Page 78 - 《渔业研究》2025年第5期

第 5 期 孔维光等： Ⅱ型草鱼呼肠孤病毒 VP4 兔源单克隆抗体的制备和鉴定 619

Preparation and identification of rabbit monoclonal antibodies against VP4 of
grass carp reovirus genotype Ⅱ (GCRV-Ⅱ)

KONG Weiguang，YUAN Xinjie，DING Guangyi，ZHANG Huanyu，XU Zhen *
(State Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture, Institute of Hydrobiology, Chinese Academy of
Sciences, Wuhan 430072, China)

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.
Key words: grass carp reovirus genotype Ⅱ (GCRV-Ⅱ); VP4 protein; single B cell technology; monoclonal
antibodies preparation