可阻断IBDV感染的抗CEF膜蛋白单克隆抗体的筛选

doi:10.3864/j.issn.0578-1752.2009.02.040

中国农业科学 ›› 2009, Vol. 42 ›› Issue (2): 701-705 .doi: 10.3864/j.issn.0578-1752.2009.02.040

可阻断IBDV感染的抗CEF膜蛋白单克隆抗体的筛选

河南省农业科学院/河南省动物免疫学重点实验室

收稿日期:2008-01-25 修回日期:2008-06-16 出版日期:2009-02-10 发布日期:2009-02-10
通讯作者: 张改平

Screening of the Monoclonal Antibodies Against the Membrane Proteins of Chicken Embryo Fibroblast Which Can Block the Infection of IBDV

河南省农业科学院/河南省动物免疫学重点实验室

Received:2008-01-25 Revised:2008-06-16 Online:2009-02-10 Published:2009-02-10
Contact: ZHANG Gai-ping

摘要/Abstract

摘要：

【目的】利用细胞融合技术及细胞免疫组化方法，筛选可特异性阻断鸡传染性法氏囊病病毒（IBDV）感染的抗鸡胚成纤维细胞（CEF）膜蛋白单克隆抗体，为进一步研究IBDV的CEF受体奠定基础。【方法】用CEF细胞免疫Balb/c小鼠，经细胞融合获得杂交瘤细胞上清。单层CEF用杂交瘤细胞上清孵育2 h后接种IBDV，病毒感染后固定细胞，先后与F22-EA6-Biotin及Streptavidin-HRP进行反应，最后用AEC染色，显微镜下计数，统计感染细胞减少的百分率以判定单抗上清阻断IBDV感染的效果。【结果】利用细胞组化的方法，共计检测了768株杂交瘤细胞上清，6株（1A5、1H11、2B12、3G1、4D10和4B8）显示有阻断IBDV感染的效果，其中4B8可完全阻断IBDV对CEF的感染，该单抗针对的CEF膜蛋白很有可能是IBDV的细胞受体。【结论】筛选到的抗CEF膜蛋白的单抗可以阻断IBDV感染CEF，表明所建立的方法具有较高的敏感性和特异性。这些单抗可以用于进一步研究IBDV的CEF细胞受体。

关键词: 鸡胚成纤维细胞, IBDV受体, Biotin Streptavidin

Abstract:

【Objective】To establish an efficient assay for screening monoclonal antibodies (McAbs) against the membrane proteins of chicken embryo fibroblast (CEF) for further studies of the cellular receptors of infectious bursal disease virus (IBDV). 【Method】 McAbs against the membrane proteins of CEF were prepared by cell fusion. The monolayer CEF pre-incubated with the CEF-specific McAbs for 2 h were infected with IBDV and incubated with F22-EA6-Biotin postinfection. Then, the cells were reacted with Streptavidin-HRP and finally stained by AEC. The inhibitory percentage of IBDV infection was calculated by counting the IBDV-infected cells to determine the inhibition efficiency of the CEF-specific McAbs. 【Result】 Compared with the control cells, the IBDV-infected cells pretreated with CEF-specific antibody significantly decreased; Supernatant fluids of total of 768 hybridomas were analyzed. The results of immunohistochemistry assays showed that six ones (1A5, 1H11, 2B12, 3G1, 4D10 and 4B8) have the abilities to block the infection of IBDV to CEF, and among which 4B8 can perfectly block the infection. 【Conclusion】 This novel method is a sensitive and specific assay for the screening of CEF membrane protein-specific McAbs which can block the infection of IBDV to CEF, and these McAbs can be used for the further investigations of the cellular receptors of IBDV.

Key words: CEF, IBDV receptor, Biotin-streptavidin

. 可阻断IBDV感染的抗CEF膜蛋白单克隆抗体的筛选[J]. 中国农业科学, 2009, 42(2): 701-705 .