Development of a monoclonal antibody specifically recognizing a linear epitope on the E1 protein of Getah virus.

Virology Pub Date : 2025-01-01 Epub Date: 2024-11-26 DOI:10.1016/j.virol.2024.110315
Muyang Liu, Tongwei Ren, Liping Zhang, Peijie Li, Zhen Zhong, Lingshan Zhou, Yifeng Qin, Kang Ouyang, Ying Chen, Weijian Huang, Zuzhang Wei
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Abstract

Getah virus (GETV) is a mosquito-borne alphavirus that can cause disease outbreaks in domesticated mammals. The E1 protein of GETV plays a crucial role in mediating the fusion of viruses and host cells. In this study, the recombinant GETV E1 protein was expressed and administered to BALB/c mice. Hybridoma cells secreting a monoclonal antibody (mAb) 7D4-2 against E1 protein were subsequently obtained using a cell fusion protocol between SP2/0 cells and splenocytes. The reactivity of mAb 7D4-2 with GETV was confirmed through Western blot analysis and indirect immunofluorescence assay (IFA). A precise B cell linear epitope, 281-DIPDTAF-287, was identified using Western blot analysis with the produced mAb 7D4-2 by constructing and expressing a series of truncated His-fused E1 proteins. Conservation analysis indicated that this epitope is highly conserved in Group III strains of GETV, but exhibits an amino acid difference (T285S) compared to Group I, Group II, and Group IV. Cross-reactivity analysis by Western blot demonstrated that the B-cell epitope containing the mutation could be recognized by mAb 7D4-2. The ability of mAb 7D4-2 to recognize the epitope carrying the T285S mutation was further confirmed using an infectious clone of GETV. This study enhances the understanding of the biological characteristics of the E1 protein and will facilitate the future development of diagnostic tests for GETV.

一种特异性识别Getah病毒E1蛋白线性表位的单克隆抗体的研制。
Getah病毒(GETV)是一种蚊媒甲病毒,可在家养哺乳动物中引起疾病暴发。GETV的E1蛋白在介导病毒与宿主细胞融合中起着至关重要的作用。本研究表达重组GETV E1蛋白,并将其应用于BALB/c小鼠。随后通过SP2/0细胞与脾细胞的细胞融合获得了分泌针对E1蛋白的单克隆抗体(mAb) 7d2 -2的杂交瘤细胞。通过Western blot分析和间接免疫荧光法(IFA)证实mAb 7D4-2与GETV的反应性。通过构建和表达一系列截断的his -融合E1蛋白,利用Western blot分析获得了精确的B细胞线性表位281-DIPDTAF-287。保守分析表明,该表位在GETV III组菌株中高度保守,但与I组、II组和IV组相比存在氨基酸差异(T285S)。Western blot交叉反应性分析表明,含有该突变的b细胞表位可以被mAb 7d2 -2识别。mAb 7D4-2识别携带T285S突变的表位的能力通过GETV的感染性克隆得到进一步证实。本研究增强了对E1蛋白生物学特性的认识,将促进GETV诊断测试的未来发展。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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