人类磷脂氢谷胱甘肽过氧化物酶的真核表达及其多克隆抗体的制备  

Eukaryotic expression of human phospholipid hydroperoxide glutathione peroxidase and the preparation of its polyclonal antibody

作　　者：赵文然[1] 钟翔宇[2] 孙辉[3] 周令望[3] 曲章义[4] 钟学宽[3] 

机构地区：[1]哈尔滨医科大学细胞生物学教研室,150081 [2]第二临床医学院普外科 [3]中国疾病预防控制中心地方病控制中心克山病研究所 [4]卫生微生物学教研室

出　　处：《中国地方病学杂志》2006年第1期39-41,共3页Chinese Jouranl of Endemiology

基　　金：国家自然科学基金资助项目(30170803);黑龙江省科技攻关课题资助项目(GB01C10601)

摘　　要：目的在真核细胞中表达人磷脂氢谷胱甘肽过氧化物酶(PHGPx)并制备抗PHGPx多克隆抗体。方法用特异引物从人睾丸组织cDNA文库中扩增出人线粒体型PHGPxcDNA,与真核表达载体pcDNA3.1/His重组,在COS-1细胞内表达PHGPx。用大肠杆菌表达的突变的PHGPx免疫动物,制备抗PHGPx抗体。结果重组体pcDNA-PHGPx转染的COS-1细胞表达了人PHGPx融合蛋白,PHGPx活性为111.5μmol·mg-1·min-1,高于对照组5倍。免疫扩散测得抗PHGPx多克隆抗血清滴度为1∶16;用于蛋白印迹时稀释倍数为1∶500,可见特异免疫沉淀条带。结论人PHGPx在COS-1细胞内获得了表达,抗PHGPx多克隆抗体有特异性,可以用于重组表达的PHGPx的鉴定。Objective To express human phospholipid hydroperoxide glutathione peroxidase （PHGPx） in eukaryotic cells and to prepare its polyclonal antibody. Methods PHGPx cDNA was amplified from a human testis library using specific primers and cloned into expression vector pcDNA3.1/His. Expression of PHGPx was performed in COS-I cells. Polyclonal antibody against PHGPx was prepared using mutated PHGPx expressed in E.coli. Results The activity of the PHGPx fusion protein expressed in COS-1 cells transfected with recombinant pcDNA-PHGPx was 111.5 μmol·mg^-1·min^-1 ,5-fold higher than that of control group. The titer of anit-PHGPx polyclonal antibody was 1 ： 16 obtained by immunoprecipitation assay,and it could be diluted into 1 ： 500 when used in Western Blotting. The specificity of the polyclonal antibody was verified by the specific immunoprecipitation straps. Conclusions Recombinant PHGPx is expressed in COS-I cells and anit-PHGPx antibody can be used to identify the recombinant PHGPx.

关 键 词：磷脂氢谷胱甘肽过氧化物酶 基因表达 抗体 硒 

分 类 号：R346[医药卫生—基础医学]