重组IkBaM因子腺病毒的生产与纯化研究
文献类型：学位论文
| 作者 | 祁丽 |
| 学位类别 | 硕士 |
| 答辩日期 | 2004 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 中国科学院过程工程研究所 |
| 导师 | 丛威 |
| 关键词 | 293细胞 灌注培养 条件优化 微载体回收 |
| 其他题名 | Production and Purification of Recombinant Adenovirus Encoding IkBaM |
| 中文摘要 | 随着基因工程技术的发展，基因治疗尤其是对癌症的基因治疗越来越多地得到人 们的关注。人们构建了各种基因治疗载体，重组腺病毒就是基因治疗的重要载体之一。 293细胞是在重组腺病毒的生产过程中常用的包装细胞之一。能否生产出大量高质量 的腺病毒载体是制约体外实验和临床实验的关键因素。为了满足体外和临床实验的需 要，本论文针对293细胞包装体系生产Ad5-IkBaM重组．腺病毒载体进行了以下几个方的研究。 1、293细胞的生长特性。实验发现293细胞贴壁能力很弱，对乳酸的耐受性很好：对搅拌速度非常敏感，所以，在微载体培养时，接种后前2h采用间歇搅拌，2h后控制搅拌速度在20r／min。 2、摸索了293细胞批式和灌注培养的生长与代谢。在方瓶和搅拌瓶批式（微载体浓度为69／L）培养条件下，检测了葡萄糖、谷氨酞胺和乳酸的代谢。在此基础上设计了间歇式灌注培养的方案，间歇灌注培养最终细胞密度达到了批式培养的三倍。根据293细胞生长特性，在Logistic模型的基础上，建立了描述批式和灌注微载体培养的293细胞生长模型，模型同实验结果符合很好。 3、对Ad5-IkBaaM的生产工艺进行了优化。确定了病毒的最佳接种量为MOI＝8，血清浓度为10％，pH＝7.2，接毒时间为对数生长中后期，接毒后4oh收获病毒。采用MUSTANG Q膜层析系统对重组腺病毒的纯化进行了初步的探讨，该方法有效的减少了纯化的步骤，从而降低了纯化过程中Ad5-IkBaM的损失。 4、为了降低贴壁依赖性细胞规模培养的成本，本论文探索了293细胞培养过程中的Cytodex-3的回收工艺。实验结果表明经回收处理后的微载体可以很好的支持293细胞的生长。比较了细胞在回收的、新的和胰酶处理后的微载体上的生长规律。发现前两者的生长代谢规律十分相似。细胞在胰酶处理后的微载体上的生长则受到了很大的影响。 |
| 英文摘要 | With the development of gene technique, gene therapy has gained a lot of attention, especially on the treatment of cancer. Replication-defective adenoviruses are main vectors of gene transfer system. The 293-human embryo kidney cells are the complementary cells mainly used for the production of recombinant adenoviruses. To meet the need of experiment and clinical application, it is necessary to produce these recombinant adenovirus vectors in large scale. The main studies of this paper are: Growth characteristics of the 293 cells. The capability of 293 cells to attach to the growth surface is weakly and it could grow well in high concentration of lactic acid. Being sensitive to the stirring speed, it is important to use the intermittent stir at the first 2h post inoculation and control the stirring speed within 20r/min after 2h during the culture on micro carriers. The growth and metabolism of 293 cells. Cell culture conditions were monitored for glucose consumption and lactate production during the batch culture in T-flasks and in the 100ml stirred bioreactor which concentration of Cytodex-3 microcarriers was 6g/L. On the basis of batch culture, the intermittent perfusion method during perfusion culture on the Cytodex-3 microcarriers in the 100ml stirred bioreactor was designed, the maximum cell density was three times of the batch culture. On the basis of Logistic model, According to the characteristics of 293 cells growth, the mathematical model was suggested and fitted the experiment very well. Optimum conditions of Ad5-kBaM proliferation on 293 cells were studied, and results were: MOI=5, serum concentration= 10%, pH=7.2, infection before the stationary period and harvest at 40h post infection. Primary research in the purification of Ad5-I K BaM was conducted by the MUSTANG Q chromatogram membrane system which can reduce purification steps and the lose of Ad5-lKBaM. 4. To reduce the cost of cell culture on microcarriers, the technics of recycling Cytodex-3 microcarriers for 293 cells culture was studied in this paper. It was found that recycled microcarriers could support the growth of 293 cells well. There was no obvious difference between recycled microcarriers and new micocarriers in supporting the growth of 293 cells. But using the trypsin could markedly affect the growth of 293 cell on such microcarriers. |
| 语种 | 中文 |
| 公开日期 | 2013-09-16 |
| 页码 | 80 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1423]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 祁丽. 重组IkBaM因子腺病毒的生产与纯化研究[D]. 中国科学院过程工程研究所. 中国科学院过程工程研究所. 2004. |
入库方式： OAI收割
来源：过程工程研究所
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