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目的构建人pEGFP-C2-PRDX3真核表达载体,并检测其在人髓母细胞瘤DAOY细胞中的表达。方法从人髓母细胞瘤DAOY细胞中提取总RNA,采用RT-PCR法对编码PRDX3的基因片段进行扩增,应用基因重组技术,将目的片段亚克隆到pEGFP-C2真核表达载体,经PCR、酶切和测序鉴定后,用脂质体法将重组质粒转染入DAOY细胞,分别采用RT-PCR法、West-ern blot法检测PRDX3在DAOY细胞中的表达。结果双酶切及测序结果验证PRDX3片段正确插入pEGFP-C2质粒,转染后的PRDX3基因mRNA及蛋白质水平均明显上调。结论成功构建了具有报告基因-增强绿色荧光蛋白(EGFP)基因的真核表达载体pEGFP-C2-PRDX3,为进一步研究PRDX3基因在人髓母细胞瘤中的生物学功能奠定了基础。
Objective To construct the eukaryotic expression vector pEGFP-C2-PRDX3 and to detect its expression in human medulloblastoma DAOY cells. Methods Total RNA was extracted from human medulloblastoma DAOY cells. The gene fragment encoding PRDX3 was amplified by RT-PCR. The recombinant plasmid was subcloned into pEGFP-C2 eukaryotic expression vector by gene recombination technique. After digestion and sequencing, the recombinant plasmid was transfected into DAOY cells by lipofectamine. The expression of PRDX3 in DAOY cells was detected by RT-PCR and West-ern blot respectively. Results Double digestion and sequencing confirmed that the PRDX3 fragment was correctly inserted into pEGFP-C2 plasmid. The mRNA and protein levels of PRDX3 gene after transfection were significantly up-regulated. Conclusion The eukaryotic expression vector pEGFP-C2-PRDX3 with reporter gene-enhanced green fluorescent protein (EGFP) gene was successfully constructed, which laid the foundation for further study on the biological function of PRDX3 gene in human medulloblastoma.