SRBSDV结构蛋白P10和非结构蛋白P9-1抗体的制备及应用  

Preparation and Application of Polyclonal Antibodies Against Structural Protein P10 and Non-structural Protein P9-1 Encoded by SRBSDV

作　　者：吴锦鸿[1] 陈晓敏[1] 林文武[1] 宛柏杰 吴祖建[1] 张洁[1] 

机构地区：[1]福建农林大学植物病毒研究所/福建省植物病毒学重点实验室,福州350002

出　　处：《农业生物技术学报》2016年第8期1190-1198,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.31301640);高等学校博士学科点专项科研基金(新教师类)项目(No.20133515120004);福建省教育厅科技项目(No.JA13103)

摘　　要：为丰富南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)的预测预报体系,本研究利用Gateway原核表达系统,将SRBSDV结构蛋白P10和非结构蛋白P9-1进行了大量表达,纯化的表达蛋白分别免疫新西兰大白兔(Oryctolagus cuniculus)获得抗血清,抗血清的间接酶联免疫吸附分析(indirect enzyme-linked immunosorbent assay,in-ELISA)效价分别为1∶6 400和1∶3 200,Western blot检测结果表明,制备的2种抗体均具特异性。利用免疫捕获RT-PCR(immunocapture RT-PCR,IC-RT-PCR)和斑点酶联免疫吸附分析(dot enzyme-linked immunosorbent assay,dot-ELISA)方法比较两种抗体检测水稻(Oryza sativa)和白背飞虱(Sogatella furcifera,WBPH)带毒率的应用效果,结果表现一致,说明SRBSDV结构蛋白P10和非结构蛋白P9-1均可用于病毒的田间检测,为病害的预测预报奠定了基础。Southern rice black-streaked dwarf virus（SRBSDV）, the genus Fijivirus in the family Reoviridae, is transmitted mainly by white backed planthopper（Sogatella furcifera, WBPH） in a persistent- propagative manner. SRBSDV encodes at least 6 putative structural proteins（P1, P2, P3, P4, P8 and P10） and 7 putative nonstructural proteins（P5- 1, P5- 2, P6, P7- 1, P7- 2, P9- 1 and P9- 2）. P10 are putative major outer capsid proteins, while P9-1 can form viroplasms which is the putative sites of viral replication. In this study, to enrich the prediction system of SRBSDV, polyclonal antibodies of P10 and P9-1 were prepared. By using a Gateway prokaryotic system, firstly, part fragment of P10（591 bp） and P9- 1（714 bp） were recombined to the p DONR221 vector, respectively. After verified by sequencing, they were recombined to the expression vector p DEST17, respectively. Then they were transformed into Escherichia coli strain Rosetta. By isopropyl- β- dthiogalactoside（IPTG） inducing, recombinant proteins of 27 k D（for P10） and 33 k D（for P9-1） were obtained.The recombinant protein was used to immunize New Zealand white rabbits（Oryctolagus cuniculus） forpreparation of polyclonal antibodies. By an indirect enzyme-linked immunosorbent assay（in-ELISA） method,the antiserum titer of P10 and P9- 1 were determined to be 1∶6 400 and 1∶3 200, respectively. Western blot analysis showed that P10 and P9-1 antibodies could capture proteins of 63 and 39.9 k D in infected rice（Oryza sativa） plants, respectively, which were consistent of the actual length of the P10 and P9-1. There were no any protein captured by the 2 antibodies in healthy rice plants. Our results revealed that both antibodies could detect the infected rice plants specifically. To compare the application effect of the 2 antibodies in the detection of infected rice plants and viruliferous percent of WBPHs, immunocapture- RT- PCR（IC- RT- PCR）and dot- ELISA assays were done. In the IC- RT- PCR assays, using infected rice pl

关 键 词：南方水稻黑条矮缩病毒(SRBSDV) 抗体 免疫捕获RT-PCR 斑点酶联免疫吸附分析(dot-ELISA) 

分 类 号：S432.41[农业科学—植物病理学]