猪急性腹泻综合征冠状病毒核衣壳蛋白单克隆抗体的制备及其抗体序列的分析

doi:10.11843/j.issn.0366-6964.2023.06.027

Abstract

Abstract: There is no commercially diagnostic kits for swine acute diarrhea syndrome coronavirus (SADS-CoV). To fill the gap, in this study, monoclonal antibodies (MAbs) against the SADS-CoV nucleocapsid (N) protein were prepared and the sequences of MAbs were analyzed. The N protein was expressed using prokaryotic expression system. The purified N protein was used as an antigen to immunize Balb/C mice. After cell fusion, screening, and subcloning, five stable secreting MAbs against N protein were obtained, and designated as 1C10, 4B10, 6G1, 6F3 and 6E8,respectively. The variable region gene sequences of MAbs were obtained by nested PCR. Indirect immunofluorescence assay (IFA) results showed that all MAbs could specifically recognize Huh7 infected with SADS-CoV. ELISA results showed that the five MAbs could react with the purified N protein, while only the MAb 6E8 could react with SADS-CoV. The results of western blot showed that the five MAbs specifically recognized with the purified N protein, as well as the native N protein in cells infected with SADS-CoV. Isotyping revealed that the MAbs 1C10, 4B10, 6G1, and 6F3 were of the IgG1 class, and 6E8 was of the IgG2a class, and which have a kappa light chain. Truncated expression analysis showed that the recognition region of N protein of the five MAbs was 1-142aa. Overall, the MAbs against SADS-CoV N protein were promising a useful tool for development of new immunodiagnostic methods and research N protein function and structure.

Key words: swine acute diarrhea syndrome coronavirus (SADS-CoV), nucleocapsid protein, monoclonal antibody, antibody sequence analysis

CLC Number:

S852.659.6

CAO Liyan, KONG Xiangyu, LI Xiangtong, SUO Xuepeng, DUAN Yueyue, YUAN Cong, SHI Lei, ZHANG Yu, MA Guoxiang, ZHENG Haixue, WANG Qi. Preparation and Sequence Analysis of Monoclonal Antibody against the Nucleocapsid Protein of Porcine Acute Diarrhea Syndrome Coronavirus[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2487-2497.

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Preparation and Sequence Analysis of Monoclonal Antibody against the Nucleocapsid Protein of Porcine Acute Diarrhea Syndrome Coronavirus

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