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以人胎盘绒毛膜为原料提纯运铁蛋白受体,是用1％Triton X-100增溶所得可溶性绒毛膜蛋白,经聚丙烯酰胺凝胶电泳(PAGE)分离,再经蛋白质硝酸纤维纸转移电泳后,以免疫酶标染色法及同位素碘标放射自显影法检测出运铁蛋白受体蛋白的区带。切取此区带,用生理盐水浸出,超滤浓缩后得到电泳纯的运铁蛋白受体。每个新鲜人胎盘可提取运铁蛋白受体蛋白约200μg,用SDS-PAGE测定其分子量(MW)为90,000。用卵清蛋白、人血清a_1-酸性蛋白、人IgG竞争抑制试验证明此受体与运铁蛋白特异性结合。用提纯的运铁蛋白受体免疫家兔,得到抗人运铁蛋白受体抗血清。采用间接免疫酶标法检测人宫颈癌培养细胞株(HeLa)及人肝癌培养细胞株(SMMC-7721),证明其细胞膜上运铁蛋白受体含量均增高。
Purification of transferrin receptor using human placental chorion as raw material is a soluble chorionic protein solubilized with 1% Triton X-100, separated by polyacrylamide gel electrophoresis (PAGE), and then subjected to protein nitrocellulose paper transfer electrophoresis The bands of transferrin receptor protein were detected by immuno-enzyme-linked immunosorbent assay and isotope iodine-labeled autoradiography. Cut this zone, leaching with saline, concentrated ultrafiltration electrotransfer pure transferrin receptor. Each fresh human placenta can extract about 200 μg of transferrin receptor protein and has a molecular weight (MW) of 90,000 as determined by SDS-PAGE. Ovalbumin, human serum a 1 - acidic protein, human IgG competition inhibition test proved that this receptor and transferrin specific binding. Rabbits were immunized with the purified transferrin receptor to obtain anti-human transferrin receptor antiserum. Indirect immunoenzyme-linked immunosorbent assay was used to detect HeLa and SMMC-7721 cells, and the levels of transferrin receptor on the cell membrane were increased.