GFP fused to the cytoplasmic tail of integrin alphaIIb allows the normal expression of alphaIIb beta3 compound in CHO cells.
- Author:
Bin FU
1
;
Gan FU
;
Fang-Ping CHEN
;
Wei LIU
;
Xi-Lian HUANG
;
Guang-Fen XIAO
Author Information
1. Department of Hematology, Xianya Hospital, Central South University, Changsha 410008, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Blotting, Western;
CHO Cells;
Cricetinae;
Cricetulus;
Endoplasmic Reticulum;
metabolism;
Golgi Apparatus;
metabolism;
Green Fluorescent Proteins;
genetics;
metabolism;
Microscopy, Confocal;
Platelet Glycoprotein GPIIb-IIIa Complex;
genetics;
metabolism;
Recombinant Fusion Proteins;
genetics;
metabolism;
Transfection
- From:
Journal of Experimental Hematology
2005;13(2):182-187
- CountryChina
- Language:Chinese
-
Abstract:
To investigate the effect of GFP fused to C terminal of integrin alpha(IIb) on the biosynthesis and expression of alpha(IIb) beta(3) compound, the alpha(IIb) GFP expression plamid, named palpha(IIb) GFP, the cDNA of alpha(IIb) was constructed from p3.1-2b and fused to pEGFP-N1 in frame. When the sequence of palpha(IIb) GFP was confirmed by sequencing it was transferred to Chinese Hamster Ovary (CHO) cells with or without p3.1-3a expressing integrin beta(3). Then the expression of alpha(IIb) GFP fusion protein was confirmed by Western blot and then its subcellular localization was determined with laser confocal scanning microscopy. The results showed that the target gene was cloned into recombinant vector by restriction analysis and sequencing. Overexpression of the fusion protein in the transfected CHO cells was identified with Western blot. Subcellular localization analysis confirmed that alpha(IIb) GFP was expressed in CHO cells and could be transferred from endoplasmic reticulum to Golgi apparatus. It is concluded that the eukaryotic expression plasmid containing alpha(IIb) GFP fusion gene is successfully constructed. GFP fused to the cytoplasmic tail of integrin alpha(IIb) allows the normal expression of alpha(IIb) beta(3) in CHO cells.
