猪丁型冠状病毒N蛋白原核表达及其多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2022.07.028

Abstract

Abstract: 【Objective】 This study was aimed to express and purify the nucleocapsid (N) protein of Porcine deltacoronavirus (PDCoV) and then prepare its polyclonal antibody (PcAb).【Method】 The full-length coding sequence of N gene of PDCoV was amplified by RT-PCR using the genomic RNA of the PDCoV CHN-HN-1601 strain as the template.The resulting amplicon was cloned into the prokaryotic expression vector pET-28a(+) to construct a recombinant plasmid pET-28a-PDCoV-N.After verification by enzyme digestion and sequencing,the recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells,which were induced by 0.5 mmol/L IPTG at 37 ℃ for 12 h.The recombinant PDCoV-N protein which carried both an N-terminal and a C-terminal 6×His tag was purified from the supernatant of the lysate of pET-28a-PDCoV-N-transformed E.coli BL21 (DE3) using Ni-NTA agarose under native conditions.The purified N protein was used to immunize New Zealand White rabbits to prepare antisera,from which the PcAb was purified by Protein A/G agarose affinity chromatography.The purified PcAb was identified by Western blotting and indirect immunofluorescence assays,and the titer of PcAb was determined by indirect enzyme-linked-immunosorbent assay (ELISA).【Result】 The results showed that the recombinant N protein was expressed in both soluble and inclusion body forms with a molecular weight of about 42 ku.The purity of the purified recombinant N protein reached 90% and the protein concentration was 0.45 mg/mL.The purified polyclonal antibody has high purity,the titer of the purified PcAb was 1∶6 400,and that the PcAb was able to specifically recognize the recombinant N protein and PDCoV,and had no cross-reaction with other important porcine enteric coronaviruses causing diarrhea in pigs,such as Porcine epidemic diarrhea virus (PEDV),porcine Transmissible gastroenteritis virus (TGEV) and Porcine rotavirus (PRoV).【Conclusion】 The successful preparation of recombinant PDCoV-N protein and its PcAb provided valuable biomaterials for the further study of the function of N protein,the development of serological detection methods,the preparation of subsequent immunochromatographic strip and the basic research of PDCoV.

Key words: Porcine deltacoronavirus (PDCoV); nucleocapsid (N) protein; prokaryotic expression; polyclonal antibody (PcAb)

CLC Number:

S852.65^＋1

LIU Ming, ZHANG Yongning. Prokaryotic Expression of Porcine Deltacoronavirus Nucleocapsid Protein and Preparation of Its Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2698-2707.

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Prokaryotic Expression of Porcine Deltacoronavirus Nucleocapsid Protein and Preparation of Its Polyclonal Antibody

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