ERp29基因的克隆及其在IEC-6细胞中的表达

Molecular cloning of mouse ERp29 and expression in IEC-6 cells

目的克隆小鼠ERp29基因并在IEC-6细胞中表达。方法采用RT-PCR方法从小肠上皮细胞克隆该基因的编码区,经T-载体克隆并测序证实后构建真核表达载体,并转染IEC-6细胞,RT-PCR分析外源基因的表达。结果从小肠上皮细胞总RNA中成功地克隆了ERp29基因,序列测定结果表明克隆的序列与数据库中序列完全一致。构建的真核表达载体与预期一致,表达质粒转染细胞后的RT-PCR分析结果表明:外源的质粒得到了有效的表达。结论成功地克隆了ERp29基因,并获得真核表达。

Objective To clone mouse ERp29 gene from intestinal epithelia and express it in IEC-6 cells. Methods The total RNAs of mouse intestinal epithelia were isolated and the coding region of mouse ERp29 gene was amplified by RT-PCR. Then the coding region was cloned to T-vector and sequenced, and the expression vector was constructed by using pSGS. Cultured IEC-6 cells were transfected with recombinant vector by lipidsome, and the expression was confirmed by RT-PCR. Results A DNA fragment about 850 bp was amplified from total RNAs of mouse intestinal epithelial cells using specific primers of ERp29. The sequencing confirmed the coding region was successfully cloned into T-vector, which was completely coincident with the sequence in GenBank. After the fragment of T-vector containing ERp29 was inserted into pSG5, the recombinant plasmid was transferred to IEC-6 cells. RT-PCR assay showed that a DNA fragment of 967 bp could be amplified, indicating the transcription of pSGS-ERp29. Conclusion Mouse ERp29 gene was successfully cloned and expressed in IEC-6 cells, which help to further study the biochemical and physiological role in radiation response of this protein.