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Design of novel polymer adsorbents for selective removal of DNA

用于选择性去除 DNA 的新型聚合物吸附剂的设计

基本信息

批准号：
12555261
负责人：
HIRAYAMA Chuichi
金额：
$ 5.44万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

To remove nucleic acids from a solution of a cell product used as a drug, animated poly(γ-metbyl L-glutamate) (PMLG-NH_2) particles and cross-linked poly(ethyleneimine) (PH) spherical particles were prepared. The particles, which had diameters of 44 to 105 μm were used as adsorbents. The adsorption of DNA and cell products to the adsorbents were determined using a batchwise method. The adsorbing activity for nucleic acid (purified DNA from salmon spermary) increased with increase in either the amino-group content or pore-size (molecular mass exclusion of polysaccharide, M_<lim>) of the adsorbent.The apparent dissociation constant (K_d) between the DNA and the PMLG-NH_2 adsorbent (amino-group content: 3.5 meq/g) decreased from 8.2 × 10^<-13> mol/l (M) to 2.0 × 10^<-13> M with increase in pore size from 2 × 10^3 to 2 × 10^5, at pH 7.0 and an ionic strength of μ = 0.05. On the other hand, the adsorbing activity of bovine serum albumin (BSA) increased with increasing M_<lim> of the particl … More es, but decreased with increasing ionic strength of the buffer. As a result, when M_<lim> was 2 × 10^3 and amino-group content was 3.5 meq/ml, the PMLG-NH_2 removed DNA from various DNA-containing samples, such as protein solutions, at pH 7.0 and ionic strength (μ) of 0.2.The K_d between the DNA and the PEI adsorbent decreased from 8.5 × 10^<-8> to 9.5 × 10^<-10> M with an increase in PEI ratio from 50 to 90 unit-mol% under physiological conditions (ionic strength of μ = 0.2, pH 7.0). On the other hand, the adsorbing activity of BSA also increased with increasing PEI ratio of the adsorbent from 70 unit-mol% or higher, but sharply decreased with increasing buffer's ionic strength. The adsorbing activity of γ-globulin increased with decreasing PEI ratio to 70 unit-mol% or lower. As a result, when the PEI adsorbent, having a PEI ratio of 80 unit-mol%, were used as the adsorbent, they only selectively removed DNA from various protein solutions at an ionic strength of μ = 0.2 and a pH of 7.0. The adsorbent decreased the concentration of DNA in each protein solution to less than 10 ng/ ml, and the recovery rate of protein was more 97% in all cases. Less

为了从用作药物的细胞产物溶液中去除核酸，制备了动态聚γ-甲基l -谷氨酸（PMLG-NH_2）颗粒和交联聚乙烯亚胺（PH）球形颗粒。采用直径为44 ~ 105 μm的颗粒作为吸附剂。采用批量法测定了吸附剂对DNA和细胞产物的吸附。随着氨基含量的增加和孔隙大小（多糖的分子质量排除，M_<lim>）的增加，吸附剂对核酸（从鲑鱼精子中纯化的DNA）的吸附活性均有所提高。在pH 7.0、离子强度μ = 0.05的条件下，PMLG-NH_2吸附剂（氨基含量为3.5 meq/g）与DNA的表观解离常数（K_d）由8.2 × 10^<-13> mol/l (M)降至2.0 × 10^<-13> M，孔径由2 × 10^3增加到2 × 10^5。另一方面，对牛血清白蛋白（BSA）的吸附活性随着颗粒M_<lim>的增加而增加，但随着缓冲液离子强度的增加而降低。结果表明，当M_<lim> = 2 × 10^3，氨基含量为3.5 meq/ml时，在pH 7.0，离子强度（μ）为0.2的条件下，PMLG-NH_2对各种含DNA样品（如蛋白质溶液）的DNA均有脱除作用。在生理条件下（离子强度μ = 0.2, pH 7.0）， PEI比从50增加到90单位摩尔%，DNA与PEI吸附剂之间的K_d从8.5 × 10^<-8> M降低到9.5 × 10^<-10> M。另一方面，随着吸附剂PEI比在70单位摩尔%以上时的增加，BSA的吸附活性也随之增加，但随着缓冲液离子强度的增加，BSA的吸附活性急剧下降。γ-球蛋白的吸附活性随着PEI比的降低而增加，PEI比在70单位摩尔%以下。结果表明，当PEI比为80单位摩尔%的PEI吸附剂作为吸附剂时，它们只能在离子强度为μ = 0.2、pH为7.0的各种蛋白质溶液中选择性地去除DNA。该吸附剂可将蛋白质溶液中的DNA浓度降低至10 ng/ ml以下，蛋白质回收率均在97%以上。少