甘蔗花叶病毒单克隆抗体的制备及其血清学检测应用  

Production and Serological Detection Application of Monoclonal Antibodies Against Sugarcane mosaic virus

作　　者：饶黎霞[1] 黄德青 周雪平[1] 钱亚娟[1] 吴建祥[1] RAO Li-Xia;HUANG De-Qing;ZHOU Xue-Ping;QIAN Ya-Juan;WU Jian-Xiang(Institute of Biotechnology,Zhejiang University / State Key Laboratory of Rice Biology,Hangzhou 310058,China)

机构地区：[1]浙江大学生物技术研究所/水稻生物学国家重点实验室,杭州310058

出　　处：《农业生物技术学报》2018年第11期1989-1995,共7页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展计划(No.2014CB138400);国家重点研发计划(No.2017YFD0201604)

摘　　要：甘蔗花叶病毒(Sugarcane mosaic virus, SCMV)是危害玉米(Zea mays)的重要病毒之一,每年造成严重的玉米产量损失。建立特异、灵敏的检测技术是防控SCMV的关键。本研究以提纯的甘蔗花叶病毒云南分离物(Sugarcane mosaic virus Yunnan isolate, SCMV-YN)病毒粒子为免疫原,利用杂交瘤技术获得8株分泌SCMV单克隆抗体的杂交瘤细胞株(12A10、10B11、6A1、19F3、15C12、22A2、21H3和21C12),并制备其单克隆抗体腹水。利用间接酶联免疫吸附试验(indirect-enzyme-linked immunosorbent assay, IndirectELISA)测得8株单克隆抗体腹水的抗体效价为10-6或10-7,单克隆抗体类型均为IgG1、κ轻链。Western blot结果显示,22A2、21H3和6A1杂交瘤细胞株分泌的抗体可识别SCMV北京分离物(Sugarcane mosaic virus Beijing isolate, SCMV-BJ)和SCMV-YN分离物的共同抗原决定簇;12A10、15C12、10B11、21C12和19F3杂交瘤细胞株分泌的抗体仅识别SCMV-YN抗原位点。以制备的单抗隆抗体作为一抗,建立检测SCMV的斑点酶联免疫吸附试验(dot enzyme-linked immunosorbent assay, dot-ELISA),其特异性分析结果显示,以22A2、21H3和6A1单克隆抗体建立的dot-ELISA方法检测SCMV-YN和SCMV-BJ分离物都呈阳性,以12A10、19F3、10B11、21C12和15C12单克隆抗体建立的dot-ELISA方法检测SCMV-YN分离物呈阳性反应,检测SCMV-BJ分离物呈阴性反应。该结果表明,上述5个单克隆抗体可用于SCMV-YN分离物的检测和株系鉴定。以不同细胞株单克隆抗体建立dot-ELISA方法检测感染SCMV的病叶,其灵敏度分析结果显示,15C12、10B11、21H3和6A1单克隆抗体检测病叶的最高稀释倍数达到1∶10 240 (W/V, g/mL),19F3和12A10单抗隆抗体检测病叶的最高稀释倍数均为1∶5 120,22A2和21C12单抗隆抗体检测病叶的最高稀释倍数为1∶2 560。本研究制备了SCMV单克隆抗体并建立了dot-ELISA血清学检测方法,为我国作物上SCMV的检测与诊断、株系鉴定、抗病育种以及科学防Sugarcane mosaic virus （SCMV）, one of the most important maize viruses, causes annually serious yield loss in maize （Zea mays） industry. Establishment of sensitive and specific virus detection technique is critical for preventing and controlling SCMV. For this purpose, 8 hybridoma lines （12A10, 10B11, 6A1, 19F3, 15C12, 22A2, 21H3, and 21C12） secreting monoclonal antibodies （MAbs） against SCMV were obtained using the purified virions of SCMV Yunnan isolate （SCMV-YN） as the immunogen and the hybridoma technology. With the obtained hybridomas, the ascitic fluids containing MAbs were produced. Using an indirect-enzyme-linked immunosorbent assay （indirect-ELISA）, the titers of 8 ascitic fluids containing MAbs were detected to be 10-6 or 10-7, and all 8 MAbs belonged to IgG1, κ light chain. Western blot assay demonstrated that 3 MAbs （22A2, 21H3 and 6A1） recognized common epitopes on capsid protein （CP） of SCMV Beijing isolates （SCMV-BJ） and SCMV-YN. MAbs 12A10, 15C12, 10B11, 21C12 and 19F3 could only recognize the specific epitopes on SCMV-YN. The dot enzyme-linked immunosorbent assay （dot-ELISA） was established by these prepared MAbs as the first antibody. Results of the specificity analyses of the developed dot-ELISAs indicated that 3 MAbs （22A2, 21H3 and 6A1） and their dot-ELISAs had a positive response when detected SCMV-BJ and SCMV-YN isolates, while other prepared 5 MAbs （12A10, 19F3, 10B11, 21C12 and 15C12） and their dot-ELISAs only had a positive response with SCMV-YN isolate, manifesting that those 5 MAbs could be used to detect and identify SCMV-YN isolate. Sensitivity analyses indicated that the dot-ELISAs based respectively on MAbs 15C12, 10B11, 21H3 or 6A1 were the most sensitive, and their sensitivities were up to 1∶10 240 （W/V, g/mL） dilution for SCMV-infected maize leaf crude extract. Detection sensitivity of the MAbs 19F3 or 12A10 was 1∶5 120 dilution, and 22A2 or 21C12 was 1∶2 560 dilution. The anti-SCMV MAbs and the developed dot-ELISA serol

关 键 词：甘蔗花叶病毒(SCMV) 单克隆抗体 斑点酶联免疫吸附试验(dot—ELISA) 血清学检测 

分 类 号：S643.6[农业科学—蔬菜学] S532[农业科学—园艺学]