| Deinococcus radiodurans is best known for its astonishing ability to resist the lethal effects of ionizing radiation, UV, desiccation, chemical mutagen and many other DNA damaging agents. The capacity for repairing ionizing radiation-induced DNA double-strand breaks is the most striking feature of D.radiodurans. It survives acute exposures to gamma radiation that exceed 5kGy levels with lethality or induced mutation. D.radiodurans can survive on 100 to 200 irradiation-induced double-strand breaks (DSBs); exceed 3,000 DNA single–strand breaks (SSBs) and over 1,000 base damage sites per haploid genome. In these DNA damages, DNA DSBs is the most fatal to cells. Unlike other extremophile organisms, D.radiodurans dose not thrive on extreme conditions. It can recover from damage inflicted by such conditions with the capacity to efficiently and accurately repair the heavy DNA damage and reconstitute a functional genome from numerous DNA fragments within hours.Recent research has revealed that D.radiodurans is is effected by a highly proficient DNA repair capacity. Meanwhile the generation of reactive oxygen species (ROS) and the hydrolytic cleavage of water which bring by ionizing radiation and UV can indirectly cause DNA damage. So the super radioresistance of D.radiodurans may have close relation with its antioxidant capacity. D.radiodurans may have a combination of non-enzymic and enzymic antioxidant defenses allow relatively conventional repair pathways to function with greater efficiency than in other bacteria. D.radiodurans has expressed superior antioxidant capacity and radio-resistance through scavenging oxygen free radicals, participating regulation of antioxidant system and effectively preventing DNA damage caused by free radicals. Study of extreme resistant and DNA damage repair mechanisms of D.radiodurans will be conducive to better research on the pathogenesis of tumor and the search for anti-radiation drug. It can be used to repair the radiation pollution of the environment.Homologous recombination (HR) is one of the primary mechanisms by which cells repair DSBs and SSBs and is important for restart of stalled DNA replication. In E.coli, there are two major recombination pathways, RecBCD and RecFOR. D.radiodurans is capable to repair of thousands DSBs though it lakes RecBCD pathway, The involvement of all three RecF, RecO, RecR proteins in HR initiation is well documented by genetic and cellular approaches, yet the biochemical fuction in the initiation process is unclear. In present study we constructed prokaryotic and eukaryotic expression system for recO and recF as the agents of RecFOR from D.radiodurans and tested the protective effect of transfection of recO and recF gene on oxidation and inflammatory injury of UVB-induced human skin fibroblasts in vitro. The purpose of this study is to experiment data, and develop a new biological sunscreen to protect human from UV.PartΙ: Construction of RecO and RecF from Deinococcus radiodurans in prokaryotic expression System in Vitro1. PCR primers were designed from the GenBank database index .D.radiodurans gene sequence of rccO (DR0819) and recF (DR1089) and the D.radiodurans genome DNA was used as the template for PCR amplification. The sizes of the PCR products were 735bp and 1080bp separately. The results cloned into the pGEM-T plasmid. Recombinant plasmids were checked for the correct insert DNA by restriction analysis and DNA sequencing.2. The PCR products were sequenced to insure the displacement of recF and recO cloned into the espress vector pET30b(+) plasmid, then constructed prokaryotic expression recombined plasmid pET30b(+)-recO and pET30b(+)-recF. To transform the recombined plasmid into the E.coli BL21(DE3).3. Optimization expression of induction conditions of RecO and RecF: Research the effects of protein expression with different concentration of IPTG,different induction time and temperature in order to determine the optimization expression of induction conditions of RecO and RecF. The results showed that the highest expression of RecF at 16℃,0.3mM IPTG, 2-3 hour; the highest expression of RecO at 37℃0.1mM IPTG, 2-3 hour.4. Initial detection discovery recO and recF gene can increase resistant ability of E.coli. Part II: Anti-radiation effects of human skin fibroblasts transfected recO and recF gene agaisnt UVB-induced damage1. Constructing the model of transfection recO and recF gene in human skin fibroblasts. Isolation of human fibroblasts using trypsin enzyme digestion method. After 3 days of the isolation, the fibroblasts were digested and passage. The bordierline of cells were clear, the shape of cell body was fusiform or polygon, nucleolus are clear. Those can be observed in the cytoplasm after HE staining with optical microscope. After tested the growth curve of fibroblasts, 4-7days after isolation were the exponential phase of growth. Cells in this period were chosen to carry out the following experiment. Human fibroblasts transfection of recombination plasmid pcDNA3.1-NT-GFP Fusion TOPO-recO and pcDNA3.1-NT-GFP Fusion TOPO-recF using liposomes method. After testing the change of proliferative activity of fibroblasts, decided to use 0,30,90,120 mJ/cm2 UVB as determination doses.2. To test the growth curve of human skin fibroblasts by MTT assay. There were seven groups in MTT assay: Group I (untransfection-irradiated), GroupII (untransfection -nonirradiated), GroupIII (empty plamid transfection irradiated ), GroupIV (recO transfection irradiated), GroupV (recO transfection nonirradiated), GroupVI ( recF transfection irradiated), GroupVII(recF transfection nonirradiated). Ater testing the growth curve of cells, GroupII, GroupV and GroupVII were non significant differences(P>0.05). After 90 mJ/cm2 UVB irradiation 4 days , the proliferative activity of GroupV and GroupVII were gradual recovery,there were significant differences(P<0.05) when compare with GroupII and GroupIII.3. Compared radiation damage morphology of recO transfection irradiated group with recF transfection irradiated group: HSF received different doses of UVB irradiation after 24 h, cells showed varying degrees of damage. After 30 mJ/cm2 UVB irradiation, the cells body shape gradually became round and swelling, and vacuole was observed. Cell debris and dead cells dramatically increased at the dose of 90 mJ/cm2 UVB. The tendency showed that at the same irradiation dose (≥90 mJ/cm2),the degree of cell damage was aggravated with obvious time-response relationship. No obvious cell morphology changes were observed in the three groups (Untransfection -nonirradiated group, recO transfection nonirradiated group and recF transfection nonirradiated group) .Group of recO transfection nonirradiated and recF cell morphology began to change that cell body shape were from fusiform or polygon to irregularity, the gap between cells widen, vacuolization of cytoplasm were obviously increased. However, no obvious cell debris and dead cells were observed.4. Tested the capacity of antiradiation damage in cells: Took the concentration of lactate dehydrogenase (LDH) in culture medium as a signal to reflect the degree of cells early damage. The LDH would leak out of cells because of the change of the membrane permeability when cells suffered stimulating factors. The concentration of LDH was tested at 24 h, 48 h and 72 h. The concentration of LDH increased in untransfection-irradiated group at low UVB irradiation,and the increase was more obvious at higher dose. The concentration of LDH of recF and recO transfection groups were non-significant difference than empty plamid gruup and untransfection group at 24 h. But recF and recO transfection groups were significantly decreased than empty plamid gruup at 48～72 h.5. Tested the capacity of antioxidant in cells: The activity of superoxide dismutase(SOD)reflected the capacity of removing oxygen free radicals in cells . Malondialdehyde(MDA)was a product of lipid peroxidation.Its content could indirectly reflect the degree of lipid peroxidation and damage of cells. The rate of cell survival and the activity of SOD significantly decresed while MDA obviously increased after 30 mJ/cm2 UVB irradiation at 24～72 h. Meanwhile,recF and recO transfection could to a certain extent help cells to remove oxygen free radicals,increase the activity of T-SOD and decrease the concentration of MDA .6. Tested the capacity of anti-inflammatory in cells: Signal transmission system was activatated by stimulating factors in human skin keratinocytes. Secretion of IL-1αby stimulating factors like UVB in keratinocytes,could induce secretion of IL-6 for many cells including hunman skin fibroblasts. TNF-αplays a important role in inducing tumor and immune response regulation, and closely correlated with skin damage inflammatory response and apoptosis. The results of tested concentration of IL-6 and TNF-αin all four groups indicated that UVB irradiation promoted the secretion of IL-6 and TNF-α. There was a dose-response relationshhip in a certain range. Secretion of IL-6 and TNF-αof recO and recF transfection groups were significantly lower than empty plamid group and untransfection group. In summary, this research cloned the recO and recF gene from Deinococcus radiodurans into pET30b(+) express vector, and made their gene expreeion under the control of transcription and translation signals of phage T7 strong promoter. Induced its express in E.coli BL21(DE3) by IPTG. Initial detection discovered that recO and recF gene could increase resistant ability of E.coli. Human fibroblasts transfection of recombination plasmid pcDNA3.1-NT-GFP Fusion TOPO-recO and pcDNA3.1-NT-GFP Fusion TOPO-recF using liposomes method. The results indicated that recO and recF gene transfection could increase the capacity of cells removing oxygen free radicals, inhibiting lipid peroxidation, and reducing cell damage caused by UVB. The capacity of cells survival,antioxidant, anti-injury of transfection recF group was significantly better than transfection recO group. Considering the complexity of protective effect, the specific mechanism shall be elucidated in the further research.
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