Objective  To construct the wt-p53’s eukaryotic expression vector pE

6

/p53/GFP that was controlled by the radiation induced promoter and research its functions.   Methods  Radiation response element E

6

was synthesized by gene synthesis. The wt-p53 cDNA sequence was prepared from pcDNA3.1(+)/p53 plasmid by PCR. IRES

2

-EGFP report gene segment was prepared from double cistron expression vector IRES

2

-E

GFP by enzyme digestion. After sequenced and identified

the recombinant plasmid was transformed into H1299(p53

-/-

) cell with Lipofectamine 2000

and the cell lines in stable expression was screened by G418. In the H1299(p53

-/-

) cell transfected with the recombinant plasmid or without

wt-p53 mRNA expression was analyzed by RT-PCR

the p53 expression by Western blot when exposed to 4 Gy 8 MV X ray for 0

3

8

12

24

36 h or when exposed to 0

1

2

4

8 Gy 8 MV X ray for 12 h. The cell cycle of H1299(p53

-/-

) cell transfected stably with the recombinant vector was analyzed by flow cytometry after exposed to 4 Gy 8 MV X ray.   Results  The recombinant pE

6

p53/GFP plasmid had been constructed correctly and the expression of p53 gene in the transfected H1299 cell lines had been determined. After 4 Gy X ray radiation

the expression of wt-p53 protein had a significant rise. The transfected H1299 cell lines stopped in G

1

stage after radiation and their cloning efficiency decreased notably.   Conclusion  We had constructed successfully the recombinant pE

6

/p53/GFP plasmid that was regulated by radiation induced response element E6. This provides experimental data for radiation-gene therapy of non-small lung cancer.