摘要
[目的]为抑制素质粒的大量提取与纯化寻找一种成本低的方法。[方法]采用碱裂解法与DEAE-纤维柱层析法提取与纯化抑制素质粒DNA,用分光光度法测定其浓度和纯度。[结果]抑制素质粒DNA的等电点为2.0～2.5,低于大部分蛋白质。pH值为8.0时,抑制素质粒DNA带负电荷,且电荷数量与RNA和蛋白质不同。用紫外分光光度计测定的柱层析后洗脱峰1、2、3在260和280 nm波长处的吸光度值分别为3.01和2.9、0.95和0.51、0.84和0.76,其OD260 nm/OD280 nm分别为1.04、1.86和1.10。3 mol/LNaCl的洗脱峰1大部分是蛋白质,0.6 mol/LNaCl的洗脱峰2是浓度为93.0 ng/ml的抑制素质粒DNA,3 mol/LNaCl的洗脱峰3是杂质。[结论]利用碱裂解法能从大肠杆菌细胞中分离出抑制素质粒DNA,DEAE-纤维柱层析法纯化的抑制素质粒DNA达到了要求的纯度。
[ Objective] The purpose was to find a low-cost method for mass extraction and purification of inhibin plasmid. [ Method ] The inhibin plasmid DNA was extracted and purified by alkaline lysis method and DEAE-fiber column chromatography and its conen, and purity were determined by spectrophotometry. [ ResUlt] The isoelectric point of inhibin plasmid DNA was 2.0 - 2.5 and it was lower than that of most proteins. When pH value was 8.0, the inhibin plasmid DNA was negatively charged and its charge quantity was different from that of RNA and protein. The absorbance values of elution peaks 1,2 and 3 from column separation determined at the wavelengths of 260 and 280 nm by UV- spectrometer were 3.01 and 2.9, 0.95 and 0. 51,0.84 and 0. 76 resp. and their ratios of OD260 nm/OD280 nm were 1.04, 1,86 and 1.10 resp. Most of elution peak 1 eluted with 3 mol/L NaCl was protein. The elution peak 2 eluted with 0.6 mol/L NaCl was inhibin plasmid DNA with concn, as 93.0 ng/ml. The elution peak 3 eluted with 3 mol/L NaCl was impurity. [ Conclusion] The inhibin plasmid DNA could be separated from Escherichia coli cells by alkaline lysis method and the purity of inhibin plasmid DNA purified by DEAE-fiber column chromatography met the requirements.
出处
《安徽农业科学》
CAS
北大核心
2009年第23期10895-10896,共2页
Journal of Anhui Agricultural Sciences
基金
江苏省教育厅自然科学研究基金(06KJD1801)
关键词
质粒
提取
纯化
碱裂解法
DEAE-纤维柱层析
Plasmid
Extraction
Purification
Alkaline lysis method
DEAE -fiber column chromatography
