Construction of conditionally replicative adenovirus vector carrying TRAIL gene and its mRNA and protein expressions induced by ionizing radiation / 吉林大学学报(医学版)

ABSTRACT

Objective To construct the conditionally replicative adenovirus vector pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K carrying early growth response gene-1 (Egr1)promoter and tumor necrosis factor related apoptosis inducing ligand (TRAIL)gene, and to observe the effects of the vector combined with 2 Gy irradiation on the TRAIL expression in MDA-MB-231 cells.Methods Egr-1 promotor sequence was cloned from pMD18 T-Egr1, TRAIL was constructed the downstream of Egr1 promoter, pShuttle-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K (CRAd.pEgr1-TRAIL)was constructed,after the adenovirus vector was packaged successfully,MDA-MB-231 cells were infected with them and irradiated with X-rays.Real time PCR method and ELISA were used to detect the expression levels of TRAIL mRNA and protein, respectively. Six groups in the experiment were set upcontrol, 2 Gy,CRAd.p,CRAd.pEgr1-TRAIL,CRAd.p + 2 Gy and CRAd.pEgr1-TRAIL + 2 Gy. Results The recombinant adenovirus vector pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K was constructed and packaged successfully.The expression level of TRAIL mRNA in MDA-MB-231 cells transfected with the vector of 5 MOI for 24 h following 2.0 Gy X-rays irradiation began to increase and arrived to the top 8 h later in various groups,then declined.The expression level of TRAIL protein in MDA-MB-231 cells began to increase 6 h after irradiation and reached to the peak 24 h later,then declined 48 h later.There were significant differences in the expression levels of TRAIL protein between CRAd.pEgr1-TRAIL + 2.0 Gy and other groups at the same time point (P<0.01). Conclusion The recombinant adenovirus vector is obtained successfully, and the TRAIL mRNA and protein expression levels in MDA-MB-231 cells can be increased significantly by the vector combined with 2.0 Gy X-rays irradiation.