通过简单步骤高产率纯化人淋巴母细胞干扰素。

Purification of human lymphoblastoid interferon by a simple procedure with high yields.

作者信息

Yonehara S, Yanase Y, Sano T, Imai M, Nakasawa S, Mori H

出版信息

J Biol Chem. 1981 Apr 25;256(8):3770-5.

Abstract

Human Namalwa cell interferon, induced by Sendai virus and composed of a single species with molecular weight of 17,000, was purified to 4.5 X 10(8) international reference units/mg of protein by a combination of salt precipitation, ion exchange chromatography, metal chelate chromatography, hydrophobic chromatography, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. By immunization of a rabbit with this purified interferon and by extensive absorption with Namalwa cells and an impurity column, highly specific antibody was obtained. Namalwa cells, treated with 5-bromo-2'-deoxyuridine, produced 10-fold more interferon upon induction by Sendai virus. Interferon in this case consisted of heterogeneous species with molecular weight ranging from 15,000 to 24,000. These heterogeneous interferon molecules were purified to 7.6 X 10(8) international reference units/mg of protein by successive chromatography using immobilized highly specific rabbit anti-interferon antibody, Blue Sepharose, and immobilized goat anti-rabbit IgG antibody. The overall recovery of interferon activity was 72%, and the purity of the final preparation was ascertained by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

摘要

人成淋巴细胞 Namalwa 细胞干扰素由仙台病毒诱导产生，为单一分子种类，分子量为 17,000。通过盐析、离子交换层析、金属螯合层析、疏水层析以及在十二烷基硫酸钠存在下的聚丙烯酰胺凝胶电泳相结合的方法，将其纯化至 4.5×10⁸国际参考单位/毫克蛋白质。用这种纯化的干扰素免疫兔子，并通过 Namalwa 细胞和杂质柱进行广泛吸附，获得了高度特异性抗体。用 5-溴-2'-脱氧尿苷处理的 Namalwa 细胞，在受到仙台病毒诱导时产生的干扰素增加了 10 倍。此时的干扰素由分子量范围为 15,000 至 24,000 的异质分子组成。通过使用固定化的高度特异性兔抗干扰素抗体、蓝色琼脂糖和固定化山羊抗兔 IgG 抗体进行连续层析，将这些异质干扰素分子纯化至 7.6×10⁸国际参考单位/毫克蛋白质。干扰素活性的总回收率为 72%，最终制剂的纯度通过在十二烷基硫酸钠存在下的聚丙烯酰胺凝胶电泳确定。