FoxM1相关重组腺病毒载体小试生产及初步质量控制研究

发布时间：2018-01-01 16:20

本文关键词：FoxM1相关重组腺病毒载体小试生产及初步质量控制研究　出处：《湖南大学》2011年硕士论文　论文类型：学位论文

【摘要】：基因治疗技术最初作为遗传病的一种治疗手段开始被人们熟知。二十多年来,随着基因治疗技术的发展进步,基因治疗已经被应用于多种疾病的治疗,尤其是肿瘤的基因治疗。据全球基因治疗临床数据库的数据显示,目前全球肿瘤基因治疗应用范例中使用最多的载体是腺病毒载体。随着基因治疗向临床转化的深入,对基于腺病毒载体的药物需求日益增大,质量要求也增高,因此大规模临床级别腺病毒载体药物的生产成为一个难题。传统纯化腺病毒的氯化铯密度梯度离心的方法已经不能满足需求,目前层析方法纯化腺病毒已成为规模生产的首选方法。重组腺病毒AdFoxM1shRNA是在FoxM1可作为抑制肿瘤生长的靶基因的基础上研发的新型重组腺病毒药物载体。本研究中我们即利用了AdFoxM1shRNA进行了腺病毒的小试生产和初步质量控制研究。通过引入生产时需构建细胞库的概念,建立了293A细胞的细胞库,确保了后续产品来源的稳定性和同一性。并运用转瓶培养体系扩增293A细胞,进行病毒感染,收集病毒扩增时的上清液。在下游纯化工艺研究中通过摸索超滤浓缩、阴离子交换层析和凝胶过滤层析等工艺获得了大量的AdFoxM1shRNA重组腺病毒,最后通过纯度、病毒滴度、复制型病毒检测三个核心指标对获得的重组腺病毒进行了初步质量控制研究,并与传统方法获得的重组腺病毒载体进行对比,确定层析方法获得的AdFoxM1shRNA重组腺病毒质量的优越性。
[Abstract]:Gene therapy has been widely known since 20 years. With the development of gene therapy, gene therapy has been used in many diseases. In particular, gene therapy for cancer. According to the global database of gene therapy clinical data shows. At present, adenovirus vector is the most widely used vector in the application of gene therapy in the world. With the further transformation of gene therapy to clinic, the demand for the drug based on adenovirus vector is increasing day by day, and the quality requirement is also increasing. Therefore, the production of large clinical grade adenovirus vector drugs has become a difficult problem. The traditional method of cesium chloride density gradient centrifugation for the purification of adenovirus can not meet the demand. At present, purification of adenovirus by chromatography has become the preferred method for large-scale production. Recombinant adenovirus AdFoxM1shRNA is a new type of adenovirus based on FoxM1 as a target gene to inhibit tumor growth. Recombinant adenovirus drug vector. In this study, we used AdFoxM1shRNA to carry out the pilot production of adenovirus and preliminary quality control study. By introducing the concept of construction of cell bank during production. The cell bank of 293A cells was established to ensure the stability and identity of the subsequent product source. 293A cells were amplified by flask culture system for virus infection. The supernatant of virus amplification was collected and concentrated by ultrafiltration in the downstream purification process. A large number of AdFoxM1shRNA recombinant adenovirus were obtained by anion exchange chromatography and gel filtration chromatography. Finally, the purity and titer of the recombinant adenovirus were obtained. The quality control of the recombinant adenovirus was studied by three core indicators of replicative virus detection and compared with the recombinant adenovirus vector obtained by the traditional method. To determine the superiority of AdFoxM1shRNA recombinant adenovirus quality obtained by chromatography.
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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