BBTV单克隆抗体的制备及其检测应用  

Production and Application of Monoclonal Antibody Against BBTV

作　　者：宋西娇 刘欢[1] 周雪平[1] 洪健[1] 吴舒艾 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

出　　处：《农业生物技术学报》2016年第2期288-294,共7页Journal of Agricultural Biotechnology

基　　金：公益性行业(农业)科研专项(No.201203076-05)

摘　　要：香蕉束顶病毒(Banana bunchy top virus,BBTV)是危害香蕉(Musa paradisiaca)生产的重要病毒。本研究旨在以制备的抗BBTV特异性单克隆抗体(monoclonal antibody,MAb)为核心建立检测BBTV的血清学方法,从而为该病毒的诊断和科学防控提供技术支撑。通过改进提纯方法获得了提纯的BBTV,以BBTV病毒粒子为抗原免疫BALB/c小鼠(Mus musculus),经细胞筛选和克隆,获得1株分泌抗BBTV单克隆抗体的杂交瘤细胞22E3,并制备其单抗腹水。用间接酶联免疫吸附试验(indirect-enzyme-linked immunosorbent assay,in-ELISA)方法测定制备的单抗腹水效价达到10-7,抗体类型及亚类为IgG1,κ轻链。Western blot检测表明,该单抗与BBTV外壳蛋白亚基有特异性反应。利用制备的单抗建立了检测BBTV的斑点酶联免疫吸附试验(dot-ELISA)。该方法检测感染BBTV香蕉植物组织粗提液呈特异性阳性反应,而和健康香蕉组织呈阴性反应,说明建立的dot-ELISA方法能特异性地检测香蕉植物组织中的BBTV。灵敏度分析表明,以22E3单抗为核心建立的dot-ELISA方法检测香蕉病组织的灵敏度达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、可靠地用于香蕉中BBTV病毒的检测。BBTV单克隆抗体的制备及其dot-ELISA血清学检测方法的建立为我国香蕉上BBTV的诊断、无毒苗的生产及科学防控提供了物质和技术支撑。Banana bunchy top virus（BBTV） is an important pathogen of banana（Musa paradisiaca）,which seriously harm banana production worldwide. The purpose of this study was to develop serological methods for detecting BBTV using prepared monoclonal antibodies（MAbs） against BBTV,and to provide technical support for the diagnosis and scientific prevention and control of BBTV in fields. BBTV- infected banana plants were identified by PCR and nucleotide sequencing. A 513 bp coat protein gene（CP） was amplified from infected banana plants,which had 99% nucleotide sequence identities with Chinese isolates of BBTV in Gen Bank. BBTV particles with a diameter of 18 nm were obtained from BBTV- infected plants by an improved purification method. Electron microscopic observation showed that concentration of purified BBTV particles was high enough to prepare MAbs against BBTV. After cell selecting and cloning,a hybridoma cell line（22E3） which secreted a MAb against BBTV was produced by fusing mouse（Mus musculus） myeloma cells（SP2/0） with spleen cells from BALB/c immunized by purified BBTV particles. The hybridoma was injected into pristane-primed BALB/c mice to prepare the ascetic fluid,which contained the MAb. The titer of this MAb in ascites determined by an indirect-enzyme-linked immunosorbent assay（in-ELISA） is up to 10- 7.Isotype and subclass of this MAb belonged to Ig G1,κ light chain. The Ig G yield of this MAb in the ascetic fluid was 8.32 mg/m L. Western blot analysis indicated that this MAb could specifically react with the 19.3 k D CP of BBTV in BBTV- infected leaf crude extracts,had a negative reaction with healthy banana leaf crude extracts. Using the MAb,dot- ELISA for detecting BBTV was established. Results of phalanx tests showed that the dilutions of 22E3 at 1∶5 000 and goat anti-mouse Ig G conjugated with alkaline phosphatase at 1∶8 000 were suitable in dot- ELISA for BBTV detection in field samples. Specificity analysis of the dot- ELISA demonstrated that this method could strongly

关 键 词：香蕉束顶病毒(BBTV) 单克隆抗体 DOT-ELISA 

分 类 号：S436.68[农业科学—农业昆虫与害虫防治]