Original Article
Expression of pprM gene from Deinococcus radiodurans in eukaryotic cells
Shuya He, Qi Yang, Rujing Wang, Fangzhu Xiao, Wuzhou Wang, Yan Tang, Yuwei Jiang, Yun Ma
Published 2017-03-25
Cite as Int J Radiat Med Nucl Med, 2017, 41(2): 103-107. DOI: 10.3760/cma.j.issn.1673-4114.2017.02.005
Abstract
ObjectiveTo enhance the radiation resistance of eukaryotic cells and to identify the mechanisms that underlie radiation resistance, the radioresistant pprM gene was amplified from Deinococcus radiodurans and used to construct a recombinant pEGFP-C1-pprM plasmid for the expression of PprM protein in 293T cells.
MethodsThe pprM gene was amplified from pGEX-6p-1-pprM via PCR and purified from agarose gel with a DNA recovery kit. The purified PCR product was digested with EcoR I and BamH I and ligated into the pEGFP-C1 plasmid. Recombinant plasmids were transfected into competent JM109 cells, which were then cultured on LB solid medium that contained kanamycin. Positive clones were identified and characterized via bacterial colony PCR, restriction enzyme digestion, and sequencing analysis. Lipofectamine 2000 reagent was used to transfect pEGFP-C1-pprM plasmids into 293T cells. Green fluorescent fusion protein was observed via fluorescence microscopy and identified by Western blot.
ResultsBacterial colony PCR and double digestion showed that the target band is approximately 400 bp in length. Sequencing results showed that the base sequence was identical to the original gene sequence, thus indicating the successful construction of the recombinant plasmid. Fluorescence photography results showed that pEGFP-C1-pprM plasmids were successfully transfected into 293T cells. Western blot results showed that fusion protein is approximately 40×103 in weight.
ConclusionsThe pEGFP-C1-pprM plasmid was transfected into 293T cells, which then successfully expressed PprM protein. This study provides the foundation for future research on the pprM gene and the effects of its products on the radiation resistance of 293T cells.
Key words:
Deinococcus radiodurans; Radiation; pprM gene; 293T cells
Contributor Information
Shuya He
Department of Biochemistry and Molecular Biology, University of South China, Hengyang 421001, China
Department of Radiation Medicine, School of Public Health, University of South China, Hengyang 421001, China
Qi Yang
Department of Biochemistry and Molecular Biology, University of South China, Hengyang 421001, China
Rujing Wang
the First People's Hospital of Hengyang, Hengyang 421002, China
Fangzhu Xiao
Department of Radiation Medicine, School of Public Health, University of South China, Hengyang 421001, China
Wuzhou Wang
Department of Biochemistry and Molecular Biology, University of South China, Hengyang 421001, China
Yan Tang
Department of Radiation Medicine, School of Public Health, University of South China, Hengyang 421001, China
Yuwei Jiang
Department of Biochemistry and Molecular Biology, University of South China, Hengyang 421001, China
Yun Ma
Department of Biochemistry and Molecular Biology, University of South China, Hengyang 421001, China
