人mdr1全长cDNA的克隆和pc-MDR1质粒的构建

Molecular cloning of a full length human mdr1 cDNA and construction of the pc-MDR1 plasmid

目的 从耐药肿瘤组织中进行mdr1cDNA的全长克隆和pc MDR1重组质粒的构建。并转染HepG2细胞 ,快速诱导其耐药 ,并筛选其抗性克隆。 方法 设计单酶切位点引物 ,利用长距离逆转录 聚合酶链反应 (LongRT PCR )技术 ,从HepG2耐药细胞扩增mdr1cDNA ,约 3 .8kb大小。将其插入至真核表达载体 pcDNA3 .0质粒中构建 pc MDR1诱导质粒 ,使其能够在真核细胞中表达P 糖蛋白 (P gp)。转染HepG2细胞 ,并用G418筛选出转染的细胞。结果 成功地扩增出3 .8kb左右的mdr1cDNA片段 ,经酶切鉴定、RT PCR扩增特异片段和序列测序结果显示质粒构建初步成功 ,用G418筛选细胞耐药抗性克隆的时间约为 14d。结论 从耐药肿瘤组织中进行mdr1cDNA全克隆和pc MDR1诱导质粒构建的成功为快速诱导细胞耐药和进一步研究肿瘤细胞的耐药机制奠定了基础。

Objective To isolate the full-length mdr1 cDNA,construct the pc-MDR1 plasmid and transfect the HepG2 cells.Methods Firstly,the single site primers were designed by the GeneFisher 1.3 soft.Sencondly,the full-length mdr1 cDNA (about 3.8 bp) was isolated from HepG2 MDR tumor on nude mice.The target segment was inserted into the expressing vector pcDNA3.0 and transfected the HepG2 cells which were screened out by G418 at last.Results The full-length cDNA was cloned successfully and the pc-MDR1 plasmid was constructed.The transfected HepG2 cells were isolated by G418 after 2 weeks.Conclusion The clone of the full-length cDNA and the construction of the plasmid will help greatly to further study the signal control of mdr1 gene.