Published online Jul 15, 2004. doi: 10.3748/wjg.v10.i14.2109
Revised: January 5, 2004
Accepted: February 1, 2004
Published online: July 15, 2004
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 EcoR I-BamH I 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.