ABSTRACT
OBJECTIVE: To construct eukaryotic expression vectors using recombinant adenovirus containing the gene fragments encoding Her2/neu extracellular first ligand-binding domain (Her2-ECD), full-length extracellular domain (Her2-ECD), and extracellular and transmembrane domain (Her2-TM). METHODS: The cDNAs were amplified by RT-PCR and inserted into shuttle pAdTrack-CMV plasmids. Viral plasmids were obtained from homologous recombination in E. coli BJ5183, and transfected into 293 cells via liposome. Formation of viral plaque and expression of green fluorescent protein were observed by fluorescence microscopy, and the target proteins were detected by Western blotting. RESULTS: The target cDNA fragments were amplified by PCR with expected lengths and the DNA sequences were confirmed against Genbank. Formation of viral plaque, expression of green fluorescent protein and the target proteins were detected in 293 cells transfected by the viral plasmids, which showed elevated expression of Her2/neu protein with the increase of multiplicity of infection (MOI). CONCLUSION: The eukaryotic expression vectors using recombinant adenovirus have been successfully constructed for expression of Her2/neu extracellular and transmembrane domains.
Subject(s)
Adenoviridae/genetics , Genetic Vectors/genetics , Receptor, ErbB-2/metabolism , Blotting, Western , Cell Line , Cloning, Molecular , Eukaryotic Cells/cytology , Eukaryotic Cells/metabolism , Gene Expression , Genetic Vectors/chemistry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Liposomes/chemistry , Microscopy, Fluorescence , Peptide Fragments/genetics , Peptide Fragments/metabolism , Receptor, ErbB-2/chemistry , Receptor, ErbB-2/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transfection/methodsABSTRACT
OBJECTIVE: To investigate the survival and cell cycle of HaCaT keratinocytes following UVB irradiation at various dosages, and observe the effect of vitamin E(Vit E) on the growth behavior of the cells in this condition. METHODS AND RESULTS: MTT assay and flow cytometry were employed to analyze the cell survival ratio and changes in cell growth behavior in various phases of the cell cycle. It was found that for a given length of time of cell incubation following UVB irradiation, the survival ratio of HaCaT keratinocytes decreased gradually as the irradiation dose increased; at a fixed UVB irradiation dose, prolonged incubation of the cells following the irradiation resulted in decreased cell survival ratio, which, however, began to increase when the minimum ratio was reached, with the exception of the cells subjected to irradiation for 15 min. As indicated by cell cycle analysis, the proliferation indexes of the cells [(S+G2)/(G1+S+G2)] subjected to irradiation for less than 5 min increased gradually as the cell incubation time was prolonged, and contrary results occurred in cells receiving UVB irradiation for longer time. Obvious protective effect of Vit E on the cells was observed against UVB irradiation-induced cell growth inhibition, with optimal dose of Vit E of 40 microg/ml. CONCLUSION: UVB irradiation for no less than 5 min can inhibit HaCaT human keratinocyte growth, and the effect is antagonized by Vit E administration.
