柑橘碎叶病毒(CTLV)单克隆抗体的制备及其检测应用  

Preparation and Application of Monoclonal Antibodies Against Citrus tatter leaf virus(CTLV)

作　　者：宋西娇 段硕[2] 洪健[1] 周常勇[2] 周雪平[1] 吴建祥[1] 

机构地区：[1]浙江大学生物技术研究所/国家水稻生物学重点实验室,杭州310058 [2]西南大学中国农业科学院柑橘研究所,重庆400712

出　　处：《农业生物技术学报》2017年第1期165-172,共8页Journal of Agricultural Biotechnology

基　　金：公益性行业(农业)科研专项"果树病毒病防控技术研究与示范"(No.201203076-05);中央高校基本科研业务费专项资金(2016FZA6014)

摘　　要：柑橘碎叶病毒(Citrus tatter leaf virus,CTLV)是严重危害世界柑橘(Citrus reticulata)产业的重要病毒,也是柑橘脱毒苗生产上的重要检疫对象。为了建立以抗CTLV特异性单克隆抗体(monoclonal antibody,MAb)为核心的检测CTLV的血清学方法。本研究以原核表达的CTLV外壳蛋白为抗原免疫BALB/c小鼠(Mus musculus),经细胞融合、筛选和克隆获得3株(6C5,14E11和15F8)分泌抗CTLV单克隆抗体的杂交瘤细胞。分别制备3株杂交瘤细胞的单抗腹水,经鉴定单抗腹水的抗体类型及亚类均为Ig G1、κ链,单抗腹水的间接酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)效价达到10-6以上。三抗体夹心酶联免疫吸附实验(triple antibody sandwich ELISA,TAS-ELISA)和Western blot分析单抗特异性表明,3株单抗均与感染CTLV柑橘病叶有特异性免疫反应,而不与柑橘衰退病毒(Citrus tristeza virus,CTV)、苹果茎沟病毒(Apple stem grooving virus,ASGV)及健康柑橘树叶反应。利用制备的单抗为核心建立能特异性地检测柑橘病叶中CTLV的斑点酶联免疫吸附实验(dot-ELISA)。灵敏度分析表明,以3株单抗为核心建立的dot-ELISA方法检测病叶组织的灵敏度达1∶640(W/V,g/m L)。田间样品检测结果表明,建立的dot-ELISA方法能准确、可靠和灵敏地用于柑橘树中CTLV病毒的检测。本研究为我国柑橘树中CTLV的诊断、无毒苗的生产及科学防控提供了技术支撑。Citrus tatter leaf virus（CTLV）, a species of the family Capillovirus, is an important citrus（Citrus reticulate） virus and a quarantine object in obtaining virus-free citrus seedlings, which causes significant losses to citrus industry worldwide. It is very important to prepare sensitive and specific monoclonal antibodies（MAbs） against CTLV for the diagnosis and detection of CTLV in citrus groves. For this purpose, the 714 bp coat protein gene（CP） of CTLV was cloned from a CTLV-infected citrus sample collected from citrus grove in Chongqing municipality, which shared 100% nucleotide sequence identity with a Chinese isolate of CTLV deposited in Gen Bank. The CTLV CP then was cloned into the His-tagged prokaryotic expression vector, p ET-28 a. The resulting expression vector was transformed into Escherichia coli BL21（DE3） strain. After inducedby isopropylthio-β-D-galactoside（IPTG）, E. coli BL21（DE3） cells harboring the recombinant vector expressed an approximately 30 k D fusion protein. The recombinant fusion protein was purified with Ni2＋-NTA agarose and used to immunize BALB/c mice（Mus musculus）. Three hybridoma cell lines（6C5, 14E11 and15F8） secreting MAbs against CTLV were prepared by fusing mouse myeloma cells（SP 2/0） with spleen cells from the immunized BALB/c mouse. The hybridomas were injected into pristine-primed BALB/c mice to prepare the ascetic fluids contained the MAbs. The titers of 3 MAbs in ascitic fluids ranged from 10-6to 10-7in indirect-ELISA. Isotypes and subclasses of all 3 MAbs belonged to Ig G1, κ light chain. The Ig G yields of 3MAbs（6C5, 14E11 and 15F8） in ascetic fluids were 9.41, 7.83 and 9.93 mg/m L respectively. Triple antibody sandwich enzyme-linked immunosorbent assay（TAS-ELISA） analysis indicated that the 3 MAbs could specifically react with CTLV-infected citrus leaf and CTLV-infected Chenopodium quinoa leaf crude extracts,had negative reactions with Citrus tristeza virus, Apple stem grooving virus, healthy citrus leaf and Chenopodiu

关 键 词：柑橘碎叶病毒(CTLV) 单克隆抗体 斑点酶联免疫吸附实验(dot-ELISA) 

分 类 号：S431[农业科学—农业昆虫与害虫防治]