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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 predict the epitope of Enterovirus 71 (EV71) nonstructural protein 3D and to display the specific anti-EV71-3D monoclonal antibody (m Ab) using HBc as a carrier. Methods: The bioinformatics method was used to predict the polypeptide fragments with high hydrophilicity and immunogenicity index of EV71 3D protein. The peptide fragments were displayed by using HBc particle protein carrier to construct the polypeptide fusion protein and immunized BALB / c female mice. The specific m Ab of anti-EV71-3D protein was prepared and purified by hybridoma technique and affinity chromatography. The properties of m Ab were preliminarily identified by indirect ELISA, ELISPOT, IFA and IHC. Results: HBc recombinant protein expressing the 34-43 amino acid residues, 61-76 amino acid residues, 151-164 amino acid residues of the 3D protein was constructed and immunized. After several rounds of cloning and screening, the anti-EV71- 3D monoclonal antibody 3E1, whose subclass is Ig G2a; immunofluorescence assay, ELISPOT assay and immunohistochemical staining showed that it could specifically bind to EV71. Conclusion: The successful preparation of monoclonal antibody 3E1 that can specifically recognize EV71 lays the foundation for the detection of virus and the further study of the function of 3D protein, and also verifies that the combination of bioinformatics technology and HBc particle carrier display peptide technology can rapidly Monoclonal antibodies are efficiently prepared.