Construction of eukaryotic expression vector of pE_6/p53/GFP and its influence on cell cycle of lung adenocarcinoma
- VernacularTitle:pE_6/p53/GFP重组真核表达载体的构建及其对肺腺癌细胞周期的影响
- Author:
Liwei MAO
;
Weidong WANG
;
Dezhi LI
;
Zhengtang CHEN
- Publication Type:Journal Article
- Keywords:
radiation-inducible promoter;
RT-PCR;
Western blot
- From:Journal of Third Military Medical University
2003;0(24):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the wt-p53's eukaryotic expression vector pE6/p53/GFP that was controlled by the radiation induced promoter and research its functions.Methods Radiation response element E6 was synthesized by gene synthesis.The wt-p53 cDNA sequence was prepared from pcDNA3.1(+)/p53 plasmid by PCR.IRES2-EGFP report gene segment was prepared from double cistron expression vector IRES2-EGFP 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 pE6-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 G1 stage after radiation and their cloning efficiency decreased notably.Conclusion We had constructed successfully the recombinant pE6/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.
