Construction of luciferase reporter gene plasmid containing hBD-2 and mutant correlated with Crohn's disease
AIM: To construct and identify luciferase reporter gene plasmid containing hBD-2 and its mutant correlated with Crohn's disease. METHODS: The reporter gene plasmid hBD-2-780-luc was constructed by genetic recombination and hBD-2-mut-luc by site-directed mutagenesis to induce the mutation of the promoter for the - 233 site(G) in hBD-2 into C. After lipofectin-mediated transient transfection of HEK-293T with plasmids including one of the promoter gene plasmids and NOD2/CARD15 eukaryotic expression vector, luciferase was quantitated with TNF-α incentive. RESULTS: Double restriction enzyme digestion and nucleotide sequencing demonstrated completion of the recombination and mutagenesis. Two plasmids were obtained, and both of which could produce luciferase in HEK-293T cells with the stimulation of TNF-α significantly more than that of empty plasmid. CONCLUSION: The recombinant plasmids hBD-2-780-luc and hBD-2-mut-luc have been successfully constructed with luciferase expression in cells.
human beta-defensin 2Crohn's diseaseluciferase reporter gene plasmidsite-directed mutagenesis
NETL
NSTL
万方数据
维普
