猪δ冠状病毒RBD蛋白多肽抗体的制备与鉴定

doi:10.16431/j.cnki.1671-7236.2023.04.029

Abstract

Abstract: 【Objective】 The purpose of this study was to design and synthesize antigen epitopes for the receptor binding domain (RBD) of Porcine deltacoronavirus (PDCoV),preparation and identification polyclonal antibodies.【Method】 In order to specifically detect PDCoV RBD antigen,bioinformatics techniques were applied to predict its potential antigenic epitopes.Match the epitope amino acid sequence with 15 other strains of delta Coronavirus and 14 other Porcine coronavirus strains were compared for similarity. The selected dominant epitope was coupled with the carrier protein keyhole limpet hemocyanin (KLH) to synthesize polypeptides and immunize mice to prepare specific antibody against PDCoV RBD.The PDCoV RBD expressed in different systems and S protein expressed in PDCoV,TGEV and PEDV -infected ST cells were identified by Western blotting,and the antibody potency was determined by indirect ELISA.【Result】 After sequence alignment,bioinformatics technology predicted that the synthetic epitope antigens had very low sequence similarity with other 15 delta Coronaviruses (0-14.28%) and other 14 Porcine coronaviruses (0-7.14%),with good conservation.And the Western blotting results showed that after 1∶500,1∶1 000 and 1∶2 000 dilutions all the prepared peptide antibodies at fold ratio dilution could specifically recognize the PDCoV RBD protein expressed by Expi293F transfected with pcDNA3.1-PDR-Strep,the PDCoV RBD protein expressed by Baculovirus infected SF9 cells.The PDCoV S protein expressed in ST cells infected with PDCoV could be detected after 1∶1 000 dilution,while the expression of specific protein was not detected in cells infected with TGEV and PEDV.ELISA detection of peptide-specific antibodies in antisera could reach a potency of 1∶12 800.【Conclusion】 PDCoV RBD protein antigenic epitopes were successfully predicted using bioinformatics techniques.The antibodies obtained after immunization had good specificity.

Key words: Porcine deltacoronavirus(PDCoV); receptor binding domain(RBD); antigenic epitope; peptide antibody

CLC Number:

S855

ZHANG Guoqing, JIANG Yuhang, ZHANG Shuang, LI Letian, HAO Jiayi, CHEN Jing, ZOU Wancheng, LI Chang, LI Taiyuan, GAO Xu, JIN Ningyi. Preparation and Identification of PDCoV RBD Protein Peptide Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1575-1583.

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Preparation and Identification of PDCoV RBD Protein Peptide Antibody

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