摘要
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。
Purpose To construct a human eukaryotic expression vector pEGFP-C2-PRDX3 and to investigate its expression in human medulloblastoma DAOY cells. Methods Total RNA was extracted from human medulloblastoma DAOY cells and then the target se- quence of human PRDX3 gene was amplified by reverse transcription-polymerase chain reaction （RT-PCR）. With the technique of gene re-arrangement, PRDX3 gene was subcloned into pEGFP-C2 plasmid. After identification with PCR, restriction enzyme digestion and sequence analysis, the recombinant piasmid was transfected into DAOY cells with the cationic loposome. RT-PCR, Western blot- ting were used to detect the PRDX3 mRNA and protein expression of DAOY cells. Results Correct construction of pEGFP-C2-PRDX3 was identified by means of double digestion and sequencing. The expression of mRNA and protein of PRDX3 gene was significantly up- regulated after transfection. Conclusions The eukaryotic expression vector pEGFP-C2-PRDX3 with a reporter gene-enhance green flu- orescent protein （EGFP） gene is successfully constructed and would contribute to the further studies on the role of PRDX3 in human medulloblastoma.
出处
《临床与实验病理学杂志》
CAS
CSCD
北大核心
2012年第12期1325-1328,共4页
Chinese Journal of Clinical and Experimental Pathology
基金
深圳市科技工贸和信息化委员会项目(201101022)
