Preparation and Identification of Chicken-derived Single-chain Variable Fragments Against the VP1-CDE Protein of Feline calicivirus
WANG Hong-Mei1, LIU Yi-Han1, LIU Chao2, ZHAO Zhen-Ping1, JIN Ya-Li1, ZHU Xin-Long1, WEI Yan-Quan1,*
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China; 2 Chongqing Hesheng Biotechnology Company Limited, Chongqing 402460, China
Abstract：Accurate diagnosis of Feline calicivirus (FCV) infection relies on specific antibody tools, and single-chain variable fragments (scFvs) show important application potential in clinical practice. In this study, the FCV VP1-CDE protein expressed and purified through prokaryotic expression was used as the immunogen to immunize chickens (Gallus gallus domesticus). A phage-displayed scFv library was constructed and positive clones were obtained by solid-phase panning. Eukaryotic expression vectors were constructed and expressed in human embryonic kidney 293F cells, followed by purification. The binding activities were verified by Western blot, indirect immunofluorescence assay (IFA), and bio-layer interferometry (BLI). The results showed that soluble VP1-CDE protein was successful obtained. The titer of chicken serum was higher than 1∶64 000 after 5 immunizations. A phage-display library with a capacity of 5.25×107 CFU/mL and good diversity was constructed. Thirty-five recombinant phage cloned with strong reactivity were obtained after panning, and 5 unique scFv sequences were acquired. Western blot analysis demonstrated that 5 clones (X1A2, X2B2, X2C1, X2E1 and X2G5) could specifically bind to the VP1-CDE protein. IFA results indicated that 3 of them (X2B2, X2E1 and X2G5) exhibited good binding activity to intact viral particles, which was further confirmed by BLI. In conclusion, this study successfully constructed a phage-displayed chicken scFv library against FCV VP1-CDE protein and obtained 3 scFv clones with high binding activity, providing valuable materials for basic research and diagnostic development of FCV.
王红梅, 刘怡涵, 刘超, 赵珍萍, 靳亚丽, 朱新龙, 魏衍全. 猫杯状病毒VP1-CDE蛋白鸡源单链抗体的制备及鉴定[J]. 农业生物技术学报, 2026, 34(11): 2463-2474.
WANG Hong-Mei, LIU Yi-Han, LIU Chao, ZHAO Zhen-Ping, JIN Ya-Li, ZHU Xin-Long, WEI Yan-Quan. Preparation and Identification of Chicken-derived Single-chain Variable Fragments Against the VP1-CDE Protein of Feline calicivirus. 农业生物技术学报, 2026, 34(11): 2463-2474.
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