Production and identification of PEDV RBD protein peptide antibody
10.16303/j.cnki.1005-4545.2025.07.01
- VernacularTitle:PEDV RBD蛋白多肽抗体的制备与鉴定
- Author:
Jiayu YU
1
;
Yuhang JIANG
;
Guoqing ZHANG
;
Lichao YI
;
Shuang ZHANG
;
Letian LI
;
Aijian QIN
;
Chang LI
Author Information
1. 扬州大学兽医学院,江苏扬州 225009;中国农业科学院长春兽医研究所病原微生物生物安全全国重点实验室/吉林省人兽共患病预防控制重点实验室,吉林长春 130122
- Publication Type:Journal Article
- Keywords:
PEDV;
RBD;
antisera;
specificity
- From:
Chinese Journal of Veterinary Science
2025;45(7):1357-1365
- CountryChina
- Language:Chinese
-
Abstract:
This study aims to screen epitope antigens targeting the receptor binding domain(RBD)of porcine epidemic diarrhea virus(PEDV)based on its amino acid sequence(GenBank accession number:AKN45969.1),prepare PEDV RBD polyclonal antibody,and perform their identification.Bioinformatics analysis software was used to predict the potential antigenic epitopes of PEDV RBD and sequence comparison with porcine coronavirus strains was performed,the selected dominant antigen epitopes were then conjugated with keyhole limpet hemocyanin(KLH),to synthesize pep-tides directly and immunize mice to generate specific antibody,Western blot technique and indirect immunofluorescence assay were utilized to identify the specificity of the antibodies,and indirect ELISA method was further applied to determine the antibody potency.Results showed the selected PEDV RBD dominant epitope sequence shared 100％similarity with 18 other PEDV strains,while exhibiting low sequence similarity with 11 TGEV strains(27.8％—29.3％)and 16 PDCoV strains(10.5％—13.4％),indicating good epitope conservation.Western blot showed that the specificity of the prepared peptide antibody specifically recognized the PEDV RED protein overexpressed in Ex-pi293F cells and overexpressed in baculovirus system,and at the same time,the antibody was still able to detect the PEDV S protein expressed in PEDV-infected Vero cells at a 1∶2 000 dilution,while it did not react with TGEV-and PDCoV-infected ST cells,indicating that the good specificity of the peptide antibody.ELISA revealed that the potency of specific antibodies in mouse serum could reach up to 1∶25 600.The above results indicate that bioinformatics techniques were suc-cessfully utilized to predict antigenic epitopes of PEDV RBD protein,and specific PEDV RBD pep-tide antibodies were prepared.
