Effect Of PprA And PprI Gene Transfer And Expression Of Protein Interaction On Radiation Resistance Of Eukaryotic Cells

Background and purpose:Deinococcus radiodurans(DR) is one of the most radiation resistant organisms in the world, ppr I is the key gene of DNA damage repair and radiation resistance, and it is the main switch on activation damage repair pathway of DR.pprA has double functions of DNA damage repair and antioxidant.pprI regulates the upstream expression of pprA though the promoter, it may enhance the radiation resistance of DR by regulating expression of recA and pprI. This experiment aims to observe the effect of pprA and pprI gene transfer and expression of protein interaction on radiation resistance of eukaryotic cells, in order to improve the anti-radiation regulation network of DR and provide a new method for the gene therapy of radioprotection.Methods: 1. To construct fluorescence expression plasmids pEGFP-N1-pprA and p DsRed1-N1-flag-pprI, pGADT-7-pprA and pet28a-pprI plasmid constructed at an earlier laboratory stage were used as the template. Lipofectamine 2000 was employed to transfect two recombinant vectors into 293 T cells. A fluorescent microscope andWestern blot was employed to observe and examine the expression of the fusion protein.2. The interaction of PprA and PprI protein was predicted by bioinformatics analysis, and the intracellular localization of them was observed by Laser scanning confocal microscope.3. MTT method was used to detect transfection cell viability. Clone formation assay was used to observe the effects of PprA and PprI on clone formation capability of Eukaryotic cells. WST-1 method,microplate test, and DCFH-DA method were used to detect SOD activity,GSH activity, MDA content, and ROS content, respectively, ?-H2 AX focus analysis was used to test the DNA repair ability.Results : 1.Double digestions and the agarose gel electrophoresis showed that target bands appeared at 4 700 bp and 1000 bp, 4800 bp and 1100 bp. No apparent frameshift mutation occurred as shown in the sequencing results. The fluorescent microscopy showed red and green phosphors; and the fluorescence are strongest in 48-72 h. the Western blot results indicated protein expression at 65 kDa and 60 kDa.2. STRING predicts that PprA interacts with the PprI protein, which confidence score is 0.665. Laser scanning confocal microscope observed that PprA and PprI were mainly distributed in the cytoplasm of 293 T cells,and colocation phenomena was existed in it.3. Compared to untransfected blank 293 T cells and transfected withempty plasmid cells, the survival rate of transfected with ppr I and pprI +ppr A cells under irradiation increased significantly, ROS and MDA content decreased significantly, GSH and SOD activity was significantly increased, the number of ?-H2 AX focus was significantly decreased(P<0.05), and transfected with pprI + pprA cells are more obvious(P<0.05).Conclusions:1. p DsRed1-N1-flag-pprI and p EGFP-N1-pprA were successfully constructed.2. The prokaryotic gene DR-pprA and DR-pprI can co-express in eukaryotic cells. PprA and PprI protein may exist interaction.3. Expression of pprI and pprI + pprA in eukaryotic cells can improve the proliferative capacity, enhance the antioxidative capacity and DNA repair ability which improve the radiation resistance of eukaryotic cells.The effect of pprI + pprA is more obvious, they may have synergistic anti-radiation effect.