猪流行性腹泻病毒N蛋白原核表达及多克隆抗体制备

Prokaryotic Expression of N Protein of Porcine Epidemic Diarrhea Virus and Preparation of Polyclonal Antibodies

【目的】利用原核表达系统表达猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)核衣壳蛋白(N),并制备具有高效价和特异性强的多克隆抗体。【方法】利用生物信息学工具分析PEDV N蛋白氨基酸序列,并预测其抗原性;利用原核表达载体pColdⅠ诱导表达重组N蛋白,并通过SDS-PAGE和Western blotting鉴定重组蛋白;使用兔多抗制备佐剂与纯化后的重组N蛋白混合后免疫新西兰白兔,收集血清制备多克隆抗体。利用间接ELISA法测定重组N蛋白多克隆抗体效价,并对多克隆抗体进行Western blotting和间接免疫荧光试验(IFA)验证。【结果】生物信息学预测结果显示,N蛋白不含信号肽和跨膜结构,具有良好的抗原性和溶解度。SDS-PAGE电泳结果显示,重组N蛋白大小约为58 ku,主要以可溶性蛋白存在;Western blotting结果显示,该蛋白能与抗His标签的鼠源单克隆抗体发生特异性反应。间接ELISA结果显示,多克隆抗体效价可达1∶204800;Western blotting结果表明,抗体稀释度为1∶5000时能特异性识别重组N蛋白及PEDV感染后的Vero细胞样品中的N蛋白;IFA结果显示,当稀释度为1∶1000时,该抗体能有效识别PEDV感染细胞样品中的N蛋白。【结论】本研究成功制备了效价高和特异性好的兔抗N蛋白多克隆抗体,为深入研究PEDV N蛋白的功能、了解PEDV复制机制和病毒-宿主细胞间的互作提供试验材料,为开发PEDV诊断和检测方法奠定基础。

【Objective】The purpose of this study was to express the N protein of Porcine epidemic diarrhea virus(PEDV)using a prokaryotic expression system,and prepare polyclonal antibody with high efficiency and specificity.【Method】Bioinformatics tools were used to analyze the amino acid sequence of PEDV N protein and predict its antigenicity.Recombinant N protein was induced by prokaryotic expression vector pColdⅠ,and the recombinant protein was identified by SDS-PAGE and Western blotting.New Zealand White rabbits were immunized with the preparation adjuvant of rabbit polyclonal antibody mixed with purified recombinant N protein,and serum was collected to prepare polyclonal antibody.The titer of recombinant N protein polyclonal antibody was determined by indirect ELISA,and the polyclonal antibody was verified by Western blotting and indirect immunofluorescence assay(IFA).【Result】The results of bioinformatics prediction show that N protein didn’t contain signal peptide and transmembrane structure,and had good antigenicity and solubility.SDS-PAGE results showed that the recombinant N protein approximately 58 ku in size,existed primarily in a soluble form.Western blotting revealed that the protein specifically react with anti-His labeled mouse monoclonal antibodies.Indirect ELISA results demonstrated that the titer of polyclonal antibody could reach 1∶204800.Western blotting results showed that recombinant N protein and N protein in PEDV infected Vero cell samples could be specifically identified when the antibody dilution was 1∶5000.IFA results showed that when the dilution was 1∶1000,the antibody could effectively identify N protein in PEDV-infected cell samples.【Conclusion】The rabbit anti-N protein polyclonal antibody with high efficiency and good specificity was successfully prepared,providing experimental materials for further study of PEDV N protein function,understanding PEDV replication mechanism and virus-host cell interaction,and laying a foundation for the development of PEDV diagnosis and detection methods.