猪流行性腹泻病毒Nsp5基因的原核表达及生物信息学分析
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摘要
旨在对猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)Nsp5基因进行原核表达,利用生物信息学软件,预测其编码蛋白的结构和功能,为了解PEDV致病机理奠定基础。本研究克隆PEDV/LY/2014/04毒株的Nsp5基因,亚克隆进pET-28a(+)原核表达载体,PCR和酶切验证重组质粒的构建,重组质粒pET-28a(+)-Nsp5转入E.coli BL21(DE3)中,SDS-PAGE检测Nsp5的表达和His band Ni+纯化情况,Vector NTI Advance等软件对Nsp5蛋白氨基酸组成、抗原表位、二级和三级结构进行预测和分析。结果表明,成功克隆并构建了重组质粒pET-28a(+)-Nsp5,表达重组蛋白大小约22 ku,且主要以包涵体形式存在,His band Ni+纯化后获得高纯度重组蛋白。Nsp5蛋白是由196个氨基酸残基组成的多肽,其分子质量的理论值为21 820.07 u,理论等电点(pI)为8.734,略偏碱;二级结构骨架中α-螺旋(h)占55.61%,β-折叠(t)占7.65%,无规则卷曲(c)占20.92%,延伸链(e)占15.82%;Nsp5蛋白质的三级结构中,中间段以α-螺旋为主作为骨架,C端多个复杂二级结构构成该蛋白的酶活性中心;B细胞抗原表位的预测,表明有15个潜在的B细胞优势表位。本研究为猪流行性腹泻病毒Nsp5蛋白质的生物学功能相关研究提供数据支持。
Porcine epidemic diarrhea virus(PEDV) nonstructural protein 5(Nsp5) gene was expressed by the prokaryotic expression system and the structure of the encoded protein was predicted by bioinformatics software for understanding the pathogenecity mechanism of PEDV. In this study, the Nsp5 gene of PEDV/LY/2014/04 strain was subcloned into prokaryotic expression vector pET-28 a, and the recombinant plasmid was identified by polymerase chain reaction(PCR) and restriction enzyme digestion. The recombinant plasmid pET-28 a(+)-Nsp5 was transformed into competent cells of Escherichia coli BL21(DE3). The expression and purification effect of Nsp5 were detected by SDS-PAGE. The Vector NTI Advance, online software was utilized to predict and analyze the amino acid composition, epitope, secondary and tertiary structure of Nsp5 protein. The results showed that the recombinant plasmid pET-28 a(+)-Nsp5 was successfully constructed, and approximately 22 ku recombinant protein was expressed and mainly existed in the form of inclusion bodies. High-purity recombinant protein was obtained after purifying by His band Ni+. The Nsp5 protein consisted of a polypeptide with 196 amino acid residues. The theoretical molecular mass of the slightly alkaline recombinant protein was 21 820.07 u, and the theoretical isoelectric point(pI) was 8.734. The secondary structure was composed of 55.61% α-helix(h), 7.65% β-fold(t), 20.92% irregular curl(c) and 15.82% extended chain(e). In the tertiary structure of the Nsp5 protein, the middle segment skeleton is mainly formed by α-helix, and a plurality of complex secondary structures at the C-end constituted the enzyme active center of the protein. The prediction of B cell epitope indicated that there were 15 potential B cell dominant epitope. This study provided data support for studying on the biological function of the porcine epidemic diarrhea virus Nsp5 protein.
Porcine epidemic diarrhea virus(PEDV) nonstructural protein 5(Nsp5) gene was expressed by the prokaryotic expression system and the structure of the encoded protein was predicted by bioinformatics software for understanding the pathogenecity mechanism of PEDV. In this study, the Nsp5 gene of PEDV/LY/2014/04 strain was subcloned into prokaryotic expression vector pET-28 a, and the recombinant plasmid was identified by polymerase chain reaction(PCR) and restriction enzyme digestion. The recombinant plasmid pET-28 a(+)-Nsp5 was transformed into competent cells of Escherichia coli BL21(DE3). The expression and purification effect of Nsp5 were detected by SDS-PAGE. The Vector NTI Advance, online software was utilized to predict and analyze the amino acid composition, epitope, secondary and tertiary structure of Nsp5 protein. The results showed that the recombinant plasmid pET-28 a(+)-Nsp5 was successfully constructed, and approximately 22 ku recombinant protein was expressed and mainly existed in the form of inclusion bodies. High-purity recombinant protein was obtained after purifying by His band Ni+. The Nsp5 protein consisted of a polypeptide with 196 amino acid residues. The theoretical molecular mass of the slightly alkaline recombinant protein was 21 820.07 u, and the theoretical isoelectric point(pI) was 8.734. The secondary structure was composed of 55.61% α-helix(h), 7.65% β-fold(t), 20.92% irregular curl(c) and 15.82% extended chain(e). In the tertiary structure of the Nsp5 protein, the middle segment skeleton is mainly formed by α-helix, and a plurality of complex secondary structures at the C-end constituted the enzyme active center of the protein. The prediction of B cell epitope indicated that there were 15 potential B cell dominant epitope. This study provided data support for studying on the biological function of the porcine epidemic diarrhea virus Nsp5 protein.
引文
[1]LI W T,LI H,LIU Y B,et al.New variants of porcine epidemic diarrhea virus,China,2011[J].Emerging Infectious Diseases,2012,18(8):1350-1353.
[2]ZHAO X Y,LI Z L,ZENG X D,et al.Sequence analysis of the spike gene of Porcine epidemic diarrhea virus isolated from South China during 2011-2015[J].Journal of Veterinary Science,2017,18(2):237-243.
[3]VAN DIEP N,SUEYOSHI M,NORIMINE J,et al.Molecular characterization of US-like and Asian non-S INDEL strains of porcine epidemic diarrhea virus(PEDV)that circulated in Japan during 2013-2016 and PEDVs collected from recurrent outbreaks[J].BMC Veterinary Research,2018,14(1):96-107.
[4]LARA-ROMERO R,GOMEZ-NUEZ L,CERRITEO-SNCHEZJ L,et al.Molecular characterization of the spike gene of the porcine epidemic diarrhea virus in Mexico,2013-2016[J].Virus Genes,2018,54(2):215-224.
[5]SUN J,LI Q J,SHAO C Y,et al.Isolation and characterization of Chinese porcine epidemic diarrhea virus with novel mutations and deletions in the S gene[J].Veterinary Microbiology,2018,221:81-89.
[6]MESQUITA J R,HAKZE-VAN DER HONING R H V,ALMEI-DA A,et al.Outbreak of porcine epidemic diarrhea virus in portugal,2015[J].Transboundary and Emerging Diseases,2015,62(6):586-588.
[7]LI Z L,ZHU L,MA J Y,et al.Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus(PEDV)field strains in south China[J].Virus Genes,2012,45(1):181-185.
[8]JUNG K,ANNAMALAI T,LU Z Y,et al.Comparative pathogenesis of US porcine epidemic diarrhea virus(PEDV)strain PC21A in conventional 9-day-old nursing piglets vs.26-dayold weaned pigs[J].Veterinary Microbiology,2015,178(1/2):31-40.
[9]ZHANG Q Z,SHI K C,YOO D.Suppression of type I interferon production by porcine epidemic diarrhea virus and degradation of CREB-binding protein by nsp1[J].Virology,2016,489:252-268.
[10]ZHANG Q Z,KE H Z,BLIKSLAGER A,et al.Type III interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp1 in IRF1 signaling[J].Journal of Virology,2017,92(4):JVI.01677-17.
[11]YE G,DENG F,SHEN Z,et al.Structural basis for the dimerization and substrate recognition specificity of porcine epidemic diarrhea virus 3C-like protease[J].Virology,2016,494:225-235.
[12]ST JOHN S E,ANSON B J,MESECAR A D.X-ray structure and inhibition of 3C-like protease from porcine epidemic diarrhea virus[J].Scientific Reports,2016,6:25961.
[13]WANG D,FANG L R,SHI Y L,et al.Porcine epidemic diarrhea virus 3C-like protease regulates its interferon antagonism by cleaving NEMO[J].Journal of Virology,2016,90(4):2090-2101.
[14]JARU-AMPORNPAN P,JENGARN J,WANITCHANG A,et al.Porcine epidemic diarrhea virus 3C-like protease-mediated nucleocapsid processing:possible link to viral cell culture adaptability[J].Journal of Virology,2017,91(2):e01660-16.
[15]ZHU X Y,WANG D,ZHOU J W,et al.Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving STAT2[J].Journal of Virology,2017,91(10):pii:e00003-17.
[16]TOMAR S,JOHNSTON M L,ST JOHN S E,et al.Ligandinduced dimerization of middle east respiratory syndrome(MERS)coronavirus nsp5 protease(3CLpro)[J].Journal of Biological Chemistry,2015,290(32):19403-19422.
[17]PERLMAN S,NETLAND J.Coronaviruses post-SARS:update on replication and pathogenesis[J].Nature Reviews Microbiology,2009,7(6):439-450.
[18]ZHU X Y,FANG L R,WANG D,et al.Porcine deltacoronavirus nsp5 inhibits interferon-βproduction through the cleavage of NEMO[J].Virology,2017,502:33-38.
[19]SHI Y J,LEI Y Y,YE G,et al.Identification of two antiviral inhibitors targeting 3C-like Serine/3C-like protease of porcine reproductive and respiratory syndrome virus and porcine epidemic diarrhea virus[J].Veterinary Microbiology,2018,213:114-122.
[20]REN Z L,YAN L M,ZHANG N,et al.The newly emerged SARS-like coronavirus HCoV-EMC also has an“Achilles'heel”:current effective inhibitor targeting a 3C-like protease[J].Protein&Cell,2013,4(4):248-250.[LinkOut]
[21]SHI J H,HAN N Y,LIM L,et al.Dynamically-driven inactivation of the catalytic machinery of the SARS 3C-like protease by the N214A mutation on the extra domain[J].PLoSComputational Biology,2011,7(2):e1001084.
[22]LEE C C,KUO C J,KO T P,et al.Structural basis of inhibition specificities of 3C and 3C-like proteases by zinc-coordinating and peptidomimetic compounds[J].Journal of Biological Chemistry,2009,284(12):7646-7655.
[23]KUMAR V,TAN K P,WANG Y M,et al.Identification,synthesis and evaluation of SARS-CoV and MERS-CoV 3C-like protease inhibitors[J].Bioorganic&Medicinal Chemistry,2016,24(13):3035-3042.
[2]ZHAO X Y,LI Z L,ZENG X D,et al.Sequence analysis of the spike gene of Porcine epidemic diarrhea virus isolated from South China during 2011-2015[J].Journal of Veterinary Science,2017,18(2):237-243.
[3]VAN DIEP N,SUEYOSHI M,NORIMINE J,et al.Molecular characterization of US-like and Asian non-S INDEL strains of porcine epidemic diarrhea virus(PEDV)that circulated in Japan during 2013-2016 and PEDVs collected from recurrent outbreaks[J].BMC Veterinary Research,2018,14(1):96-107.
[4]LARA-ROMERO R,GOMEZ-NUEZ L,CERRITEO-SNCHEZJ L,et al.Molecular characterization of the spike gene of the porcine epidemic diarrhea virus in Mexico,2013-2016[J].Virus Genes,2018,54(2):215-224.
[5]SUN J,LI Q J,SHAO C Y,et al.Isolation and characterization of Chinese porcine epidemic diarrhea virus with novel mutations and deletions in the S gene[J].Veterinary Microbiology,2018,221:81-89.
[6]MESQUITA J R,HAKZE-VAN DER HONING R H V,ALMEI-DA A,et al.Outbreak of porcine epidemic diarrhea virus in portugal,2015[J].Transboundary and Emerging Diseases,2015,62(6):586-588.
[7]LI Z L,ZHU L,MA J Y,et al.Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus(PEDV)field strains in south China[J].Virus Genes,2012,45(1):181-185.
[8]JUNG K,ANNAMALAI T,LU Z Y,et al.Comparative pathogenesis of US porcine epidemic diarrhea virus(PEDV)strain PC21A in conventional 9-day-old nursing piglets vs.26-dayold weaned pigs[J].Veterinary Microbiology,2015,178(1/2):31-40.
[9]ZHANG Q Z,SHI K C,YOO D.Suppression of type I interferon production by porcine epidemic diarrhea virus and degradation of CREB-binding protein by nsp1[J].Virology,2016,489:252-268.
[10]ZHANG Q Z,KE H Z,BLIKSLAGER A,et al.Type III interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp1 in IRF1 signaling[J].Journal of Virology,2017,92(4):JVI.01677-17.
[11]YE G,DENG F,SHEN Z,et al.Structural basis for the dimerization and substrate recognition specificity of porcine epidemic diarrhea virus 3C-like protease[J].Virology,2016,494:225-235.
[12]ST JOHN S E,ANSON B J,MESECAR A D.X-ray structure and inhibition of 3C-like protease from porcine epidemic diarrhea virus[J].Scientific Reports,2016,6:25961.
[13]WANG D,FANG L R,SHI Y L,et al.Porcine epidemic diarrhea virus 3C-like protease regulates its interferon antagonism by cleaving NEMO[J].Journal of Virology,2016,90(4):2090-2101.
[14]JARU-AMPORNPAN P,JENGARN J,WANITCHANG A,et al.Porcine epidemic diarrhea virus 3C-like protease-mediated nucleocapsid processing:possible link to viral cell culture adaptability[J].Journal of Virology,2017,91(2):e01660-16.
[15]ZHU X Y,WANG D,ZHOU J W,et al.Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving STAT2[J].Journal of Virology,2017,91(10):pii:e00003-17.
[16]TOMAR S,JOHNSTON M L,ST JOHN S E,et al.Ligandinduced dimerization of middle east respiratory syndrome(MERS)coronavirus nsp5 protease(3CLpro)[J].Journal of Biological Chemistry,2015,290(32):19403-19422.
[17]PERLMAN S,NETLAND J.Coronaviruses post-SARS:update on replication and pathogenesis[J].Nature Reviews Microbiology,2009,7(6):439-450.
[18]ZHU X Y,FANG L R,WANG D,et al.Porcine deltacoronavirus nsp5 inhibits interferon-βproduction through the cleavage of NEMO[J].Virology,2017,502:33-38.
[19]SHI Y J,LEI Y Y,YE G,et al.Identification of two antiviral inhibitors targeting 3C-like Serine/3C-like protease of porcine reproductive and respiratory syndrome virus and porcine epidemic diarrhea virus[J].Veterinary Microbiology,2018,213:114-122.
[20]REN Z L,YAN L M,ZHANG N,et al.The newly emerged SARS-like coronavirus HCoV-EMC also has an“Achilles'heel”:current effective inhibitor targeting a 3C-like protease[J].Protein&Cell,2013,4(4):248-250.[LinkOut]
[21]SHI J H,HAN N Y,LIM L,et al.Dynamically-driven inactivation of the catalytic machinery of the SARS 3C-like protease by the N214A mutation on the extra domain[J].PLoSComputational Biology,2011,7(2):e1001084.
[22]LEE C C,KUO C J,KO T P,et al.Structural basis of inhibition specificities of 3C and 3C-like proteases by zinc-coordinating and peptidomimetic compounds[J].Journal of Biological Chemistry,2009,284(12):7646-7655.
[23]KUMAR V,TAN K P,WANG Y M,et al.Identification,synthesis and evaluation of SARS-CoV and MERS-CoV 3C-like protease inhibitors[J].Bioorganic&Medicinal Chemistry,2016,24(13):3035-3042.
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