呈现埃博拉病毒包膜糖蛋白抗原表位重组病毒样颗粒的构建
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摘要
目的构建呈现埃博拉病毒(Ebola virus,EBOV)包膜糖蛋白(glycoprotein,GP)抗原表位的病毒样颗粒(viruslike particles,VLPs)。方法通过EBOV GP抗原性预测,并结合其功能结构域分析获得潜在的B细胞表位,经基因重组将该抗原表位插入至乙肝核心抗原(HBcAg)的优势表位区域,SDS-PAGE法分析重组HBcAg抗原表位嵌合蛋白的表达。重组菌体破碎上清经硫酸铵沉淀和蔗糖密度梯度离心纯化后,通过电镜观察VLPs形态。将VLPs与Alum佐剂按1∶1的比例混合,经背部皮下免疫小鼠。ELISA及Western blot法检测小鼠血清的特异性。结果重组HBcAg抗原表位嵌合蛋白的相对分子质量约19 300,纯化后蛋白浓度为1.244 mg/ml,镜下可见其以VLPs形式存在。VLPs免疫小鼠血清可与GP发生特异性免疫反应。结论成功构建了呈现EBOV GP抗原表位的VLPs,其可有效诱导小鼠产生特异性免疫应答。本实验为EBOV基因重组疫苗的研究及特异性抗体的制备奠定了基础。
Objective To construct virus-like particles(VLPs) embedding an antigenic epitope of envolope glycoprotein(GP) of Zaire Ebola virus(EBOV). Methods Potential B cell epitopes were predicted by analyzing the antigenicity and domain of GP peptides. Oligonucleotides encoding for the predicted antigenic peptides were synthesized and inserted into plasmid vector p Thio His A-HBcAg through gene recombination. The expression of chimeric HBcAg/GP peptide was analyzed by SDS-PAGE. After purification by ammonium sulfate precipitation and sucrose density gradient centrifugation,the protein samples were observed by electron microscopy for the morphology of VLPs. BALB/c mice were immunized with the purified VLPs mixed with alum adjuvant at a ratio of 1 ∶ 1, of which the specific antibody in sera was measured by ELISA and Western blot. Results Recombinant HBcAg antigenic epitope chimeric protein, with a relative molecular mass of about 19 300, reached a concentration of 1. 244 mg/ml and existed in a form of VLPs under microscope. The sera of mice immunized with the VLPs showed specific reaction with GP. Conclusion The VLPs embedding GP epitope of EBOV induced specific immune response in mice, which laid a foundation of study on recombinant EBOV vaccine and preparation of specific antibody.
Objective To construct virus-like particles(VLPs) embedding an antigenic epitope of envolope glycoprotein(GP) of Zaire Ebola virus(EBOV). Methods Potential B cell epitopes were predicted by analyzing the antigenicity and domain of GP peptides. Oligonucleotides encoding for the predicted antigenic peptides were synthesized and inserted into plasmid vector p Thio His A-HBcAg through gene recombination. The expression of chimeric HBcAg/GP peptide was analyzed by SDS-PAGE. After purification by ammonium sulfate precipitation and sucrose density gradient centrifugation,the protein samples were observed by electron microscopy for the morphology of VLPs. BALB/c mice were immunized with the purified VLPs mixed with alum adjuvant at a ratio of 1 ∶ 1, of which the specific antibody in sera was measured by ELISA and Western blot. Results Recombinant HBcAg antigenic epitope chimeric protein, with a relative molecular mass of about 19 300, reached a concentration of 1. 244 mg/ml and existed in a form of VLPs under microscope. The sera of mice immunized with the VLPs showed specific reaction with GP. Conclusion The VLPs embedding GP epitope of EBOV induced specific immune response in mice, which laid a foundation of study on recombinant EBOV vaccine and preparation of specific antibody.
引文
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[10]URATA S,NADA T,KAWAOKA Y,et al.Interaction of Tsg-101 with Marburg virus VP40 depends on the PPPY motif,but not the PT/SAP motif as in the case of Ebola virus,and Tsg-101 plays a critical role in the budding of Marburg virus-like particles induced by VP40,NP,and GP[J].J Virol,2007,81(9):4895-4899.
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[12]SUN P Y,LI K,LIU C B,et al.Co-expression of Ebola virus GP and VP40 proteins and virus-like particles assembly in mammalian cells[J].China Biotechnol,2016,36(12):66-71.(in Chinese)孙鹏艳,黎奎,刘存宝,等.埃博拉病毒GP和VP40蛋白在哺乳动物细胞中的共表达及病毒样颗粒装配[J].中国生物工程杂志,2016,36(12):66-71.
[13]VOLCHKOV V E,FELDMANN H,VOLCHKOVA V A,et al.Processing of the Ebola virus glycoprotein by the proprotein convertase furin[J].Proc Natl Acad Sci USA,1998,95(10):5762-5767.
[14]LEE J E,FUSCO M L,HESSELL A J,et al.Structure of the Ebola virus glycoprotein bound to an antibody form a human survivor[J].Nature,2008,454(7201):177-182.
[15]SULLIVAN N,PETERSON M,YANG Z Y,et al.Ebola virus glycoprotein toxicity is mediated by a dynamin-dependent protein-trafficking pathway[J].J Virol,2005,79(1):547-553.
[16]YANG Z,DUCKERS H J,SULLIVAN N J,et al.Identification of the Ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury[J].Nat Med,2000,6(8):886-889.
[2]WHO.Ebola Response Roadmap Situation Report[OL].(2014-10-15)[2014-10-16].http//www.who.int/csr/disease/ebola/situation-reports/archive/en/.
[3]WONG G,KOBINGER G P,QIU X.Characterization of host immune responses in Ebola virus infections[J].Expert Rev Clin Immunol,2014,10(6):781-790.
[4]WARFIELD K L,BOSIO C M,WELCHER B C,et al.Ebola virus-like particles protect from lethal Ebola virus infection[J].Proc Natl Acad Sci USA,2003,100(26):15889-15894.
[5]DOWLING W,THOMPSON E,BADGER C,et al.Influences of glycosylation on antigenicity,immunogenicity,and protective efficacy of ebola virus GP DNA vaccines[J].J Virol,2007,81(4):1821-1837.
[6]MARTINS K A,WARREN T K,BAVARI S.Characterization of a putative filovirus vaccine:virus-like particles[J].Virol Sin,2013,28(2):65-70.
[7]SCHILLER J T,LOWY D R.Papillomavirus-like particle vaccines[J].J Natl Cancer Inst Monogr,2000,5(28):50-54.
[8]ULRICH R,NASSAL M,MEISEL H,et al.Core particles of hepatitis B virus as carrier for foreign epitopes[J].Adv Virus Res,1998,50(1):141-182.
[9]BEYER T,HERRMANN M,REISER C,et al.Bacterial carriers and virus-like-particles as antigen delivery devices:role of dendritic cells in antigen presentation[J].Curr Durg Targets Infect Disord,2001,1(3):287-302.
[10]URATA S,NADA T,KAWAOKA Y,et al.Interaction of Tsg-101 with Marburg virus VP40 depends on the PPPY motif,but not the PT/SAP motif as in the case of Ebola virus,and Tsg-101 plays a critical role in the budding of Marburg virus-like particles induced by VP40,NP,and GP[J].J Virol,2007,81(9):4895-4899.
[11]WANG Y,LIU Z,DAI Q.A highly immunogenic fragment derived from Zaire Ebola virus glycoprotein elicits effective neutralizing antibody[J].Virus Res,2014,189(8):254-261.
[12]SUN P Y,LI K,LIU C B,et al.Co-expression of Ebola virus GP and VP40 proteins and virus-like particles assembly in mammalian cells[J].China Biotechnol,2016,36(12):66-71.(in Chinese)孙鹏艳,黎奎,刘存宝,等.埃博拉病毒GP和VP40蛋白在哺乳动物细胞中的共表达及病毒样颗粒装配[J].中国生物工程杂志,2016,36(12):66-71.
[13]VOLCHKOV V E,FELDMANN H,VOLCHKOVA V A,et al.Processing of the Ebola virus glycoprotein by the proprotein convertase furin[J].Proc Natl Acad Sci USA,1998,95(10):5762-5767.
[14]LEE J E,FUSCO M L,HESSELL A J,et al.Structure of the Ebola virus glycoprotein bound to an antibody form a human survivor[J].Nature,2008,454(7201):177-182.
[15]SULLIVAN N,PETERSON M,YANG Z Y,et al.Ebola virus glycoprotein toxicity is mediated by a dynamin-dependent protein-trafficking pathway[J].J Virol,2005,79(1):547-553.
[16]YANG Z,DUCKERS H J,SULLIVAN N J,et al.Identification of the Ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury[J].Nat Med,2000,6(8):886-889.