PDF(Pubmed)
Abstract：
The porcine epidemic diarrhea virus (PEDV) infection inflicted substantial economic losses upon the global pig-breeding industry. This pathogen can infect all pigs and poses a particularly high fatality risk for suckling piglets. The S1 subunit of spike protein is a crucial target protein for inducing the particularly neutralizing antibodies that can intercept the virus-host interaction and neutralize virus infectivity. In the present study, the HEK293F eukaryotic expression system was successfully utilized to express and produce recombinant S1 protein. Through quantitative analysis, five monoclonal antibodies (mAbs) specifically targeting the recombinant S1 protein of PEDV were developed and subsequently evaluated using enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and flow cytometry assay (FCA). The results indicate that all five mAbs belong to the IgG1 isotype, and their half-maximal effective concentration (EC50) values measured at 84.77, 7.42, 0.89, 14.64, and 7.86 pM. All these five mAbs can be utilized in ELISA, FCA, and IFA for the detection of PEDV infection. MAb 5-F9 exhibits the highest sensitivity to detect as low as 0.3125 ng/mL of recombinant PEDV-S1 protein in ELISA, while only 0.096 ng/mL of mAb 5-F9 is required to detect PEDV in FCA. The results from antigen epitope analysis indicated that mAb 8-G2 is the sole antibody capable of recognizing linear epitopes. In conclusion, this study has yielded a highly immunogenic S1 protein and five high-affinity mAbs specifically targeting the S1 protein. These findings have significant implications for early detection of PEDV infection and provide a solid foundation for further investigation into studying virus-host interactions.
摘要：
猪流行性腹泻病毒（PEDV）感染给全球养猪业造成了巨大的经济损失。这种病原体可以感染所有猪，并对哺乳仔猪造成特别高的死亡风险。刺突蛋白的S1亚基是诱导可以拦截病毒与宿主相互作用并中和病毒感染性的特别中和抗体的关键靶蛋白。在本研究中,利用HEK293F真核表达系统成功表达和生产重组S1蛋白。通过定量分析,开发了五种特异性靶向PEDV重组S1蛋白的单克隆抗体（mAb），随后使用酶联免疫吸附测定（ELISA）进行评估，间接免疫荧光分析(IFA)，和流式细胞术测定(FCA)。结果表明，所有五种mAb都属于IgG1同种型，以及在84.77、7.42、0.89、14.64和7.86pM时测得的它们的半最大有效浓度（EC50）值。所有这5种单克隆抗体均可用于ELISA，FCA,和IFA用于检测PEDV感染。MAb5-F9在ELISA中检测低至0.3125ng/mL的重组PEDV-S1蛋白的灵敏度最高，而在FCA中检测PEDV只需要0.096ng/mL的mAb5-F9。来自抗原表位分析的结果表明mAb8-G2是能够识别线性表位的唯一抗体。总之,这项研究产生了一种高免疫原性的S1蛋白和5种特异性靶向S1蛋白的高亲和力mAb.这些发现对于PEDV感染的早期检测具有重要意义，并为进一步研究病毒-宿主相互作用提供了坚实的基础。
