人CD1d分子胞外区的原核表达及其多克隆抗体的制备

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.

Prokaryotic expression of the extracellular region of human CD1d and preparation of its polyclonal antibody

AIM: To explore the prokaryotic expression of the extracellular region of human CD1d (hCD1d) and prepare its polyclonal antibody. METHODS: The gene encoding the extracellular region of hCD1d was amplified by PCR and cloned into prokaryotic expression vector pET28, then expressed in E.coli BL21(DE3) with IPTG induction. The recombinant protein was purified by Ni(2+)-NTA agarose column and then used as immunogen to immunize the mouse. The generated polyclonal antibody was evaluated by ELISA, Western blot and immunohistochemical staining (IHC), respectively. RESULTS: The recombinant extracellular region of hCD1d was successfully expressed and purified. The polyclonal antibody with high titer and high specificity was obtained, which could recognize the native hCD1d in the human small intestinal tissues. CONCLUSION: The recombinant extracellular region of hCD1d has been obtained. The antibody with high titer and high specificity against the extracellular region of hCD1d from the mouse has been successfully prepared, which lays a foundation for further research into the detection and functional study of CD1d.