摘要
为制备猪流行性腹泻病毒(PEDV)纤突糖蛋白(S)单克隆抗体,首先采用切向流超滤系统和凝胶过滤层析相结合的方法纯化PEDV CH/Hubei/2016毒株,免疫6~8周龄BALB/c小鼠,将血清抗体效价较高的小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,通过免疫过氧化物酶单层细胞试验(IPMA)对杂交瘤细胞株进行筛选,对阳性杂交瘤细胞进行亚克隆获得PEDV单克隆抗体杂交瘤细胞株。利用PEDV S1区域重组蛋白对单克隆抗体进行酶联免疫吸附试验和Western-blot鉴定,经间接免疫荧光试验(IFA)和激光共聚焦扫描显微术(CLSM)进一步验证单克隆抗体与病毒的反应原性。结果显示,所获得PEDV S1蛋白单克隆抗体共有4株,分别命名为9B9H6、10G11、17H6B10和18E9G6。通过小鼠体内诱生法制备腹水,抗体的IPMA效价在2.56×10^(-4)至1.024×10^(-5)之间。IFA结果表明,4株单克隆抗体均可与PEDV感染的Vero细胞发生特异性反应。进一步通过CLSM发现,4株单克隆抗体可以指示PEDV感染Vero细胞过程中病毒新合成的S蛋白在内质网、内质网-高尔基体中间区域、高尔基体的亚细胞定位。PEDV S蛋白特异性单克隆抗体的成功制备,为PEDV感染机制研究和病原检测方法建立奠定了重要的分子基础。
In order to prepare monoclonal antibodies(m Abs)against porcine epidemic diarrhea virus(PEDV)spike(S)glycoprotein,tangential flow system and gel filtration chromatography were applied to purify PEDV CH/Hubei/2016 virions,which were utilized as immunogen to immunize the BALB/c mice aged 6to 8 weeks.The spleen cells from the mouse with higher serum antibody titers were fused with SP2/0 myeloma.The hybridoma cell lines were screened by immunoperoxidase monolayer assay(IPMA),and the positive cells were subcloned to obtain PEDV m Abs.The m Abs were identified by enzyme-linked immunosorbent assay and Western-blot using recombinant S1 protein.Reactivities of the m Abs were characterized by indirect immunofluorescence assay(IFA)and confocal laser scanning microscopy(CLSM).The results showed that four m Abs against PEDV S1 protein were identified and named as 9B9H6,10G11,17H6B10 and 18E9G6,respectively.Ascites were prepared via in vivo induction in mice,and their IPMA titers were from 2.56×10^(-4)to 1.024×10^(-5).IFA showed that the four m Abs were reacted with PEDV-infected Vero cells.CLSM further showed the subcellular localization of S protein in endoplasmic reticulum(ER),ER-Golgi intermediate com part-ment or Golgi apparatus during PEDV infection in Vero cells using the m Abs.The successful preparation of the specific m Abs against PEDV S protein laid an important molecular basis for research on PEDV infection and establishment of effective PEDV diagnostic methods.
作者
温颖
李青梅
乔松林
王丽
李睿
张改平
WEN Ying;LI Qing-mei;QIAO Song-lin;WANG Li;LI Rui;ZHANG Gai-ping(College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China;Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2022年第6期720-728,共9页
Chinese Veterinary Science
基金
国家现代农业产业技术体系项目(CARS-35)
河南省现代农业产业技术体系项目(S2012-06)
河南省农业科学院基本科研业务费项目(2021ZC75)。
作者简介
温颖(1995-),女,河南南阳人,硕士生,研究方向为分子病原学与免疫学,E-mail:2941015617@qq.com。;通讯作者:李睿(1986-),男,河南郑州人,副研究员,博士,主要从事猪繁殖与呼吸综合征病毒致病机制研究,E-mail:1irui860620@sina.com;通讯作者:张改平(1960-),男,河南内黄人,主要从事动物免疫学及重大疫病快速检测技术研究,E-mail:zhanggaip@126.com。
