Aboagye-Mathiesen G, Toth F D, Juhl C, Nørskov-Lauritsen N, Petersen P M, Ebbesen P
Danish Cancer Society, Department of Virus and Cancer, Aarhus.
Prep Biochem. 1991;21(1):35-51. doi: 10.1080/10826069108021514.
Human placental trophoblast challenged with Sendai virus induced IFNs mainly of the beta-type (75%) and relatively low levels of the alpha-type (25%). A two-step high performance liquid chromatographic procedure ("two-dimensional HPLC") has been developed for the complete purification of the placental trophoblast interferon beta (tro-IFN-beta) from serum-containing culture supernatant. The method involved a combination of high performance liquid affinity chromatography (HPLAC) on Cibacron Blue 3GA immobilized on an activated pressure stable macroporous synthetic polymer, 2-hydroxyethyl methacrylate vinyl sulphone (HEMA-BIO 1000 VS), as the first dimension and reversed-phase high performance liquid chromatography (RP-HPLC) on Separon SGX C-18 as the second. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot experiments showed that the tro-IFN-beta was present as a 24 kDa protein. Densitometric scanning analysis of Coomassie-stained gel revealed the purity of the final preparation to be greater than 99%. The purified tro-IFN-beta had a specific activity of 1.03 x 10(8) IU/mg of protein and the overall recovery was 81% of the total IFN-beta activity in the crude preparation and 61% of the total IFN activity.
受到仙台病毒攻击的人胎盘滋养层细胞诱导产生的干扰素主要为β型（75%），α型水平相对较低（25%）。已开发出一种两步高效液相色谱法（“二维高效液相色谱法”），用于从含血清的培养上清液中完全纯化胎盘滋养层细胞干扰素β（tro-IFN-β）。该方法包括将固定在活化的压力稳定大孔合成聚合物甲基丙烯酸2-羟乙酯乙烯砜（HEMA-BIO 1000 VS）上的汽巴蓝3GA进行高效液相亲和色谱（HPLAC）作为第一维，以及在Separon SGX C-18上进行反相高效液相色谱（RP-HPLC）作为第二维。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹实验表明，tro-IFN-β以24 kDa的蛋白质形式存在。考马斯亮蓝染色凝胶的光密度扫描分析显示最终制剂的纯度大于99%。纯化的tro-IFN-β的比活性为1.03×10⁸ IU/mg蛋白质，总回收率为粗制品中总IFN-β活性的81%，总IFN活性的61%。
