1株猪δ冠状病毒N蛋白单克隆抗体制备与鉴定

Preparation and identification of a monoclonal antibody against N protein of porcine deltacoronavirus

摘要:

【背景】猪δ冠状病毒(porcine deltacoronavirus，PDCoV)是一种新发现的猪肠道冠状病毒，主要引起以急性水样腹泻、呕吐和脱水等特征的胃肠道疾病。在PDCoV的4个结构蛋白中，核衣壳蛋白(nucleocapsid protein，N)为高度保守的结构蛋白，在病原诊断方面具有重要意义。【目的】以纯化的重组PDCoV N蛋白为抗原，制备抗N蛋白的单克隆抗体并进行特性鉴定。【方法】利用已构建的pET-28a-N表达菌，经IPTG诱导表达获得具有免疫原性的重组N蛋白，并将纯化的重组N蛋白免疫BALB/c小鼠制备杂交瘤细胞，经3次亚克隆筛选，选取抗体效价最高的一株杂交瘤细胞注射小鼠腹腔并制备腹水单克隆抗体，利用Protein G亲和层析柱纯化。通过腹水单克隆抗体效价测定、抗体亚型鉴定、Western blot鉴定其特性；利用细胞间接免疫荧光试验、组织石蜡切片荧光试验、免疫组化、流式细胞荧光分选技术鉴定其诊断应用价值。【结果】经筛选后得到一株杂交瘤细胞命名为4E88，其腹水单克隆抗体效价为1:105，亚型为IgG1型，轻链为κ链，Western blot试验表明该单克隆抗体与重组N蛋白和超速离心纯化的PDCoV全病毒反应形成特异性条带。此外，单克隆抗体4E88可以用于猪δ冠状病毒检测的间接免疫荧光试验、免疫组化染色和流式细胞荧光分选技术。【结论】研究获得的单克隆抗体4E88具有较好的反应性，为后续开展PDCoV鉴定、诊断和N蛋白功能研究等奠定基础。

Abstract:

[Background] Porcine deltacoronavirus (PDCoV) is a newly discovered porcine enteric coronavirus, which causes gastrointestinal disease characterized with acute watery diarrhea, vomiting and dehydration, etc. Among the four structural proteins of PDCoV, nucleocapsid protein (N) is a highly conserved structural protein, which plays an important role in pathogen diagnosis. [Objective] A monoclonal antibody (MAb), based on purified recombinant PDCoV N protein was prepared and its characteristics were systemically identified. [Methods] The constructed recombinant pET-28a-N bacteria was induced by IPTG for N protein expression, and BALB/c mice were immunized with purified N protein to prepare hybridoma cells. After three rounds of subcloning screening, one hybridoma cell with the highest antibody titer was selected for the subsequent production of mice ascitic fluid. The mice asctic fluid was purified by Protein G affinity chromatography column. The monoclonal antibody were systematically identified by titer determination, subtype identification and Western blot. The diagnostic application potential of the monoclonal were evaluated by using cell indirect immunofluorescence assay, tissue paraffin section fluorescence experiments, immunohistochemistry, and flow cytometry. [Results] A hybridoma cell was screened and named as 4E88, the titer of the ascites the MAb was 1:105, the subtype was IgG1 and the light chain was κ chain. Western blot analysis showed that the MAb could specifically reacted with recombinant N protein and ultracentrifuge purified PDCoV whole virus. In addition, the MAb 4E88 can be used for indirect immunofluorescence assay, immunohistochemistry and flow cytometry for PDCoV detection. [Conclusion] The MAb 4E88 has good reactivity in this study which laid a good foundation for applied research associated with PDCoV identification, diagnosis and N protein function.