Construction and identification of hGPx-1-198Leu expression vector and its expression in rat H9C2 cells

Objective: To construct and identify a recombinant hGPx-1-198Leu cDNA eukaryotic expression vector in order to lay foundation for studying the function of the mutated human GPx-1 gene. Methods: First, we synthesized the mutated human GPx-1 cDNA. Meanwhile, two restriction endonucleases BamHI and NotI sites were introduced in 5'and 3' ends. Second, the GPx-1 cDNA and pEGFP-N3 plasmid were digested by BamHI and NotI restriction endonucleases, and then the GPx-1 cDNA was directly inserted into the downstream of the human cytomegalovirus promoter of the pEGFP-N3 plasmid by T4 DNA ligase. Third, the ligation products were transformed into DH5α; the positive colonies that survived in the LB+ Kanamycin medium were selected, and then plasmid DNA was abstracted and identified by polymerase chain reaction (PCR) and sequencing. The hGPx-1-198Leu eukaryotic expression vector was transfected into HEK293 cells, and the transfection efficiency was observed by fluorescence microscope. H9C2 cells were also transfected with hGPx-1-198Leu construct and then interfered with sodium selenite. Results: The total length of the synthesized gene products was 869bp. Both PCR and sequencing analysis showed that the mutated GPx-1 gene was successfully inserted into the pEGFP-N3 plasmid, which laid foundation for further studying the function of Pro198Leu polymorphism of GPx-1 gene. The expression level of GPx-1 in the H9C2 cells transfected with hGPx-1-198Leu construct was significantly higher than that of the other two group cells; the higher the concentration of selenium, the higher the level of GPx-1 protein. Conclusion: The hGPx-1-Pro198Leu eukaryotic expression vector was successfully constructed and the selenocystenine insertion sequence was also included in the vector, which can make GPx-1 express successfully.