MERS-CoV S1蛋白的表达及鉴定
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摘要
目的真核表达具有天然构象的中东呼吸综合征冠状病毒(MERS-CoV)S1蛋白,并进行免疫反应性鉴定。方法以pcDNA3.1-S重组质粒为模板,PCR扩增S1基因,连接至pFastBac1质粒,构建重组质粒pFB1-S1。将pFB1-S1转化至DH10Bac感受态中,构建重组杆粒BpFB1-S1,然后转染sf9细胞获得重组杆状病毒rpFB1-S1并连续传代。将第四代rpFB1-S1接种sf9细胞后悬浮培养96h,取上清,用PEG8000浓缩并进行免疫亲和层析纯化,采用SDS-PAGE和Western blot鉴定其分子质量及免疫反应性;另取感染rpFB1-S1 48h的sf9细胞进行IFA鉴定。结果 S1基因PCR扩增产物大小为2 212bp,与预期相符。重组质粒pFB1-S1经酶切和测序分析鉴定构建正确。重组杆粒BpFB1-S1经PCR鉴定构建正确。IFA试验可见感染rpFB1-S1的sf9细胞出现特异性绿色荧光,即有目的蛋白表达。SDSPAGE显示纯化的重组S1为单一100×103(Mr)蛋白条带,Western blot检测该蛋白能被鼠抗S-RBD蛋白单克隆抗体识别。结论成功表达了具有免疫反应性的MERS-CoV S1蛋白,为MERS-CoV快速诊断方法的建立及候选疫苗的构建奠定了基础。
Objectives To express the MERS-CoV S1 protein with natural conformations using a baculovirus/insect cell system and to determine its immunoreactivity. Methods The plasmid pcDNA3.1-S was used as a template to amplify the S1 gene using PCR.After double-enzyme digestion,the S1 gene and pFastBac1 plasmid were ligated to construct the recombinant plasmid pFB1-S1.The pFB1-S1 plasmid was transformed into DH10 Bac competent cells to construct the recombinant bacmid BpFB1-S1.The recombinant baculovirus rpFB1-S1 was rescued by transfecting the BpFB1-S1 bacmid into sf9 cells and was passaged in succession.A suspension of sf9 cells was cultured for 96 hafter infection with the fourth passage of rpFB1-S1.The supernatant was concentrated with PEG8000 and purified using immunoaffinity chromatography.The molecular mass of the purified protein was determined with SDS-PAGE and its immunoreactivity was determined with Western blotting.sf9 cells infected with rpFB1-S1 for 48 hwere identified using an IFA. Results The PCR product was 2 212 bp in length.The pFB1-S1 plasmid was verified as correct using restriction analysis and DNA sequencing.The BpFB1-S1 bacmid was verified using PCR.In an IFA,specific green fluorescence was observed in sf9 cells infected with rpFB1-S1,i.e.the protein of interest was expressed.SDS-PAGE indicated that the purified S1 protein was a single 100×103(Mr)protein band.Western blotting indicated that the purified protein was recognized by mouse anti-S-RBD monoclonal antibody. Conclusion Immunoreactive MERS-CoV S1 protein was successfully expressed.This has laid the foundation for the development of methods to rapidly diagnose MERS-CoV and to create potential vaccines.
Objectives To express the MERS-CoV S1 protein with natural conformations using a baculovirus/insect cell system and to determine its immunoreactivity. Methods The plasmid pcDNA3.1-S was used as a template to amplify the S1 gene using PCR.After double-enzyme digestion,the S1 gene and pFastBac1 plasmid were ligated to construct the recombinant plasmid pFB1-S1.The pFB1-S1 plasmid was transformed into DH10 Bac competent cells to construct the recombinant bacmid BpFB1-S1.The recombinant baculovirus rpFB1-S1 was rescued by transfecting the BpFB1-S1 bacmid into sf9 cells and was passaged in succession.A suspension of sf9 cells was cultured for 96 hafter infection with the fourth passage of rpFB1-S1.The supernatant was concentrated with PEG8000 and purified using immunoaffinity chromatography.The molecular mass of the purified protein was determined with SDS-PAGE and its immunoreactivity was determined with Western blotting.sf9 cells infected with rpFB1-S1 for 48 hwere identified using an IFA. Results The PCR product was 2 212 bp in length.The pFB1-S1 plasmid was verified as correct using restriction analysis and DNA sequencing.The BpFB1-S1 bacmid was verified using PCR.In an IFA,specific green fluorescence was observed in sf9 cells infected with rpFB1-S1,i.e.the protein of interest was expressed.SDS-PAGE indicated that the purified S1 protein was a single 100×103(Mr)protein band.Western blotting indicated that the purified protein was recognized by mouse anti-S-RBD monoclonal antibody. Conclusion Immunoreactive MERS-CoV S1 protein was successfully expressed.This has laid the foundation for the development of methods to rapidly diagnose MERS-CoV and to create potential vaccines.
引文
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[15]Du L,Tai W,Zhou Y,et al.Vaccines for the prevention against the threat of MERS-CoV[J].Expert Rev Vaccines,2016,15(9):1123-34.
[16]Chi H,Zheng X,Wang X,et al.DNA vaccine encoding Middle East Respiratory Syndrome coronavirus S1protein induces protective immune responses in mice[J].Vaccine,2017,35(16):2069-75.
[17]Kim E,Okada K,Kenniston T,et al.Immunogenicity of an adenoviral-based Middle East Respiratory Syndrome coronavirus vaccine in BALB/c mice[J].Vaccine,2014,32(45):5975-82.
[18]AL-Amri SS,Abbas AT,Siddiq LA,et al.Immunogenicity of candidate MERS-CoV DNA vaccines based on the spike protein[J].Sci Rep,2017,7(1):44875.
[19]Iatrou K.Baculovirus expression vectors:A laboratory manual[J].Cell,1993,74(1):7-8.
[2]Bermingham A,Chand MA,Brown CS,et al.Severe respiratory illness caused by a novel coronavirus,in a patient transferred to the United Kingdom from the Middle East,September 2012[J].Euro Surveill,2012,17(40):20290.
[3]Zaki AM,Van Boheemen S,Bestebroer TM,et al.Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia[J].N Engl J Med,2012,367(19):1814-20.
[4]Chan JF,Liks M,To kk W,et al.Is the discovery of the novel human betacoronavirus 2cEMC/2012(HCoV-EMC)the beginning of another SARS-like pandemic?[J].J Infect,2012,65(6):477-89.
[5]Chan JF,Lau SK,To KK,et al.Middle East respiratory syndrome coronavirus:another zoonotic betacoronavirus causing SARS-like disease[J].Clin Microbiol Rev,2015,28(2):465-522.
[6]Cheng VC,Lau SK,Woo PC,et al.Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection[J].Clin Microbiol Rev,2007,20(4):660-94.
[7]Woo PC Y,Lau SK,Yuen KY.Infectious diseases emerging from Chinese wet-markets_zoonotic origins of severe respiratory viral infections[J].Curr Opin Infect Dis,2006,19(5):401-7.
[8]Graham RL,Donaldson EF,Baric RS.A decade after SARS:strategies for controlling emerging coronaviruses[J].Nat Rev Microbiol,2013,11(12):836-48.
[9]Josset L,Menachery VD,Gralinski LE,et al.Cell host response to infection with novel human coronavirus EMC predicts potential antivirals and important differences with SARS coronavirus[J].MBio,2013,4(3):e00165-13.
[10]Van Boheemen S,de Graaf M,Lauber C,et al.Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans[J].MBio,2012,3(6):e00473-12.
[11]Qian Z,Dominguez SR,Holmes KY,et al.Role of the spike glycoprotein of human middle east respiratory syndrome coronavirus(MERS-CoV)in virus entry and syncytia formation[J].PLoS One,2013,8(10):e76469.
[12]Raj VS,Mou H,Smits SL,et al.Dipeptidyl peptidase 4is a functional receptor for the emerging human coronavirus-EMC[J].Nature,2013,495(7440):251-4.
[13]Modjarrad K.MERS-CoV vaccine candidates in development:The current landscape[J].Vaccine,2016,34(26):2982-7.
[14]Kumar V,Jung YS,Liang PH.Anti-SARS coronavirus agents:apatent review(2008-present)[J].Expert Opin Ther Pat,2013,23(10):1337-48.
[15]Du L,Tai W,Zhou Y,et al.Vaccines for the prevention against the threat of MERS-CoV[J].Expert Rev Vaccines,2016,15(9):1123-34.
[16]Chi H,Zheng X,Wang X,et al.DNA vaccine encoding Middle East Respiratory Syndrome coronavirus S1protein induces protective immune responses in mice[J].Vaccine,2017,35(16):2069-75.
[17]Kim E,Okada K,Kenniston T,et al.Immunogenicity of an adenoviral-based Middle East Respiratory Syndrome coronavirus vaccine in BALB/c mice[J].Vaccine,2014,32(45):5975-82.
[18]AL-Amri SS,Abbas AT,Siddiq LA,et al.Immunogenicity of candidate MERS-CoV DNA vaccines based on the spike protein[J].Sci Rep,2017,7(1):44875.
[19]Iatrou K.Baculovirus expression vectors:A laboratory manual[J].Cell,1993,74(1):7-8.