可阻断IBDV感染的抗CEF膜蛋白单克隆抗体的筛选

【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。

Screening for Monoclonal Antibodies Against the Membrane Proteins of Chicken Embryo Fibroblast Blocking the Infection of IBDV

This article aims 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）. 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-horseradish peroxidase （HRP） and finally stained by 3-amino-9-ethylcarbazole （AEC）. The inhibitive percentage of IBDV infection was calculated by counting the IBDV-infected cells to determine the inhibition efficiency of the CEF-specific McAbs. Compared with the control cells, the IBDV-infected cells pretreated with CEF-specific antibody significantly decreased; supernatant fluids of a total of 768 hybridomas were analyzed. The results of immunohistochemistry assays showed that six of them （1A5, 1H11, 2B 12, 3G1, 4D10, and 4B8） have the abilities to block the infection of IBDV to CEF, among which 4B8 can perfectly block the infection. 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.

IBDV感染SPF雏鸡法氏囊中TLR3表达的动态变化

为了研究鸡Toll样受体3(ch TLR3)基因在传染性法氏囊病病毒(IBDV)感染的雏鸡法氏囊内的表达情况,试验用IBDV感染14日龄SPF雏鸡,采用荧光定量PCR技术及间接免疫荧光技术检测法氏囊中TLR3基因及蛋白的表达情况。结果表明:感染组雏鸡法氏囊中TLR3 mRNA及蛋白表达均呈先升高后降低的趋势。说明TLR3参与雏鸡抵抗IBDV入侵的过程。

鸡Toll样受体在IBDV免疫中的作用研究

为了揭示鸡先天性免疫反应在IBDV疫苗过程中的作用,将40只14日海兰褐蛋鸡随机分成两组,实验组接种IBDV活疫苗,对照组注射生理盐水,隔离饲养。于免疫前和免疫后12h、24h、48h、72h、130h各取脾脏和法氏囊分离淋巴细胞并提取总RNA,用实时荧光定量检测方法检测各组中chTLR3、chTLR7、chTLR21表达的动态变化。结果显示,在免疫后36h,鸡法氏囊和脾脏中chTLR3表达变化最显著,在免疫后48h,鸡脾脏和法氏囊中chTLR21表达变化也较为明显。提示chTLR3在鸡对IBDV的免疫应答中起主要作用。