烟草花叶病毒<i>P</i>54基因的原核表达与蛋白纯化

李聪; 田培洁; 张宇; 张德咏; 刘勇; 张松柏

doi:10.19303/j.issn.1008-0384.2021.02.011

烟草花叶病毒P54基因的原核表达与蛋白纯化

Prokaryotic Expression and Purification of Tobacco Mosaic Virus Specific P54 Protein

摘要

摘要:
目的烟草花叶病毒（Tobacco mosaic virus，TMV）编码的与复制相关通读P183基因中，编码一个P54基因，其功能及其作用分子机制还鲜见报道。为获得P54蛋白纯化样品开展本研究，以期为P54蛋白的功能及其作用分子机制研究奠定基础。
方法采用RT-PCR技术，从感染TMV的三生烟（Nicotiana tabacum var. Samsun NN）cDNA中扩增获得片段大小为1 425 bp的烟草花叶病毒P54基因，并克隆到原核表达载体pEASY-Blunt E1，转化E coli BL21（DE3）中进行诱导表达，包涵体透析复性后采用Ni-NTA柱层析纯化，Western-blotting进行鉴定。
结果原核表达的TMV P54蛋白，主要以包涵体的形式存在；复性后经Ni-NTA柱层析纯化，SDS-PAGE表明纯化蛋白为一分子量约为60 kDa的单一条带。Western-blotting结果证实纯化蛋白为P54重组蛋白。
结论本研究纯化得到了重组P54蛋白，为进一步深入研究P54蛋白的功能奠定基础。

Abstract:
Objective Pathogenicity and molecular mechanisms of tobacco mosaic virus (TMV), a typical member belonging to Tobamovirus of Vigaviridae that infect more than 400 species in 36 families of plants causing 20%-30% reduction or complete loss on crop yield, were studied.
Methods Sequence of the 1 425 bp TMV specific P54 protein was amplified by RT-PCR from the cDNA of Nicotiana tabacum var. Samsun NN infected with TMV and cloned into prokaryotic expressing plasmid, pEASY^®-Blunt E1, followed by expressing in E. coli BL21 (DE3) by IPTG induction. Subsequently, the expression products were retrieved and purified by Ni-NTA chromatography and confirmed by western-blotting identification.
Results The genome of TMV was a positive-sense single-stranded RNA of 6 400 bp that encoded one structural protein and two nonstructural proteins. The amplified 1 425 bp P54 was inserted in the virus replication associated P183 gene. The prokaryotic expressed recombinant P54 was insoluble. It was retrieved and purified to show a molecular weight of approximately 60 kDa and verified by means of western blot.
Conclusions TMV P54 protein was successfully expressed and purified for further study on the devastating diseases on plants caused by TMV.

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