Construction of ICAM-1-GFP and its binding with Molt-4 cells.
- Author:
Wei-Hua CHEN
1
;
Wan-Ming DA
;
Chun-Ji GAO
Author Information
1. Department of Hematology, PLA General Hospital, Beijing, China.
- Publication Type:Journal Article
- MeSH:
Animals;
CHO Cells;
Cricetinae;
Cricetulus;
DNA, Complementary;
genetics;
Genetic Vectors;
Green Fluorescent Proteins;
genetics;
Humans;
Intercellular Adhesion Molecule-1;
genetics;
Recombinant Proteins;
genetics;
Transfection
- From:
Journal of Experimental Hematology
2009;17(3):650-655
- CountryChina
- Language:English
-
Abstract:
This study was aimed to clone human intercellular adhesion molecule-1 (ICAM-1) gene, to transfect the constructed eukaryotic expression vector ICAM-1-GFP into CHO cells, as well as to detect ICAM-1-GFP expression in CHO cells binding with Molt-4 cells. ICAM-1 cDNA gene was amplified by RT-PCR and inserted in PMD(18)-T vector. Then ICAM-1 cDNA from pMD18-ICAM-1 vector was subcloned into eukaryotic expression vector pEGFP-C1 to construct recombinant ICAM-1-pEGFP-C1 vector. Restriction analysis and DNA sequencing were used to confirm the recombinant vector. After stable transfection of CHO-K1 cells with the recombinant vector, the expression and subcellular localization of ICAM-1-GFP were detected by RT-PCR, flow cytometry and fluorescence microscopy. The function of ICAM-1-GFP fusion protein was assessed by the binding of ICAM-1-GFP/CHO cells to Molt-4 cells. The results showed that 1622 bp full-length ICAM-1 cDNA obtained and was successfully ligated with pMD(18)-T-vector, subcloned to construct recombinant ICAM-1-pEGFP-C1 vector. Restriction analysis and DNA sequencing indicated that recombinant ICAM-1-GFP was successfully constructed and ICAM-1-GFP was expressed stably in CHO cells. ICAM-1-GFP expression was only observed in the cytoplasm of ICAM-1-GFP/CHO cells by fluorescence microscopy. The ICAM-1-GFP/CHO cells were bound to PMA-treated Molt-4 cells. The expression of MEM-148 was very weak in PMA-treated Molt-4 cells. It is concluded that the ICAM-1-GFP eukaryotic expression vector has been constructed successfully and expresses stably in CHO cells. PMA can increase the binding of Molt-4 cells to ICAM-1-GFP/CHO cells by inducing specialized form of ICAM-1 clustering.
