用染料配体亲和色谱纯化DNA聚合酶α  

Purification of DNA Polymerase α by Dye Ligand Affinity Chromatography

作　　者：徐德明[1] 李碧羽 陈石根[1] 

机构地区：[1]贵阳医学院分子生物学研究室,复旦大学生物系生化学教研室

出　　处：《贵阳医学院学报》1994年第4期323-326,共4页Journal of Guiyang Medical College

摘　　要：将７７１２系正常小鼠的胸腺、肝、脾等制成匀浆，经３７０００×ｇ离心，所得上清液用平衡缓冲液透析后，通过三嗪蓝色染料－琼脂糖（Ｓｅｐｈａｒｏｓｅ４Ｂ）亲和色谱，用氯化钠溶液梯度洗脱，获得较纯的ＤＮＡ聚合酶α，比活性为３４０Ｕ／ｍｇ，提纯倍数为１８．７，回收率为６９．２％，表明：采用染料配体亲和色谱从匀浆中分离纯化ＤＮＡ聚合酶α，其所得产率高，方法简便快速，色谱柱可反复使用。Normal cells were collected from thymus,liver and spleen of 7712 mice,then homogenized with ultrasonication.The homogenate was centrifugalized in 37 000×g and collected the supernatant dialysed against the buffer. Afterwards, the solution was treated with affinitychromatograph)in Cibacron blue dextran-Sepharose 4B column.The activity of DNA polymerase αin the supernatant and the eluant were measured with tritiated deoxyadenosine triphosphate(3HdATP),i.e,radioisotopic enzyme assay.At the same time,the protein in the supernatant and the eluant were determined by ultraviolet absorption spectrometry. Separately, specific activity in the supernatant is 18.2 U/mg and in the eluant is 340 U/mg. Purifing multiple is 18.7,yield is 69.2%. The above-mentioned method is simple, quick and practical to extraction and purification of DNA plolymerase α.

关 键 词：聚合酶Ⅱ类 亲和色谱法 DNA 染料 

分 类 号：Q559.103[生物学—生物化学] R362[医药卫生—病理学]