MAGE-3基因真核表达载体构建及在小鼠黑色素瘤细胞中的表达

目的　扩增人黑色素瘤抗原 3(Melanomaantigen 3,MAGE 3)基因 ,构建真核表达载体 ,并在小鼠黑色素细胞瘤B16中进行表达。方法　采用PCR扩增MAGE 3基因 ,连接到真核表达载体pIRES2 EGFP中 ,构建真核表达载体pIRES2 EGFP MAGE 3,脂质体法转染小鼠黑色素瘤B16细胞。G4 18筛选阳性克隆 ,荧光显微镜和RT PCR分别检测阳性克隆中增强型绿色荧光蛋白 (Enhancedgreenfluorescentprotein ,EGFP)的表达和MAGE 3mRNA的表达。结果　从pUC19 MAGE 3重组质粒中扩增获得一条约 95 0bp的片段 ,成功构建了真核表达载体pIRES2 EGFP MAGE 3,转染B16细胞后筛选得到阳性克隆 ,可见融合蛋白表达产生明亮的绿色荧光 ,RT PCR检测到MAGE 3mRNA的表达。结论　成功构建了真核表达载体pIRES2 EGFP MAGE 3,获得了稳定表达该载体的小鼠黑色素瘤B16细胞系 ,为研究MAGE 3在肿瘤免疫治疗中的应用奠定了基础。

Construction of MAGE-3 gene eukaryotic expression vector and its expression in mouse melanoma cells

Objective To amplify human melanoma antigen 3 (MAGE-3) gene and construct the eukaryotic expression vector that can be expressed in B16 mouse melanoma cells. Methods The MAGE-3 gene was amplified by PCR, then cloned into the vector pIRES2-EGFP for constructing the pIRES2-EGFP-MAGE-3 plasmid. The plasmid was transfected into the B16 mouse melanoma cells under mediation of lipofectamine and the positive clones were selected. The expression of enhanced green fluorescent protein (EGFP) and MAGE-3 mRNA in positive clones were detected by fluorescence microscopy and RT-PCR, respectively. Results The full length of MAGE-3 was amplified by PCR. The pIRES2-EGFP-MAGE-3 plasmid was constructed and transfected into B16 cell successfully. Green fluorescence of fused protein expression was found and MAGE-3 mRNA expressions were detected in the positive clones. Conclusion The eukaryotic expression plasmid pIRES2-EGFP-MAGE-3 was constructed successfully. The B16 cell lines that stably transfected MAGE-3 is obtained, which contribute to the research of MAGE-3 in the immunotherapy of tumor.