DNA疫苗生产工艺的研究
发布时间：2018-10-14 14:26
【摘要】： 近年来DNA疫苗使用质粒DNA作为目的基因载体进入靶细胞成为研究热点。DNA疫苗可使外源基因在靶细胞中表达,诱导宿主产生体液和细胞免疫应答。但质粒DNA在靶细胞中表达效率比较低,所以如何大规模生产符合药用标准的质粒DNA成为关注的焦点。 本论文对工程菌培养基的优化、发酵工艺各参数的影响、碱裂解的优化、层析纯化路线及质粒DNA质量控制进行深入的研究。 (1)利用响应曲面法的Plackett-Burman法和中心组合设计对培养基进行了优化,我们确定了发酵培养基配方。(2)通过实验确定了发酵各参数对质粒DNA拷贝数影响,优化发酵工艺。(3)设计了碱裂解反应器,使碱裂解操作变成自动、连续化的过程,适用于工业化生产。(4)确定质粒DNA层析纯化路线为离子交换色谱、疏水色谱、分子排阻色谱。(5)建立与本工艺相符质量检测平台,对工艺各阶段能实施质量检测和质量控制。 本工艺路线质粒DNA总回收率为46%。依据“DNA疫苗临床用药质量指导原则”进行各项检验,结果符合DNA疫苗临床用药标准。结果表明本工艺适用于工业化生产符合药用标准的质粒DNA。
[Abstract]:In recent years, plasmid DNA has been used as a target gene vector for DNA vaccine to enter target cells. DNA vaccine can induce the expression of exogenous genes in target cells and induce humoral and cellular immune responses of the host. However, the expression efficiency of plasmid DNA in target cells is relatively low, so how to produce plasmid DNA in large scale according to medical standards has become the focus of attention. In this paper, the optimization of the culture medium of engineering bacteria, the influence of fermentation parameters, the optimization of alkali cracking, The purification route of chromatography and the quality control of plasmid DNA were studied. (1) the culture medium was optimized by using the Plackett-Burman method of response surface method and the center combination design. We determined the formula of fermentation medium. (2) determined the effect of fermentation parameters on the copy number of plasmid DNA, and optimized the fermentation process. (3) designed the alkaline cracking reactor to make the alkaline lysis operation automatic and continuous. It is suitable for industrial production. (4) the purification routes of plasmid DNA chromatography are determined as ion exchange chromatography, hydrophobic chromatography and molecular exclusion chromatography. (5) the quality detection platform consistent with this process is established, and the quality detection and quality control can be carried out in all stages of the process. The total recovery rate of plasmid DNA was 46%. According to the guiding principles of Clinical Drug use of DNA Vaccine, the results were in accordance with the standard of clinical use of DNA vaccine. The results show that this process is suitable for the industrial production of plasmid DNA. which meets the medical standard.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：Q789;R392
本文编号：2270746
[Abstract]:In recent years, plasmid DNA has been used as a target gene vector for DNA vaccine to enter target cells. DNA vaccine can induce the expression of exogenous genes in target cells and induce humoral and cellular immune responses of the host. However, the expression efficiency of plasmid DNA in target cells is relatively low, so how to produce plasmid DNA in large scale according to medical standards has become the focus of attention. In this paper, the optimization of the culture medium of engineering bacteria, the influence of fermentation parameters, the optimization of alkali cracking, The purification route of chromatography and the quality control of plasmid DNA were studied. (1) the culture medium was optimized by using the Plackett-Burman method of response surface method and the center combination design. We determined the formula of fermentation medium. (2) determined the effect of fermentation parameters on the copy number of plasmid DNA, and optimized the fermentation process. (3) designed the alkaline cracking reactor to make the alkaline lysis operation automatic and continuous. It is suitable for industrial production. (4) the purification routes of plasmid DNA chromatography are determined as ion exchange chromatography, hydrophobic chromatography and molecular exclusion chromatography. (5) the quality detection platform consistent with this process is established, and the quality detection and quality control can be carried out in all stages of the process. The total recovery rate of plasmid DNA was 46%. According to the guiding principles of Clinical Drug use of DNA Vaccine, the results were in accordance with the standard of clinical use of DNA vaccine. The results show that this process is suitable for the industrial production of plasmid DNA. which meets the medical standard.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：Q789;R392
【引证文献】
相关期刊论文 前1条
1 宋振忠;徐志光;黄炯;薛英;王延;许礼义;张读朴;鲁立柱;蔡宏;呼西旦;;牛分支杆菌DNA疫苗工程菌的高密度培养研究[J];动物医学进展;2010年05期
相关硕士学位论文 前3条
1 宋振忠;牛结核病三价DNA疫苗制备工艺研究及免疫效果初步评价[D];新疆农业大学;2010年
2 韩依辰;基因治疗用质粒DNA生产及产物纯化的研究[D];长春工业大学;2010年
3 朱麟;携带IBV基因真核表达质粒的大肠杆菌工程菌发酵工艺研究[D];四川农业大学;2008年
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