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

Abstract

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

CLC Number:

S855.3

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.

References

[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.

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

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