分子伴侣辅因子热休克蛋白70羧基末端相互作用蛋白真核表达载体pDsRed2-N1(+)/CHIP构建及在COS-7细胞中的表达

Expression of human carboxyl terminus of the Hsc70-interacting protein(pDsRed2-N1(+)/CHIP) in COS-7 cells following its eukaryotic expression vectors construction

背景:热休克蛋白70羧基末端相互作用蛋白与许多神经退行性疾病的相关蛋白质存在相互作用,并促进这些蛋白质的降解,因此对热休克蛋白70羧基末端相互作用蛋白功能的研究具有非常重要的意义。目的:构建分子伴侣辅因子热休克蛋白70羧基末端相互作用蛋白真核表达载体pDsRed2-N1(+)/CHIP,检测其转染COS-7细胞后的表达。设计、时间及地点:单一样本观察,于2007-05/2008-02在广东省人民医院医学研究中心完成。材料:大肠杆菌Ecoli DH5α,质粒pDsRed2-N1(+)为广东省人民医院医学研究中心肖定璋主任馈赠;人cDNA文库为中南大学湘雅医院神经内科馈赠。方法:用聚合酶链反应方法从人cDNA文库DNA中扩增热休克蛋白70羧基末端相互作用蛋白全长cDNA,该基因片段两端设计了HindⅢ和BamHⅠ限制性酶切位点。将所得片段连接到T载体上测序证实。利用重组DNA技术将热休克蛋白70羧基末端相互作用蛋白的cDNA构建到真核表达载体pDsRed2-N1(+)上,酶切鉴定。通过脂质体介导的转染技术将所构建的载体导入COS-7细胞中,体外培养,于转染48h后利用反转录-聚合酶链反应和Western Blotting方法检测目的基因的表达情况。主要观察指标:①热休克蛋白70羧基末端相互作用蛋白cDNA扩增产物检测。②pDsRed2-N1(+)/CHIP重组体酶切鉴定。③反转录-聚合酶链反应。④免疫荧光及Western-blotting。结果:经琼脂糖凝胶电泳及测序证实,聚合酶链反应获得的片段与GenBank中登记的热休克蛋白70羧基末端相互作用蛋白序列完全相同;pDsRed2-N1/CHIP酶切片段的长度与理论大小相符;转染48h后的COS-7细胞在mRNA和蛋白质水平均可检测到热休克蛋白70羧基末端相互作用蛋白的表达。结论:热休克蛋白70羧基末端相互作用蛋白真核表达载体构建成功,在COS-7细胞中可获得表达。

BACKGROUND： The carboxyl terminuses of the Hsc70-interacting protein （CHIP） associate with many proteins in neurodegenerative diseases, which can promote the degradation of these proteins. Therefore, it is of a great significance to study the function of CHIP. OBJECTIVE： To construct eukaryotic expression vector of human carboxyl terminus of the Hsc70-interacting protein （pDsRed2-N1 （＋）/CHIP） and verify CHIP expression in pDsRed2-N1 （＋）/CHIP transfected COS-7 cells. DESIGN, TIME AND SETTING： A single sample observation was performed at the Medicine Research Center of Guangdong General Hospital between May 2007 and February 2008. MATERIALS： E coil DH5a, and pDsRed2-N1 （＋） was supplied by Xiao Ding-zhang, the director of Medicine Research Center, Guangdong General Hospital. Human cDNA library was provided by Department of Neurology of Xiangya Hospital, Central South University. METHODS： The cDNA fragment encoding the human CHIP gene was amplified by PCR method from human cDNA library DNA. After nucletide sequencing, this cDNA fragment was inserted into a eukaryotic expression vector pDsRed2-N1 （＋） using gene cloning and DNA recombination. The recombinant was then tranferred into COS-7 cells by liposome. The expression of the target molecule was assayed by RT-PCR and Western blotting. MAIN OUTCOME MEASURES： Detection of cDNA amplification products; enzyme identification of pDsRed2-N1 （＋）/CHIP recombinant; RT-PCR; immunofluorescence and Western-blotting. RESULTS： The sequence of DNA fragment amplified by PCR was consistent with that published in Genbank. Length of recombinant plasmid fragments was conformity with expected size. CHIP was expressed and synthesised in COS-7 cells at 48 hours after transfection. CONCLUSION： A eukaryotic expression plasmid containing human CHIP gene was successfully constructed, which is expressed in COS-7 cells.