通过对质粒提取过程进行分析，探讨质粒提取过程中的影响因素，分析影响质粒质量的因素，以及优化提取工艺流程，并建立质控标准来控制质粒提取过程中带来质量和数量损失，从而制备出高纯度的质粒DNA。温度诱导可以增加质粒DNA的产量，在大肠杆菌对数生长中期进行温度诱导，从37~43 ℃，每间隔1 ℃度进行诱导，结果表明经42 ℃诱导12 h，质粒DNA的产量可以增加66%以上，为降低成本，大规模生产重组质粒提供良好保障。另外对碱裂解粗提质粒的方法和流程进行改进，采取LiCl去除细胞碎片和杂蛋白；PEG8000沉淀质粒DNA从而去除经RNAase消化的RNA；之后利用少量的酚氯仿混合液去除蛋白质；不仅蛋白质，RNA去除干净，而且在实验时间安排上以及试剂使用量上进行控制，达到了贵重试剂使用量少，去除杂质干净的效果，更为后续的精提做好准备。利用凝胶层析，亲和层析，阴离子交换层析依次去除残余在质粒DNA中的蛋白质、RNA、非超螺旋DNA以及内毒素，达到了制备高纯度质粒DNA要求，并且达到了可以在动物实验中进行转染的质量要求。

The influential factors in the plasmid DNA extraction on the quality of the plasmid DNA was investigated, the extraction conditions of plasmid DNA were optimized and the quality-controlling standards were set in order to produce the high-purity plasmid DNA. Effect of temperature inducement on the production of plasmid DNA was studied by increasing the temperature from 37 ℃ to 43 ℃ at intervals of 1 ℃. After 12 hours of cultivation at 42 ℃, the plasmid yield was significantly increased by 66%, which demonstrated that the new method could be used for economically preparation of recombinant plasmids. Moreover, the standard alkaline lysis method for preparation of plasmid DNAs was modified and the optimized procedure was as follows: removing the cell fragments and the epactal proteins by LiCl; precipitating the plasmid DNA by PEG8000 to wipe off the digested RNA; and removing the reliquus protein by Phe-nol-CH3Cl3.In order to obtain the high-purity plasmid DNA, the crude plasmid DNA was further purified by different kinds of chromatography.

国家自然科学基金重大研究计划项目(NO90412015)；国家自然科学基金项目（2005DKA64001）；广东省科技攻关计划重大项目（200521-E4023）