摘要
目的构建p33ING1b核定位序列(nuclearlocatingsequence,NLS)绿荧光蛋白融合表达载体,将其转染到人胚肺纤维母细胞系MRC5,建立稳定表达该融合蛋白的细胞模型。方法应用逆转录PCR获得p33ING1b的NLS序列,然后将NLS序列插入绿荧光蛋白融合表达载体pEGFP C1的多克隆位点,构建pEGFP C1NLS绿荧光蛋白融合表达载体,再用此载体转染MRC5细胞系,观察活细胞绿荧光蛋白的亚细胞定位。结果成功构建了pEGFP C1NLS绿荧光蛋白融合表达载体,由该载体表达的绿荧光蛋白NLS肽段融合蛋白产生的绿色荧光信号全部定位于胞核部位,而空载体转染的细胞表达的绿色荧光蛋白,绿色荧光信号定位于细胞浆中。结论在活细胞内,生理情况下P33ING1b完全定位于细胞核,并且在其亚细胞定位的转运过程中,NLS肽段起着决定性作用。
Objective To construct the NLS（ING1）-GFP vector, transfer it into MRC-5 cells and establish a cell model expressing NLS （ ING1 ）-GFP fusion protein. Methods Firstly, c-DNA fragment of nuclear locating sequence （NLS） of inhibitor of growth-1 gene （1NG1）was gained by RT-PCR and inserted into multi-clone site of pEGFP-C1 to construct the NLS （1NGI） -GFP expression vector. Then the vector was used to transfect the MRC-5 cells to observe the subcellular signal locsdization of green fluorescence protein （GFP）. Results We successfully constructed the expressing vector of NLS （1NG1） -GFP fusion protein. After transferring the fusion expressing vector into MRC-5 cells, we observed that green fluorescence signal located in the cell nucleus. However, the green fluorescence signal located in the cytoplasm in MRC-5 cells transfected with pEGFP-C1 control only expressing GFP. Conclusion In living cells, physiolngically p33^ING1b locates absolutely in nucleus. The p33^ING1b NLS plays a decisive role in the transporting process of subcellular localization.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
2006年第3期330-332,共3页
Chinese Journal of Medical Genetics
基金
天津市高等学校科技发展基金(2004ZD06)
天津医科大学博士论文课题创新研究基金~~
