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...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-PCRand 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.展开更多
基金Supported by the National Natural Science Foundation of China,No.30230360
文摘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-PCRand 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.
