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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2023, Vol. 49 Issue (5): 744-754    DOI: 10.3785/j.issn.1008-9209.2022.08.011
动物科学与动物医学     
猫冠状病毒非结构蛋白3的多克隆抗体制备及亚细胞定位
王子仪1(),金子安1,2,卢辰赫1,乔治3,王圣文1,颜焰1,周继勇1,郑肖娟1()
1.浙江大学动物医学中心, 农业农村部动物病毒学重点实验室, 浙江 杭州 310058
2.浙江大学海南研究院, 海南 三亚 572025
3.浙江大学-帕特诺尔联合研发中心, 浙江 杭州 310058
Preparation of polyclonal antibodies and subcellular localization of non-structural protein 3 encoded by feline coronavirus
Ziyi WANG1(),Zi’an JIN1,2,Chenhe LU1,Zhi QIAO3,Shengwen WANG1,Yan YAN1,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.Hainan Institute of Zhejiang University, Sanya 572025, Hainan, China
3.ZJU-Partner Joint Research Center, Hangzhou 310058, Zhejiang, China
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摘要：

冠状病毒的非结构蛋白3（non-structural protein 3, Nsp3）是复制/转录复合体的组成部分，也是重要的潜在抗病毒靶标之一。本研究在对Nsp3进行跨膜区、信号肽和抗原表位预测的基础上，通过聚合酶链反应扩增Ⅱ型猫冠状病毒（feline coronavirus, FCoV）代表毒株（WSU 79-1683）Nsp3蛋白中抗原性较好的区段（第50—550位氨基酸），随后将其亚克隆到pCOLD-TF原核表达载体上。在低温条件下，经异丙基硫代-β-D-半乳糖苷诱导，成功表达分子量约为130 kDa的His-Nsp3重组融合蛋白。在非变性条件下，采用镍柱亲和层析获得了纯度较高的目的重组蛋白。以纯化的重组蛋白为抗原免疫BALB/c小鼠，制备Nsp3多克隆抗血清（多抗血清），蛋白质印迹法（Western blotting, WB）和间接免疫荧光试验（indirect immunofluorescence assay, IFA）结果显示，Nsp3多抗血清能特异性识别FCoV感染细胞中的Nsp3蛋白。采用免疫荧光双标法结合激光共聚焦显微镜对FCoV感染细胞中Nsp3蛋白的亚细胞定位进行分析，结果发现，FCoV感染细胞中Nsp3发生聚集现象，并与内质网存在共定位现象。Nsp3蛋白的特异性抗体制备以及亚细胞定位研究为进一步开展Nsp3蛋白的生物学功能分析奠定了基础。
关键词： 猫冠状病毒;  非结构蛋白3;  原核表达;  多克隆抗体;  亚细胞定位    
Abstract:

The non-structural protein 3 (Nsp3) of coronavirus, a component of the replication and transcription complex, is one of the potentially important antiviral targets. In this study, the transmembrane region, signal peptide, and epitope of Nsp3 were predicted, and then the region with better antigenicity (50-550 amino acids) of Nsp3 protein in a representative strain (WSU 79-1683) of type Ⅱ feline coronavirus (FCoV) was amplified by polymerase chain reaction. Subsequently, it was subcloned into pCOLD-TF prokaryotic expression vector. Under the low-temperature condition, the recombinant fusion protein His-Nsp3 with a molecular weight of about 130 kDa was successfully induced by isopropylthio-β-D-galactoside. The targeted recombinant protein His-Nsp3 was purified using a non-denaturing nickel affinity column, and the purified protein was used as an antigen to immunize BALB/c mice for preparing Nsp3 polyclonal antiserum. Western blotting (WB) and indirect immunofluorescence assay (IFA) results showed that Nsp3 polyclonal antiserum could specifically recognize Nsp3 protein in FCoV-infected cells. The subcellular localization of Nsp3 protein in FCoV-infected cells was studied by double-labeling IFA combined with laser confocal microscopy. The results showed that Nsp3 protein aggregated in FCoV-infected cells and co-localized with the endoplasmic reticulum. The specific antibody preparation and subcellular localization study of Nsp3 protein provided an important basis for further analysis of the biological function of Nsp3 protein.
Key words: feline coronavirus    non-structural protein 3    prokaryotic expression    polyclonal antibody    subcellular location
收稿日期: 2022-08-01 出版日期: 2023-10-25
CLC:  S855.3  
基金资助: 浙江大学-帕特诺尔联合研发中心项目(K20211327)
通讯作者: 郑肖娟     E-mail: 21917102@zju.edu.cn;zhengxiaojuan@zju.edu.cn
作者简介: 王子仪（https://orcid.org/0000-0002-2346-9421），E-mail：21917102@zju.edu.cn
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王子仪
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引用本文:

王子仪,金子安,卢辰赫,乔治,王圣文,颜焰,周继勇,郑肖娟. 猫冠状病毒非结构蛋白3的多克隆抗体制备及亚细胞定位[J]. 浙江大学学报（农业与生命科学版）, 2023, 49(5): 744-754.

Ziyi WANG,Zi’an JIN,Chenhe LU,Zhi QIAO,Shengwen WANG,Yan YAN,Jiyong ZHOU,Xiaojuan ZHENG. Preparation of polyclonal antibodies and subcellular localization of non-structural protein 3 encoded by feline coronavirus. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 744-754.

链接本文:

https://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2022.08.011    或    https://www.zjujournals.com/agr/CN/Y2023/V49/I5/744

图1  Nsp3蛋白的序列比对分析A. 56条FCoV Nsp3的核苷酸序列比对；B.部分Nsp3的核苷酸序列比对（400～580 bp）。nt：核苷酸；Ubl1：泛素样结构域1；Ac：酸性残基；PLP1：木瓜样蛋白酶1；Ubl2：泛素样结构域2；PLP2：木瓜样蛋白酶2；TM1：跨膜结构域1；TM2：跨膜结构域2；Y：未知功能的Y结构域。
图2  WSU 79-1683 Nsp3蛋白的跨膜区、信号肽和抗原表位预测A.跨膜结构预测；B.信号肽预测；C.抗原表位预测（黄色方框为截取的第50—550位氨基酸区域）。
图3  Nsp3重组蛋白的诱导表达、亲和纯化与鉴定M：蛋白质梯度标志物。泳道1代表诱导后菌体蛋白，泳道2代表诱导前菌体蛋白，泳道3～7依次代表经20、30、40、50、60 mmol/L咪唑溶液洗脱后的蛋白质样品。
图4  Nsp3多抗血清的蛋白质印迹法（A）和间接免疫荧光试验（B）反应性结果M：蛋白质梯度标志物。泳道1代表未感染的CRFK细胞，泳道2代表WSU 79-1683感染的CRFK细胞。
图5  Nsp3在FCoV感染CRFK细胞中的亚细胞定位N：核衣壳；ER：内质网；Mito：线粒体；Golgi：高尔基体；Act：微丝骨架；Mem：细胞膜。
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