[Construction of GFP-AWP1 fusion gene vector and its expression in 293 cells].
OBJECTIVE
To construct green fluorescent protein (GFP)-AWP1 (a novel human protein associated with protein kinase C-related kinase 1) fusion gene vector for observing the expression and localization of AWP1 in 293 cells.


METHODS
The coding region in AWP1 cDNA was amplified by RT-PCR from human endothelial cell line ECV304 and recombined into pEGFP-C2 plasmid expressing GFP. After identification with restriction endonucleases and sequence analysis, the recombinant plasmid was transfected into 293 cells with the cationic liposome DOTAP as the transfection reagent. The expression and localization of AWP1 were observed under a fluorescence microscope.


RESULTS
Restriction endonuclease assay and sequence analysis verified the successful construction of the recombinant vector pEGFP-C2/AWP1, and GFP-AWP1 fusion protein was highly efficiently expressed in 293 cells. Under fluorescent microscope, green fluorescence was seen homogeneously distributed in the entire cell body of the cells transfected by the empty vector pEGFP-C2, but diffusely in the cytoplasm of the cells transfected by the recombinant vector pEGFP-C2/AWP1.


CONCLUSIONS
GFP-AWP1 fusion gene vector is successfully constructed and the fusion protein expressed in the cytoplasm of 293 cells.
Cao
Yong-Kuan
YK
Department of Pathophysiology and Key Laboratory of Shock and Microcirculation of PLA, Southern Medical University, Guangzhou 510515, China.
Mo
Yong-Yan
YY
Tian
Fu-Zhou
FZ
Liu
Ya-Wei
YW
Deng
Peng
P
Qin
Qing-He
QH
Jiang
Yong
Y
D002460:Cell Line; D004622:Embryo, Mammalian / Q000166:cytology; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C