人重组pMAGE-A1/EGFP真核表达质粒的构建与表达

目的　特异克隆MAGE A1开放读码框基因,构建以绿色荧光蛋白(EGFP)为报告基因的包含MAGE A1的真核表达载体并转染人PBMC来源DC细胞。方法　根据MAGE A1特征序列设计PCR扩增引物,从Mel 5 2 6中扩增MAGE A1基因开放读码框,并克隆至pIRES EGFP中,构建人重组pMAGE A1 EGFP真核表达质粒。利用电转染方法将该重组质粒转入人外周血来源DC细胞并检测MAGE A1和EGFP的表达情况。结果　成功构建真核表达质粒pMAGE A1 EGFP ,转染人PBMC来源DC细胞并检测到MAGE A1和EGFP表达,两者表达率分别为(8.8±0 .9) %和(8.4 7±0 .78) % ,无统计学差异。结论　成功构建人重组真核表达质粒pMAGE A1 EGFP ,为开展MAGE A1为基础的免疫治疗提供有利的分子生物学工具。

Construction and expression of recombinant human eukaryotic expression vector pMAGE-A1/EGFP

Objective To clone MAGE-A1 gene open reading frame and construct eukaryotic expression vector with green fluorescent protein, and to transfect human PBMC-derived DCs. Methods MAGE-A1 open reading frame was amplified from Mel-526 by RT-nest PCR with specific primers, and then cloned into eukaryotic expression vector with EGFP. After construction of recombinant plasmid pMAGE-A1/ EGFP, human PBMC-derived DCs were transfected with pMAGE-A1/EGFP by electroporation, and protein expression was identified by fluorescent microscopy. Results The pMAGE-A1/EGFP eukaryotic expression vector was constructed successfully. MAGE-A1 and EGFP were expressed after transfection into DCs. Conclusion The pMAGE-A1/EGFP eukaryotic expression vector will provide a new modality of tumor immunotherapy targeting MAGE-A1 gene.