猪脑心肌炎病毒2A蛋白的原核表达与纯化

doi:10.16431/j.cnki.1671-7236.2020.11.005

中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (11): 3454-3459.doi: 10.16431/j.cnki.1671-7236.2020.11.005

猪脑心肌炎病毒2A蛋白的原核表达与纯化

韩若婵^1,2,3, 梁瑞英¹, 左玉柱⁴, 萧飒², 崔尚金¹

1. 中国农业科学院北京畜牧兽医研究所, 北京 100193;
2. 西北农林科技大学动物医学院, 杨凌 712100;
3. 保定职业技术学院畜牧兽医系, 保定 071001;
4. 河北农业大学动物医学院, 保定 071001

收稿日期:2020-05-27 出版日期:2020-11-20 发布日期:2020-11-20
通讯作者: 萧飒, 崔尚金 E-mail:saxiao@nwafu.edu.cn;cuishangjin@caas.cn
作者简介:韩若婵(1981-),女,河北保定人,博士生,研究方向:动物传染病致病机理,E-mail:hanruochan@126.com
基金资助:
国家自然科学基金面上项目"猪脑心肌炎病毒2A蛋白调控宿主细胞凋亡分子机制研究"（31873019）；河北省重点研发计划项目（19226622D）

Prokaryotic Expression and Purification of 2A Protein of Porcine Encephalomyocarditis Virus

HAN Ruochan^1,2,3, LIANG Ruiying¹, ZUO Yuzhu⁴, XIAO Sa², CUI Shangjin¹

1. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
2. College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China;
3. Department of Animal Husbandry and Veterinary Medicine, Baoding Vocational and Technical College, Baoding 071001, China;
4. College of Veterinary Medicine, Hebei Agricultural University, Baoding 071001, China

Received:2020-05-27 Online:2020-11-20 Published:2020-11-20

摘要/Abstract

摘要： 本试验对猪脑心肌炎病毒（encephalomyocarditis virus，EMCV）2A蛋白进行原核表达和纯化。采用大肠杆菌原核表达系统，将EMCV 2A基因插入原核表达载体pET-28a-sumo中构建重组原核表达质粒pET28a-EMCV-2A，经PCR和测序鉴定无误。将重组原核表达质粒转化至大肠杆菌BL21（DE3）感受态细胞中，超声破碎后，2A蛋白的表达主要以包涵体形式存在。通过优化蛋白表达条件，使目的蛋白能够实现可溶性表达。利用磁珠纯化方法对重组蛋白进行纯化，并以SDS-PAGE和Western blotting进行双重鉴定。结果显示，本试验成功构建2A的重组表达质粒；重组蛋白分子质量约为25 ku，与预期大小一致；IPTG浓度1.0 mmol/L、16 ℃低温诱导16 h为最佳诱导条件，约有50%的蛋白呈现可溶性表达；纯化后获得2A重组蛋白。本试验通过对EMCV 2A蛋白的原核表达及纯化，成功获得纯度较高的EMCV 2A蛋白，为进一步阐明EMCV的分子致病机制以及研发基因疫苗和抗病毒药物奠定基础。

关键词: 脑心肌炎病毒(EMCV); 2A蛋白; 原核表达; 纯化

Abstract: In this study,encephalomyocarditis virus 2A protein was prokaryotic expressed and purified.E.coli was used to express foreign proteins,the EMCV 2A gene was inserted into the prokaryotic expression vector pET-28a-sumo to obtain the recombinant plasmid pET28a-EMCV-2A and confirmed by PCR and sequencing.The plasmid was transformed into E.coli BL21(DE3).After ultrasound fragmentation,the expression of 2A protein was mainly in the form of inclusion bodies.By optimizing the protein expression conditions,the soluble expression of the target protein could be achieved.The recombinant protein was purified by beads and identified by SDS-PAGE and Western blotting.The results showed that the recombinant plasmid was successfully constructed.The molecular weight of the recombinant 2A protein was about 25 ku.The best condition was 16 ℃ with 1.0 mmol/L IPTG,about 50% of the proteins were soluble.The purified recombinant protein was double-stained by SDS-PAGE and Western blotting,and indicated that the purified protein was the recombinant protein.The study provided the basic theoretical basis for elucidating the molecular pathogenesis of EMCV and preparation of gene vaccines and antiviral drugs.

Key words: encephalomyocarditis virus (EMCV); 2A protein; prokaryotic expression; purification

中图分类号:

S855.3

韩若婵, 梁瑞英, 左玉柱, 萧飒, 崔尚金. 猪脑心肌炎病毒2A蛋白的原核表达与纯化[J]. 中国畜牧兽医, 2020, 47(11): 3454-3459.

HAN Ruochan, LIANG Ruiying, ZUO Yuzhu, XIAO Sa, CUI Shangjin. Prokaryotic Expression and Purification of 2A Protein of Porcine Encephalomyocarditis Virus[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(11): 3454-3459.

参考文献

[1] GELMETTI D,MERONI A,BROCCHI E,et al.Pathogenesis of encephalomyocarditis experimental infection in young piglets:A potential animal model to study viral myocarditis[J].Veterinary Research,2006,37(1):15-23.
[2] PAPAIOANNOU N,BILLINIS C,PSYCHAS V,et al.Pathogenesis of encephalomyocarditis virus (EMCV) infection in piglets during the viraemia phase:A histopathological,immunohistochemical and virological study[J].Journal of Comparative Pathology,2003,129:161-168.
[3] KOENEN F,VANDERHALLEN H.Comparative study of the pathogenic properties of a Belgian and a Greek encephalomyocarditis virus (EMCV) isolate for sows in gestation[J].Zentralbl Veterinarmed B,1997,44(5):281-286.
[4] CAROCCI M,BAKKALI-KASSIMI L.The encephalo-myocarditis virus[J].Virulence,2012,3:351-367.
[5] AMINEV A G,AMINEVA S P,PALMENBERG A C.Encephalomyocarditis viral protein 2A localizes to nucleoli and inhibits cap-dependent mRNA translation[J].Veterinary Research,2003,95(1-2):45-57.
[6] GROPPO R,PALMENBERG A C.Cardiovirus 2A protein associates with 40S but not 80S ribosome subunits during infection[J].Journal of Virology,2007,81(23):13067-13074.
[7] AGOL V I,GMYL A P.Viral security proteins:Counteracting host defences[J].Nature Reviews Microbiology,2010,8(12):867-878.
[8] GROPPO R,BROWN B A,PALMENBERG A C.Mutational analysis of the EMCV 2A protein identifies a nuclear localization signal and an eIF4E binding site[J].Virology,2011,410(1):257-267.
[9] CAROCCI M,CORDONNIER N,HUER H,et al.Encephalomyocarditis virus 2A protein is required for viral pathogenesis and inhibition of apoptosis[J].Journal of Virology,2011,85(20):10741-10754.
[10] WEI J,ZHANG H,LI X,et al.Transcriptional profiling of host cell responses to encephalomyo-carditis virus (EMCV)[J].Virology Journal,2017,14(1):45.
[11] BAZZONE L E,KING M,MACKAY C R,et al.A disintegrin and metalloproteinase 9 domain (ADAM9) is a major susceptibility factor in the early stages of encephalomyocarditis virus infection[J].American Society for Microbiology,2019,10(1):e02734-18.
[12] LI L,FAN H,SONG Z,et al.Encephalomyocarditis virus 2C protein antagonizes interferon-β signaling pathwaythrough interaction with MDA5[J].Antiviral Research,2019,161:70-84.
[13] OHGUCHI A,YAMAUCHI H,DOI K,et al.Activation of type Ⅰ interferon signaling in the parotid and exorbital lachrymal glands during the acute phase of encephalomyocarditis (EMC) virus infection in mice[J].Experimental and Molecular Pathology,2016,100(3):434-440.
[14] ROOS F C,ROBERS A M,HWANG I I L,et al.Oncolytic targeting of renal cell carcinoma via encephalomyocarditis virus[J].EMBO Molecular Medicine,2010,2(7):275-288.
[15] MORRISON J M,RACANIELLO V R.Proteinase 2A pro is essential for enterovirus replication in type Ⅰ interferon-treated cells[J].Journal of Virology,2009,83(9):4412-4422.
[16] VISSER L J,LANGEREIS M A,RABOUW H H,et al.Essential role of enterovirus 2A protease in counteracting stress granule formation and the induction of type Ⅰ interferon[J].Journal of Virology,2019,93(10):e00222-19.
[17] NIX W A,KHETSURIANI N,PENARANDA S,et al.Diversity of picornaviruses in rural Bolivia[J].Journal of General Virology,2013,94(Pt 9):2017-2028.
[18] SONG Q Q,LU M Z,SONG J,et al.Coxsackievirus B32A protease promotes encephalomyocarditis virus replication[J].Veterinary Research,2015,208:22-29.
[19] NAPTHINE S,LING R,FINCH LK,et al.Protein-directed ribosomal frameshifting temporally regulates gene expression[J].Nature Communications,2017,8:15582.
[20] YU H,HUANG L,ZHANG Y,et al.An attenuated EMCV-HB10 strain acts as a live viral vector delivering a foreign gene[J].Journal of General Virology,2016,97:2280-2290.
[21] BAI J,LI L,GAO Y,et al.Inhibition of encephalomyocarditis virus replication by shRNA targeting 1C and 2A genes in vitro and in vivo[J].Veterinary Microbiology,2020,244:108664.

猪脑心肌炎病毒2A蛋白的原核表达与纯化

Prokaryotic Expression and Purification of 2A Protein of Porcine Encephalomyocarditis Virus

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