烟草丛顶病毒ORF3的克隆及原核表达

利用RT-PCR方法,自烟草丛顶病毒(Tobacco bush topvirus,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714 bp,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1.0 mmol/L的IPTG在37℃诱导培养4 h时,该融合蛋白表达量最高.融合蛋白经Ni2+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35 000,与预计的相对分子质量大小相一致.

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

The ORF3 was amplified from RNA of Tobacco bush top virus(TBTV) Longling isolate(TBTV-YLLi)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.