Abstract:

Abstract:Objective To construct the vector of specific expression of the enhanced green fluorescent protein(EGFP) regulated by oxygen dependent degradation domain(ODDD) and detect the activity of ODDD in cells at limiting oxygen conditions. Methods The encoding sequences of ODDD403-601aa,ODDD549-575aa,and 2ODDD549-575aa were inserted into pEGFP-C1 respectively.Transient expression of EGFP in φA cells and relative EGFP expression were measured by RT-PCR,Western blotting and flow cytometry.Results ODDD/EGFP specific expression vector was cloned and  the function in cells was analyzed.The ODDD could specially regulate the expression of EGFP in φA cells under hypoxia conditions detected by Western blotting and the activity of ODDD was controlled by proteasome in cells.The fluorescence intensities of EGFP in φA cells under conditions of normoxia in pEGFP-ODDD401-603,pEGFP-ODDD549-575 and pEGFP-ODDD2(549-575)  groups were （6.06±1.97）％,（11.99±1.13）％,and （23.16±2.79）％,respectively,and they were lower than that of samples at limiting oxygen conditions (P<0.05). Conclusion Luciferase reporter vectors regulated by ODDD are successfully constructed.

Key words: hypoxia；enhanced green fluorescent protein；oxygen dependent degradation domain；gene therapy