稳定表达人MAGE-1基因的小鼠细胞系的建立与鉴定

目的：克隆人黑素瘤抗原-1（MAGE-1）基因，构建真核表达载体，建立稳定表达人MAGE-1的小鼠细胞株。方法：提取人肝癌细胞的总RNA，RT-PCR法获得人MAGE-1cDNA，将测序正确的MAGE-1基因克隆至真核表达载体pcDNA3中，进行酶切鉴定和序列测定。将重组质粒pcDNA／MAGE和空载体pcDNA3分别转染入小鼠骨髓瘤细胞SP2／0中，经G418筛选获得稳定表达株，用RT-PCR和Western印迹检测MAGE-1基因的转录及蛋白表达。结果：从肝癌细胞中成功克隆到人MAGE-1基因，经测序证明基因正确，酶切和序列测定表明，人MAGE-1基因的真核表达质粒已成功构建。将此重组质粒转入的SP2／0细胞可稳定表达人MAGE-1基因。结论：成功构建可稳定表达人MAGE-1基因的小鼠转基因细胞，为MAGE-1的免疫治疗研究奠定了基础。

Establishment and identification of SP2/0 cell line stably expressing human MAGE-1 gene

Objective:To construct an eukaryotic expression vector and estab lish a transgen ic SP2 /0 cell line stab ly ex-pressing hum an MAGE-1 gene.M ethod s:The hum an MAGE-1 gene was amp lified by RT-PCR from BEL-7402 cell line and c loned into eukaryotic expression vector pcDNA3.The recomb inatnt expression vector and pcDNA3 were transfected in-to SP2 /0 by L ipofectin respectively.The positive c lones were selected via G418.The SP2 /0 cell line stab ly expressing hu-m an MAGE-1 was confirm ed by RT-PCR and W estern b lot.R esu lts:Hum an MAGE-1 gene was amp lified by RT-PCR and c loned into eukaryotic expression vector pcDNA3 successfu lly.RT-PCR andW estern b lot showed that SP2 /0 cell line stab ly expressingMAGE-1 gene was successfu lly estab lished.C onc lu sion:The transgen ic SP2 /0 cell line stab ly expressing hu-m an MAGE-1 is estab lished successfu lly and it can be used as an experim entalmodel for anti-tumor immunotherapy.