中国兽医科学2024，Vol.54Issue(2)：217-225,9.DOI:10.16656/j.issn.1673-4696.2024.0017
猪圆环病毒2型Cap蛋白单克隆抗体的制备与鉴定
Preparation and identification of monoclonal antibodies of porcine circovirus type 2 capsid protein
摘要
Abstract
This study aims to prepare monoclonal antibody(mAb)to capsid(Cap)protein of porcine circovirus type 2(PCV2),and to the develop and apply immunological diagnostic methods.First,PCV2 Cap protein was expressed in Escherichia coli,and the purified Cap protein was assembled into virus-like particles(VLPs)in vitro.BALB/c mice were immunized with VLPs.Hybridoma cell technology was used to screen hybridoma cell lines that could secrete monoclonal antibodies against PCV2 Cap protein statically.Monoclonal ascites were further prepared and purified,and the specificity of monoclonal antibodies was identified by Western-blot and IFA methods.Forty-five short peptides covering the full length of PCV2 Cap protein were synthesized by stepwise interception method,and the epitope regions recognized by monoclonal antibodies were identified by indirect ELISA.Two strains of mAbs were used to detect and identify PCV2 in lung tissues of clinically infected pigs by immunohistochemistry(IHC).Twom Abs(1A6 and 1A8)were obtained,and mAb1A6 and 1A8 belong to IgG2b and IgG1 subclass,respectively,both had κ light chain.The titers of these two mAbs were 1∶656 100,respectively.IFA results showed that both mAbs could react specifically with PCV2-infected PK-15 cells.Western-blot results showed that both strains of mAbs reacted specifically with PCV2 Cap protein,and did not cross-react with PCV3 and PCV4 Cap protein.Both mAbs could recognize the 211-225 aa peptide at C-terminal of PCV2 Cap protein.IHC results showed that both mAbs could specifically recognize PCV2-infected clinical samples.In this study,2 mAbs(1A6 and 1A8)were successfully obtained,both of which can recognize PCV2 Cap with good specificity,providing important biological materials for the establishment of PCV2 immunological diagnostic method and the study of biological function of PCV2 Cap protein.关键词
猪圆环病毒2型/Cap蛋白/原核表达系统/病毒样颗粒/单克隆抗体Key words
porcine circovirus type 2/Cap protein/prokaryotic expression system/virus-like par-ticle/monoclonal antibody分类
农业科技引用本文复制引用
张英婕,王东亮,周文锋,邹亚文,白一涵,蒋一凡,湛洋,王乃东,杨毅,杜红梅..猪圆环病毒2型Cap蛋白单克隆抗体的制备与鉴定[J].中国兽医科学,2024,54(2):217-225,9.基金项目
国家自然科学基金项目(32172844) （32172844）
湖南省自然科学基金项目(2023JJ40134) （2023JJ40134）
湖南省技术攻关"揭榜挂帅"项目(2021NK1030) （2021NK1030）
