【作者】 张泉；

【导师】 孙怀昌； 朱鸿飞；

【作者基本信息】 扬州大学 ， 预防兽医学， 2006， 博士

【摘要】 基因治疗和基因免疫是目前生物医药领域研究的热点之一,其中以表达目的基因重组质粒为基础的非病毒基因转移策略具有安全性好等突出优点,更加受到研究者的青睐。目前实验室制备重组质粒DNA不仅技术较为成熟,而且有众多的商品化试剂盒可供选用,但这些试剂盒不仅价格昂贵,而且不能满足大规模制备质粒DNA的需要。大规模制备既符合相关质量要求,价格又便宜的重组质粒DNA,特别是动物用重组质粒DNA,仍是制约核酸疫苗和基因治疗研究的瓶颈。目前,大规模制备质粒DNA的方法主要有阴离子交换、分子排阻、疏水色谱等层析方法,这些技术不仅需要用苯酚等有毒化学物质和具有潜在病原污染的动物源RNA酶对质粒DNA进行前处理,而且具有耗时、产量低和成本昂贵等缺点。为了解决动物基因免疫或基因治疗用重组质粒DNA的规模化生产工艺,本研究在筛选出具有价格优势的重组菌高密度发酵培养基配方的基础上,研究了不同重组质粒DNA转化大肠杆菌的发酵培养条件,发酵培养过程中宿主菌生长动态与质粒DNA复制的关系,并以150L发酵罐为标准,研究了不同发酵培养条件下重组质粒DNA的产量。结果表明,DH5α和JM109重组菌在筛选的发酵培养基中的生长动态及单位湿菌重比在进口试剂配制的标准LB培养基中要高,在生长对数中后期对重组菌进行42℃诱导,可显著提高质粒产量,其中JM109宿主菌较DH5α宿主菌的质粒产量约高20%。规模化生产重组质粒DNA的另一重要环节是重组菌的破碎处理和质粒DNA的纯化。目前常用的细菌破碎处理方法包括热裂解和碱溶解法,前者需要专门设计的仪器设备,裂解条件不易控制,后者不需专门设备,但生产成本较高。本研究对标准的重组大肠杆菌碱溶解法进行了改进,使其在不影响质粒产量和质量的前提下,制备成本明显下降。进而在公开发表的十六烷基三甲基溴化铵(CTAB)更多还原

【Abstract】 Gene therapy and gene immunization are hot research spots in current biomedical field. Among the gene transfer strategies under investigation, the non-viral gene transfer using the recombinant plasmids expressing the gene of interest has the overt advantages of good safety and ease of preparation over viral-mediated vectors. In terms of plasmid DNA preparation, currently-available commercial kits have the disadvantages of small-scale preparation, high cost and requirement of pretreatment using toxic chemicals, which are unsuitable for large-scale preparation of recombinant plasmids for animal use. As to other methods for large-scale production of recombinant plasmids such as anion-exchange chromatography, size-exclusion chromatography and hydrophobic chromatography, pretreatments using toxic chemicals such as phenol and chloroform and animal-derived RNase A are required, as well as time-consuming and high cost. To establish a suitable method for large-scale preparation of plasmid DNA for animal gene immunization/therapy, cost-effective culture media and the fermentation conditions were established for high-density fermentation of recombinant E.coli. Furthermore, the relationship between the plasmid replication and host bacterial growth was investigated in a 150-liter fermentator. The results showed that the growth state and the wet weight of recombinant DH5αand JM109 E.coli in the modified medium were better than that in standard LB medium. By shifting the culture temperature to 42℃after mid-log growth phase, the plasmid yield could be greatly improved.Other bottlenecks in the industrial production of plasmid DNA include disruption更多还原