Expression vector for the inhibitor of growth-1 gene is constructed and the NLS-GFP fusion protein expresses in MRC-5 cells.
- Author:
Zhao-ming SUN
1
;
Shi-zhu YU
;
Wen-zhi ZHANG
;
Chun-sheng KANG
;
Hong-xu ZHOU
;
Hui-ling HUANG
;
Tong-ling AN
Author Information
- Publication Type:Journal Article
- MeSH: Base Sequence; Cell Line; Genetic Vectors; genetics; Green Fluorescent Proteins; genetics; metabolism; Humans; Inhibitor of Growth Protein 1; Intracellular Signaling Peptides and Proteins; genetics; metabolism; Microscopy, Fluorescence; Molecular Sequence Data; Nuclear Proteins; genetics; metabolism; Recombinant Fusion Proteins; genetics; metabolism; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Transfection; Tumor Suppressor Proteins; genetics; metabolism
- From: Chinese Journal of Medical Genetics 2006;23(3):330-332
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct the NLS(ING1)-GFP vector, transfer it into MRC-5 cells and establish a cell model expressing NLS (ING1)-GFP fusion protein.
METHODSFirstly, cDNA fragment of nuclear locating sequence (NLS) of inhibitor of growth-1 gene (ING1) was gained by RT-PCR and inserted into multi-clone site of pEGFP-C1 to construct the NLS (ING1)-GFP expression vector. Then the vector was used to transfect the MRC-5 cells to observe the subcellular signal localization of green fluorescence protein (GFP).
RESULTSWe successfully constructed the expressing vector of NLS (ING1)-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.
CONCLUSIONIn living cells, physiologically p33 ING1b locates absolutely in nucleus. The p33(ING1b) NLS plays a decisive role in the transporting process of subcellular localization.
