烟草丛顶病毒ORF3的克隆及原核表达  

Molecular Cloning and Prokaryotic Expression of the ORF3 of Tobacco bush top virus

作　　者：龙亚芹[1] 左瑞娟[1] 李凡[1] 赵秀兰 李玲[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,云南昆明650201 [2]沧源县农业局植保站,云南临沧677400

出　　处：《华南农业大学学报》2010年第2期32-35,共4页Journal of South China Agricultural University

基　　金：国家自然科学基金(30660101)

摘　　要：利用RT—PCR方法，自烟草丛顶病毒（Tobacco bush top virus，TBTV）龙陵分离物（TBTV—YLLi）的RNA中扩增出ORF3目的片段，并克隆到pMD18-T载体上进行序列分析．结果表明，TBTV-YLLi的ORF3全长714bp，与TBTV其他分离物的核苷酸相似性和氨基酸相似性分别为98．3％～98．6％和95．4％～95．8％；与同属的花生丛簇病毒（Groundnut rosette virus，GRV）、豌豆耳突花叶病毒2号（Pea enation mosaic virus-2，PEMV-2）和胡萝卜拟斑驳病毒（Carrot mottle mimic virus，CMoMV）的核苷酸相似性分别为65．1％、61．6％和49．7％，氨基酸相似性分别为36．4％、34．6％和16．5％．将ORF3克隆到原核表达载体pET28a（＋）上，获得的重组子pET28-ORF3转化大肠杆菌BL21-plysS，使用终浓度为1．0mmol／L的IPTG在37℃诱导培养4h时，该融合蛋白表达量最高．融合蛋白经Ni^2＋亲和柱层析纯化后，SDS—PAGE电泳表明，融合蛋白相对分子质量约为35000，与预计的相对分子质量大小相一致．The ORF3 was amplified from RNA of Tobacco bush top virus （TBTV） Longling isolate （TBTVYLLi） by RT-PCR, and the amplified cDNA was then cloned into pMD18-T. The results of sequence analysis indicated that the ORF3 of TBTV-YLLi was consisted of 714 bp, and shared 98.3% to 98.6% nucleotide identities and 95.4% to 95.8% amino acid identities with other TBTV isolates, respectively. Compared with the ORF3 of TBTV with other umbraviruses,the results demonstrated that TBTV had 61.6%, 65.1% and 49.7% nucleotide identities and 34.6% ,36.4% and 16.5% amino acid with Pea enation mosaic virus-2 （ PEMV-2 ）, Carrot mottle mimic virus （CMoMV） and Groundnut rosette virus （ GRV ）, respectively. The ORF3 was then cloned into pET28a（ ＋ ）, and the recombinant plasmid of pET-ORF3 was then transformed into E. coli B121-plysS and induced by IPTG. The fusion protein reached the highest expression level when induced with 1.0 mmol/L IPTG for 4 h at 37 %. The expressed protein was then purified through Ni^2＋ affinity chromatography column. SDS-PAGE analysis revealed that the expected 35 000 fusion protein was expressed successfully.

关 键 词：烟草丛顶病毒 ORF3 序列分析 原核表达 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]