硫酸铵和氯化钙对质粒DNA提取的影响+.pdfVIP

硫酸铵和氯化钙对质粒DNA提取的影响 1,2 2 李渊 ，孙彦 （1 天津工业大学应用化学系，天津300160，中国；2 天津大学生物化工系，天津300072，中国） 摘要：随着基因治疗及基因疫苗的不断发展，质粒的需求量迅速增加。本文以 pcDNA3 为目的产物，考察了在 其提取过程中硫酸铵和氯化钙的加入对 pcDNA3 收率的影响和对杂质的去除效果。实验发现：硫酸铵和氯化钙 的加入都可以明显降低质粒提取液中蛋白质、RNA杂质的含量；但是氯化钙在降低蛋白质和RNA含量的同时也 降低了质粒DNA的收率。硫酸铵是去除蛋白质、RNA杂质的良好试剂，同时能够保持较高的质粒DNA收率。当 质粒提取液中硫酸铵浓度加入到 2.5 M 时，质粒的纯化因子可达到10.2，而收率在90% 以上。 关键词：基因治疗；基因疫苗；质粒；纯化；硫酸铵；氯化钙 The Effect of Ammonium Sulfate and Calcium Chloride in the Purification of Plasmid DNA Yuan Li, Yan Sun (1 Department of Applied Chemistry, Tianjin Polytechnic University,Tianjin 300160, China; 2 Department of Biochemistry, Tianjin University, Tianjin 300072 , China) Abstract: Interest in producing large quantities of plasmid DNA has recently increased as a result of the rapid evolution of gene therapy and DNA vaccines. In this paper, the effect calcium chloride and ammonium sulfate were invested on the yield of pcDNA3 and on the removal of RNA and protein in the plasmid purification process. It was found that RNA and protein be mainly removed by the addition of calcium chloride and ammonium sulfate. However, the addition of calcium chloride will reduce the yield of plasmid. In contrast to calcium chloride, ammonium sulfate was a better precipitant for RNA and protein. When the concentration of ammonium sulfate was 2.5 M, the yield of plasmid was up to 90% while the purification factor was 10.2. Key words: Gene therapy, DNA vaccine, Plasmid, Purification, Calcium chloride, Ammonium sulfate 近些年来，随着基因工程、基因治疗和基因疫苗的不断发展，质粒的需求量迅速增加[1]。从菌体中 得到质粒粗品的方法有煮沸法、碱裂解法、SDS 裂解法等，其中碱裂解法是提取质粒最常用的方法。为 了达到药用标准，要对质粒粗品进行进一步纯化，其中色谱分离是纯化质粒的最有效方法 [2]。为了降低 色谱操作的负荷,，提高色谱分离的效率，在色谱操作之前往往要先对质粒提取液进行初步的纯化，降低 杂质的含量。RNA 和蛋白质是质粒提取液中的两种主要的杂质。在常规的分子生物学实验中RNA 的去 除，一般先用 RNA 酶对其进行降解，然后再采用一些方法去除降解后得到的核酸