摘要
采用增强型绿色荧光蛋白（EGFP）示踪的方法,研究人DJ-1蛋白在真核细胞中的定位及其对刺激的反应.将克隆在pGEX-KG上的DJ-1亚克隆到载体pEGFP-C2上,对阳性克隆进行PCR、酶切和测序鉴定,用脂质体转染NIH3T3细胞;并用荧光显微镜观察pEGFP-DJ-1在细胞内的定位以及在血清刺激时的移位;探讨在氧化应激时DJ-1对细胞的保护作用,以及其在细胞内定位的变化.重组质粒在NIH3T3细胞中得到高效表达,绿色荧光弥散分布于胞质、胞核中,但以胞质居多;血清刺激后,细胞中的绿色荧光从胞质移位到胞核;在200～600 μmol/L H2O2刺激下,DJ-1能有效保护细胞抵抗氧化应激,并且也能从胞质移位到胞核.上述研究结果为进一步研究DJ-1蛋白的功能提供了一个重要依据.
To study the location of human DJ-1 protein in eukaryotic cells and it＇s response to stimulus with GFP（green fluorescence protein） trace method, DJ-1 with GST tag in pGEX-KG expression vector was subcloned into the green fluorescent protein vector pEGFP-C2. The positive clone was identified by PCR, enzyme digestion and sequencing. The successful construction of recombinant vector was then transfected into mouse NIH3T3 cells by lipofectin, followed by observation of the cells with fluorescent microscope; discussing the protective effects of DJ-1 on cells and the translocation of DJ-1 in cells after oxidative stress. The recombinant vector was highly expressed in NIH3T3 cells. The green fluorescence was observed both in the cell cytoplasm and nuclei, but mainly located in the cytoplasm. After serum stimulation, the green fluorescence of some NIH3T3 cells translocated from cytoplasm to neiclei. With 200 ～600 μmol/L H2O2 stimulation, DJ-1 effectively protected cells from oxidative stress, and it also translocated from cytoplasm to neiclei. All these data help to further study the function of DJ-1 protein.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2006年第7期530-534,共5页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家重点基础研究发展规划(973规划)项目(No.2002CB513005)
广东省科技计划项目(No.A1090202)
广州市科技计划项目(No.2001-z-035-01-1)
广东省自然科学基金重点项目(No.13058)资助~~
关键词
DJ-1蛋白
增强型绿色荧光蛋白
双氧水
血清
转染
human DJ- 1 protein
enhanced green fluorescence protein
H2O2
serum
transfection
作者简介
姜勇,联系人,Tel：020-61648231;Fax：020-61648231;E-mail：yjiang@fimmu.com。
