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

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (3): 1344-1354.doi: 10.11843/j.issn.0366-6964.2025.03.033

非洲猪瘟病毒H108R蛋白的制备及其免疫原性评价

张越¹(), 茹毅¹, 郝荣增¹, 杨锐², 赵陇和¹, 李亚军², 杨洋¹, 张荣², 蒋成辉², 郑海学¹^,*()

1. 中国农业科学院兰州兽医研究所, 兰州大学动物医学与生物安全学院, 动物疫病防控全国重点实验室, 兰州 730000
2. 中农威特生物科技股份有限公司, 兰州 730046

收稿日期:2024-04-28 出版日期:2025-03-23 发布日期:2025-04-02
通讯作者: 郑海学 E-mail:1969962334@qq.com;zhenghaixue@caas.cn
作者简介:张越(1995-)，男，甘肃华池人，博士生，主要从事动物疫病防控与检疫研究，E-mail: 1969962334@qq.com
基金资助:
国家重点研发计划(2021YFD1801302-3)；甘肃省自然科学基金重点项目(23YFNA0011)；中央高校基本科研业务费专项(lzujbky-2022-ct02)；兰州市青年科技人才创新项目(2023-QN-3)；中央级公益性科研院所基本科研业务费专项(CAAS-ZDRW202409)；国家生猪产业技术体系(CARS-35)；甘肃省科技重大专项(23ZDKA0002)

Preparation and Immunogenicity Evaluation of African Swine Fever Virus H108R Protein

ZHANG Yue¹(), RU Yi¹, HAO Rongzeng¹, YANG Rui², ZHAO Longhe¹, LI Yajun², YANG Yang¹, ZHANG Rong², JIANG Chenghui², ZHENG Haixue¹^,*()

1. State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730000, China
2. China Agricultural Vet. Bio. Science and Technology Co., Ltd., Lanzhou 730046, China

Received:2024-04-28 Online:2025-03-23 Published:2025-04-02
Contact: ZHENG Haixue E-mail:1969962334@qq.com;zhenghaixue@caas.cn

摘要/Abstract

摘要：

本研究旨在制备ASFV H108R蛋白，通过免疫小鼠评价该蛋白的免疫原性，为该蛋白作为疫苗候选抗原的研究提供理论依据。利用生物信息学工具分析H108R蛋白的结构及基本理化性质，选择编码蛋白33~108 AA的基因序列串联融合到原核表达载体，转化后经IPTG诱导表达，用镍亲和层析纯化目标蛋白质。免疫小鼠利用流式细胞术检测脾脏活化的CD4⁺和CD8⁺T淋巴细胞，分离小鼠血清测定特异性抗体滴度，利用Western blot和IFA鉴定多克隆抗体的特异性。并用试剂盒检测血清中细胞因子含量，将血清与ASFV-GFP病毒孵育评估抗体是否能抑制病毒的复制。生物信息学分析表明H108R蛋白为亲水性蛋白质，有跨膜区，无信号肽，蛋白二三级结构中存在较多α-螺旋。SDS-PAGE、Western blot显示IPTG诱导后，成功表达纯化H108R蛋白。蛋白免疫小鼠后能够诱导小鼠脾脏T细胞的活化，刺激机体产生更高水平的细胞因子。免疫制备的多克隆抗体效价为1∶128 000，能与ASFV-GFP病毒特异性结合，与病毒孵育后能明显抑制病毒的复制。本研究成功表达制备了ASFV H108R蛋白，免疫能够诱导脾脏T淋巴细胞活化，引起细胞因子水平升高，制备的多克隆抗体能够明显抑制病毒的复制，具有较好的免疫原性，为深入研究H108R蛋白的生物学功能及疫苗研究奠定基础。

关键词: 非洲猪瘟病毒(ASFV), H108R蛋白, 多克隆抗体, 免疫原性

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

中图分类号:

S852.659.1

张越, 茹毅, 郝荣增, 杨锐, 赵陇和, 李亚军, 杨洋, 张荣, 蒋成辉, 郑海学. 非洲猪瘟病毒H108R蛋白的制备及其免疫原性评价[J]. 畜牧兽医学报, 2025, 56(3): 1344-1354.

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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非洲猪瘟病毒H108R蛋白的制备及其免疫原性评价

Preparation and Immunogenicity Evaluation of African Swine Fever Virus H108R Protein

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