可阻断IBDV感染的抗CEF膜蛋白单克隆抗体的筛选  

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

作　　者：杨艳艳[1] 朱礼倩[1,2] 杨继飞[1] 滕蔓[1] 柴书军[1] 王丽[1] 罗俊[1] 邓瑞广[1] 张改平[1] 

机构地区：[1]河南省农业科学院舸南省动物免疫学重点实验室,郑州450002 [2]扬州大学兽医学院,江苏扬州225009

出　　处：《中国农业科学》2009年第2期701-705,共5页Scientia Agricultura Sinica

基　　金：国家自然科学基金资助项目(30671561);国家“973”计划(2005CB523203);国家科技支撑计划(2006BAD06A04-6)

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2h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。[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 1BDV 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 oflBDV 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.

关 键 词：鸡胚成纤维细胞 IBDV受体 BIOTIN STREPTAVIDIN 

分 类 号：S855[农业科学—临床兽医学]