人线粒体DNA聚合酶γ表达纯化及活性分析  

Expression,purification,and activity analysis of human mitochondrial DNA polymerase gamma

作　　者：盛彦敏 肖丽娟[1] 杨亚兰 刘禹佳 齐英 沈鑫 李庆奇 SHENG Yanmin;XIAO Lijuan;YANG Yalan;LIU Yujia;QI Ying;SHEN Xin;LI Qingqi(Guangzhou Huaxia Vocational College,Guangzhou 510900,China;Changchun Normal University,Changchun 130032,China)

机构地区：[1]广州华夏职业学院,广州510900 [2]长春师范大学,长春130032

出　　处：《长春中医药大学学报》2024年第12期1342-1346,共5页Journal of Changchun University of Chinese Medicine

基　　金：吉林省预算内基本建设资金(创新能力建设)计划项目(2020C034-5)。

摘　　要：目的表达纯化人线粒体DNA聚合酶γ(Polγ),并分析其活性,以应用于核苷(酸)类抗病毒药的毒副作用检测与筛选。方法以质粒PCMV-SPORT6-POLG为模板,PCR扩增人线粒体DNA聚合酶γ基因(POLG),并克隆至pPIC9K载体中,构建高效表达载体pPIC9K-POLG,重组表达载体经酶切线性化后采用电击转化法转化GS115毕赤酵母细胞,30℃1%甲醇诱导表达。表达蛋白经SDS-PAGE和Western Blot鉴定和纯化,并利用掺入法对比商品Polγ酶分析其活性。结果PCR扩增的POLG基因大小约3.7 kb,测序分析结果与Gen Bank中公布的序列一致,表达的Polγ重组蛋白为可溶性蛋白,其分子量约为138 KD,重组Polγ酶具有比商品酶略低的活性。结论构建了重组高效表达载体pPIC9K-POLG,成功在酵母细胞中表达了重组Polγ酶,建立了Polγ酶的体外真核表达体系。重组酶活力约为8U·µL-1,可应用于核苷(酸)类抗病毒药物的筛选和检测。Objective To express and purify human mitochondrial DNA polymerase gama(Polγ),and to analyze its activity for the detection and screening of toxic side effects of nucleoside(acid)antiviral drugs.Methods The human mitochondrial DNA polymerase gamma gene(POLG)was amplified by PCR using the plasmid PCMV-SPORT6-POLG as a template,and cloned into the pPIC9K vector to construct the efficient expression vector pPIC9K-POLG.After enzymatic linearization of the recombinant expression vector,GS115 Pichia pastoris cells were transformed by electroporation transformation method and induced to express at 30℃with 1%methanol.After purification,the expressed protein was identified by SDS-PAGE and Western Blot,and its activity was analyzed using the incorporation method to compare with commercial Polγenzyme.Results The size of the POLG gene amplified by PCR was approximately 3.7kb,and the result of the sequencing analysis was consistent with the sequence published in Gen Bank.The expressed Polγrecombinant protein was a soluble protein with a molecular weight of approximately 138 KD.The recombinant Polγenzyme had slightly lower activity than commercial enzymes.Conclusion The recombinant efficient expression vector pPIC9K POLG is constructed,and the recombinant Polγenzyme is successfully expressed in yeast cells,establishing an in vitro eukaryotic expression system for Polγenzyme.The specific activity of the recombinant enzyme is about 8 U·μL-1,which can be used in the screening and detection of nucleoside(acid)antiviral drugs.

关 键 词：人线粒体DNA聚合酶γ GS115毕赤酵母 表达纯化 活性分析 

分 类 号：R392[医药卫生—免疫学]