天津医药

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重组hIFN-α-2b-BCG的冻干制备及生物特性的研究

孙二琳1,范晓东2,韩瑞发3,赵杰4   

  1. 1. 天津医科大学第二医院 天津市泌尿外科研究所
    2. 天津医大二院泌尿外科
    3. 天津市泌尿外科研究所
    4. 天津医科大学第二医院
  • 收稿日期:2010-04-16 修回日期:2010-08-16 出版日期:2010-11-15 发布日期:2010-11-15
  • 通讯作者: 韩瑞发

Lyophilization-Preparation of recombinant IFN-α-2b-BCG and Study of its bioactivity

  • Received:2010-04-16 Revised:2010-08-16 Published:2010-11-15 Online:2010-11-15

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摘要： 摘要：目的：研究重组hIFN-α-2b -BCG-真空冷冻干燥的制备，探索制备工艺参数，研究冻干重组BCG的生物特性，为临床提供生物免疫活性稳定、安全有效的冻干制剂。方法：采用真空冷冻干燥法制备重组IFN-α-2b -BCG，绘制冷冻干燥曲线，筛选最佳工艺参数。用平板计数法比较冷冻干前后的活菌数，计算存活率。比较冻干前后rBCG的抗酸染色特征与形态。连续测定冻干前、后重组BCG的OD值，绘制生长曲线。PCR和抗性培养分析冻干前后的质粒的稳定性。ELISA测定rBCG表达IFN-α-2b水平。结果：冻干后重组BCG为 6.5×106cfu/ml，液态（冻干前）为1×107cfu/ml，存活率为65%左右。冻干前后重组BCG的形态、生长率及抗酸染色特征无明显差异。质粒插入基因稳定率为91.7%，丢失率为8.3%。冻干前后分泌IFN-α-2b的水平无显著差别(p>0.05)。结论：成功制备了hIFN-α-2b重组BCG冻干制剂，筛选出制备参数。冻干后达到BCG生物免疫治疗所需活菌>100万cfu/ml的生物免疫标准，冻干后rBCG-hIFN-α-2b的形态、生长、表达外源性蛋白—人干扰素-α-2b无显著性差异。冷冻干燥过程对hIFN-α-2b质粒插入基因的遗传表达稳定，均可耐受冷冻、低温与真空干燥过程中的不利影响。

关键词: 重组BCG, IFN-α-2b, 膀胱肿瘤, 冷冻干燥

Abstract: Objective: To explore the preparation process of lyophilization of recombinant IFN-α-2b-BCG(rBCG), and study the biological characteristics of lyophilized rBCG for clinical immunological activity to provide biological stability, safe and effective freeze-dried formulations.. Method: Vacuum-freeze-drying of recombinant IFN-α-2b-BCG, drawing the curve of lyophilization process, screening the best processing parameters. Comparing the number of viable cells with the plate count, before and after lyophilization, to calculate the survival rate. The acid-fast staining characteristics and morphology of the lyophilized rBCG was analyzed. Continuous measurement of the OD value of freeze-dried rBCG, drawing the growth curve. The plasmid stability of lyophilized rBCG was analysed by PCR and kanamycin-resistance. The level of IFN-α-2b expressed by rBCG was determined with ELISA. Results: The number of viable rBCG reached 6.5×106cfu/ml after lyophilization, the value was 1×107cfu/ml before freeze-drying, so survival rate was about 65%. The morphology, growth rate and acid-fast staining characteristics of lyophilized rBCG was not significantly different, from that of un-lyophilized. The stability rate of gene insertion（hIFN-α-2b）was 91.7%, loss rate 8.3%. The level of secretion of rIFN-α-2b by rBCG had no significant difference after lyophilization (p>0.05). Conclusion：Freeze-dried powder of hIFN-α-2b BCG was successfully prepared. After lyophilization, viable rBCG achieved 1×107cfu/ml, which is the standard of bio-immune-therapy needed. There was no significant difference in morphology, growth and expression of exogenous IFN-α-2b,comparing before with after lyophilization. Adverse effects of freezing, low temperature and vacuum drying of lyophilization process has on significant effects on hIFN-α-2b expression, plasmid insert, genetic stability of rBCG.

Key words: recombinant BCG, IFN-α-2b, bladder tumor, lyophilization