Neame P J, Acton R T
Adv Exp Med Biol. 1984;172:269-79. doi: 10.1007/978-1-4615-9376-8_16.
We report a methodology suitable for the large scale production and partial purification of human lymphoblastoid interferon with a minimum expense and reasonable (10%) degree of purity of product. The cells used were Namalwa lymphoblastoid cells, which have the advantage of being both easy to grow and well characterized. They were grown in RPMI 1640 containing 10% fetal calf serum to a density of 1.5 - 2 X 10(6) cells/ml and then diluted 50% by the addition of serum free medium containing 2mM sodium butyrate. After 48 hours, the medium was removed and the cells induced to produce interferon by Sendai virus. Typical initial interferon titres were in the region of 4 - 4.8 log units/ml, while specific activities were in the region of 10(6) units/mg protein. The initial stage in the purification involved batchwise adsorption of the interferon to Procion red- HE7B Sepharose CL6B . The Sepharose was then packed into a column, washed with 0.5M KC1 and the interferon eluted with 2M KC1. The interferon was further purified by gel filtration to give an activity of approximately 10(7) units/mg protein. Yields were between 30-50% of the initial interferon in a volume of 25 ml. Further separation of the components of the heterogeneous alpha interferon could be obtained on Procion blue-HER Sepharose, or by utilizing reverse phase HPLC.
我们报告了一种适用于大规模生产和部分纯化人淋巴母细胞干扰素的方法，该方法成本最低，产品纯度合理（10%）。所用细胞为Namalwa淋巴母细胞，其优点是易于生长且特性明确。将它们在含有10%胎牛血清的RPMI 1640培养基中培养至密度为1.5 - 2×10⁶个细胞/毫升，然后加入含有2mM丁酸钠的无血清培养基将其稀释50%。48小时后，去除培养基，并用仙台病毒诱导细胞产生干扰素。典型的初始干扰素滴度在4 - 4.8对数单位/毫升范围内，而比活性在10⁶单位/毫克蛋白范围内。纯化的初始阶段包括将干扰素分批吸附到Procion红-HE7B琼脂糖凝胶CL6B上。然后将琼脂糖凝胶装入柱中，用0.5M KCl洗涤，并用2M KCl洗脱干扰素。通过凝胶过滤进一步纯化干扰素，得到的活性约为10⁷单位/毫克蛋白。产量为初始干扰素的30 - 50%，体积为25毫升。在Procion蓝-HER琼脂糖凝胶上，或利用反相高效液相色谱法可进一步分离异质性α干扰素的各组分。
