significant difference in humoral immune levels between rSVP2-B and rSVP2-BT immunized mice with purification protein. Both of them induced strong specific IgG and IgG1 antibodies, and mainly induced humoral immune response biased towards Th2 type. The results of virus neutralization test showed that the neutralizing antibody titers induced by the two recombinant proteins ranged from 1∶64 to 1∶128. Meanwhile, mice immunized rSVP2-B and rSVP2-BT also produced strong cellular immunity. The mice spleen lymphocytes proliferation levels and interleukin-2 (IL-2) and interferon- γ (IFN- γ) expression levels in the mice vaccinated with the rSVP2-BT protein group was significantly higher than those in the rSVP2-B group. In conclusion, the recombinant tandem multi-epitope proteins rSVP2-B and rSVP2-BT with good immunogenicity were successfully prepared, and the immunogenicity of rSVP2-BT was superior to that of rSVP2-B. This study provides a theoretical basis for the development of SVA multi-epitope vaccine.
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