猪轮状病毒VP4单克隆抗体的制备及抗原表位鉴定

doi:10.3864/j.issn.0578-1752.2026.01.016

Abstract

Abstract:

【Background】Porcine Rotavirus (PoRV) is one of the key pathogens causing viral diarrhea in newborn and young piglets, and its infection can lead to severe gastrointestinal dysfunction in the host, with clinical manifestations, including dehydration, diarrhea, and even death, resulting in huge economic losses to the global pig industry. The VP4 protein is one of the key structural proteins on the surface of PoRV virions. The VP4 protein is cleaved by trypsin to generate two functional subunits, such as VP8 and VP5, which play a core role in the initial stage of viral infection of host cells, mediating the adsorption of the virus to host cell receptors and the subsequent membrane penetration process. Meanwhile, the VP4 protein is also an important target antigen that elicits the host immune response. However, current research on specific monoclonal antibodies (mAbs) against the PoRV VP4 protein is relatively scarce, which limits the development of related diagnostic methods and new vaccines. 【Objective】This study aimed to prepare specific monoclonal antibodies (mAbs) against the VP4 protein of Porcine Rotavirus (PoRV), and on this basis, comprehensive biological characterization of these mAbs were analyzed, including reactivity, antigenic epitope types (conformational or linear), and subtype identification, and key neutralizing activities were evaluated too. The aim was to precisely identify the antigenic epitope regions with important functional significance on the VP4 protein, thereby providing support for the accurate diagnosis of PoRV infection and the design of novel vaccines. 【Method】BALB/c mice were immunized with purified VP4*P23 recombinant protein, and hybridoma cell lines were screened using splenocyte fusion technology. The reactivity of monoclonal antibodies was identified by Western blot, indirect immunofluorescence assay (IFA), and immunoperoxidase monolayer assay (IPMA). The conformational sensitivity of monoclonal antibodies was evaluated by indirect ELISA. VP5 truncated proteins were constructed to determine the antigenic epitope regions, and the neutralizing ability of VP5 monoclonal antibodies was assessed by in vitro neutralization assay. 【Result】A total of 26 hybridoma cell lines stably secreting antibodies were successfully obtained. The antibody subtypes included multiple types such as IgG1, IgG2a, IgG2b and IgM, among which the light chain type was mainly κ chain. Among the obtained 26 mAbs, 15 of them were confirmed to specifically react with natural PoRV virions (detected by IFA and IPMA). Indirect ELISA detection showed that mAb11, 14, 15 and 23 were conformation-insensitive mAbs, while mAb16, 17, 18, 19, 21, 24, 25 and 26 were conformation-sensitive mAbs. It was worth noting that the reactivity of three mAbs (mAb1, 2 and 22) was enhanced after antigen denaturation. Western blot analysis further focused on mAbs recognizing linear epitopes (mAb11, 14, 15), and the results showed that they could all specifically recognize the linear epitope within the amino acid region of about 300-360 on the VP5 protein, possibly targeting the same antigenic epitope. However, in vitro neutralization assay evaluation showed that these three mAbs had no neutralizing effect on PoRV strains. 【Conclusion】Multiple mAbs targeting the PoRV VP4 protein were successfully prepared, among which 15 mAbs exhibited the ability to bind to natural viruses. Through systematic characterization, not only the subtype distribution and light chain type of mAbs were clarified, but more importantly, the antigen recognition characteristics of mAbs were deeply analyzed: conformation-sensitive and conformation-insensitive mAbs were successfully distinguished, and the linear antigenic epitope regions recognized by mAb11, 14, and 15 were precisely located. This study provided key antibody resources for the optimization of PoRV diagnostic reagents and the development of subunit vaccines, and laid a foundation for in-depth research on the immunological functions and antiviral mechanisms of the VP4 protein.

Key words: porcine rotavirus, VP4 protein, monoclonal antibody, antigenic epitope, diagnosis

ZHAO JiaLi, BIAN XianYu, SONG JiaPeng, WANG Chen, TANG XueChao, LI YunChuan, ZHOU JinZhu, ZHU XueJiao, TAO Ran, DONG HaiLong, ZHANG XueHan, LI Bin. Preparation of Monoclonal Antibody to Porcine Rotavirus VP4 and Preliminary Characterization of Antigenic Epitope[J].Scientia Agricultura Sinica, 2026, 59(1): 220-232.

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抗体编号 Antibody number	亚型 Antibody subtype	抗体编号 Antibody number	亚型 Antibody subtype
NO.01	IgG1,κ	NO.14	IgG1,κ
NO.02	IgG1,λ	NO.15	IgG1,κ
NO.03	IgG1,λ	NO.16	IgG2a,κ
NO.04	IgM,κ	NO.17	IgG2a,κ
NO.05	IgG1,κ	NO.18	IgG1,λ
NO.06	IgG1,κ	NO.19	IgG1,κ
NO.07	IgG1,κ	NO.20	IgG2b,κ
NO.08	IgG1,κ	NO.21	IgG1,κ
NO.09	IgG1,κ	NO.22	IgG2b,λ
NO.10	IgG1,κ	NO.23	IgG2a,κ
NO.11	IgG1,κ	NO.24	IgG2a,κ
NO.12	IgG1,κ	NO.25	IgG2a,κ
NO.13	IgM,κ	NO.26	IgG2a,κ

Preparation of Monoclonal Antibody to Porcine Rotavirus VP4 and Preliminary Characterization of Antigenic Epitope

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