犬瘟热病毒F蛋白融合信号肽单克隆抗体的制备及其表位的鉴定  

Preparation and antigenic epitope screening of monoclonal antibody against canine distemper virus F protein corresponding author fusion signal peptide

作　　者：崔赛赛 李博韬[1] 刘家森[1] 康洪涛 杨鸣发 姜骞[1] 曲连东[1] CUI Sai-sai;LI Bo-tao;LIU Jia-sen;KANG Hong-tao;YANG Ming-fa;JIANG Qian;QU Lian-dong(Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,State Key Laboratory of Veterinary Biotechnology,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/宠物疫病创新团队,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2023年第5期523-528,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划项目(2017YFD0501600)。

摘　　要：为制备犬瘟热病毒(CDV)F蛋白融合信号肽(FSP)的单克隆抗体(MAb),本研究通过原核表达系统表达CDV HLJ1-13株重组FSP蛋白(rFSP)。将其免疫BALB/c小鼠,通过间接ELISA方法筛选到1株能够稳定分泌抗FSP蛋白MAb的杂交瘤细胞株G8D6E3E5,亚类鉴定结果显示该株MAb重链为Ig G2a型,轻链为κ型。构建一系列表达部分重叠的重组FSP片段,经western blot鉴定该株MAb识别的抗原区域为aa1~aa55,通过合成一系列重叠多肽,经ELISA进一步鉴定该株MAb识别的抗原表位为^(21)QQHSTRSTET^(30)。采用western blot检测该株MAb与r FSP的反应原性;采用间接免疫荧光试验(IFA)检测该株MAb与天然FSP的反应原性。Western blot结果显示,CDV Snyder Hill和HLJ1-13株r FSP蛋白样品在34 ku左右均出现特异性条带;IFA结果显示,感染CDV Snyder Hill株48 h后的vero细胞出现红色荧光。以上结果表明,该株MAb既能够与原核表达的CDV Snyder Hill和HLJ1-13株的r FSP蛋白反应,也能够与天然表达的CDV Snyder Hill株FSP蛋白反应。上述研究结果为CDV的检测以及FSP生物学功能的研究奠定了坚实基础。To prepare monoclonal antibody(MAb)against canine distemper virus(CDV)F protein fusion signal peptide(FSP),the recombinant FSP(rFSP)of CDV HLJ1-13 strain was expressed by prokaryotic expression system.BALB/c mice were immunized with r FSP and one hybridoma cell line G8D6E3E5 that could stably secretion of MAb against FSP was screened by indirect ELISA.The subtype identification results showed that the heavy chains type of the MAb was Ig2a,and the light chains type was theκtype.A series of recombinant FSP fragments with partially overlapping were constructed and prepared,and the antigenic region recognized by the MAb of this strain was identified by western blot.The results showed that the region recognized by the MAb was aa1-aa55.A series of synthetic overlapping polypeptide were used to further identify the epitope recognized by the MAb by ELISA.The results showed that the epitope recognized by the MAb was^(21)QQHSTRSTET^(30).Western blot was used to detect the reactivity of the MAb with r FSP protein.Indirect immunofluorescence assay(IFA)was used to detect the reactivity of the MAb with FSP protein naturally expressed in vero cell.Western blot results showed that the MAb could recognize the r FSP proteins of CDV Snyder Hill and HLJ1-13 strains.IFA results showed that red fluorescence appeared in vero cell infected with CDV Snyder Hill strain.The above results indicated that the MAb could not only react with r FSP proteins of CDV Snyder Hill and HLJ1-13 strains,but also react with naturally expressed FSP proteins of CDV Snyder Hill strain.This study have laid a solid research foundation for the diagnosis of CDV and the study of biological function of FSP.

关 键 词：犬瘟热病毒 F蛋白融合信号肽 单克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]