Expression In Yeast And Anti-radiation Effect And Mechanism Of PprI Gene From Deinococcus Radiodurans

Objective:The gene pprI is about970bps long in Deinococcus radiodurans(DR).It is a switch gene resposing to radiation in DR. pprI can regulated theexpression of recA and pprA that is the most important Radiation repair gene in DR, itcan also enhance the ability of cells to remove free radicals. DNA repair and theprotective effect mediated by pprI gene is one of the most important reasons that DRcould survive and maintain genomic integrity in strong radiation. Not only can itEnhanced radiation resistance of Escherichia coli,but anti-eversion force and salttolerance after transformation into Escherichia coli.The antiradiation effect of pprI is realized through its protein function.PprI isunique only in prokaryotic, it is necessary to obtain Bioactive protein througheukaryotic expression system in order to Study on the effects in eukaryotic organisms,especially in mammals and cells in vitro. It has not been reported at home and abroadthat the pprI gene was transformed into and stable expressed in eukaryotic organisms,obtaining bioactive PprI protein. It has not been reported either that the purified PprIprotein from eukaryotic for animal experiment.The main purpose of this research is to transformed pprI gene into yeast, applyprotein purification in animal experiment, Observate anti radiation function of PprIprotein in yeast and mammals. The successful expression of biologically active PprIprotein will be helpful to further study of its anti radiation mechanism, and may be greathelpful to the application of nuclear radiation protection. This study will providetheoretical and experimental data for further clinical application in radiation injury.Materials and methods:It is successful that pprI gene was transformed intoGS115yeast strains by vector pHBM-pprI It is confirmed that the PprI protein was secreted expression in yeast by the mass spectrometry analysis. Radiation resistant testwas carried out on transformation strain and parent strain is not converted and theantioxidant enzymes SOD, CAT activity assay in yeast was tested. Radiation repairprotein Rad24and Rad51in yeast were detected by western blot and semi quantitativeanalysis.In order to study the anti radiation effect of PprI protein on mammals and cells invitro, the expression vector pPICZαA-pprI was constructed with (His)6and TAT. Aftertransformation the PprI protein was secretory expressed in yeast and concentratedreaching0.34mg/ml by nickel column chromatography purification.The purified PprI protein was applied in animal experiment. First,it is observedthat the effect of muscle and intraperitoneal injection on the mortality of mice afterradiation;second, the changes in peripheral blood and bone marrow cell proliferation ofmice after radiation were counted;then, it was detected the SOD, CAT activity andRad17and Rad51expression in the mice organ;last, by culturing human peripheralblood in vitro, the effect of PprI protein on the lymphocyte chromosome aberration ratewas observed.Result:1. Synthesis of pprI gene according to Pichia pastoris codon preference wasproved to be effective in vector construction and transformation of yeast. It is confirmedthat PprI from DR was successfully secreted expression in Pichia pastoris strainGS115By mass spectrometry analysis.2. Ectopic expression of pprI gene improved the radiation resistance of yeast, afterradiation, clone formation rate of yeast was significantly higher than that of controlgroup（P <0.05）. Ectopic expression of pprI increasing the expression of Rad24、Rad51and SOD、CAT activity in yeast（P <0.05）.3. Eukaryotic expression vector pPICZαA–pprI constructed from PCMV-HA-pprIwas successfully transformed into Pichia pastoris strain X33and was secretedexpressed verificated by Western blot. The concentration of the protein at0.34mg/mlwas obtained by nickel column chromatography purification fromThe fermentated supernatant of the yeast.4. Given intramuscular and intraperitoneal injection of PprI protein in one hour canmake mice mortality decreased from50%to20%after irradiation,. PprI Protein injectedintraperitoneal into mice increased peripheral blood erythrocytes, platelets, percentageof lymphocytes,Bone marrow cell clone formation rate（P <0.05）5． PprI protein in culture medium in vitro had no significant effect on humanlymphocyte chromosome aberration.Conclusion:1. The research group had successfully synthesized new pprI gene codingsequence, constructed the eukaryotic expression vector pPICZαA–pprI For the firsttime and Ectopic expressed and purificated in yeast.2. Radiation resistant of the yeast pprI transformated significantly increased,antioxidant capacity and radiation repair protein expression increasing maybe one of themolecular mechanism of it’s radiation resistance.3. Injection of PprI protein can improve the anti radiation ability of mice,performance for the mortality of mice significantly reduced; Bone marrow cell cloneformation rate rised and Mouse peripheral blood erythrocyte, platelet and lymphocytepercentage increase.4.The study found that PprI Protein can improve the activity of SOD in CAT of redblood cells, plasma, liver, bone marrow cells in mice,but has no effect on myocardialcells, renal tissues.It can also Increase the expression of Rad17、 Rad51protein in liver,kidney, spleen, heart tissue, expression time extensing.These conclusions will be valueable to further clinical application of PprI proteinon anti radiation.