HO-1真核表达质粒的构建及在肾小管上皮细胞中的表达

CONSTRUCTION OF EUKARYOTIC EXPRESSION PLASMID OF HEME OXYGENASE-1 GENE AND ITS EXPRESSION IN RAT RENAL TUBULAR EPITHELIAL CELLS

[目的]构建HO-1基因真核表达质粒,转染大鼠肾小管上皮细胞,检测其在细胞中的表达效果。[方法]从大鼠肾组织中提取总RNA,通过逆转录PCR(RT-PCR)扩增大鼠HO-1基因片段,将其克隆入真核表达载体pcDNA3.1(+)中,运用脂质体将重组质粒pcDNA 3.1(+)-HO-1转染大鼠肾小管上皮细胞,通过RT-PCR和Western blot分析HO-1基因细胞转染及表达的效果。[结果]成功构建了HO-1基因真核表达重组质粒pcDNA3.1(+)-HO-1。RT-PCR及Western blot分析显示,HO-1基因能转染大鼠肾小管上皮细胞并在细胞内有效表达。[结论]HO-1基因真核表达重组质粒pcDNA 3.1(+)-HO-1的成功构建及其在肾小管上皮细胞中的有效表达为深入研究其生物学作用奠定了基础。

[Objective] To construct HO-1 gene recombinant eukaryotic expression plasmid and to detect the effects of its transfecting into rat renal tubular epithelial cells（RRTECs）and the expression of target protein.Methods Total RNA from rat kidney was extracted.The HO-1 gene was amplified using reverse transcription-polymerase chain reaction（RT-PCR）and cloned into the eukaryotic expression vector pcDNA 3.1（＋）by polymerase of recombinant gene technology.Afterwards,the recombinant plasmid pcDNA 3.1（＋）-HO-1 was transfected into RRTECs by using the liposome.The effects of transfection and the expressin of HO-1 gene were detected by RT-PCR and Western blot analysis.Results As indentified with double restriction endonuclease digestion and sequence analysis,the DNA sequence of cloned HO-1 gene was correct and the eukaryotic expression recombinant plamid pcDNA 3.1（＋）-HO-1 was successfully constructed.RT-PCR and Western bolt analysis showed that HO-1 was transfected successfully into RRTECs and efficiently expressed at both mRNA and protein level.Conclusion The successful construction of eukaryotic expression recombinant plasmid of HO-1 gene and the efficient expression of HO-1 protein in RRTECs lay the foundation for further study on its biological function.