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目的 :摸索一种适合于中试生产的点突变重组人 β干扰素 ( 17Ser IFN β)发酵与纯化工艺。 方法 :研究不同培养基和热诱导时间对17Ser IFN β表达的影响 ;观察pH、蛋白浓度和层析条件对17Ser IFN β纯化的影响。 结果 :17Ser IFN β工程菌在含甘油、酪蛋白水解物及微量元素的M9培养基中 ,17Ser IFN β表达量明显增加。灰度扫描显示热诱导 4h后的表达量最高 ,目的蛋白可达菌体总蛋白的 2 0 %以上。17Ser IFN β可以相对特异性地被仲丁醇抽提至有机相中 ,纯度达 80 % ,经S2 0 0凝胶过滤柱层析后纯度达 90 % ,用G2 5柱转换缓冲液 ,使缓冲液pH值 >8,同时去除缓冲液中的还原剂DTT ,将复性后的样品用C4反相柱层析进行精制 ,最终获得纯度 >98%和比活性 >3× 10 7U/mg的17Ser IFN β制品。 结论 :成功地建立了注射用新型重组人 β 干扰素的中试生产工艺 ,为随后进行的临床前研究及临床试验打下基础。
Objective: To explore a point mutation recombinant human interferon (17Ser IFN β) fermentation and purification process suitable for pilot production. Methods: The effects of different media and heat induction time on the expression of 17Ser IFN-beta were studied. The effects of pH, protein concentration and chromatographic conditions on the purification of 17Ser IFN-beta were observed. Results : The 17Ser IFN β engineered bacteria significantly increased the expression of 17Ser IFN β in M9 medium containing glycerol, casein hydrolysates and trace elements. Gray-scale scanning showed that the expression level was highest after heat induction for 4 h, and the target protein reached more than 20 % of total bacterial proteins. 17Ser IFN beta can be specifically extracted by secondary butanol to the organic phase with a purity of 80% and a purity of 90% after S2 0 0 gel filtration column chromatography. The buffer is converted by G2 5 column to buffer. The pH value of the solution is higher than 8 and the reducing agent DTT in the buffer is removed. The refolded sample is purified by C4 reversed-phase column chromatography to finally obtain 17Ser with a purity of >98% and a specific activity of >3×10 7 U/mg. IFN beta products. Conclusion : The pilot production process of a novel recombinant human interferon beta for injection was successfully established, laying the foundation for the subsequent preclinical studies and clinical trials.