马立克病病毒单克隆抗体库构建及gB蛋白特异性单抗的筛选与鉴定

doi:10.11843/j.issn.0366-6964.2023.11.025

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (11): 4712-4723.doi: 10.11843/j.issn.0366-6964.2023.11.025

马立克病病毒单克隆抗体库构建及gB蛋白特异性单抗的筛选与鉴定

李林燕^1,2,3, 滕蔓^2,3, 刘金玲^2,3, 郑鹿平^2,3, 柴书军^2,3, 丁轲¹, 余祖华^1*, 罗俊^2,3*

1. 河南科技大学动物科技学院, 功能微生物与畜禽健康实验室, 洛阳 471003;
2. 河南省农业科学院动物免疫学重点实验室, 农业农村部动物免疫学重点实验室, 河南省动物免疫学重点实验室, 郑州 450002;
3. 河南省农业科学院, 中英禽病国际研究中心, 郑州 450002

收稿日期:2023-02-23 出版日期:2023-11-23 发布日期:2023-11-26
通讯作者: 罗俊,主要从事动物病毒学研究,E-mail:luojun593@aliyun.com;余祖华,主要从事动物分子免疫学研究,Tel:0379-64280348,E-mail:yzhd05@163.com
作者简介:李林燕(1997-),女,河南安阳人,硕士生,主要从事动物疫病防控技术研究,E-mail:lly97623@163.com
基金资助:
国家重点研发计划(2023YFE0106100);国家自然科学基金(U21A20260);河南省杰出青年科学基金(232300421009);河南省农业科学院自主创新项目(2023ZC089)

Development of a Monoclonal Antibody Pool for Marek’s Disease Virus and Identification of a Specific mAb against the Glycoprotein gB

LI Linyan^1,2,3, TENG Man^2,3, LIU Jinling^2,3, ZHENG Luping^2,3, CHAI Shujun^2,3, DING Ke¹, YU Zuhua^1*, LUO Jun^2,3*

1. Laboratory of Functional Microbiology and Animal Health, College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, China;
2. Key Laboratory of Animal Immunology, Ministry of Agriculture and Rural Affairs of China & Henan Province Key Laboratory of Animal Immunology, Henan Academy of Agriculture Sciences, Zhengzhou 450002, China;
3. UK-China Centre of Excellence for Research on Avian Diseases, Henan Academy of Agriculture Sciences, Zhengzhou 450002, China

Received:2023-02-23 Online:2023-11-23 Published:2023-11-26

摘要/Abstract

摘要： 马立克病(MD)是由马立克病病毒(MDV)感染引起的危害最严重的一种家禽免疫抑制病与肿瘤病,制备MDV单克隆抗体可为后续的基因功能、致病机制及诊断技术研究提供关键试剂。本研究利用NE-PER^TM细胞核和细胞质提取试剂,分别制备了vvMDV(MDV超强毒株)标准毒株Md5感染的鸡胚成纤维细胞(CEF)核蛋白和浆蛋白,通过切向流超滤浓缩后制备免疫原,分别免疫6~8周龄雌性BALB/c小鼠,常规细胞融合方法制备单克隆抗体杂交瘤细胞。通过间接免疫荧光试验(IFA)筛选,建立了一个容量为 31株纯化的MDV单克隆抗体杂交瘤细胞库,包括27株稳定分泌MDV-1特异性抗体和4株稳定分泌MDV-1、MDV-2和火鸡疱疹病毒(HVT)保守抗体的杂交瘤细胞株。经过293T细胞真核表达gB蛋白并结合IFA染色,其中1株单克隆抗体J-1F3-C6被鉴定为MDV糖蛋白gB的特异性单克隆抗体,其单抗腹水IFA效价为1∶25 600,轻链型为Kappa,IgG亚型为IgG2a。进一步IFA染色表明,J-1F3-C6可特异性识别MDV-1、MDV-2和HVT毒株表达的gB蛋白,但在蛋白质免疫印迹(Western blot)分析中J-1F3-C6只与MDV-1和HVT发生特异性反应。综上,本研究建立了一个MDV单克隆抗体杂交瘤细胞库,并从中筛选鉴定了1株gB蛋白特异性的单克隆抗体,为后续研究奠定了重要基础。

关键词: 马立克病, 单克隆抗体, 糖蛋白gB, 间接免疫荧光试验, 共聚焦分析, 蛋白质免疫印迹

Abstract: Mare’k disease (MD) is caused by the infection of Marek’s disease virus (MDV) and is regarded as one of the most serious immunosuppressive and neoplastic diseases of the poultry. Development of specific monoclonal antibodies (mAbs) against MDV is crucial for providing key reagents for the studies on gene functions, pathogenesis and diagnostic techniques. The NE-PER^TM Nuclear and Cytoplasmic Extraction Reagents was used to prepare the nuclear and plasma proteins from the chicken embryo fibroblasts (CEF) cultures infected with vvMDV (very virulent MDV) strain Md5, and then were concentrated by tangential flow ultrafiltration to make the immunogen and immunize female BALB/c mice at age of 6 to 8 weeks. The hybridomas were produced by conventional cell fusion. Through the screening of positive supernatants by indirect immunofluorescence assay (IFA), a total of 31 purified monoclonal hybridomas stably secreting mAbs specific for MDV were obtained, of which 27 hybridomas secret MDV-1 specific mAbs and the other four hybridomas secret conserved mAbs recognizing MDV-1, MDV-2 and herpesvirus of turkey (HVT). Furthermore, the J-1F3-C6 was identified as a specific mAb against MDV glycoprotein B (gB) thorough the IFA staining of 293T cells over-expressing gB protein. The titer of mAb J-1F3-C6 in ascites is 1∶25 600 determined by IFA, and the light chain and IgG subtype was characterized as Kappa and IgG2a, respectively. The cross reaction experiments have shown that the J-1F3-C6 specifically recognized the gB proteins from all the tested strains of MDV-1, MDV-2 and HVT, but for Western blot analysis it can only recognize MDV-1 and HVT strains. In conclusion, we have developed a pool of mAbs against MDV and identified a mAb specific for gB protein, providing a solid foundation for future research.

Key words: Marek’s disease, monoclonal antibody, glycoprotein gB, IFA, confocal analysis, Western blot

中图分类号:

S852.659.1

李林燕, 滕蔓, 刘金玲, 郑鹿平, 柴书军, 丁轲, 余祖华, 罗俊. 马立克病病毒单克隆抗体库构建及gB蛋白特异性单抗的筛选与鉴定[J]. 畜牧兽医学报, 2023, 54(11): 4712-4723.

LI Linyan, TENG Man, LIU Jinling, ZHENG Luping, CHAI Shujun, DING Ke, YU Zuhua, LUO Jun. Development of a Monoclonal Antibody Pool for Marek’s Disease Virus and Identification of a Specific mAb against the Glycoprotein gB[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4712-4723.

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马立克病病毒单克隆抗体库构建及gB蛋白特异性单抗的筛选与鉴定

Development of a Monoclonal Antibody Pool for Marek’s Disease Virus and Identification of a Specific mAb against the Glycoprotein gB

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