猪流行性腹泻病毒N蛋白原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2024.10.032

Abstract

Abstract: 【Objective】 The purpose of this study was to express the N protein of Porcine epidemic diarrhea virus (PEDV) using a prokaryotic expression system，and prepare polyclonal antibody with high efficiency and specificity.【Method】 Bioinformatics tools were used to analyze the amino acid sequence of PEDV N protein and predict its antigenicity. Recombinant N protein was induced by prokaryotic expression vector pColdⅠ，and the recombinant protein was identified by SDS-PAGE and Western blotting. New Zealand White rabbits were immunized with the preparation adjuvant of rabbit polyclonal antibody mixed with purified recombinant N protein，and serum was collected to prepare polyclonal antibody. The titer of recombinant N protein polyclonal antibody was determined by indirect ELISA，and the polyclonal antibody was verified by Western blotting and indirect immunofluorescence assay (IFA).【Result】 The results of bioinformatics prediction show that N protein didn’t contain signal peptide and transmembrane structure，and had good antigenicity and solubility. SDS-PAGE results showed that the recombinant N protein approximately 58 ku in size，existed primarily in a soluble form. Western blotting revealed that the protein specifically react with anti-His labeled mouse monoclonal antibodies . Indirect ELISA results demonstrated that the titer of polyclonal antibody could reach 1∶ 204 800. Western blotting results showed that recombinant N protein and N protein in PEDV infected Vero cell samples could be specifically identified when the antibody dilution was 1∶5 000. IFA results showed that when the dilution was 1∶1 000，the antibody could effectively identify N protein in PEDV-infected cell samples.【Conclusion】 The rabbit anti-N protein polyclonal antibody with high efficiency and good specificity was successfully prepared，providing experimental materials for further study of PEDV N protein function，understanding PEDV replication mechanism and virus-host cell interaction，and laying a foundation for the development of PEDV diagnosis and detection methods.

Key words: Porcine epidemic diarrhea virus (PEDV); N protein; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65

WU Jie, DU Jing, FAN Fan, REN Jinyang, LU Huipeng, ZHANG Li, LEI Xinnuo, CAO Shinuo, WU Zhi, ZHU Rui, ZHU Shanyuan. Prokaryotic Expression of N Protein of Porcine Epidemic Diarrhea Virus and Preparation of Polyclonal Antibodies[J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(10): 4522-4530.

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Prokaryotic Expression of N Protein of Porcine Epidemic Diarrhea Virus and Preparation of Polyclonal Antibodies

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