血清Ⅲ型马立克病病毒单克隆抗体的制备及在疫苗蚀斑计数中的应用  

Preparation of serotype-Ⅲspecific monoclonal antibody against MDV and its preliminary applications in plaque counting of vaccines

作　　者：李文超 刘丹[2] 杨承槐[2] 李启红[2] 李欣[1] 张莹 毛娅卿[2] 李俊平[2] LI Wenchao;LIU Dan;YANG Chenghuai;LI Qihong;LI Xin;ZHANG Ying;MAO Yaqing;LI Junping(College of Life Engineering,Shenyang Institute of Technology,Fushun,Liaoning 113122,China;China Institute of Veterinary Drug Control,Beijing 100081,China)

机构地区：[1]沈阳工学院生命工程学院,辽宁抚顺113122 [2]中国兽医药品监察所,北京100081

出　　处：《中国兽医学报》2024年第2期258-263,共6页Chinese Journal of Veterinary Science

基　　金：辽宁省教育厅2021年度科学研究经费面上基金资助项目(LJKZ1339);兽药行业公益性重点专项基金资助项目(GY202105)。

摘　　要：通过制备马立克病病毒(Marek’s disease virus,MDV)血清Ⅲ型特异性单克隆抗体,并对其特性进行鉴定,为建立鸡马立克病(Marek’s disease,MD)二价活疫苗间接免疫荧光蚀斑计数方法奠定基础。以纯化的MDV血清Ⅲ型HVT Fc-126株免疫BALB/c小鼠,采用常规单克隆抗体技术,经间接免疫荧光方法筛选杂交瘤细胞株;选取1株制备MDVⅢ型单克隆抗体,建立MD二价活疫苗的间接免疫荧光蚀斑计数方法。结果筛选出2株稳定分泌抗MDV血清Ⅲ型单克隆抗体的杂交瘤细胞株,并选择1D2株单抗建立了检测MD二价活疫苗的间接免疫荧光蚀斑计数方法,可快速准确地计数二价活疫苗中血清Ⅲ型病毒的蚀斑形成单位数(Plaque forming unit,PFU),与琼脂覆盖法相比,敏感性和特异性大大提高。因此,制备的MDV单克隆抗体具有良好的血清型特异性,可用于MD二价活疫苗的间接免疫荧光蚀斑计数。In order to prepare and characterize serotype-Ⅲspecific monoclonal antibody(McAb)against Marek's disease virus(MDV),and then utilized it for establishing plaque counting method by indirect immunofluorescent staining.Purified MDV serotype-ⅢHVT Fc-126 strains were used to immunize BALB/c mice.The hybridoma cultured supernatants were screened by indirect immunofluorescent assay with HVT Fc-126 infected chicken embryo fibroblast cells(CEF).The hybridoma clones were selected and employed for preparation of McAb.Plaque counting method by indirect immunofluorescent staining was established with the McAb to detect the MDV serotype-Ⅲplaque forming units(PFU)in Marek's Disease bivalent live vaccines.Two hybridoma clones that constitutively secrete McAb against MDV serotype-Ⅲwere screened.High purified McAb was prepared with 1D2 hybridoma clone.Compared with agarose plaque assay,the indirect immunofluorescent staining method characterized higher sensitivity,specificity,and more rapidity.Significantly,the indirect immunofluorescent staining method could accurately count PFU of serotype-Ⅲviruses in bivalent vaccine containing two serotype viruses.Hence,serotype-Ⅲspecific McAb against MDV has been prepared successfully,and could be applied in plaque counting of bivalent vaccines by indirect immunofluorescent staining method.

关 键 词：马立克病病毒 血清型 单克隆抗体 二价疫苗 蚀斑计数 

分 类 号：S852.65[农业科学—基础兽医学]