Expression of pprM gene from Deinococcus radiodurans in eukaryotic cells

Abstract

Objective To 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. Methods The 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. Results Bacterial 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. Conclusions The 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