【摘要】： 近年來,抗體廣泛地用于疾病診斷和治療中,需求量大,對(duì)純度要求高。本文以提高抗體純度為目標(biāo),結(jié)合疏水電荷誘導(dǎo)色譜和抗體分離的特點(diǎn),篩選出一種小分子配基。通過烯丙基溴活化方法將篩選出的配基偶聯(lián)在凝膠介質(zhì)上,獲得了修飾密度為90μmol/mL wet resin的新型色譜介質(zhì)。以γ球蛋白和白蛋白(BSA)為模型蛋白,通過考察不同離子強(qiáng)度、pH值、添加劑和鹽種類條件下模型蛋白的動(dòng)靜態(tài)吸附性能,來研究模型蛋白與配基之間的作用機(jī)制。研究結(jié)果表明,γ球蛋白主要通過疏水作用與配基結(jié)合,具有耐鹽特性,動(dòng)態(tài)吸附量維持在較高的水平;而BSA主要通過靜電作用與配基結(jié)合,在高鹽條件下吸附量很低。同時(shí),由于γ球蛋白和BSA等電點(diǎn)的差異,二者在pH5條件下的解吸性能也存在顯著差異。 利用新型色譜介質(zhì)從蛋白質(zhì)混合物和成牛血清中純化抗體,研究結(jié)果表明該介質(zhì)能夠選擇性地吸附γ球蛋白,并且有效地排除白蛋白的共吸附和共洗脫。純化蛋白質(zhì)混合物時(shí),在pH5條件下獲得了純度高達(dá)90%以上的γ球蛋白;從成牛血清中純化抗體時(shí),在pH4.5條件下回收抗體的純度為80%,純化因子達(dá)到37.2。除此以外,使用新型色譜介質(zhì)純化抗體能夠在較溫和的條件下獲得高收率的產(chǎn)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 緯 -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 緯 -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 緯 globulin and BSA isoelectric point, the desorption properties of 緯 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 緯 globulin and effectively exclude the co-adsorption and coelution of albumin. When the protein mixture was purified, the purity of 緯 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.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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