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| Optimization of Purification Conditions for Classical swine fever virus E2 Protein Derived from Rice and Study on the Immunological Activity of E2 Vaccine Under Different Stable Conditions |
| PAN Shi-Yuan1, CHENG Wen-Bo1, ZHANG Jiang-Nan1, CHEN Heng-Yao1, KOU Shao-Kang1, KOU Zi-Qian1, ZHANG Er-Qin1,2,*, ZHANG Gai-Ping1,2,* |
1 College of Veterinary Medicine/International Joint Research Center of National Animal Immunology, Henan Agricultural University, Zhengzhou 450046, China;
2 Longhu Laboratory of Advanced Immunology, Zhengzhou 450046, China |
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Abstract Classical swine fever (CSF) is an acute, febrile and contact infectious disease of swine caused by Classical swine fever virus (CSFV), which belongs to the genus Pestivirus within the family Flaviviridae. The E2 protein is an envelope glycoprotein on the surface of CSFV and also the major protective antigen that induces the production of neutralizing antibodies in the host. Based on previous research, to reduce the downstream processing cost of rice (Oryza sativa)-derived E2 vaccine and improve vaccine stability, this study optimized the purification conditions of rice-derived E2 protein and evaluated the stability of the vaccine under different conditions. According to the physicochemical properties of E2 protein and rice endosperm proteins, the purification conditions of E2 protein were optimized by screening anion and cation chromatography resins. Purified E2 protein (10 μg) was then used to prepare vaccines under different formulations and storage conditions to immunize 5~7 week old female BALB/c mice (Mus musculus). Antibody blocking values in mice were detected using an IDEXX antibody blocking kit, antibody neutralizing titers were determined by microneutralization assay, and the levels of IL-2, IFN-γ, IL-4 and IL-10 cytokines were measured by ELISA. The results showed that the purity of E2 protein reached approximately 80% after purification with two cation-exchange resins, High-S and Sulfate-650F. After immunization, the antibody blocking values of all groups were ≥40% at 28 days post-immunization except for the PBS control group. At 35 days post-immunization, the antibody blocking values of Group 6 (buffer: PB; adjuvant: 50V; temperature: 4 ℃; formaldehyde concentration: 0.1%), Group 10 (buffer: Tris; adjuvant: 201VG; temperature: 4 ℃; formaldehyde concentration: 0.05%), Group 12 (buffer: Tris; adjuvant: 50V; temperature: 4 ℃; formaldehyde concentration: 0.05%) and the inactivated vaccine group were ≥80%. The neutralizing antibody titer of Group 6 reached 2?, while those of Group 12 and the inactivated vaccine group were 27. The levels of IL-2, IL-4 and IL-10 in Group 6, Group 12 and the inactivated vaccine group peaked at 35 days post-immunization. The content of IFN-γ remained relatively high in Groups 4, 6, 7, 8, 9, 10, 12 and the inactivated vaccine group. This study optimized the purification method of E2 protein and screened the stability conditions of the vaccine, providing basic data and references for subsequent research and development of rice-derived E2 vaccine.
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Received: 03 February 2026
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| Fund:河南省高校重点科研项目(25A230003); 河南省自然科学基金(252300423063) |
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Corresponding Authors:
*zhangerqin76@163.com; zhanggaiping2003@163.com
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