Preparation and Identification of Monoclonal Antibodies against the Bovine Coronavirus

doi:10.13560/j.cnki.biotech.bull.1985.2025-0838

Abstract

Abstract:

Objective Bovine coronavirus (BCoV) is a major pathogen causing diarrhea in calves and winter dysentery in adult cattle, and respiratory diseases in cattle of all ages. The diseases caused by BCoV are widespread and lead to serious harm in the livestock industry. There are limitations in traditional diagnostic and control methods. The development of BCoV-specific monoclonal antibodies aims to facilitate precise diagnostic detection and provide critical experimental materials for coronavirus research. Method BALB/c mice were immunized with purified virus, and hybridoma cells were prepared using cell fusion technology. Monoclonal cell lines stably secreting specific antibodies were screened. The monoclonal antibodies were identified and subclassified by indirect immunofluorescence assay (IFA) and indirect enzyme-linked immunosorbent assay (ELISA). Concurrently the eukaryotic expression plasmids for the full-length S protein and S1, S2 subunits of cpCoV were constructed and then transfected into 293T cells. The viral proteins recognized by monoclonal antibodies were identified by IFA and Western-blot techniques. Result Monoclonal antibody 1E1 was successfully obtained through monoclonal screening. The ascites titer of antibody was >1∶81 000, and the subtype was identified as IgG1. The antibody specifically reacted with both BCoV and cpCoV. Through gene cloning and eukaryotic expression, it was verified that the antigenic epitopes recognized by 1E1 antibody were located in the S1 domain of the S protein. Conclusion By preparing BCoV monoclonal antibodies, the obtained monoclonal antibody 1E1 can be used for identification of both BCoV and cpCoV. The antigenic epitopes recognized by the antibody are verified, and analysis reveals that the homology of S1 protein sequence is 95.4%-99.6%, and has significant differences between BCoV and cpCoV. The result provides a new method for the detection of the two coronaviruses and a new tool for the basic research of BCoV and cpCoV.

Key words: bovine coronavirus, caprine coronavirus, monoclonal antibody, antigen epitope, identification

LIU Yu, LIU Xiao-xiao, ZHENG Jian-hao, LI Bin, HE Hong-hong, MAO Li. Preparation and Identification of Monoclonal Antibodies against the Bovine Coronavirus[J]. Biotechnology Bulletin, 2026, 42(1): 67-75.

Figures/Tables 7

Table 1 Primer sequences

引物名称 Primer	寡核苷酸序列 Oligonucleotide sequence（5'-3'）	基因长度 Gene length （bp）
cpCoV-S-F	ATGTTTTTGATACTTTTAATTTC	4 092
cpCoV-S-R	GTCGTCATGTGAGGTTTT	4 092
cpCoV-S1-F	ATGTTTTTGATACTTTTAATTTC	2 304
cpCoV-S1-R	TCTACGACTTCGTCTTTTTG	2 304
cpCoV-S2-F	AGACGAAGTCGTAGATCG	1 803
cpCoV-S2-R	GTCGTCATGTGAGGTTTT	1 803

Fig. 1 Measurement of serum antibody titer in immunized mice

Fig. 2 IFA analysis of the reactivity of mAb 1E1 against BCoV and cpCoV

Fig. 3 Identification results of plasmids of S geneA: PCR amplification of pCAGGS-cpCoV-S-HA. B: PCR amplification of pCAGGS-cpCoV-S1-HA. C: PCR amplification of pCAGGS-cpCoV-S2-HA. M: DNA molecular weight standard. 1, 2: RT-PCR products of the target gene

Fig. 4 Expression and identification of S proteinA: IFA test results. B: Western-blot test results. M: Protein molecular weight standard. 1: Positive plasmid control of HA-BCoV-HE. 2, 3: Western-blot test results of HA-cpCoV-S recombinant plasmid. 4, 5: Western-blot test results of HA-cpCoV-S1 recombinant plasmid. 6, 7: Western-blot test results of HA-cpCoV-S2 recombinant plasmid. 8: Empty pCAGGS-HA; 9: 293T cells control

Fig. 5 IFA identification results of monoclonal antibodies 1E1 and 6D5

Fig. 6 Analysis results of homology and differential proteins of BCoV and cpCoV S1 proteinsA: Pairwise genetic distance heatmap based on the complete amino acid sequences of the S1 fragments of BCoV and cpCoV. Black triangle: Isolate BCoV-NXWZ2301. B: Comparison of the S1 protein sequences of BCoV and cpCoV

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