【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1×106，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（porcinetransmissiblegastroenteritisvirus,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。更多还原

【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 ⅠFCo V(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.更多还原