Construction of pcDNA 3.1(+)-HRE-Egr-1-Dbait and Its Radiosensitization Effect on Human Cervical Cancer HeLa Cells in Hypoxia Response

Abstract

Objective To construct recombined eukaryotic expressional vector pcDNA 3.1(+ )-HRE-Egr- 1-Dbait by hypoxia and radiation dual-induced promoter and to observe its radiosensitivity effect on HeLa cells in hypoxia response.Methods Egr-1 promoter elements were amplificated from C57BL/6 nude mice by PCR,Dbait oligonucleotide and HRE enhancer elements were gained by chemical synthesis.The recombined plasmid pcDNA 3.1(+ )-HRE-Egr-l-Dbait was constructed by gene reconstruction.pcDNA 3.1(+ )-HRE-Egr-l-Dbait was transfected into HeLa cells.Colony Formation Assay was used to the detect the radiosensitivity effect transfectants.Results The recombinant plasmids pcDNA 3.1(+ )-HRE-Egr -1-Dbait was constructed and identified by enzyme digestion and sequencing.The D_0,Dq,SF2,α/βvalue of HeLa cells in normal oxygen were 1.98,0.93,0.52,11.12,respectively.The D_0,Dq,SF2 value in hypoxia were 1.74,1.46,0.43,15.82.OER value was 0.88.Conclusion We successfully constructed the combined eukaryotic expressional plasmid vector pcDNA3.1(+ )-HRE-Egr-l-Dbait characterized by radiation and hypoxia dual-induced promoter.And this expression vector could significantly increase the radiosensitivity of human cervical cancer HeLa cells in hypoxic response.