Zhang Xiu-min, Sui Yan-fang, Si Shao-yan, Li Xia, Huang Yang, Hu Pei-zhen, Ge Wei
Department of Pathology, Fourth Military Medical University, Xi'an 710032, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2006 Jul;22(4):450-1, 453.
To construct the eukaryotic expression vector of tumor antigen MAGE-3 and establish human hepatocellular carcinoma cell line (HHCC) expressing MAGE-3.
The MAGE-3 gene was amplified by PCR and cloned into the eukaryotic expression vector pIRES2-EGFP to construct the pIRES2-EGFP-MAGE-3 plasmid. The recombinant plasmid pIRES2-EGFP-MAGE-3 was transfected into HHCC cells by lipofectamine, and then the positive clones were screened by G418. The expression of enhanced green fluorescent protein (EGFP) and MAGE-3 mRNA in positive clones were detected by fluorescence microscope and RT-PCR, respectively.
The eukaryotic expression vector pIRES2-EGFP-MAGE-3 was successfully constructed. The expression of EGFP was found by fluorescence microscope detection and MAGE-3 mRNA transcription was detected by RT-PCR in the positive clones.
The stable MAGE-3-transfected HHCC cell line is successfully established, which will provide experimental basis for further study on immunotherapy for hepatocellular carcinoma using MAGE-3 as target antigen.
构建肿瘤抗原MAGE-3的真核表达载体，并建立表达MAGE-3的人肝癌细胞系（HHCC）。
通过PCR扩增MAGE-3基因，并将其克隆到真核表达载体pIRES2-EGFP中，构建pIRES2-EGFP-MAGE-3质粒。用脂质体将重组质粒pIRES2-EGFP-MAGE-3转染到HHCC细胞中，然后用G418筛选阳性克隆。分别用荧光显微镜和RT-PCR检测阳性克隆中增强绿色荧光蛋白（EGFP）的表达和MAGE-3 mRNA的表达。
成功构建了真核表达载体pIRES2-EGFP-MAGE-3。通过荧光显微镜检测发现阳性克隆中有EGFP表达，通过RT-PCR检测到阳性克隆中有MAGE-3 mRNA转录。
成功建立了稳定转染MAGE-3的HHCC细胞系，这将为进一步以MAGE-3为靶抗原进行肝癌免疫治疗的研究提供实验依据。
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