渔业研究2025，Vol.47Issue(5)：609-619,11.DOI:10.14012/j.jfr.2025070
Ⅱ型草鱼呼肠孤病毒VP4兔源单克隆抗体的制备和鉴定
Preparation and identification of rabbit monoclonal antibodies against VP4 of grass carp reovirus genotype Ⅱ(GCRV-Ⅱ)
摘要
Abstract
[Objective]Grass carp reovirus genotype Ⅱ(GCRV-Ⅱ)poses a serious threat to the healthy develop-ment of grass carp(Ctenoparyngodon idellus)aquaculture in China.This study aims to generate genetically engi-neered antibodies targeting the VP4 protein of GCRV-Ⅱ by integrating single B cell antibody technology with mammalian expression systems.[Methods]The VP4 gene of GCRV-Ⅱ was cloned into a eukaryotic expres-sion vector and expressed in a mammalian system to produce recombinant VP4 protein.New Zealand white rabbits were immunized with the purified protein to elicit antigen-specific memory B cells.Splenic B cells were isol-ated and subjected to polymerase chain reaction(PCR)amplification of immunoglobulin variable(VH/VL)and constant region genes.Recombinant monoclonal antibodies(mAbs)were expressed in HEK293F cells through transient transfection.Antigen-binding characteristics were systematically evaluated using enzyme-linked im-munosorbent assay(ELISA),Western blot,and immunofluorescence assay(IF).[Results]Through screening the supernatants of isolated single B cells via ELISA,we identified three B-cell clones(1D5,1F9,and 1H1)with strong binding affinity to the VP4 protein.Their ability to bind VP4 antigens in virus-infected tissues was further confirmed by IF assays.Subsequently,we recombinantly expressed these antibodies and obtained three anti-VP4 monoclonal antibodies(1D5,1F9,and 1H1),all exhibiting ELISA titers of up to 1∶128 000.Western blot analysis demonstrated that the 1D5,1F9,and 1H1 monoclonal antibodies specifically recognized a 65.4 kDa protein band in GCRV-YX246-infected brain cells,consistent with the theoretical molecular weight of the viral VP4 protein.IF assays further verified that these antibodies could specifically detect viral antigens in GCRV-YX246-infected head kidney tissue sections of grass carp.[Conclusion]The study successfully gener-ate and characterize VP4-specific recombinant monoclonal antibodies with high affinity and specificity.The res-ults of the study lays the foundation for the development of rapid diagnostic kits for GCRV-Ⅱ and functional studies of the VP4 protein.关键词
Ⅱ型草鱼呼肠孤病毒/VP4蛋白/单个B细胞技术/单克隆抗体制备Key words
grass carp reovirus genotype Ⅱ(GCRV-Ⅱ)/VP4 protein/single B cell technology/monoclonal antibodies preparation分类
农业科技引用本文复制引用
孔维光,袁新婕,丁广义,张环宇,徐镇..Ⅱ型草鱼呼肠孤病毒VP4兔源单克隆抗体的制备和鉴定[J].渔业研究,2025,47(5):609-619,11.基金项目
国家自然科学基金(32225050、U24A20461、32303053) （32225050、U24A20461、32303053）
