聚乙二醇化重组人白细胞介素6的放射性标记

分别用常规氯氨T和改进的双相氯氨T法对PEG-rhIL-6进行放射性碘标记,用柱层析法和离心超滤法对标记物分离纯化,同时用三氯乙酸沉淀法和SDS-PAGE电泳法鉴定纯化后的标记化合物放射化学纯度,采用rhIL-6依赖细胞7TD1,用MTT比色法测定标记物的生物学活性。结果表明:1.改进的双相氯氨T法标记率为74.5%,高于常规氯氨T法的62.3%,放射性比活度分别为5.513×105和4.610×105Bq/μg。2.经柱层析法和离心超滤法对标记物分离纯化后,用三氯乙酸沉淀法测得放射化学纯度均能达到99%以上。3.用SDS-PAGE电泳法检测两种标记方法所得的标记物与非标记物的结果表明,常规氯氨T法标记物比非标记物多1条高分子量蛋白带,认为是标记过程中蛋白质受到部分损伤;改进的双相氯氨T的标记物与非标记物蛋白质条带一致,认为蛋白质标记后基本没有受到损伤。4.生物活性检测表明改进的双相氯氨T标记物与非标记物活性之间无显著差异,常规氯氨T法标记物活性略低于双相氯氨T的标记物活性。

RADIO-LABELING OF POLYETHYLENE GLYCOL MODIFICATION OF RECOMBINANT HUMAN INTERLEUKIN-6

Iodine-125 was used as labeling nuclide,and the PEG-rhIL-6 was labeled by the common used chloramines-T and the two-phase chloramines-T,respectively.The labeled compound was purified by both methods of gel filtration and ultrafiltration respectively.The purity of the labeled PEG-rhIL-6 was determined by both trichloroacetic acid(TCA) and SDS-PAGE,and the biological activity was determined by MTT method.The results demonstrated that the labeling rate and specific radioactivity were 74.5% and 5.513×10~5 Bq/μg for PEG-rhIL-6 by the two-phase chloramines-T method,higher than that by the common used chloramines-T method,which was 62.3% and 4.610×10~5 Bq/μg respectively.The purity of labeled PEG-rhIL-6,purified by both gel filtration and ultrafiltration methods,was over 99% with TCA method.The labeled PEG-rhIL-6 by two-phase chloramines-T method showed two bands,which was identical to that of standard PEG-rhIL-6 though SDS-PAGE,but the labeled PEG-rhIL-6 by common used chloramines-T method had one more band compared with standard PEG-rhIL-6.When determined by MTT,it shown that the biological activity of PEG-rhIL-6 iodinated by common used chloramines-T method was lower than that by two-phase chloramines-T method.