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Objective To establish a technical route for the efficient expression and purification of PprI protein from Deinococcus radiodurans R1 by using eukaryotic Pichia pastoris.Methods The encoding sequence of the Deinococcus radiodurans pprI gene was modified according to the preference of Pichia pastoris' codon.Modified pprI gene was fully synthesized with PCR and a 6 × His tag was added at its Nterminal.The PCR products were purified and then cloned into Pichia pastoris expression vector pHBM-905A.After utilizing Cop I and Not I double enzyme digestion and retrievering linear objective fragment,new pprI gene was transformed to the GS115 strain of Pichia pastoris.The obtained Pichia pastoris transformants were induced to express.Culture supernatants were detected by SDS-PAGE,Western blot,and mass spectrometry.A Ni-NTA column was uesd to purify the target protein and the BCA method was used to determine protein concentration.Results The coding sequence of new synthetic Deinococcus radiodurans pprI gene was correct.The purpose protein band of a molecular weight of 43 000 was detected in the culture supernatant of transformed Pichia pastoris strains by SDS-PAGE and Western blot.The mass spectrometry confirmed that it was the Deinococcus radiodurans PprI protein.When the concentration of imidazole was 250 mmol/L,the elution rate of PprI protein was the highest.The purified protein concentration was 0.35 mg/ml measured by BCA method.Conclusions This study has successfully constructed a new pprI gene and the recombinant strain of Pichia pastoris secreting PprI protein,and established a technical route for the efficient expression and purification of PprI protein.
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Objective To investigate the radioresistant effects of pprI gene of Deinococcus radiodurans on BALB/c mice.Methods Male BALB/c mice in SPF level were applied for this work.The pEGFP-c1 plasmid and pEGFP-c1-pprI gene recombinant plasmid were transferred into anterolateral muscle of mice with in vivo electroporation technology.The mice were irradiated by 6 Gy 60Co γ-rays in whole body and the mortality of mice was observed within 30 days after irradiation.In addition,the mouse were irradiated with 4 Gy γ-rays and then the peripheral blood cell number,apoptosis rates of thymocyte cells,spleen cells and bone marrow cells were observed in the days of 1,7,14,28 and 35 after irradiation while the histopathological changes of lung and testis were observed in the days 7 and 28 after γ-ray irradiation.Results The highest gene transfection efficiency of muscle cells was obtained in a Plasmid injection amount of 50 μg/50 μl and electric field strength of 200 V/cm.The acute radiation mortality of pEGFP-c1-pprI gene recombinant plasmid transfer group was 30％,lower than that of irradiation group (60.0％) and pEGFP-c1 plasmid transfer group (63.3％) after 6 Gy γ-ray irradiation (x2 =4.90,6.24,P ＜ 0.05).Compared with the irradiation group and pEGFP-c1 plasmid transfer group,the WBC count of pEGFP-c1-pprI gene recombinant plasmid group in peripheral blood of mice was significantly higher in the days of 1,7,14 and 28 (F =16.26,8.10,6.37,10.74,P ＜0.05),PLT count was significantly higher in days of 7 and 14 (F =7.36,5.71,P ＜ 0.05),meanwhile the lymphocyte percentage was increased significantly on the 7th day (F =18.43,P ＜ 0.05) after irradiation.On the other hand,the apoptosis rates of thymocyte cells and bone marrow cells were significantly decreased in the days of 1,7,14,28 and 35 (F =3.88,14.91,14.14,39.86,5.65,P ＜0.05 and F=53.70,11.75,21.78,41.40,4.54,P ＜0.05) while the apoptosis rate of spleen cells was significantly decreased in the days of 1,7,14 and 28 (F =97.95,56.61,33.55,14.71,P ＜0.05) after irradiation.Finally,the radiation histopathological changes of lung and testis of the pEGFP-c1-pprI gene recombinant plasmid group were slight and easy to recover.Conclusions Transfection of pprI gene of Deinococcus radiodurans by in vivo electroporation has significant protective effect on the acute radiation injury in BALB/c mice,which may have important clinical applications.
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Objective To construct the eukaryotic expression vector of pprI gene from Deinococcus radiodurans R1 and investigate its radioresistant effects in eukaryotic cells.Methods A recombinant vector pEGFP-c1-pprI was constructed by DNA recombinant technique.The empty vector pEGFP-c1 and the pEGFP-c1-pprI were transferred into human lung epithelial cells Beas-2B by LipofectamineTM 2000,respectively.Then the infected cells were screened in order to develop a cell line with stable expression of pprI gene.Cell survival rate was tested by clone-forming assay.Cell cycle distribution and apoptosis were detected by a flow cytometry.The fluorescence intensity of reactive oxygen species (ROS) was observed by a fluorescent microscope.γ-H2AX foci in the irradiated cell was detected by immunofluorescence.Results The eukaryotic expression plasmid of pprI prokaryotic gene was constructed and PprI fusion protein was expressed in human lung epithelial cells successfully,and the cell line (2BG) with a stable pprI gene expression was established.After irradiation,the cell survival fraction of 2BG cells was significantly higher than Beas-2B cells so that the value of D0 、Dq and N of the survival curve were increased.Moreover,the fluorescence intensity of ROS and the number of γ-H2AX foci in 2BG cells were also lower than those of B eas-2B cells(F =16.73,19.47,6.94,P ＜ 0.05).Between these two cell lines,the apoptosis rate and cell cycle G2 arrest also had significant difference (F =139.73,237.92,P ＜ 0.05).Conclusions The pprI gene from Deinococcus radiodurans RI can be stably expressed in the eukaryotic cells and it allows the transferred cells to have a radioresistant function.
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Objective To investigate the mechanism of Deinococcus radiodurans pprI gene in enhancing mice radioresistance to γ-rays by transfecting it in vivo.Methods The male Kunming mice were randomly divided into control group,irradiated group,pCMV-HA transfected group and pCMV-HA-pprI transfected group.The pCMV-HA-pprI plasmid contained pprI gene was injected into the muscle of mice which were exposed to total 6 Gy of γ-ray irradiation.After injection,the in vivo gene electroporation technology was used to transfect the pprI gene into the cells,and Western blot was used to identify the PprI protein,mammalian homolog protein Rad51 corresponding to recA gene downstream of pprI,and protein Rad52.Results In the muscle of the mice of transfected pCMV-HA-pprI group,the protein PprI expressed significantly at 1 d post-irradiation,but there was no expression of pprI gene 7 d post-irradiation and in other groups.In the mice of transfected with pCMV-HA-pprI,the expression of Rad51 protein was significantly increased in the lungs at 1,7 and 14 d post-irradiation,and significantly increased in the liver at 1 and 28 d post-irradiation and increased in the kidneys at 1 and 14 d post-irradition.However,there was no obvious change of Rad52 protein expression in the lungs and livers of mice in all groups.Conlusions The prokaryotic gene pprI could act on the mammalian homologisation analogues rad51 gene downstream of recA gene and then increase the expression level of protein Rad51 which results in the enhancement of radioresistance.
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Objective To investigate the dose response relationship for K562 cells' loss of proliferation ability induced by 60Co γ ray irradiation. K562 cell morphological change and Egr-1 expression level are also investigated. Methods 3H-TdR incoporation assay, Giemsa Staining, RT-PCR were used in this study.Results It was noted that the proliferation inhibition is positively dose-dependent. K562 cells showed extensive changes in both morphology and Giemsa staining characteristics after radiation exposure. The irradiated cells acquired characteristics which the high differentiated cells had only. In the early stage after exposure to 5 Gy 60Co γ ray irradiation. A transient transcriptional upregulation of Egr-1 gene which was related to apoptosis and differentiation was observed.The transcriptional level reached its top on time point of 0.5 ～1 hours after exposure. Eight hours after exposure, the transcriptional level was below normal.Conclusions The proliferation inhibition of K562 cell is positively dose dependent. Egr-1 gene can be rapidly activated after exposure to 5 Gy 60Coγ ray. After exposure to 60Co γ ray irradiation, K562 cells entered into a more mature stage on differentiation of morphological
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With students as the teaching center,the teaching quality was improved through improving teacher's quality,making use of various teaching methods,stirring up the student's experiment interest and constructing excellent study atmosphere.
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Objective To investigate the effects of low doses 147Pm internal irradiation against hereditary mutation in mouse cells induced by subsequent high dose of 147Pm irradiation. Methods The frequencies of micronucleated polychromatic erythrocytes (mn-PCE) micronucleated normochromatic erythrocytes (mn-NCE) and chromosome aberration in bone marrow and peripheral blood cells were observed. Results The frequencies of nm-PCE、mn-NCE and chromosome aberration induced by high dose of 147Pm irradiation showed a highly significant increase over the control at 24 h after irradiation (P＜0.05 or 0.01).However, the frequencies in cells pre-exposed to 0.37-37 Bq/g of 147Pm were decreased significantly (P＜0.05 or 0.01),as compared with those of the group without pre-exposure. Conclusion Low doses of in vivo 147Pm β-irradiation can induce a radioadaptive response of the lymphocytes and erythroblasts.The induction of the adaptive response has a wide dose range.The mn-PCE and mn-NCE are more sensitive indexes for revealing biological effects of radiation as compared with the chromosome aberration.