抗A群猪轮状病毒VP7蛋白208—222aa结构域单克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2025.12.034

摘要/Abstract

摘要： 【目的】制备抗A群猪轮状病毒(Porcine rotavirus,PoRV)VP7蛋白第208—222位氨基酸处(208—222aa)结构域单克隆抗体，并探究其免疫原性，为建立A群PoRV检测方法提供参考。【方法】利用AlphaFold3软件预测4种主要流行G基因型(G3、G4、G5和G9)PoRV VP7蛋白三聚体结构，对其208—222aa区域的特征性抗原表位进行比对和拟合。设计截短VP7蛋白，并分别与GST、MBP标签融合。通过原核表达系统分别表达重组蛋白GST-VP7和MBP-VP7，利用亲和层析纯化重组蛋白。利用纯化的MBP-VP7重组蛋白免疫小鼠，制备杂交瘤细胞株，采用人工合成208—222aa多肽进行筛选，获得特异性分泌抗体的细胞株。【结果】208—222aa区域结构分析显示，该结构域在不同基因型PoRV VP7蛋白中具有较高的构象保守性。通过原核表达系统，成功获得高纯度可溶性表达的重组蛋白GST-VP7和MBP-VP7。通过208—222aa多肽筛选，获得2株可分泌针对该结构域特异性单克隆抗体的杂交瘤细胞株：10F11G和5F6B。间接免疫荧光检测显示，10F11G和5F6B抗体可有效识别天然PoRV颗粒。【结论】本研究成功获得了2株可稳定分泌针对PoRV VP7蛋白208—222aa结构域特异性单克隆抗体的杂交瘤细胞株。试验结果为A群PoRV相关抗体制备和检测方法开发提供了基础材料。

关键词: A群猪轮状病毒; VP7蛋白; 单克隆抗体; 杂交瘤细胞; 原核表达

Abstract: 【Objective】 The aim of this experiment was to prepare monoclonal antibody against the structural domain of 208-222 amino acid (208-222aa) in VP7 protein of group A Porcine rotavirus (PoRV),and investigate its immunogenicity,in order to provide a reference for the establishment of a detection method for group A PoRV. 【Method】 AlphaFold3 software was used to predict the structure of the VP7 trimeric proteins of the four major popular G genotypes (G3,G4,G5 and G9),and the characteristic antigenic epitopes in the 208-222aa region of the VP7 proteins were compared and fitted.The truncated VP7 protein was designed and fused with GST and MBP tags,respectively.The recombinant proteins GST-VP7 and MBP-VP7 were expressed using the Escherichia coli expression system,respectively.Purification of recombinant protein by affinity chromatography.The purified MBP-VP7 recombinant protein was used to immunize mice,and hybridoma cell lines were prepared and screened using synthetic 208-222aa peptide to obtain cell lines that specifically secreted antibodies. 【Result】 Structural analysis of the 208-222aa region showed that this structural domain had high conformational conservation in different genotypes of PoRV VP7 proteins.Through the prokaryotic expression system,highly pure soluble expressed recombinant proteins GST-VP7 and MBP-VP7 were successfully obtained.Through the screening of the 208-222aa peptide,two hybridoma cell lines capable of secreting specific monoclonal antibodies against this domain were obtained:10F11G and 5F6B.Indirect immunofluorescence assay showed that the antibodies 10F11G and 5F6B could effectively recognize natural PoRV particles. 【Conclusion】 This study successfully obtained two hybridoma cell lines that could stably secrete monoclonal antibodies specific to the 208-222aa domain of VP7 protein of PoRV.This results provided basic materials for the preparation of group A PoRV related antibodies and the development of detection methods.

Key words: group A Porcine rotavirus; VP7 protein; monoclonal antibody; hybridoma cells; prokaryotic expression

中图分类号:

S852.65⁺1

刘博, 叶乐添, 康桦华, 贾晗铎, 李春玲, 周霞, 李艳, 蒋智勇, 勾红潮, 楚品品, 卞志标, 臧莹安, 翟少伦. 抗A群猪轮状病毒VP7蛋白208—222aa结构域单克隆抗体的制备[J]. 中国畜牧兽医, 2025, 52(12): 5901-5909.

LIU Bo, YE Letian, KANG Huahua, JIA Handuo, LI Chunling, ZHOU Xia, LI Yan, JIANG Zhiyong, GOU Hongchao, CHU Pinpin, BIAN Zhibiao, ZANG Yingan, ZHAI Shaolun. Preparation of Monoclonal Antibody Against the 208-222aa Structural Domain of VP7 Protein of Group A Porcine Rotavirus[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(12): 5901-5909.

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