Construction of eukaryotic expression vector carrying pprI gene of Deinococcus radiodurans and its radioresistant effect
10.3760/cma.j.issn.0254-5098.2014.08.002
- VernacularTitle:耐辐射球菌pprI基因真核表达载体的构建及其抗辐射作用
- Author:
Ling WEN
;
Yi SHI
;
Lili REN
;
Ying CONG
;
Zhanshan YANG
- Publication Type:Journal Article
- Keywords:
Deinococcus radiodurans R1;
pprI gene;
Acute radiation injury;
Radiation resistance
- From:
Chinese Journal of Radiological Medicine and Protection
2014;34(8):563-568
- CountryChina
- Language:Chinese
-
Abstract:
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.
