喉癌来源黑色素瘤抗原A3在小鼠黑色素瘤细胞模型的表达

Melanoma antigen A3 sourced from laryngeal cancer was expressed in mouse melanoma cell model

目的:建立pIRES2-EGFP/MAGE-A3真核质粒在小鼠黑色素瘤B16细胞稳定表达的肿瘤细胞模型。方法:将喉癌来源的黑色素瘤抗原A3(Melanoma-associated antigen A3,MAGE-A3)构建成真核质粒pIRES2-EGFP/MAGE-A3,脂质体法将pIRES2-EGFP/MAGE-A3转染小鼠黑色素瘤B16细胞,G418筛选阳性克隆,荧光显微镜检测阳性克隆中增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)的表达,荧光定量PCR(qRT-PCR)检查MAGE-A3在B16细胞中的转录。结果:pIRES2-EGFP/MAGE-A3真核质粒转染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,qRT-PCR可检测到MAGE-A3 mRNA的转录。结论:pIRES2-EGFP/MAGE-A3真核质粒通过脂质体法能有效转染,并在B16稳定表达,成功建立了MAGE-A3肿瘤细胞模型。

Objective：To construct tumor cell model by determination of the plRES2-EGFP/MAGE-A3 eukaryotic expression plasmid expressing steadily in mouse melanoma B16 cells. Methods： The plRES2-EGFP/MAGE-A3 eukaryotic expression plasmid being constructed from the melanoma-associated antigen A3 genes sourcing laryngocarcinoma in advance was translated into the mouse melanoma B16 ceils under the mediation of lipofectamine, and the positive clones were detected with G418. The expression of enhanced green fluorescent protein（EGFP） and MAGE-A3 mRNA in positive clones were detected by fluorescence microscopy and fluorescence quantitative PCR （qRT-PCR）assay, respectively. Results： The pIRES2-EGFP/MAGE-A3 eukaryotic expression plasmid has been transfected into B16 cells successfully, the green fluorescence of fusion protein expression was found, and MAGE-A3 mRNA transcription in Bl6 cells expressions were detected in positive clones. Conclusion： The plRES2-EGFP/MAGE-A3 eukaryotic expression plasmid has been transfected effectively and expressed stably by liposome method in the B16 cells. The expression of MAGE- A3 tumor cell model has been successfully established,which provide data for the study of laryngocarcinoma immunotherapy.