中国兽医杂志2025，Vol.61Issue(10)：25-32,8.DOI:10.20157/j.cnki.zgsyzz.2025.10.004
猫传染性腹膜炎病毒S蛋白纳米抗体的筛选与鉴定
Screening and Identification of Nanobodies against FIPV S Protein
摘要
Abstract
To obtain nanobodies specific to feline infectious peritonitis virus(FIPV),a eukaryotic expression plasmid(pCAGGS-FIPV-RS)encoding the recombinant trimeric spike(RS)protein of FIPV was constructed.The FIPV RS protein was expressed and purified using human embryonic kidney 293 suspension cells(HEK293F).The purified RS protein was used as the coating antigen for phage display screening,and nanobody genes with different sequences were obtained from a natural phage display library.These genes were inserted into the eukaryotic expression vector pCAGGS-VHH-Fc and transiently expressed in HEK293T cells.Western blot and indirect ELISA were used to identify nanobodies that were correctly expressed and had high affinity.High-affinity nanobodies were further expressed and purified using HEK293F cells,and their reactivity with the RS protein was validated by indirect immunofluorescence assay(IFA).The results showed that the FIPV RS protein was successfully expressed and purified.After three rounds of biopanning,41 positive monoclonal colonies were randomly selected,and 10 unique nanobody sequences were identified through sequencing.The recombinant eukaryotic plasmid pCAGGS-VHH-Fc was successfully constructed.Western blot and ELISA results demonstrated that seven nanobodies exhibited good reactivity with the FIPV RS protein.SDS-PAGE confirmed that these nanobodies were highly expressed and successfully purified in HEK293F cells.IFA further showed that five nanobodies specifically bound to the FIPV RS protein.This study provides valuable materials for the diagnosis,treatment,and pathogenic mechanism research of FIPV.关键词
猫传染性腹膜炎病毒(FIPV)/重组三聚体刺突(RS)蛋白/真核表达/噬菌体展示技术/纳米抗体Key words
feline infectious peritonitis virus(FIPV)/recombinant trimeric spike(RS)protein/eukaryotic expression/phage display technology/nanobody分类
农业科技引用本文复制引用
储丽宇,杨姗姗,王丽丽,李封赛,马增军,宋涛,薛希彤,苗云博,唐国瑞,文晓晓,王学富,赵海鹏,侯江,芮萍..猫传染性腹膜炎病毒S蛋白纳米抗体的筛选与鉴定[J].中国兽医杂志,2025,61(10):25-32,8.基金项目
现代农业产业技术体系河北省创新团队项目(HBCT2024220208) （HBCT2024220208）
河北省高等学校科学技术研究项目(BJK2023012) （BJK2023012）
秦皇岛科学技术局项目(202302B021) （202302B021）
