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  

Soluble expression of nucleocapsid protein of feline coronavirus and reactivity identification of its polyclonal antibody

作　　者：张丹[1] 卢舒婷 卢辰赫 王子仪 徐丽华 陈伊玄[1] 王圣文 金子安 倪成章 周继勇 郑肖娟[1] ZHANG Dan;LU Shuting;LU Chenhe;WANG Ziyi;XU Lihua;CHEN Yixuan;WANG Shengwen;JIN Zi’an;NI Chengzhang;ZHOU Jiyong;ZHENG Xiaojuan(Key Laboratory of Animal Virology,Ministry of Agriculture and Rural Affairs,Center for Veterinary Sciences,Zhejiang University,Hangzhou 310058,Zhejiang,China;Institute of Animal Husbandry and Veterinary Science,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,Zhejiang,China;Hainan Institute of Zhejiang University,Sanya 572025,Hainan,China)

机构地区：[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]浙江省农业科学院畜牧兽医研究所,浙江杭州310021 [3]浙江大学海南研究院,海南三亚572025

出　　处：《浙江大学学报（农业与生命科学版）》2023年第3期424-434,共11页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：浙江大学大学生科研训练计划(SRTP)项目(Y34462)。

摘　　要：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c DNA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1×10^(6),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。To prepare the specific antibodies against nucleocapsid(N)protein of feline coronavirus(FCoV),the full-length cDNA of N gene was amplified from typeⅠFCoV(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 1×10^(6),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.

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 

分 类 号：S855.3[农业科学—临床兽医学]