新型配基的筛选及其用于抗体纯化的研究
发布时间：2018-08-04 19:18
【摘要】： 近年来,抗体广泛地用于疾病诊断和治疗中,需求量大,对纯度要求高。本文以提高抗体纯度为目标,结合疏水电荷诱导色谱和抗体分离的特点,筛选出一种小分子配基。通过烯丙基溴活化方法将筛选出的配基偶联在凝胶介质上,获得了修饰密度为90μmol/mL wet resin的新型色谱介质。以γ球蛋白和白蛋白(BSA)为模型蛋白,通过考察不同离子强度、pH值、添加剂和盐种类条件下模型蛋白的动静态吸附性能,来研究模型蛋白与配基之间的作用机制。研究结果表明,γ球蛋白主要通过疏水作用与配基结合,具有耐盐特性,动态吸附量维持在较高的水平;而BSA主要通过静电作用与配基结合,在高盐条件下吸附量很低。同时,由于γ球蛋白和BSA等电点的差异,二者在pH5条件下的解吸性能也存在显著差异。 利用新型色谱介质从蛋白质混合物和成牛血清中纯化抗体,研究结果表明该介质能够选择性地吸附γ球蛋白,并且有效地排除白蛋白的共吸附和共洗脱。纯化蛋白质混合物时,在pH5条件下获得了纯度高达90%以上的γ球蛋白;从成牛血清中纯化抗体时,在pH4.5条件下回收抗体的纯度为80%,纯化因子达到37.2。除此以外,使用新型色谱介质纯化抗体能够在较温和的条件下获得高收率的产品。
[Abstract]:In recent years, antibody is widely used in disease diagnosis and treatment. In order to improve the purity of antibodies, a small ligand was screened by combining the characteristics of hydrophobic charge-induced chromatography and antibody separation. A new chromatographic medium with modification density of 90 渭 mol/mL wet resin was obtained by coupling the selected ligand to gel medium by the method of activation of allyl bromide. Using 纬 -globulin and albumin (BSA) as model proteins, the interaction mechanism between model proteins and ligands was studied by investigating the dynamic and static adsorption properties of model proteins under different ionic strength pH values, additives and salts. The results show that 纬 -globulin mainly combines with ligands through hydrophobic interaction, which has the characteristics of salt tolerance, and the dynamic adsorption capacity is maintained at a higher level, while the adsorption capacity of BSA is very low under high salt conditions mainly through electrostatic interaction and ligand binding. At the same time, due to the difference of 纬 globulin and BSA isoelectric point, the desorption properties of 纬 globulin and BSA were also significantly different under pH5 conditions. The antibody was purified from protein mixture and bovine serum by a new chromatographic medium. The results showed that the medium could selectively adsorb 纬 globulin and effectively exclude the co-adsorption and coelution of albumin. When the protein mixture was purified, the purity of 纬 globulin was more than 90% under the condition of pH5, and the purity of the antibody recovered under the condition of pH4.5 was 80 and the purification factor was 37.2 when the antibody was purified from bovine serum. In addition, high yield products can be obtained under mild conditions by using new chromatographic media to purify antibodies.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
本文编号：2164883
[Abstract]:In recent years, antibody is widely used in disease diagnosis and treatment. In order to improve the purity of antibodies, a small ligand was screened by combining the characteristics of hydrophobic charge-induced chromatography and antibody separation. A new chromatographic medium with modification density of 90 渭 mol/mL wet resin was obtained by coupling the selected ligand to gel medium by the method of activation of allyl bromide. Using 纬 -globulin and albumin (BSA) as model proteins, the interaction mechanism between model proteins and ligands was studied by investigating the dynamic and static adsorption properties of model proteins under different ionic strength pH values, additives and salts. The results show that 纬 -globulin mainly combines with ligands through hydrophobic interaction, which has the characteristics of salt tolerance, and the dynamic adsorption capacity is maintained at a higher level, while the adsorption capacity of BSA is very low under high salt conditions mainly through electrostatic interaction and ligand binding. At the same time, due to the difference of 纬 globulin and BSA isoelectric point, the desorption properties of 纬 globulin and BSA were also significantly different under pH5 conditions. The antibody was purified from protein mixture and bovine serum by a new chromatographic medium. The results showed that the medium could selectively adsorb 纬 globulin and effectively exclude the co-adsorption and coelution of albumin. When the protein mixture was purified, the purity of 纬 globulin was more than 90% under the condition of pH5, and the purity of the antibody recovered under the condition of pH4.5 was 80 and the purification factor was 37.2 when the antibody was purified from bovine serum. In addition, high yield products can be obtained under mild conditions by using new chromatographic media to purify antibodies.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
【参考文献】
相关期刊论文 前3条
1 任军,贾凌云;亲和色谱仿生配基的筛选和设计[J];分析化学;2005年09期
2 余晓,赵睿,方梅,熊少祥,苏天升,刘国诠;以寡聚组氨酸为配基的高效亲和色谱研究[J];高等学校化学学报;2000年04期
3 孙维敏;抗体纯化中亲和色谱配体的研究进展[J];离子交换与吸附;2005年03期
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