RESUMO
Objective:To obtain highly purified recombinant human IGF-Ⅰ(rhIGF-Ⅰ) and identify it.Methods:rhIGF-Ⅰ Was purified through ion-exchange chromatography and gel filtration chromatography after the inclusion bodies of rhIGF-Ⅰ were extracted from Escherichia coli. The recombinant protein was characterized through molecular weight assay, Western-blot, and fluorescent chromatography. The renaturation and biological assay of rhIGF-Ⅰ were investigated. Results and Conclusions: The purity of rhIGF-Ⅰ was higher than 99%. The analysis of molecular weight, Western-blot, fluorescent chromatography and sequences of NH2-terminal 15 amino acids were same as those anticipated. 3-10 mg/ml was the concentration of renatured rhIGF-Ⅰ to support half-maximal stimulation of cell proliferation with BALB/c 3T3 cells.
RESUMO
Objective: Using the property of (?-estrogen receptor, Fas/?-estrogen receptor fusion gene was constructed which can lead to hormone-induced apoptosis after transfected into glioma cells. Methods: The transmembrane domain, cytoplasmic domain of human Fas gene was fused with the HBD gene fragment of human (?-estrogen receptor by PCR and gene recombination techniques, and was then inserted into eukaryotic vector pcDNA3. Human glioma cells BT325 were transfected with the recombinant plasmid by lipofectamine-rnediated gene transfection. Results: After selection with C418 (or six weeks, transformants expressing the fusion gene were selected out and identified by Western blot. MTT detection showed that (?-estradiol had cytoxic effect on the transformants with IC_(50) of about 10~(-9) mol/L. DNA Ladder detection showed that the transformants could be effectively induced to apoptosis. Conclusion: Fas/?-estrogen receptor fusion gene transfected glioma cells can be induced to apoptosis in a tight estrogen indepent manner.