期刊
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
卷 175, 期 8, 页码 3737-3749出版社
HUMANA PRESS INC
DOI: 10.1007/s12010-015-1541-2
关键词
VEGF-111; Real-time PCR; Dot blotting; ELISA; Intron 4/5
资金
- Graduate Office of the University of Isfahan
- Iranian Council of Stem Cell Technology [5656]
Among all VEGF-A isoforms, VEGF-111 is particularly important in molecular biology research owing to its potent angiogenic properties and its remarkable resistance to proteolysis. These features make it a potential candidate for therapeutic use in ischemic diseases. VEGF-111 is not expressed in normal cells, but expression is induced by UV-B irradiation and exposure to genotoxic agents. Here, to increase expression at the transcriptional and translational levels, we synthesized and cloned recombinant VEGF-111 cDNA. Two fragments encoding exons 1-4 and intron 4/5 plus exon 8a were amplified and cloned into the pBud.CE4.1 vector using a class IIs restriction enzyme-based method. The expression of VEGF-111 in CHO-dhfr - and HEK 293 cell lines was evaluated with real-time PCR, dot blotting, and ELISA. VEGF expression was increased about 10- and 18-fold in transfected CHO-dhfr - and HEK 293 cells, respectively. Dot blotting and ELISA confirmed successful production of VEGF-111 in both cell lines.
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