烟草丛顶病毒(TBTV)ORF3和ORF4的原核表达、抗血清制备及应用  

Prokaryotic Expression,Antiserum Preparation and Application of ORF3 and ORF4 from Tobacco bush top virus(TBTV)

作　　者：龙亚芹[1,2] 王万东[2] 左瑞娟[1] 李凡[1] 尹朝先[1] 陈海如[1] 

机构地区：[1]云南农业大学农业生物多样性与病虫害控制教育部重点实验室,昆明650201 [2]云南省农业科学院热带亚热带经济作物研究所,保山678025

出　　处：《农业生物技术学报》2011年第2期356-362,共7页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.30660101)资助

摘　　要：为制备烟草丛顶病毒(TBTV)ORF3和ORF4多克隆抗体,采用RT-PCR方法克隆了TBTV保山龙陵分离物(TBTV-BSLLi)的ORF3和ORF4,亚克隆至pMD18-T载体中,经测序正确后,将该基因插入原核表达载体pET28a(+)中,通过热击转化大肠杆菌(Escherichia coli)BL21(plysS),以IPTG诱导6×His融合蛋白的表达,并经Ni2+亲和柱层析纯化,成功地克隆了TBTV的ORF3和ORF4,建立了其原核表达和表达产物的纯化体系,获得了高纯度的ORF3、ORF4蛋白。用纯化的蛋白免疫大白兔,获得高效价的特异性抗血清。DAS-ELISA检测结果表明,制备的抗血清可用于田间烟草(Nicotiana tabacumL.)样品及传播介体的检测。本研究为用血清学方法检测该病毒提供了基础条件。In order to preparate polyclonal antibodies of ORF3 and ORF4 of Tobacco bushy top virus（TBTV）,ORF3 and ORF4 fragments were cloned from TBTV-BSLLi isolate by RT-PCR,and sub-cloned into pMD18-T.After correct sequencing,the ORF3 and ORF4 fragments were inserted into prokaryotic expression vector pET28a（＋）.Then the recombinant vector was transformed into Esherichia coli BL21（plysS） through heat shock,and 6×His-tagged ORF3 and ORF4 expression were induced by IPTG.The high purity protein of ORF3 and ORF4 were obtained through Ni affinity chromatography column.Their high titer specific antiserum were obtained by immunizing rabbit using the purity proteins.The DAS-ELISA results indicated that the antiserum could be used for specific detection of TBTV from field samples of tobacco（Nicotiana tabacum L.） and transmission vector.It provides the basic condition for serological detection of TBTV.

关 键 词：烟草丛顶病毒(TBTV) ORF3和ORF4 原核表达 抗血清制备 

分 类 号：S432.41[农业科学—植物病理学]