水稻矮缩病毒非结构蛋白Pns6和外壳蛋白P8多克隆抗体的制备及其应用  

Preparation and application of polyclonalantibodies against non-structural protein Pns6 and coat protein P8 of Rice dwarf virus

作　　者：宛柏杰 林文武[1] 吴锦鸿[1] 陈晓敏[1] 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,福建福州350002

出　　处：《福建农林大学学报（自然科学版）》2016年第1期42-47,共6页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：高等学校博士学科点专项科研基金(新教师类)(20133515120004);国家自然科学基金(31301640);福建省教育厅科技项目(JA13103)

摘　　要：利用RT-PCR的方法从感染水稻矮缩病毒(Rice dwarf virus,RDV)的水稻中克隆该病毒的运动蛋白基因S6和外壳蛋白基因S8,通过Gateway系统进行原核表达,获得表达载体p DEST17-Pns6和p DEST17-P8,将表达载体转化E.coli Rosetta,经IPTG诱导表达后获得分子质量约为59和46 ku含HIS标签的融合蛋白.以诱导表达的目的蛋白为抗原,免疫注射新西兰大白兔制备多克隆抗体.结果表明,Western blot检测制备的Pns6和P8抗体可特异性检测RDV,间接ELISA测定Pns6和P8抗体的效价均约为6 400倍,并建立了可靠、灵敏、特异的Dot-blot ELISA方法检测RDV.以上研究表明,RDV运动蛋白和外壳蛋白抗体均可应用于该病毒在田间的大规模调查和检测.A full length c DNA of Rice dwarf virus( RDV) segment S6 encoding movement protein and segment S8 encoding coat protein were cloned from infected rice samples by RT-PCR. Vectors of p DEST17-Pns6 and p DEST17-P8 were expressed via Gateaway system and followed by being transformed into E. coli Rosetta. Then the 59 and 46 ku HIS-tag fusion protein,obtained with the induction of IPTG from E. coli Rosetta cells,were used to immunize New Zealand Rabbits as an antigen for polyclonal antibodies production. Both Western blot and ELISA method proved that both antibodies could detect infected rice plants specifically,indirect ELISA method detection of antiserum titer of Pns6 and P8 were 6 400 times. To summarize,Dot-blot ELISA was established for reliable,sensitive and specificfor RDV detection. Both antibodies against non-structural protein and coat protein of Rice dwarf virus could be applied to fast virus detection at field-scale.

关 键 词：水稻矮缩病毒 非结构蛋白Pns6 外壳蛋白P8 多克隆抗体 DOT-BLOT ELISA 

分 类 号：S435.111.49[农业科学—农业昆虫与害虫防治]