Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, Qingdao, 266003, China.
Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, Qingdao, 266003, China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266071, China.
Fish Shellfish Immunol. 2022 Aug;127:508-520. doi: 10.1016/j.fsi.2022.06.042. Epub 2022 Jun 26.
White spot syndrome virus (WSSV) is extremely pathogenic and causes huge economic losses in the shrimp farming industry. Neutralizing antibodies against WSSV is expected to be an effective means of preventing infection with the virus. In the present study, eight monoclonal antibodies (mAbs) against VP28 were developed by immunizing BALB/c mice with WSSV-VP28 recombinant protein. Among them, three mAbs named 3B7, 2G3 and 5D2 were determined to be able to delay the mortality of WSSV-infected shrimp in vivo neutralization assay, suggesting their neutralizing ability against WSSV infection. Immunoblotting results showed that the three mAbs reacted specifically with native VP28 of WSSV, and could also recognize the virions in the gills of WSSV-infected shrimp by IFA. Furthermore, the single chain variable fragment (scFv) genes specific for WSSV-VP28 were cloned from the three hybridoma cells and expressed in Escherichia coli. After purification and refolding, three biologically active scFv recombinant proteins were all capable of recognizing the native VP28 of WSSV and delayed the mortality of WSSV-infected shrimp, indicating their neutralizing capacity against WSSV. Subsequently, the eukaryotic expression plasmids of three scFv genes were constructed and the transcriptional properties of expression vectors in shrimp were analyzed. Animal experiments also proved that the scFv eukaryotic expression plasmids were able to partially neutralize WSSV infection. Thus, the production of neutralizing mAb and recombinant scFv antibodies against WSSV has a promising therapeutic potential in prevention and treatment of white spot disease of shrimp.
白斑综合征病毒（WSSV）具有极强的致病性，给虾养殖业造成了巨大的经济损失。针对 WSSV 的中和抗体有望成为预防病毒感染的有效手段。本研究通过用 WSSV-VP28 重组蛋白免疫 BALB/c 小鼠，制备了 8 株针对 VP28 的单克隆抗体（mAbs）。其中，3B7、2G3 和 5D2 这 3 株 mAb 能够在体内中和试验中延迟 WSSV 感染虾的死亡率，表明它们具有中和 WSSV 感染的能力。免疫印迹结果显示，这 3 株 mAb 与 WSSV 天然 VP28 特异性反应，并且可以通过 IFA 识别 WSSV 感染虾的病毒粒子。此外，还从 3 株杂交瘤细胞中克隆了针对 WSSV-VP28 的单链可变片段（scFv）基因，并在大肠杆菌中表达。经过纯化和复性，3 种具有生物活性的 scFv 重组蛋白均能识别 WSSV 天然 VP28，延迟 WSSV 感染虾的死亡率，表明其具有中和 WSSV 的能力。随后，构建了 3 种 scFv 基因的真核表达质粒，并分析了表达载体在虾中的转录特性。动物实验也证明了 scFv 真核表达质粒能够部分中和 WSSV 感染。因此，针对 WSSV 产生的中和 mAb 和重组 scFv 抗体在预防和治疗虾白斑病方面具有广阔的治疗潜力。
