| 文玲,施怡,任丽丽,等.耐辐射球菌pprI基因真核表达载体的构建及其抗辐射作用[J].中华放射医学与防护杂志,2014,34(8):563-568.Wen Ling,Shi Yi,Ren Lili,et al.Construction of eukaryotic expression vector carrying pprI gene of Deinococcus radiodurans and its radioresistant effect[J].Chin J Radiol Med Prot,2014,34(8):563-568 |
| 耐辐射球菌pprI基因真核表达载体的构建及其抗辐射作用 |
| Construction of eukaryotic expression vector carrying pprI gene of Deinococcus radiodurans and its radioresistant effect |
| 投稿时间：2014-03-17 |
| DOI：10.3760/cma.j.issn.0254-5098.2014.08.002 |
| 中文关键词: 耐辐射球菌 pprI基因 急性放射损伤 辐射抗性 |
| 英文关键词:Deinococcus radiodurans R1 pprI gene Acute radiation injury Radiation resistance |
| 基金项目:国家自然科学基金（81372922）；国防基础科研资助项目（A3820060138）；江苏省高校优势学科建设工程资助项目（PAPD） |
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| 中文摘要: |
| 目的 研究耐辐射球菌pprI原核基因的真核表达载体的构建及其转染对离体真核细胞急性放射损伤的防护作用。方法 应用DNA重组技术构建pEGFP-c1-pprI质粒；采用脂质体转染技术分别将空载体质粒pEGFP-c1和重组质粒pEGFP-c1-pprI转入人肺上皮细胞系Beas-2B细胞，筛选稳定转染细胞系；应用集落形成实验检测受照转染细胞的生存能力；流式细胞仪检测细胞周期和凋亡率的变化；荧光显微镜观察受照细胞ROS荧光强度；免疫荧光实验观察受照细胞不同时间γ-H2AX焦点数目。结果 成功地构建了pprI原核基因的真核表达载体，并在Beas-2B细胞中表达了PprI融合蛋白，建立了稳定转染细胞系。与空白对照组和空载体组相比，细胞克隆生存曲线显示转染了pprI基因的Beas-2B细胞经不同剂量辐射，其存活分数SF增加，该曲线参数D0、Dq、N增高；转染了pprI基因的Beas-2B细胞受照后的ROS的荧光强度和不同时间（1、2、4 h）γ-H2AX的焦点数均显著减低（F=16.73、19.47、6.94，P<0.05）；此外，该细胞G2期阻滞（F=139.73、237.92，P<0.05）和凋亡率显著减低（F=626.02、2 052，P<0.05）。结论 耐辐射球菌pprI原核基因可在离体真核细胞中稳定表达，并且显著提高受照真核细胞的辐射抗性。 |
| 英文摘要: |
| 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 Beas-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 R1 can be stably expressed in the eukaryotic cells and it allows the transferred cells to have a radioresistant function. |
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