MAGE-1基因真核表达载体的构建及表达

目的 构建MAGE-1基因重组真核表达质粒并在细胞系NIH-3T3中高效表达。方法用RT-PCR方法从人肝细胞肝癌（HCC）组织全RNA中扩增出MAGE-1基因cDNA序列，克隆至真核表达载体pcDNA3．1（＋），构建pcDNA3．1-MAGE-1重组质粒．重组质耗用脂质体转染NIH-3T3细胞，经RT-PCR和Western-blot鉴定转化细胞中MAGE-1的表达。结果RT-PCR获得长度为927bp的MAGE-1基因，经T载体和pcDNA3．1（＋）真核表达载体克隆、酶切鉴定及序列分析后，证实重组质粒构建正确，并存转化细胞中检测到了MAGE-1的表达。结论 真核表达质粒pcDNA3．1-MAGE-1构建成功，并建立了稳定表达人MAGE-1的NIH-3T3细胞株。

Construction of pcDNA3.1-MAGE-1 Expression Vector

Objective To construct the pcDNA3.1-MAGE-1 expression vector and express MAGE-1 in NIH-3T3 cell line.Methods The mRNA of MAGE-1 gene was isolated from HCC tissue,and the cDNA was amplified by means of RT-PCR.After cloning with pGEM-T easy vector and sequencing,the MAGE-1 gene was inserted into the eukaryotic expression vector pcDNA3.1(+).NIH-3T3 cell was transfected with the recombinant.SDS-PAGE and Western-blot were used to detect the MAGE-1 protein in transfected NIH-3T3 cell.(Results )The MAGE-1 gene that was obtained through PT-PCR well matched with the known MAGE-1 sequence(GenBank~(TM) M77481).After dual enzymes cutting,the MAGE-1 gene was inserted into pcDNA3.1(+) vector.The result of PCR and Western-blot showed that there was eukaryotic expression vector pcDNA3.1(+)-MAGE-1 which can express MAGE-1 protein in transfected NIH-3T3 cell.Conclusion The pcDNA3.1-MAGE-1 vector has been constructed successfully,which can express in NTH-3T3 cell line stably.