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

摘要/Abstract

摘要： 【目的】表达与纯化猪丁型冠状病毒（Porcine deltacoronavirus，PDCoV）的核衣壳（nucleocapsid，N）蛋白，并制备其多克隆抗体（polyclonal antibody，PcAb）。【方法】以PDCoV CHN-HN-1601分离株基因组RNA为模板，利用RT-PCR扩增PDCoV全长的N基因编码序列，将其克隆至原核表达载体pET-28a（+）中构建重组质粒pET-28a-PDCoV-N。经酶切和测序鉴定后，将重组质粒转化大肠杆菌BL21（DE3）感受态细胞，利用0.5 mmol/L IPTG于37 ℃诱导表达12 h。在非变性条件下，利用Ni-NTA琼脂糖树脂从菌体裂解液上清中纯化N-端和C-端均携带6×His标签的重组N蛋白，并将其免疫新西兰白兔制备抗血清。利用Protein A/G琼脂糖树脂从免疫兔的抗血清中亲和层析纯化多克隆抗体，并对多克隆抗体进行Western blotting和间接免疫荧光试验（IFA）鉴定及抗体效价的间接ELISA测定。【结果】重组PDCoV-N蛋白以可溶性和包涵体两种形式表达，分子质量约为42 ku；上清可溶性N蛋白的纯化纯度可达90%、蛋白浓度为0.45 mg/mL；纯化后的多克隆抗体纯度较高，ELISA方法检测其效价为1∶6 400，能特异性地识别重组N蛋白和PDCoV，与可引起猪腹泻的猪流行性腹泻病毒（PEDV）、猪传染性胃肠炎病毒（TGEV）、猪轮状病毒（PRoV）无交叉反应。【结论】本研究成功制备重组PDCoV-N蛋白及其多克隆抗体，为后续深入研究N蛋白的功能、PDCoV优化血清学检测方法、免疫层析试纸条的制备及开展PDCoV相关基础研究提供参考。

关键词: 猪丁型冠状病毒(PDCoV); 核衣壳(N)蛋白; 原核表达; 多克隆抗体(PcAb)

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)

中图分类号:

S852.65^＋1

刘铭, 张永宁. 猪丁型冠状病毒N蛋白原核表达及其多克隆抗体的制备[J]. 中国畜牧兽医, 2022, 49(7): 2698-2707.

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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