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浙江大学学报（农业与生命科学版）
Journal of Zhejiang University (Agriculture and Life Sciences)  2023, Vol. 49 Issue (3): 424-434    DOI: 10.3785/j.issn.1008-9209.2021.05.072
Animal sciences & veterinary medicines     
Soluble expression of nucleocapsid protein of feline coronavirus and reactivity identification of its polyclonal antibody
Dan ZHANG1(),Shuting LU1(),Chenhe LU1,Ziyi WANG1,Lihua XU1,2,Yixuan CHEN1,Shengwen WANG1,Zi’an JIN1,3,Chengzhang NI1,Jiyong ZHOU1,Xiaojuan ZHENG1()
1.Key Laboratory of Animal Virology, Ministry of Agriculture and Rural Affairs, Center for Veterinary Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China
2.Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, Zhejiang, China
3.Hainan Institute of Zhejiang University, Sanya 572025, Hainan, China
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Abstract  

To prepare the specific antibodies against nucleocapsid (N) protein of feline coronavirus (FCoV), the full-length cDNA of N gene was amplified from type ⅠFCoV (ZJU1709) and subcloned into pCold TF prokaryotic expression vector. The recombinant N protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) at low temperature and affinity-purified using nickel column. The rabbit polyclonal antiserum was prepared using the purified recombinant N protein as antigen. Finally, the enzyme-linked immunosorbent assay (ELISA), Western blotting (WB) and indirect immunofluorescence assay (IFA) were applied to identify the reactivity of rabbit polyclonal antiserum. The results showed that the soluble recombinant N protein with a molecular weight of about 100 kDa was successfully induced and further purified under non-denaturing condition at a concentration of 1.4 mg/mL. The ELISA titer of the prepared rabbit polyclonal antiserum of N protein can reach 1×106, and it can react with the recombinant Flag-N eukaryotic protein and N protein in FCoV-infected cells by WB and IFA. Cross-reactivity analysis showed that the polyclonal antibody can react with N proteins of alphacoronavirus, including various subtypes of FCoV, as well as canine coronavirus (CCoV), porcine transmissible gastroenteritis virus (TGEV), and porcine epidemic diarrhea virus (PEDV), but failed to react with N proteins of betacoronavirus of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and gammacoronavirus of infectious bronchitis virus (IBV). In summary, the soluble recombinant N protein expressed by using pCold TF prokaryotic system showed strong immunogenicity. The rabbit polyclonal antiserum against N protein of FCoV can recognize N proteins of multiple species from alphacoronavirus, but showed no cross-reactivity with N proteins of betacoronavirus and gammacoronavirus. This antibody facilitates to study the replication mechanism of FCoV and develop efficient methods for antigen or antibody detection.

Key words： feline coronavirus      nucleocapsid (N) protein      soluble expression      polyclonal antibody      cross-reactivity     
Received: 07 May 2021      Published: 25 June 2023
CLC:  S855.3  
Corresponding Authors: Xiaojuan ZHENG     E-mail: zhangdan9395@163.com;21717092@zju.edu.cn;zhengxiaojuan@zju.edu.cn
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Dan ZHANG
Shuting LU
Chenhe LU
Ziyi WANG
Lihua XU
Yixuan CHEN
Shengwen WANG
Zi’an JIN
Chengzhang NI
Jiyong ZHOU
Xiaojuan ZHENG
Cite this article:

Dan ZHANG,Shuting LU,Chenhe LU,Ziyi WANG,Lihua XU,Yixuan CHEN,Shengwen WANG,Zi’an JIN,Chengzhang NI,Jiyong ZHOU,Xiaojuan ZHENG. Soluble expression of nucleocapsid protein of feline coronavirus and reactivity identification of its polyclonal antibody. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(3): 424-434.

URL:

https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2021.05.072     OR     https://www.zjujournals.com/agr/Y2023/V49/I3/424


猫冠状病毒核衣壳蛋白的可溶性表达与多克隆抗体反应性鉴定

为制备猫冠状病毒（feline coronavirus, FCoV）核衣壳（nucleocapsid, N）蛋白的特异性抗体，本研究将Ⅰ型FCoV（ZJU1709）的N蛋白基因全长cDNA亚克隆到pCold TF原核表达载体上，通过低温诱导表达、镍柱亲和纯化获得可溶性重组N蛋白；进一步以纯化的重组N蛋白为免疫原制备兔多克隆抗血清（多抗血清），再通过酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）、蛋白质印迹法（Western blotting, WB）、间接免疫荧光试验（indirect immunofluorescence assay, IFA）等多种方法对兔多抗血清进行反应性鉴定。结果显示：经异丙基-β-D-硫代半乳糖苷（isopropyl-β-D-thiogalactopyranoside, IPTG）和低温诱导，成功表达出分子量约为100 kDa的可溶性重组N蛋白，在非变性条件下亲和纯化获得的重组N蛋白质量浓度达到1.4 mg/mL；N蛋白兔多抗血清的ELISA效价可达1×106，与真核表达的重组蛋白Flag-N以及FCoV感染细胞中的N蛋白均具有良好的WB和IFA反应性。交叉反应性结果显示：该多克隆抗体能与α属冠状病毒中不同亚型的FCoV以及犬冠状病毒（canine coronavirus, CCoV）、猪传染性胃肠炎病毒（porcine transmissible gastroenteritis virus, TGEV）、猪流行性腹泻病毒（porcine epidemic diarrhea virus, PEDV）的N蛋白发生反应，但不与β属冠状病毒中的严重急性呼吸综合征冠状病毒2（severe acute respiratory syndrome-related coronavirus 2, SARS-CoV-2）和γ属冠状病毒中的传染性支气管炎病毒（infectious bronchitis virus, IBV）的N蛋白发生反应。综上所述，以pCold TF原核系统表达的可溶性重组N蛋白具有良好的免疫原性，以该蛋白制备的FCoV N蛋白兔多抗血清能识别多种来自不同物种的α属冠状病毒N蛋白，但与β、γ属冠状病毒N蛋白没有交叉反应性，这为进一步研究FCoV复制机制以及开发高效的抗原和抗体检测技术奠定了基础。


关键词： 猫冠状病毒,  核衣壳蛋白,  可溶性表达,  多克隆抗体,  交叉反应性 

引物名称

Primer name

引物序列（5′→3′）

Primer sequence (5′→3′)

限制性酶切位点

Restriction site

Flag-TGEV-N-XhoⅠ-FCCGCTCGAGGGATGGCCAACCAGGGACAAXhoⅠ
Flag-TGEV-N-NotⅠ-RATAAGAATGCGGCCGCTTAGTTCGTTACCTCATCNotⅠ
Flag-PEDV-N-XhoⅠ-FCCGCTCGAGGGATGGCTTCTGTCAGTTTTCXhoⅠ
Flag-PEDV-N-NotⅠ-RATAAGAATGCGGCCGCTTAATTTCCTGTATCGAAGNotⅠ
Flag-791683-N-EcoRⅠ-FGGAATTCCGATGGCCACACAGGGACAAEcoRⅠ
Flag-791683-N-XhoⅠ-RCCGCTCGAGTTAGTTCGTTACCTCATCGAXhoⅠ
Flag-1617-N-EcoRⅠ-FGGAATTCCGATGGCCACACAGGGACAAEcoRⅠ
Flag-1617-N-XhoⅠ-RCCGCTCGAGTTAGTTCGTAACCTCATCAAXhoⅠ
Flag-1709-N-EcoRⅠ-FGAGGTACCATGGCCACGCAGGGACEcoRⅠ
Flag-1709-N-SalⅠ-RGCGTCGACTTAGTTCGTAACCTCATCAATSalⅠ
Flag-SARS-CoV-2-N-XhoⅠ-FCCGCTCGAGGGATGTCTGATAATGGACCCCAXhoⅠ
Flag-SARS-CoV-2-N-NotⅠ-RATAAGAATGCGGCCGCTTAGGCCTGAGTTGAGTCAGNotⅠ
CCoV-N-Part-FGACTCAAGGAGGAAAGCA
Flag-CCoV-N-XhoⅠ-FCCGCTCGAGGGATGGCCAACCAGGGACAAXhoⅠ
Flag-CCoV-N-NotⅠ-RATAAGAATGCGGCCGCTTAGTTCGTTACCTCATCNotⅠ
Flag-1919-N-EcoRⅠ-FGGAATTCCGATGGCCACACAGGGACAAEcoRⅠ
Flag-1919-N-XhoⅠ-RCCGCTCGAGTTAGTTCGTAACCTCATCAAXhoⅠ
Table 1 Specific primers used for amplification of full-length genes for N proteins of different coronaviruses
Fig. 1 Phylogenetic analysis of amino acid sequences of N proteins of different coronaviruses
 
Fig. 3 Induced expression, soluble analysis and affinity purification of recombinant N proteinM: Protein marker. Lanes 1-4 represent pre-induction, post-induction, supernatant and precipitation samples after ultrasonication, respectively; lanes 5-6 represent pre-column samples of N protein; lanes 7-16 represent the eluting protein samples using 10, 20, 30, 40, 60, 80, 100, 150, 200, 300 mmol/L imidazole, respectively; lanes 17-18 represent WB identification of the purified protein using anti-His tag monoclonal antibody.
Fig. 4 ELISA titers of rabbit polyclonal antiserum against ZJU1709-N proteinN-T1, N-T2, and N-T3 indicate three independent detections of rabbit polyclonal antiserum
Fig. 5 WB reactivity of rabbit polyclonal antiserum against ZJU1709-N protein
Fig. 6 IFA reactivity of the rabbit polyclonal antiserum against ZJU1709-N protein and FCoV-infected A72 cells under different dilution ratios
Fig. 7 Cross-reactivity of rabbit polyclonal antiserum against ZJU1709-N protein and different coronavirus N proteins in WB analysis and their sequence homologiesA. Cross-reactivity of rabbit polyclonal antiserum against ZJU1709-N protein with different coronavirus N proteins; B. Sequence homologies of different coronavirus N proteins.
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