Abstract:

Abstract:Objective To construct secreting type human TRAIL(shTRAIL) gene vector pcDNA3.1-HRE/Egr1-shTRAIL mediated by hypoxia/radiation double sensitive promoter,and  observe the effect of  hypoxia and radiation on shTRAIL. Methods HRE upper and lower strands  were gotten by chemical synthesis,double strands HRE was gotten by PCR;pMD19T-Egr1 was digested by SacⅠ and Hind Ⅲ,then Egr1 was obtained,pshuttle-shTRAIL was digested by Kpn Ⅰ and BamH Ⅰ,then shTRAIL was obtained;HRE/Egr1 double sensitive promoter mediated shTRAIL expression vector pcDNA3.1-HRE/Egr1-shTRAIL was constructed by gene recombination technique,it was identified correctlly by enzyme digestion,PCR and sequencing.A549 cells were divided into normal,hypoxia(0.1%),irradiation(6 Gy) and hypoxia + irradiation groups.Results After enzyme digestion by BamH Ⅰ and Sma Ⅰ,the fragments which lengthes were 1284 bp and 4 998 bp,2 292 bp and 3 990 bp were obtained;the vector was amplified by PCR with Egr1 and shTRAIL primer,the products which lengthes were 469 bp and 820 bp were obtained;pcDNA3.1-HRE/Egr1-shTRAIL was sequenced,the result was same to designed,this demonstrated that the construction was right.The vectors were transfected into A549 cells of adenocarcinoma of lung,the expression levels of  shTRAIL mRNA and protein were increased after treated with hypoxia and radiation,it had statistically significant differences compared with normal group (P<0.05),and when they  were combinated,the effect was more obvious.Conclusion Secreting type human TRAIL gene vector pcDNA3.1-HRE/Egr1-shTRAIL mediated by hypoxia/radiation double sensitive promoter is constructed successfully,and hypoxia and radiation could increase the  expression of TRAIL,and they have synergetic effect.

Key words: hypoxia/radiation, tumor necrosis factor related apoptosis inducing ligand, gene recombination