Construction of GPC3 green fluorescent protein eukaryotic expression vector and its influence on growth promoting effect of growth factors in SK-Hep-1 cells
10.3724/SP.J.1008.2008.01162
- Author:
Bing WANG
1
Author Information
1. Department of General Surgery
- Publication Type:Journal Article
- Keywords:
Eukaryotic expression vector;
GPC3 gene;
Green fluorescent protein;
Growth factor;
SK-Hep-1 gene
- From:
Academic Journal of Second Military Medical University
2010;29(10):1162-1165
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To construct a GPC3 green fluorescent protein eukaryotic expression vector pEGFP-N2-G-PC3, and analyze its effects on the growth promoting effect of growth factors (fibroblast growth factor-2, FGF2; insulin-like growth factor-2, IGF2; transforming growth factor-β1, TGF-β1; and bone morphogenetic protein-4,BMP4) in human hepatoma cell line GPC3-SK-Hep-1. Methods: A eukaryotic expression vector for GPC3 genes (pEGFP-N2-GPC3) was constructed by recombinant DNA technique and was transfected into SK-Hep-1 cells by Lipofectamine™ 2000; the cells stably expressing GPC3 were screened out by G418 (600 μg/ml). The mRNA expression of GPC3 was detected by RT-PCR method and the protein expression of GPC3 by Western blotting and fluorescence microscope. Effects of GPC3 gene on the growth promoting effects of the above growth factors were examined by MTT. Results: The recombinant plasmid was verified to be correctly constructed by restriction endonuclease analysis and DNA sequencing. The green fluorescence was detected in the transfected SK-Hep-1 cells under fluorescence microscope. RT-PCR and Western blotting both confirmed that GPC3 was successfully expressed in SK-Hep-1 cells. FGF2-induced cell proliferation was significantly decreased by GPC3 gene, whereas the growth promoting effects of IGF2, TGF-β1 and BMP4 were not altered by GPC3 gene. Conclusion: We have successfully obtained the synthetic GPC3 protein, which has the same amino acid sequence as that of human GPC3 protein. The eukaryotic expression vector pEGFP-N2-GPC3 has been correctly constructed and GPC3 protein has been successfully expressed in SK-Hep-1 cells. GPC3 may negatively modulate FGF2 signaling pathway.
