越南水稻黄矮病毒多克隆血清抗体的制备（英文）  

Production of polyclonal antisera for diagnosis of rice yellow stunt virus(RYSV)in Vietnam

作　　者：何越强 陈氏懦花 陈阮河 杜新勇 阮德辉 韦善富[3] 覃武[3] 吕荣华[4] HA Viet-cuong;TRAN Thi-nhu-hoa;TRAN Nguyen-ha;DO Tan-dung;NGUYEN Duc-huy;WEI Shan-fu;QINWu;LYU Rong-hua(Department of Plant Pathology,Faculty of Agronomy,Vietnam National University of Agriculture,Hanoi 131000,Vietnam;Research Center for Tropical Plant Diseases,Vietnam National University of Agriculture,Hanoi 131000,Vietnam;Plant Protection Research Institute,Guangxi Academy of Agricultural Sciences,Nanning 530007,China;Division of International Cooperation,Guangxi Academy of Agricultural Sciences,Nanning 530007,China)

机构地区：[1]越南农业大学农学系植物病理部门,河内131000 [2]越南农业大学农学系热带植物病害研究中心,河内131000 [3]广西农业科学院植物保护研究所,南宁530007 [4]广西农业科学院国际合作处,南宁530007

出　　处：《南方农业学报》2019年第7期1472-1482,共11页Journal of Southern Agriculture

基　　金：Guangxi Science and Technology Plan Project(Guike AD16380018);Ministry of Education and Training of Vietnam(B2013-11-27);Ministry of Science and Technology of Vietnam(NN_NDT2014/01)Biography

摘　　要：【目的】制备用于检测水稻黄矮病毒(RYSV)的多克隆抗体,为水稻和黑尾叶蝉上的病毒诊断提供技术支持。【方法】利用两种不同来源的RYSV抗原免疫家兔,一种是从受感染的水稻病叶组织纯化获得RYSV病毒粒子;另一种是从越南RYSV分离株克隆出完整N蛋白编码基因,然后将其连接至pET-28a载体上,在大肠杆菌Rosetta(DE3)菌株中进行诱导表达获得N蛋白抗原。其中,N蛋白抗原又以两种形式(洗脱纯化N蛋白和N蛋白条带聚丙烯酰胺切片匀浆)对家兔进行免疫。最后,采用PTA-ELISA分析评估家兔抗体血清(多克隆抗体)对水稻叶片和黑尾叶蝉RYSV的特异性和敏感性。【结果】分离自越南RYSV分离株的N基因由1542个核苷酸组成,与来自我国和日本RYS分离株N基因序列进行比对,其核苷酸序列同源性分别为98.1%和97.9%,对应的推导氨基酸序列同源性为83.4%和99.4%。以RYSV病毒粒子、洗脱纯化N蛋白和N蛋白条带聚丙烯酰胺切片匀浆3种抗原免疫家兔获得的多克隆抗体均能有效检测出水稻植株中的RYSV,其中,感病植株的OD405分别为1.449、2.337和1.649,健康植株的OD405分别为0.375、0.294和0.283。PTA-ELISA检测结果表明,获得的多克隆抗体能从单个带病毒黑尾叶蝉中检测出RYSV,且该结论在RT-PCR检测中得到进一步验证。【结论】制备获得的多克隆抗体对RYSV具有较高特异性和敏感性,可用于水稻和黑尾叶蝉上的病毒诊断,同时表明以含有病毒植物蛋白的聚丙烯酰胺切片直接注射免疫模型动物制备多克隆抗体具有可行性。【Objective】Polyclonal antibodies specific to rice yellow stunt virus(RYSV)was prepared to provide technique support for diagnosis of this virus in rice plants and green leafhopper vectors(Nephotettix spp.).【Method】Two sources of the RYSV antigens were prepared for the rabbit immunization.The first one was the RYSV virion purified from the infected rice leaf tissue.The second one was the complete N protein of one RYSV isolate from Vietnam,which was cloned in the pET28a vector and expressed in Escherichia coli strain Rosetta(DE3).The N protein antigen was used in two forms,the eluted fraction of purified N protein and the homogenate of polyacrylamide slices containing N protein band.The specificity and sensitivity of the antisera and antibodies for detection of RYSV from rice leaves and rice green leafhopper vector individuals were evaluated by a plate trapped antigen enzyme linked immunosorbent assay(PTA-ELISA)method.【Result】In this study the N gene of the RYSV isolate from Vietnam was composed of 1542 nucleotides,shared sequence identities of 98.1%and 97.9%(nucleotide level)and 83.4%and 99.4%(amino acid level)with the two RYSV isolates from China and Japan,respectively,available in the GenBank.All three antisera produced in rabbits immunized with the virion,purified N protein and homogenate of polyacrylamide slices containing N protein band were able to detect RYSV in infected rice plants with the PTA-ELISA absorbance values(OD405)were 1.449,2.337 and 1.649,respectively,while those values in healthy plants were 0.375,0.294 and 0.283,respectively.The antisera were able to detect RYSV in a single viruliferous green leafhopper vector in PTA-ELISA test as confirmed by reverse transcriptase PCR(RT-PCR)test.【Conclusion】In conclusion,the rabbit polyclonal antisera produced in this study had high specificity and sensitivity again RYSV,and therefore they can be used for diagnosis of this virus from rice plants and green leafhopper vectors.This study also indicates that the polyacrylamide slices containing

关 键 词：水稻黄矮病毒 多克隆抗体 酶联免疫吸附测定(ELISA) N蛋白 表达 病毒纯化 

分 类 号：S435.111.49[农业科学—农业昆虫与害虫防治]