非洲猪瘟病毒H108R蛋白的制备及其免疫原性评价

doi:10.11843/j.issn.0366-6964.2025.03.033

Abstract

Abstract:

The aim of this study was to prepare the ASFV H108R protein, to evaluate the immunogenicity of the protein by immunization of mice and to provide a theoretical basis for the investigation of this protein as a candidate antigen for vaccines. The structure and basic physicochemical properties of the H108R protein were analyzed using bioinformatics tools, and the gene sequence encoding the protein 33-108 AA was selected for tandem fusion into a prokaryotic expression vector, transformed and induced to express by IPTG, and the target protein purified by nickel affinity chromatography. Immunized mice were examined by flow cytometry to detect spleen-activated CD4⁺ and CD8⁺T lymphocytes, mouse serum was isolated to determine specific antibody titres, and the specificity of polyclonal antibodies was determined by Western blot and IFA. The cytokine content of the serum was determined using the kit, and the serum was incubated with ASFV-GFP virus to assess whether the antibody could inhibit virus replication. Bioinformatics analysis showed that the H108R protein is a hydrophilic protein with a transmembrane region, no signal peptide, and more α-helices exist in the protein's ditertiary structure. SDS-PAGE and Western blot showed that the purified H108R protein was successfully expressed after IPTG induction. The protein was able to induce activation of mouse splenic T cells and stimulate the body to produce higher levels of cytokines after immunization of mice. The immunized polyclonal antibody with a potency of 1:128 000 was able to specifically bind to the ASFV-GFP virus and significantly inhibit viral replication after incubation with the virus. In this study, ASFV H108R protein was successfully expressed and produced, and immunization was able to induce activation of splenic T lymphocytes leading to an increase in cytokine levels, and the produced polyclonal antibody was able to inhibit virus replication, which had a good immunogenicity, and it lays a foundation for the in-depth study of the biological function of H108R protein and the research of vaccine.

Key words: African swine fever virus (ASFV), H108R protein, polyclonal antibodies, immunogenicity

CLC Number:

S852.659.1

ZHANG Yue, RU Yi, HAO Rongzeng, YANG Rui, ZHAO Longhe, LI Yajun, YANG Yang, ZHANG Rong, JIANG Chenghui, ZHENG Haixue. Preparation and Immunogenicity Evaluation of African Swine Fever Virus H108R Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1344-1354.

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氨基酸 Amino acids	数量/个 Number	频率/% Frequency	氨基酸 Amino acids	数量/个 Number	频率/% Frequency
丙氨酸Ala (A)	4	3.7%	赖氨酸Lys (K)	6	5.6%
精氨酸Arg (R)	1	0.9%	甲硫氨酸Met (M)	3	2.8%
天冬酰胺Asn (N)	9	8.3%	苯丙氨酸Phe (F)	5	4.6%
天冬氨酸Asp (D)	3	2.8%	脯氨酸Pro (P)	6	5.6%
谷氨酰胺Gln (Q)	5	4.6%	丝氨酸Ser (S)	8	7.4%
谷氨酸Glu (E)	8	7.4%	苏氨酸Thr (T)	10	9.3%
甘氨酸Gly (G)	2	1.9%	色氨酸Trp (W)	3	2.8%
组氨酸His (H)	3	2.8%	酪氨酸Tyr (Y)	6	5.6%
异亮氨酸Ile (I)	11	10.2%	缬氨酸Val (V)	6	5.6%
亮氨酸Leu (L)	9	8.3%	/	/	/

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Preparation and Immunogenicity Evaluation of African Swine Fever Virus H108R Protein

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