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

doi: 10.19303/j.issn.1008-0384.2021.02.011

李聪^1,,
田培洁²,
张宇³,
张德咏^{1, 3},
刘勇^{1, 3},
张松柏^{1, 3, ,}

1.
湖南大学研究生院隆平分院，湖南　长沙　410125
2.
湖南农业大学植物保护学院，湖南　长沙　410120
3.
湖南省农业科学院植物保护研究所，湖南　长沙　410125

基金项目: 国家自然科学基金项目（31701765）；国家大宗蔬菜产业体系项目（CARS-23-D-02）

详细信息

作者简介:
李聪（1996−），男，硕士生，主要从事植物病毒致病分子机制研究（E-mail：erzongli@hnu.edu.cn）

通讯作者:
张松柏（1980−），博士，研究员，主要从事蔬菜病害致害机制及绿色综合防控技术研究（E-mail：zsongb@hunaas.cn）

中图分类号: S 435
计量
- 文章访问数: 1192
- HTML全文浏览量: 224
- PDF下载量: 32
- 被引次数: 0
出版历程
- 收稿日期: 2020-10-27
- 修回日期: 2020-11-20
- 网络出版日期: 2021-02-08
- 刊出日期: 2021-02-28

Prokaryotic Expression and Purification of Tobacco Mosaic Virus Specific P54 Protein

LI Cong^1
,,
TIAN Peijie²,
ZHANG Yu³,
ZHANG Deyong^{1, 3},
LIU Yong^{1, 3},
ZHANG Songbai^{1, 3
, ,}

1.
Longping Branch, Graduate College, Hunan University, Changsha, Hunan　410125, China
2.
Plant Protection College, Hunan Agricultural University, Changsha, Hunan　410120, China
3.
Plant Protection Institute, Hunan Academy of Agricultural Science, Changsha, Hunan　410125, China

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。
- 烟草花叶病毒 /
- P54基因 /
- 原核表达 /
- 蛋白纯化
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.
- Tobacco mosaic virus /
- P54 gene /
- prokaryotic expression /
- protein purification

HTML全文

图 1 原核表达载体pEASY^®- Blunt E1::P54凝胶电泳分析

注：A：TMV P54基因PCR扩增产物；M：DNA分子量标准DL2000；1～2：TMV-P54基因PCR扩增产物；B：重组pEASY®-Blunt E1::P54菌落PCR产物；1：重组载体pEASY®-Blunt E1::P54，2：CK

Figure 1. Electrophoresis of recombinant prokaryotic expressing plasmid, pEASY-Blunt E1::P54

Note: A: PCR products of TMV P54; M: DNA ladder DL2000; 1-2: PCR products of TMV P54; B: PCR products from colonies containing recombinant plasmid, pEASY-Blunt E1::P54; 1: Recombinant plasmid, pEASY-Blunt E1::P54; 2: CK.

下载: 全尺寸图片幻灯片

图 2 P54蛋白诱导表达产物SDS-PAGE分析

注：M：蛋白分子量标准；1：含重组pEASY^®-Blunt E1::P54载体E coli未诱导沉淀；2：含pEASY^®-Blunt E1载体E coli诱导沉淀；3：含pEASY^®-Blunt E1载体E coli未诱导沉淀；4：含重组pEASY^®- Blunt E1::P54载体E coli诱导沉淀；5：含重组pEASY^®- Blunt E1::P54载体E coli未诱导上清；6：含pEASY^®- Blunt E1载体E coli诱导上清；7：含pEASY®- Blunt E1载体E coli未诱导上清；8：含重组pEASY^®- Blunt E1::P54载体E coli诱导上清

Figure 2. SDS-PAGE analysis on induced expression products of P54

Note: M: Protein ladder; 1: Precipitates from E. coli containing recombinant pEASY^®- Blunt E1::P54 without IPTG; 2: Precipitates from E. coli containing pEASY^®- Blunt E1 with IPTG; 3: Precipitates from E. coli containing pEASY®- Blunt E1 without IPTG; 4: Precipitates from E. coli containing pEASY^®- Blunt E1::P54 with ITPG; 5: Supernatants from E. coli containing pEASY^®- Blunt E1::P54 without IPTG; 6: Supernatants from E. coli containing pEASY^®- Blunt E1with IPTG; 7: Supernatants from E. coli containing pEASY^®- Blunt E1 without IPTG; 8: Supernatants from E. coli containing pEASY^®- Blunt E1::P54 with IPTG.

下载: 全尺寸图片幻灯片

图 3 TMV P54基因表达条件优化（A：28 ℃，B：37 ℃）

注：M：蛋白分子量标准；1～4：37 ℃, 0.1～ 1.2 mmol·L⁻¹ IPTG诱导表达上清液；5～8：37 ℃, 0.1 ～ 1.2 mmol·L⁻¹ IPTG 诱导表达沉淀；9～12：37 ℃，0.1 ～ 1.2 mmol·L⁻¹ IPTG诱导表达上清液；13～16：37 ℃，0.1 ～ 1.2 mmol·L⁻¹ IPTG诱导表达沉淀

Figure 3. SDS-PAGE analysis on prokaryotic expressing of TMV P54 (A: 28 ℃; B: 37 ℃)

Note: M: Protein ladder; 1-4: Supernatants of expressing products induced by 0.1-1.2 mmol·L⁻¹ IPTG at 28 ℃; 5-8: Precipitates of expressing products induced by 0.1-1.2 mmol·L⁻¹ IPTG at 28 ℃; 1-4: Supernatants of expressing products induced by 0.1-1.2 mmol·L⁻¹ IPTG at 37 ℃; 1-4: Precipitates of expressing products induced by 0.1-1.2 mmol·L⁻¹ IPTG at 37 ℃.

下载: 全尺寸图片幻灯片

图 4 纯化TMV P54重组蛋白SDS-PAGE分析

注：M：蛋白分子量标准；1～3：纯化P54重组蛋白

Figure 4. SDS-PAGE analysis on purified TMV P54

Note: M: Protein ladder; 1-3: Purified recombinant P54 protein.

下载: 全尺寸图片幻灯片

图 5 纯化TMV P54重组蛋白Western blotting分析

注：M：蛋白分子量标准；1～3：纯化P54重组蛋白；4～5：CK-

Figure 5. Western blot on purified TMV P54

Note: M: Protein ladder; 1-3: Purified recombinant P54 protein; 4-5: CK.

下载: 全尺寸图片幻灯片

参考文献(18)

[1]	BOS L. Crop losses caused by viruses [J]. Crop Protection, 1982, 1(3): 263−282.
[2]	ZIMMERN D. The 5′ end group of tobacco mosaic virus RNA is m7G5′ ppp5′ Gp [J]. Nucleic Acids Research, 1975, 2(7): 1189−1201.
[3]	HULL R, DAVIES J W. Genetic engineering with plant viruses, and their potential as vectors [J]. Advances in Virus Research, 1983, 28: 1−33.
[4]	CREAGER A N H, SCHOLTHOF K B G, SCHOLTHOF C H B. Tobacco mosaic virus: pioneering research for a century [J]. Tanpakushitsu Kakusan Koso Protein Nucleic Acid Enzyme, 1999, 11(3): 301−308.
[5]	贺振, 甘海锋, 陈雯, 等. 雀麦花叶病毒外壳蛋白基因原核表达及抗血清制备 [J]. 植物保护学报, 2020, 47(2):455−456. HE Z, GAN H F, CHEN W, et al. Prokaryotic expression of Brome mosaic virus coat protein gene and preparation of antiserum [J]. Journal of Plant Protection, 2020, 47(2): 455−456.(in Chinese)
[6]	BRUENING G, BEACHY R N, SCALLA R, et al. In vitro and in vivo translation of the ribonucleic acids of a cowpea strain of tobacco mosaic virus [J]. Virology, 1976, 71(2): 498−517.
[7]	PANG J, WU Y, LI Z, et al. Targeting of the human F8 at the multicopy rDNA locus in Hemophilia a patient-derived iPSCs using TALENickases [J]. Biochemical and Biophysical Research Communications, 2016, 472(1): 144−149.
[8]	MARZOCCHI E, GRILLI S, DELLA CIANA L, et al. Chemiluminescent detection systems of horseradish peroxidase employing nucleophilic acylation catalysts [J]. Analytical Biochemistry, 2008, 377(2): 189−194.
[9]	OSMAN T A M, BUCK K W. Identification of a region of the Tobacco mosaic virus 126- and 183-kilodalton replication proteins which binds specifically to the viral 3′-terminal tRNA-like structure [J]. Journal of Virology, 2003, 77(16): 8669.
[10]	DAS P P, MACHARIA M W, LIN Q S, et al. In planta proximity-dependent biotin identification (BioID) identifies a TMV replication co-chaperone NbSGT1 in the vicinity of 126 kDa replicase [J]. Journal of Proteomics, 2019, 204: 103402.
[11]	KONAKALLA N C, KALDIS A, BERBATI M, et al. Exogenous application of double-stranded RNA molecules from TMV p126 and CP genes confers resistance against TMV in tobacco [J]. Planta, 2016, 244(4): 1−9.
[12]	KURIHARA Y, INABA N, KUTSUNA N, et al. Binding of Tobamovirus replication protein with small RNA duplexes [J]. The Journal of General Virology, 2007, 88(Pt 8): 2347−2352.
[13]	邵碧英, 吴祖建, 林奇英, 等. 烟草花叶病毒复制酶介导抗性的研究进展 [J]. 生物技术通讯, 2003, 14(5):416−418. doi: 10.3969/j.issn.1009-0002.2003.05.022 SHAO B Y, WU Z J, LIN Q Y, et al. Research advances in tobacco mosaic virus replicase mediated resistance [J]. Letters in Biotechnology, 2003, 14(5): 416−418.(in Chinese) doi: 10.3969/j.issn.1009-0002.2003.05.022
[14]	RODRÍGUEZ-NEGRETE E, LOZANO-DURÁN R, PIEDRA-AGUILERA A, et al. Geminivirus Rep protein interferes with the plant DNA methylation machinery and suppresses transcriptional gene silencing [J]. The New Phytologist, 2013, 199(2): 464−475.
[15]	韩学波, 许崇波, 曾瑾. 大肠杆菌中外源基因的表达调节 [J]. 生物技术通讯, 2006, 17(3):418−420. doi: 10.3969/j.issn.1009-0002.2006.03.034 HAN X B, XU C B, ZENG J. Expressional regulation of heterologous gene in E. coli [J]. Letters in Biotechnology, 2006, 17(3): 418−420.(in Chinese) doi: 10.3969/j.issn.1009-0002.2006.03.034
[16]	陈明, 陈文静. 几种微生物表达系统的比较 [J]. 安徽农学通报, 2011, 17(3):68−71. doi: 10.3969/j.issn.1007-7731.2011.03.029 CHEN M, CHEN W J. Comparisons of some microbial expression systems [J]. Auhui Agricultural Science Bulletin, 2011, 17(3): 68−71.(in Chinese) doi: 10.3969/j.issn.1007-7731.2011.03.029
[17]	黄传臻, 刘香利, 曹汝菲, 等. 小麦CWI-B1的原核表达、纯化与多克隆抗体制备 [J]. 农业生物技术学报, 2017, 25(7):1102−1110. HUANG C Z, LIU X L, CAO R F, et al. Prokaryotic expression, purification antibody for wheat (Triticum aestivum) and preparation of polyclonal CWI-B1 [J]. Journal of Agricultural Biotechnology, 2017, 25(7): 1102−1110.(in Chinese)
[18]	VENTURA S. Microbial cell factories biomed central review sequence determinants of protein aggregation: tools to increase [J]. Journal of Clinical Oncology, 2005, 31(18): 2233−5.