以乙肝病毒核心蛋白为载体制备肠道病毒71型非结构蛋白3D单克隆抗体
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摘要
目的:预测肠道病毒71型(EV71)非结构蛋白3D的表位,以HBc蛋白为载体展示多肽,制备并鉴定抗EV71-3D的特异性单克隆抗体(m Ab)。方法:应用生物信息学方法分析预测出EV71 3D蛋白亲水性和免疫原性指数较高的多肽片段,并运用HBc颗粒型蛋白载体展示肽段,构建多肽融合蛋白,免疫BALB/c雌鼠,通过杂交瘤技术和亲和层析技术制备和纯化抗EV71-3D蛋白的特异性m Ab,用间接ELISA、ELISPOT、IFA和IHC对m Ab的性质进行初步鉴定。结果:构建表达分别嵌合3D蛋白34~43位氨基酸残基、61~76位氨基酸残基、151~164位氨基酸残基的HBc重组蛋白,免疫并经过多轮克隆化筛选,获得抗EV71-3D单克隆抗体3E1,其亚类为Ig G2a;免疫荧光试验、ELISPOT法和免疫组织化学染色结果显示其可与EV71特异性结合。结论:成功制备可特异性识别EV71的单克隆抗体3E1,为病毒的检测及进一步研究3D蛋白的功能奠定了基础,同时还验证了生物信息学技术与HBc颗粒型载体展示多肽技术相结合可快速高效地制备单克隆抗体。
Objective: To prepare and preliminarily identify the monoclonal antibodies( m Abs) specifically against 3D protein of Enterovirus 71( EV71),using bioinformatics to predict the epitopes of 3D,with HBc protein as a carrier. Methods: Artificial screening of 3D protein epitope sequences by bioinformatic method,inserted into the major immunodominant region( MIR) area of Hepatitis B virus core protein( HBc),to construct the recombinant protein. BALB/c mice were immunized with the recombinant virus like particles( VLPs),to prepare the m Abs against 3D protein of EV71. Affinity chromatography technology was used to purify the m Ab. The indirect ELISA,ELISPOT,immunofluorescence and immunohistochemistry staining methods were used to identify the characteristic of the m Ab. Results: We displayed 3D( aa34-43),3D( aa61-76) and 3D( aa151-164) epitopes by constructing fusion protein using HBc VLPs as a vector,after hybridization,one positive hybridoma cell line( 3E1) was selected by ELISA. The isotype of 3E1 was Ig G2 a. The results of immunofluorescence and immunohistochemistry staining assay showed that the m Ab 3E1 could specifically recognize EV71. Conclusion: The prepared m Ab 3E1 can specifically recognizes the EV71,which laid the foundation for the detection of virus and further study on 3D protein,and verified the bioinformatics technology combined with HBc carrier displaying peptides could prepare m Ab quickly and efficiently.
Objective: To prepare and preliminarily identify the monoclonal antibodies( m Abs) specifically against 3D protein of Enterovirus 71( EV71),using bioinformatics to predict the epitopes of 3D,with HBc protein as a carrier. Methods: Artificial screening of 3D protein epitope sequences by bioinformatic method,inserted into the major immunodominant region( MIR) area of Hepatitis B virus core protein( HBc),to construct the recombinant protein. BALB/c mice were immunized with the recombinant virus like particles( VLPs),to prepare the m Abs against 3D protein of EV71. Affinity chromatography technology was used to purify the m Ab. The indirect ELISA,ELISPOT,immunofluorescence and immunohistochemistry staining methods were used to identify the characteristic of the m Ab. Results: We displayed 3D( aa34-43),3D( aa61-76) and 3D( aa151-164) epitopes by constructing fusion protein using HBc VLPs as a vector,after hybridization,one positive hybridoma cell line( 3E1) was selected by ELISA. The isotype of 3E1 was Ig G2 a. The results of immunofluorescence and immunohistochemistry staining assay showed that the m Ab 3E1 could specifically recognize EV71. Conclusion: The prepared m Ab 3E1 can specifically recognizes the EV71,which laid the foundation for the detection of virus and further study on 3D protein,and verified the bioinformatics technology combined with HBc carrier displaying peptides could prepare m Ab quickly and efficiently.
引文
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[15]Jiang H,Weng L,Zhang N,et al.Biochemical characterization of enterovirus 71 3D RNA polymerase[J].Biochimica Biophys Acta,2011,1809(3):211-219.
[16]Xu L,He D,Yang L,et al.A broadly cross-protective vaccine presenting the neighboring epitopes within the VP1 GH loop and VP2 EF loop of enterovirus 71[J].Sci Rep,2015,5:12973.
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[19]Li Z,Xu L,He D,et al.In vivo time-related evaluation of a therapeutic neutralization monoclonal antibody against lethal enterovirus 71 infection in a mouse model[J].PLo S One,2014,9(10):e109391.
[20]He LL,Zhu J.Computational tools for epitope vaccine design and evaluation[J].Curr Opin Virol,2015,11:103-112.
[21]刘洪波,阳广菲,欧维琳,等.柯萨奇病毒A6型VP1蛋白的生物信息学分析[J].中国免疫学杂志,2016,32(4):536-541.
[2]白靓,成岩,曹瑞珍.乙型肝炎病毒核心蛋白病毒样颗粒作为疫苗载体的研究进展[J].中国生物制品学杂志,2015,28(12):1353-1357,1360.
[3]Zlotnick A,Cheng N,Stahl S,et al.Localization of the C terminus of the assembly domain of hepatitis B virus capsid protein:implications for morphogenesis and organization of encapsidated RNA[J].Proc Natl Acad Sci U S A,1997,94(18):9556-9561.
[4]Lange M,Fiedler M,Bankwitz D,et al.Hepatitis C virus hypervariable region 1 variants presented on hepatitis B virus capsid-like particles induce cross-neutralizing antibodies[J].PLo S One,2014,9(7):e102235.
[5]Fiers W,De Filette M,El Bakkouri K,et al.M2e-based universal influenza A vaccine[J].Vaccine,2009,27(45):6280-6283.
[6]Deng L,Cho KJ,Fiers W,et al.M2e-based universal influenza Avaccines[J].Vaccines,2015,3(1):105-136.
[7]Arora U,Tyagi P,Swaminathan S,et al.Virus-like particles displaying envelope domain III of dengue virus type 2 induce virus-specific antibody response in mice[J].Vaccine,2013,31(6):873-878.
[8]花艳红,洪渊东,吕进,等.以乙型肝炎病毒核心蛋白颗粒为载体的流感通用疫苗的构建[J].中国生物制品学杂志,2010,23(10):1075-1079.
[9]Zhu R,Liu J,Chen CY,et al.A highly conserved epitope-vaccine candidate against varicella-zoster virus induces neutralizing antibodies in mice[J].Vaccine,2016,34(13):1589-1596.
[10]杨海杰,陈敏,何水珍,等.基于乙型肝炎病毒核心蛋白的颗粒性肽展示载体的构建[J].厦门大学学报(自然科学版),2004,43(4):436-440.
[11]Chen C,Wang YX,Shan C,et al.Crystal structure of enterovirus71 RNA-dependent RNA polymerase complexed with its protein primer vpg:implication for a trans mechanism of VPg uridylylation[J].J Virol,2013,87(10):5755-5768.
[12]Sun YN,Wang YX,Shan C,et al.Enterovirus 71 VPg uridylation uses a two-molecular mechanism of 3d polymerase[J].J Virol,2012,86(24):13662-13671.
[13]Oberste MS,Penaranda S,Maher K,et al.Complete genome sequences of all members of the species Human enterovirus A[J].JGen Virol,2004,85:1597-1607.
[14]Wu Y,Lou ZY,Miao Y,et al.Structures of EV71 RNA-dependent RNA polymerase in complex with substrate and analogue provide a drug target against the hand-foot-and-mouth disease pandemic in China[J].Protein Cell,2010,1(5):491-500.
[15]Jiang H,Weng L,Zhang N,et al.Biochemical characterization of enterovirus 71 3D RNA polymerase[J].Biochimica Biophys Acta,2011,1809(3):211-219.
[16]Xu L,He D,Yang L,et al.A broadly cross-protective vaccine presenting the neighboring epitopes within the VP1 GH loop and VP2 EF loop of enterovirus 71[J].Sci Rep,2015,5:12973.
[17]Luo WX,Zhang J,Yang HJ,et al.Construction and application of an Escherichia coli high effective expression vector with an enhancer[J].Sheng Wu Gong Cheng Xue Bao,2000,16(5):578-581.
[18]Xu LF,He DL,Li ZQ,et al.Protection against lethal enterovirus71 challenge in mice by a recombinant vaccine candidate containing a broadly cross-neutralizing epitope within the VP2 EF loop[J].Theranostics,2014,4(5):498-513.
[19]Li Z,Xu L,He D,et al.In vivo time-related evaluation of a therapeutic neutralization monoclonal antibody against lethal enterovirus 71 infection in a mouse model[J].PLo S One,2014,9(10):e109391.
[20]He LL,Zhu J.Computational tools for epitope vaccine design and evaluation[J].Curr Opin Virol,2015,11:103-112.
[21]刘洪波,阳广菲,欧维琳,等.柯萨奇病毒A6型VP1蛋白的生物信息学分析[J].中国免疫学杂志,2016,32(4):536-541.