Prokaryotic Expression and Polyclonal Antibody Preparation of Porcine Deltacoronavirus（PDCoV）N Protein

doi:10.13560/j.cnki.biotech.bull.1985.2019-1196

Abstract

Abstract: This work aims to express and purify the porcine deltacoronavirus（PDCoV）N protein and to prepare its polyclonal antibody. The PDCoV N gene was amplified by RT-PCR and the recombinant prokaryotic expression vector pET28-N was transformed into Escherichia coli Transetta（DE3）,from which the fusion protein was identified by SDS-PAGE. The purified N protein was used to inoculate rabbit and the polyclonal antibody was prepared. The specificity of the serum antibody was determined by Western blot and the serum antibody titer was detected by indirect ELISA. The diagnostic application potential of the polyclonal antibody was evaluated by using indirect immunofluorescence assay（IFA）,immunofluorescence staining（IF）and flow cytometry（FCM）. Recombinant N protein was soluble with a molecular weight of 44 kD. The titer of the antibody was about 1∶204 800. IFA and FCM tests confirmed that the antibody specifically bound to PDCoV and had no cross-reactivity with PEDV and TGEV. IF tests showed that the polyclonal antibody could be used to detect PDCoV in small intestine tissue.

Key words: porcine deltacoronavirus, N protein, prokaryotic expression, polyclonal antibody

CHEN Rui, FU Jia-yu, LIU Hao-yu, LI Cheng, ZHAO Yu-jia, HU Jing-fei, QU Huan, CAO San-jie, WEN Xin-tian, WEN Yi-ping, ZHAO Qin, WU Rui, HUANG Xiao-bo. Prokaryotic Expression and Polyclonal Antibody Preparation of Porcine Deltacoronavirus（PDCoV）N Protein[J]. Biotechnology Bulletin, 2020, 36(8): 104-110.

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