猪δ冠状病毒RBD蛋白多肽抗体的制备与鉴定  

Preparation and Identification of PDCoV RBD Protein Peptide Antibody

作　　者：张国庆 姜宇航 张爽 李乐天 郝嘉翼 陈竞 邹万成 李昌 李太元[1] 高旭[1] 金宁一 ZHANG Guoqing;JIANG Yuhang;ZHANG Shuang;LI Letian;HAO Jiayi;CHEN Jing;ZOU Wancheng;LI Chang;LI Taiyuan;GAO Xu;JIN Ningyi(Yanbian University,Yanji 133002,China;Changchun Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Changchun 130122,China)

机构地区：[1]延边大学,延吉133002 [2]中国农业科学院长春兽医研究所,长春130122

出　　处：《中国畜牧兽医》2023年第4期1575-1583,共9页China Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发项目(2021YFD1801103-6)。

摘　　要：【目的】设计合成猪δ冠状病毒(Porcine deltacoronavirus,PDCoV)受体结合结构域(receptor binding domain,RBD)的抗原表位,制备并鉴定多克隆抗体。【方法】为了特异性检测PDCoV RBD抗原,应用生物信息学技术预测其潜在抗原表位,将表位氨基酸序列与δ冠状病毒属中的其他15株病毒株以及14株其他猪冠状病毒株进行相似性比对,将筛选的优势抗原表位与载体蛋白血蓝蛋白(keyhole limpet hemocyanin,KLH)偶联,合成多肽并免疫小鼠,制备PDCoV RBD特异性抗体。通过Western blotting试验对不同系统表达的PDCoV RBD以及PDCoV、TGEV和PEDV感染ST细胞表达的S蛋白进行鉴定,通过间接ELISA方法测定抗体效价。【结果】经序列比对,利用生物信息学技术预测合成的表位抗原与δ冠状病毒属中的其他15株病毒株(0~14.28%)以及14株其他猪冠状病毒株(0~7.14%)序列相似性非常低,具有良好的保守性;Western blotting结果显示,制备的多肽抗体1∶500、1∶1000、1∶2000倍比稀释后均能特异性识别转染pcDNA3.1-PDR-Strep的Expi293F表达的PDCoV RBD蛋白、杆状病毒感染SF9细胞表达的PDCoV RBD蛋白,1∶1000稀释后能够检测到PDCoV感染ST细胞表达的PDCoV S蛋白,而在TGEV和PEDV感染的细胞中未检测到特异性蛋白的表达;ELISA检测抗血清中多肽特异性抗体的效价可以达到1∶12800。【结论】利用生物信息学技术成功预测PDCoV RBD蛋白抗原表位,免疫后获得的抗体具有较好的特异性。【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∶1000 and 1∶2000 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∶1000 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∶12800.【Conclusion】PDCoV RBD protein antigenic epitopes were successfully predicted using bioinformatics techniques.The antibodies obtained after immunization had good specificity.

关 键 词：猪δ冠状病毒(PDCoV) 受体结合结构域(RBD) 抗原表位 多肽抗体 

分 类 号：S855[农业科学—临床兽医学]