Effects On The Radiation Resistance Of Eukaryotic 293T Cells Transfected With Deinococcus Radiodurans PprM And PprI Gene

Background and Objective: Deinococcus radiodurans(DR)is one of the most resistant organisms on the earth.It could survive from ionizing radiation of more than 15 kGy,which is 3000 times higher than most vertebrates.pprI is a key gene involved in DNA repair after ionizing radiation and it is a general switch responsible for extreme radioresistance of DR.pprM is a specific radiation response gene in DR,the deletion mutant of pprM is more sensitive to?-radiation than the wild.The aims of this study were to investigate the effects on the radiation resistance of 293 T cells transfected with pprM and pprI gene and provide a new idea for radiation injury treatment.Methods: 1.pprM gene was amplified from pGEX-6p-1-pprM to construct pEGFP-C1-pprM plasmid.2.pEGFP-C1-ppr M?pDsRed1-N1-flag-ppr I plasmids were transfected into 293 T cells.The fusion proteins and their locations were observed by inverted fluorescence microscope and laser scanning confocal microscope.The proteins were identified by Western blot.3.The survival rates of 293 T cells were determined by MTT.Using fluorescent probe DCFH-DA to detect reactive oxygen species(ROS)content in cells.WST-1 method and microplate test were used to detect superoxide dismutase(SOD)activity and malondialdehyde(MDA)content,glutathione(GSH)activity,respectively.Results:1.The sequence and Blast comparison results showed that the constructed vector sequence was consistent with the template base.The pEGFP-C1-pprM recombinant plasmid was constructed successfully.2.The observed proteins of red andgreen fluorescent indicated that PprI and PprM were successfully expressed in 293 T cells.The results observed by laser scanning confocal microscope showed that PprI and PprM have co-localizations in 293 T cells.Western blot results showed that PprI and PprM fusion proteins were found at about 65 kD and 40 kD.3.There was no significant difference between non-transfectedgroup and thegroup transfected with empty plasmid.However,the survival rate?SOD andgSH activity ofgroups transfected with pprM?pprI and pprM + pprI were significantly increased(P <0.05).The contents of ROS and MDA were significantly decreased(P <0.05).Conclusion:1.The PprI and PprM proteins are successfully expressed in 293 T cells and they have co-localizations in cells.2.The successful expression of DR-pprM,pprI and pprM + pprIgenes in 293 T cells can enhance their survival rate and antioxidant capacity.3.DR-pprM + pprI transfectedgroup effects is more notablely than pprM and pprI transfected respectively,there may be synergistic anti-radiation effects between pprM and pprI gene.