College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China.
Molecule Biology Laboratory, Zhengzhou Normal University, Zhengzhou 450044, China.
Viruses. 2022 Apr 8;14(4):772. doi: 10.3390/v14040772.
Porcine deltacoronavirus (PDCoV) mainly causes severe diarrhea and intestinal pathological damage in piglets and poses a serious threat to pig farms. Currently, no effective reagents or vaccines are available to control PDCoV infection. Single-chain fragment variable (scFv) antibodies can effectively inhibit virus infection and may be a potential therapeutic reagent for PDCoV treatment. In this study, a porcine phage display antibody library from the peripheral blood lymphocytes of piglets infected with PDCoV was constructed and used to select PDCoV-specific scFv. The library was screened with four rounds of biopanning using the PDCoV N protein, and the colony with the highest affinity to the PDCoV N protein was obtained (namely, N53). Then, the N53-scFv gene fragment was cloned into plasmid pFUSE-hIgG-Fc2 and expressed in HEK-293T cells. The scFv-Fc antibody N53 (namely, scFv N53) was purified using Protein A-sepharose. The reactive activity of the purified antibody with the PDCoV N protein was confirmed by indirect enzyme-linked immunosorbent assay (ELISA), western blot and indirect immunofluorescence assay (IFA). Finally, the antigenic epitopes that the scFv N53 recognized were identified by a series of truncated PDCoV N proteins. The amino acid residues GELPPNDTPATTRVT of the PDCoV N protein were verified as the minimal epitope that can be recognized by the scFv-Fc antibody N53. In addition, the interaction between the scFv-Fc antibody N53 and the PDCoV N protein was further analyzed by molecule docking. In conclusion, our research provides some references for the treatment and prevention of PDCoV.
猪德尔塔冠状病毒（PDCoV）主要引起仔猪严重腹泻和肠道病理损伤，对猪场构成严重威胁。目前，尚无有效的试剂或疫苗可用于控制 PDCoV 感染。单链片段可变（scFv）抗体可有效抑制病毒感染，可能是 PDCoV 治疗的潜在治疗试剂。本研究构建了感染 PDCoV 的仔猪外周血淋巴细胞的猪噬菌体展示抗体文库，并用于筛选 PDCoV 特异性 scFv。该文库使用 PDCoV N 蛋白进行了四轮生物淘选筛选，获得了与 PDCoV N 蛋白亲和力最高的克隆（即 N53）。然后，将 N53-scFv 基因片段克隆到质粒 pFUSE-hIgG-Fc2 中，并在 HEK-293T 细胞中表达。使用 Protein A-sepharose 纯化 scFv-Fc 抗体 N53（即 scFv N53）。通过间接酶联免疫吸附试验（ELISA）、western blot 和间接免疫荧光试验（IFA）确认纯化抗体与 PDCoV N 蛋白的反应活性。最后，通过一系列截断的 PDCoV N 蛋白鉴定 scFv N53 识别的抗原表位。验证了 PDCoV N 蛋白的氨基酸残基 GELPPNDTPATTRVT 是 scFv-Fc 抗体 N53 可识别的最小表位。此外，通过分子对接进一步分析了 scFv-Fc 抗体 N53 与 PDCoV N 蛋白之间的相互作用。综上所述，本研究为 PDCoV 的治疗和预防提供了一些参考。
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