Zhou Yao, Xie Zhi-Gang
College of Life Sciences and Agro-forest, Qiqihar University, Qiqihar 161000, PR China.
J Virol Methods. 2015 Jul;219:18-22. doi: 10.1016/j.jviromet.2015.03.012. Epub 2015 Mar 23.
Viral hemorrhagic septicaemia (VHS) is an economically important disease that affects salmon and trout worldwide. In this study, a recombinant single chain variable fragment (scFv) antibody library derived from rainbow trout immunized with purified viral hemorrhagic septicaemia virus (VHSV) was constructed. The library was subjected to three rounds of screening by flow cytometry (FCM) against VHSV through a bacteria display technology, resulting in the enrichment of scFv. Four scFv clones with different fluorescence intensity were obtained by colony pick up at random following three rounds of screening. The isolated scFv antibodies were expressed and purified. Relative affinity assay showed the four clones had different sensitivity to VHSV, in accordance with FCM. The potential use of the selected VHSV-specific scFv antibodies was demonstrated by the successful application in Western blotting assay, ELISA and immunofluorescence antibody test (IFAT), and one of the isolated scFv molecular showed excellent in vitro and in vivo blocking activities against VHSV. scFv isolated in this study can be promising diagnostic and/or therapeutic reagents for VHS. This study provides powerful strategies for screening antibodies against new diseases.
病毒性出血性败血症（VHS）是一种在全球范围内影响鲑鱼和鳟鱼的具有经济重要性的疾病。在本研究中，构建了一个源自用纯化的病毒性出血性败血症病毒（VHSV）免疫的虹鳟鱼的重组单链可变片段（scFv）抗体文库。该文库通过细菌展示技术，针对VHSV进行了三轮流式细胞术（FCM）筛选，从而实现了scFv的富集。三轮筛选后，通过随机挑取菌落获得了四个具有不同荧光强度的scFv克隆。对分离出的scFv抗体进行了表达和纯化。相对亲和力测定表明，这四个克隆对VHSV具有不同的敏感性，与FCM结果一致。所选的VHSV特异性scFv抗体在蛋白质印迹分析、酶联免疫吸附测定（ELISA）和免疫荧光抗体试验（IFAT）中的成功应用证明了其潜在用途，并且其中一个分离出的scFv分子在体外和体内均表现出对VHSV的优异阻断活性。本研究中分离出的scFv有望成为VHS的诊断和/或治疗试剂。该研究为筛选针对新疾病的抗体提供了有力策略。
