稳定表达人MAGE-1基因小鼠黑色素瘤细胞系的建立

Establishment of mouse melanoma B16 cell lines which stably express human MAGE-1

目的:构建真核表达载体,并在小鼠黑色素瘤B16细胞中稳定表达。方法:用PCR方法扩增质粒pUC19-MAGE-1中目的基因MAGE-1,连接到真核表达载体pCDNA3.1中,构建真核表达pCDNA3.1-MAGE-1,脂质体法转染小鼠黑色素瘤B16细胞。G418筛选阳性克隆(B16-MAGE-1),用RT-PCR和Western blot检测阳性克隆中mRNA和蛋白质的表达;分别以2×105个B16细胞和B16-MAGE-1接种于C57BL/6小鼠右侧背部皮下,观察B16-MAGE-1细胞的致瘤能力。结果:用PT-PCR与Western bolt分别在B16-MAGE-1细胞中检测到人MAGE-1基因的mNRA和蛋白质的表达,两组共20只小鼠均皮下成瘤,且肿瘤大小没有明显差异。结论:成功构建了真核表达载体pCDNA3.1-MAGE-1,获得了稳定表达人MAGE-1基因的小鼠黑色素瘤B16细胞系,并保持很好的致瘤能力,为研究MAGE家族在肿瘤免疫治疗中的应用奠定了基础。

Objective：To construct the eukaryotic expression vector which can be expressed in B16 cells. Methods： The MAGE - 1 gene was amplified by PCR from pUC19 - MAGE - 1 ,then cloned into pCDNA3.1 to construct the pCDNA3.1 - MAGE - 1 plasmid. Under the mediation of lipofectamine, the plasmid was transfected into the mouse melanoma B16 cells, and then the positive clones were selected by G418. The expression of MAGE - 1 mRNA and protein were respectively detected by RT- PCR and Western Blot. Mice （10 per group） were challenged with the B16 cells and B16 -MAGE -1 cells （2 × 10^5 cells/mouse, respectively） in the fight back. Results： The plasmid was constructed and transfected into B16 cells successfully. The expression of mRNA and protein of MAGE - 1 were approved in the positive clones, two groups both grow melanoma. Conclusion： The eukaryotic expression plasmid pCDNA3.1 - MAGE - 1 was constructed successfully. The B16 cell line that stably expressed MAGE - 1 is obtained, which will contribute to the research of MAGE family in the immunotherapy of tumor.