DNA聚合酶γ的提取、纯化和鉴定  

Isolation,purification and identification of DNA polymerase gamma

作　　者：张颖慧[1] 林菊生[1] 李岩[1] 高琳琳[1] 王晓燕[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院肝病研究所,湖北省武汉市430030

出　　处：《世界华人消化杂志》2007年第35期3715-3721,共7页World Chinese Journal of Digestology

基　　金：国家自然科学基金;No.30330680~~

摘　　要：目的:提取并纯化人宫颈癌细胞(HeLa)的线粒体DNA聚合酶γ(DNA polymeraseγ,Polγ),鉴定其纯度和活性.方法:运用离子交换层析等方法提取纯化HeLa细胞的线粒体Polγ,并用Bradford法检测蛋白浓度.经SDS-PAGE检测蛋白纯度和相对分子量,Western blotting验证蛋白.用α-^(32)P- dTTP掺入法,液体闪烁计数器进行放射性测量以确定Polγ的活性.结果:成功提取并纯化HeLa细胞的线粒体Polγ,经SDS-PAGE鉴定,有一个大约140 kDa的亚基单位,Western blotting证实为Polγ.对其进行150倍纯化,收得率为6%,酶的总活力为4.81 U,比活力为36.17 U/mg.结论:HeLa细胞的线粒体Polγ通过离子交换层析法提取和纯化后活性较高,可用于体外药物线粒体毒性的检测.AIM： To purify and identify the mitochondrial DNA polymerase gamma （polymerase γ, Pol γ） from HeLa cells. METHODS： Ion exchange chromatography was used to isolate Pol γ from HeLa cells. Protein concentration was measured using the Bradford method. SDS-PAGE was performed to determine the molecular weights of the subunits of Pol γ. Following the incorporation of α-^32p-dTTP, the activity of Pol γ was counted using a liquid scintillation spectrometer. RESULTS： Pol γ was purified by 150-fold to apparent homogeneity, with a 6% yield. SDSPAGE indicated the presence of one subunit of 140 kDa, and Western blotting identified the specificity. Total activity and specific activity of Pol T were determined to be 4.81 U and 36.17 U/mg, respectively, by chromatography. CONCLUSION： Pol γ, can be purified by ion exchange chromatography. It can then be activated and used as a target to detect the toxicity of some compounds to mitochondria in vitro.

关 键 词：人宫颈癌细胞 DNA聚合酶γ 线粒体 离子交换层析 

分 类 号：R346[医药卫生—基础医学]