Dame M C, Pierce E A, Prahl J M, Hayes C E, DeLuca H F
Biochemistry. 1986 Aug 12;25(16):4523-34. doi: 10.1021/bi00364a011.
Monoclonal antibodies to different domains of the porcine intestinal 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor have been produced. A nuclear extract enriched in the 1,25-(OH)2D3 receptor was prepared from small intestinal mucosa of young pigs. The receptor was purified an additional 6600-fold by chromatography on DNA-cellulose, ammonium sulfate precipitation, gel filtration high-performance liquid chromatography, and DEAE-Sepharose chromatography, with an overall yield of 23% and an average purity of 24%. A BALB/c mouse immunized with this material developed serum polyclonal antibodies to the 1,25-(OH)2D3 receptor, as demonstrated by a change in sedimentation of the porcine receptor on sucrose gradients. Spleen cells from this animal were fused with mouse myeloma cells (P3-NSI/1-Ag4-1, SP2/0-Ag14), and 24 hybridomas secreting antibodies to the 1,25-(OH)2D3 receptor were identified by both a radiometric immunosorbent assay and an immunoprecipitation assay. Twenty-one hybridoma lines were cloned by limiting dilution and further characterized as subclass IgG1 antibodies with the exception of one which is an IgA. All but two of the antibodies cross-react with the 1,25-(OH)2D3 receptor from both mammalian (human, monkey, and rat) and avian (chicken) intestine; two antibodies recognize only porcine intestinal receptor. All antibodies are unreactive to the vitamin D serum transport protein. Eight of the antibodies bind denatured receptor on an immunoblot. A solid-phase competition assay was used to identify four groups of antibodies that bind to distinct epitopes on the 1,25-(OH)2D3 receptor. One antibody from each of the four groups was used to examine the effect of antibody binding on the DNA-binding activity of the receptor-hormone complex. One antibody completely inhibited the binding of the 1,25-(OH)2D3 receptor complex to DNA-cellulose, suggesting that the epitope for this antibody may be located in the polynucleotide binding domain of the protein. Antibodies from two additional groups only slightly perturbed DNA binding, while one had no effect, suggesting that these antibodies bind to receptor epitopes distant from the region of the polypeptide directly involved in polynucleotide binding. These antibodies that are directed to several different binding sites on the 1,25-(OH)2D3 receptor provide important new tools to probe the biochemistry and topology of the 1,25-(OH)2D3 receptor and to investigate its role in mediating target tissue response to hormone.
已制备出针对猪肠道1,25 - 二羟基维生素D3 [1,25-(OH)2D3]受体不同结构域的单克隆抗体。从小猪小肠黏膜制备了富含1,25-(OH)2D3受体的核提取物。通过DNA - 纤维素柱层析、硫酸铵沉淀、凝胶过滤高效液相色谱和DEAE - 琼脂糖柱层析，该受体进一步纯化了6600倍，总产率为23%，平均纯度为24%。用这种材料免疫的BALB/c小鼠产生了针对1,25-(OH)2D3受体的血清多克隆抗体，这通过猪受体在蔗糖梯度上沉降的变化得以证明。将该动物的脾细胞与小鼠骨髓瘤细胞（P3 - NSI/1 - Ag4 - 1，SP2/0 - Ag14）融合，通过放射免疫吸附测定和免疫沉淀测定鉴定出24个分泌针对1,25-(OH)2D3受体抗体的杂交瘤。通过有限稀释法克隆了21个杂交瘤细胞系，并进一步鉴定为亚类IgG1抗体，只有一个是IgA。除两个抗体外，所有抗体都与哺乳动物（人、猴和大鼠）和禽类（鸡）肠道的1,25-(OH)2D3受体发生交叉反应；两个抗体仅识别猪肠道受体。所有抗体对维生素D血清转运蛋白均无反应。其中8个抗体在免疫印迹上能结合变性受体。采用固相竞争测定法鉴定出四组与1,25-(OH)2D3受体上不同表位结合的抗体。从这四组中各选取一个抗体来检测抗体结合对受体 - 激素复合物DNA结合活性的影响。一种抗体完全抑制了1,25-(OH)2D3受体复合物与DNA - 纤维素的结合，这表明该抗体的表位可能位于该蛋白的多核苷酸结合结构域。另外两组的抗体仅轻微干扰DNA结合，而有一组抗体无影响，这表明这些抗体结合在远离直接参与多核苷酸结合的多肽区域的受体表位上。这些针对1,25-(OH)2D3受体上几个不同结合位点的抗体为探究1,25-(OH)2D3受体的生物化学和拓扑结构以及研究其在介导靶组织对激素反应中的作用提供了重要的新工具。
