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Cloning and expression of mouse peroxiredoxin I in IEC-6 Cells
Bo Zhang1, Yong-Ping Su, Tao Wang
1Institute of Combined Injury of PLA, State Key Laboratory of Trauma, Third Military Medical University, Gaotanyan Street 30, Chongqing 400038, China.
Aim:
To clone and express mouse peroxiredoxin I in IEC-6 cells.
Methods:
Total RNAs were isolated from cultured IEC-6 cells, and the coding region of peroxiredoxin I was amplified by RT-PCR. After it was cloned into T-vector and sequenced, pSG5 was used to transiently express peroxiredoxin I in IEC-6 by liposome-mediated transfection, and the expression of peroxiredoxin I was evaluated by RT-PCR and Western blot.
Results:
A DNA fragment about 750 bp was amplified from total RNAs of IEC-6 cells using specific primers of peroxiredoxin I. The sequencing confirmed the coding region was successfully cloned into T-vector, which was completely coincident with the sequence in GeneBank. After the EcoRI-BamHI fragment of T-vector containing peroxiredoxin I was inserted into pSG5, the recombinant plasmid was transferred to IEC-6 cells. RT-PCR assay showed that a DNA fragment of 930 bp could be amplified, which indicated the transcription of pSG5-Prx. Western blot confirmed the expression of peroxiredoxin I in IEC-6 cells.
Conclusion:
Mouse peroxiredoxin I can be successfully expressed in IEC-6 cells.
