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目的:原核细胞表达人CD1d分子胞外区及制备其多克隆抗体。方法:用RT-PCR法扩增人CD1d分子胞外区基因,将其克隆入原核表达载体pET28中,转化大肠杆菌BL21(DE3),用IPTG诱导重组蛋白的表达,用亲和层析法纯化重组蛋白,以之为免疫原免疫小鼠制备多克隆抗体,并以ELISA、Western blot及免疫组织化学法检测抗体。结果:在原核细胞中高效表达和纯化了人CD1d分子胞外区蛋白,用其免疫小鼠,获得了效价高、特异性较好的多克隆抗体,免疫组织化学检测显示该抗体可识别人小肠组织中的天然CD1d分子。结论:成功制备了人CD1d分子胞外区重组蛋白及鼠抗人CD1d分子胞外区抗体,为进一步建立人CD1d分子的免疫学检测方法及其生物学功能的深入研究奠定了基础。
OBJECTIVE: Prokaryotic cells express the extracellular domain of human CD1d and prepare their polyclonal antibodies. Methods: The extracellular region of human CD1d gene was amplified by RT-PCR and cloned into prokaryotic expression vector pET28. The recombinant plasmid was transformed into E. coli BL21 (DE3). The recombinant protein was induced by IPTG and purified by affinity chromatography The recombinant protein was used as the immunogen to immunize mice to prepare polyclonal antibodies. The antibodies were detected by ELISA, Western blot and immunohistochemistry. Results: The extracellular region of human CD1d molecule was highly expressed and purified in prokaryotic cells. The immunized mice were used to obtain polyclonal antibody with high titer and specificity. Immunohistochemistry showed that the antibody could recognize human Natural CD1d molecules in intestinal tissue. Conclusion: The recombinant human CD1d extracellular domain protein and the anti-human CD1d extracellular domain antibody were successfully prepared, which laid the foundation for the further study of the immunological detection of human CD1d molecule and its biological functions.