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目的:制备并鉴定口蹄疫病毒(FMDV)非结构蛋白3D的单克隆抗体(mAb)。方法:以纯化的原核表达3D蛋白免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞融合,杂交瘤细胞经间接ELISA法筛选,有限稀释法克隆,直至所克隆的细胞能够100%的分泌抗体,用Western blot以及间接免疫荧光法对mAbs的特异性等进行鉴定。结果:免疫的BALB/c小鼠经过多次融合筛选,共获得5株抗pET28a-3D的mAb,其亚类鉴定3株为IgG1,2株为IgG2b,初步判定所得5株mAb识别2个不同表位。Western blot显示这些mAbs与重组3D蛋白和FMDVO/China99感染的BHK-21细胞中的3D抗原均特异性结合,免疫荧光结果显示这5株mAb能够特异结合FMDV感染细胞中的3D蛋白。结论:成功获得了能识别自然3D蛋白的特异性mAb,为进一步研究3D蛋白的结构和功能以及诊断方法奠定基础。
OBJECTIVE: To prepare and identify monoclonal antibodies (mAbs) for foot-and-mouth disease virus (FMDV) non-structural protein 3D. Methods: BALB / c mice were immunized with purified prokaryotic 3D protein. The spleen cells were fused with mouse myeloma cells. The hybridoma cells were screened by indirect ELISA and cloned by limiting dilution until the cloned cells could be 100% The secreted antibodies were identified by Western blot and indirect immunofluorescence on the specificity of mAbs. Results: Five BALB / c mice were screened by multiple rounds of fusion. Five anti-pET28a-3D mAbs were obtained, three of which were IgG1 in subclass and two were IgG2b. The five mAbs identified were different gauge. Western blot showed that these mAbs specifically bind to the 3D antigen in both recombinant 3D protein and BHK-21 cells infected with FMDVO / China99. The results of immunofluorescence showed that these 5 mAbs could specifically bind to the 3D protein in FMDV infected cells. Conclusion: The specific mAb that can recognize natural 3D protein was successfully obtained, which laid the foundation for further study of the structure and function of 3D protein and diagnosis method.