齿兰环斑病毒外壳蛋白的原核表达、抗体制备及检测应用  

Prokaryotic expression of the coat protein gene of Odontoglossum ringspot virus,preparation and application of its polyclonal antibodies.

作　　者：章颉[1] 孟春梅[1] 荣松 张超 洪健[1] 吴建祥[1] 

机构地区：[1]浙江大学农业与生物技术学院生物技术研究所,浙江杭州310029 [2]浙江传化生物技术有限公司,浙江杭州311231

出　　处：《浙江大学学报（农业与生命科学版）》2010年第4期375-380,共6页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家自然科学基金资助项目(30670087);杭州市重大科技创新专项资助项目(20080212A10)

摘　　要：齿兰环斑病毒(Odontoglossumringspot virus,ORSV)是感染兰花的主要病毒之一.用RT-PCR方法从感染ORSV兰花中克隆到该病毒的477 bp外壳蛋白基因(CP),外壳蛋白基因再亚克隆到原核表达载体pET-32a中构建重组原核表达载体pET-32a-CP;将重组表达载体转化大肠杆菌BL21(DE3),经IPTG诱导,Ni2+-NTA亲和柱纯化获得分子量约为37 ku含硫氧还蛋白的融合蛋白.以纯化的重组蛋白为抗原免疫新西兰大白兔制备ORSV外壳蛋白的多克隆抗体,并用制备的多抗建立了可靠、灵敏、特异的检测ORSV的免疫捕获RT-PCR及dot-blot ELISA方法,为该兰花病毒病的诊断、兰花抗病育种和脱毒苗的建立打下了基础.Odontoglossum ringspot virus（ORSV） is one of the most prevalent orchid viruses.The 477 bp coat protein gene（CP） of ORSV was cloned from the virus infected orchid samples using RT-PCR,and subcloned into a prokaryotic expression vector pET-32a.The recombinant prokaryotic expression vector（pET-32a-CP） was used to transform Escherichia coli BL21（DE3）.An about 37 ku thioredoxin A（TrxA） fusion protein was obtained with IPTG induction and Ni2＋-NTA affinity column purification.The purified recombinant protein was used to immunize rabbits for production of polyclonal antibodies against the coat protein of ORSV.Using the polyclonal antibodies,an immunocapture RT-PCR and a dot-blot ELISA were established for reliable,sensitive and specific detection of ORSV.The two detection methods utilizing the prepared polyclonal antibodies provide a base for the diagnosis of the ORSV disease,the production of virus-free seedlings and antivirus breeding of the orchid.

关 键 词：齿兰环斑病毒 原核表达 多克隆抗体 DOT-BLOT ELISA 免疫捕获RT-PCR 

分 类 号：Q78[生物学—分子生物学] S432.4[农业科学—植物病理学]