pcDNA3-MAGE-1真核表达载体的构建及其在Hepa1-6细胞中的稳定表达

Construction of eukaryotic expression vector pcDNA3-MAGE-1 and its stable expression in mouse Hepa1-6 cells

目的:构建真核表达载体,并在小鼠肝癌Hepa1-6细胞中稳定表达,观察转染细胞的增殖能力和致瘤能力。方法:用PCR方法扩增SMMC-7721人肝癌细胞株MAGE-1全长序列,连接到真核表达载体pcDNA3中,构建真核表达pcDNA3-MAGE-1,脂质体法转染小鼠Hepa1-6细胞。G418筛选阳性克隆(Hepa1-6-MAGE-1),用RT-PCR和Western blotting检测阳性克隆中mRNA和蛋白质的表达;MTT法检测Hepa1-6细胞和Hepa1-6-MAGE-1细胞生长和增殖的变化;分别以Hepa1-6细胞和Hepa1-6-MAGE-1细胞接种于C57BL/6j小鼠右侧背部皮下,观察Hepa1-6-MAGE-1细胞的致瘤能力。结果:构建MAGE-1的真核表达载体转染Hepa1-6细胞,PT-PCR与Western boltting检测分别在Hepa1-6-MAGE-1细胞中检测到人MAGE-1基因的mNRA和蛋白质的表达,两组细胞增殖无统计学差异;两组各10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-MAGE-1,获得了稳定表达人MAGE-1基因的小鼠肝癌Hepa1-6细胞株,并具有很好的增殖和致瘤能力。

Objective To construct the eukaryotic expression vector and express in mouse liver cancer cell Hepa1-6, and observe reproductive activity and tumorigenesis ability of transfected cells. Methods The MAGE-1 total length sequence was amplified from SMMC-7721 by PCR and linked to pcDNA3 to construct pcDNA3-MAGE-1. The expression vector was transfected into Hepa1-6 cells by lipofectamine, and then the positive clones were screened by G418. The expressions of mRNA and protein in positive clones were detected by RT-PCR and Western blotting. Cell growth and proliferation in Hepa1-6 cells and Hepa1-6- MAGE-1 cells were detected by MTT, then they were inoculated in the right back subcutaneous of mice. Results pcDNA3-MAGE-1 was constructed and transfected into Hepa1-6 cells, the expressions of MAGE-1 mRNA and protein were detected in Hepa1-6-MAGE-1 cells by RT- PCR and Western blotting, there was no statistical difference in cell proliferation between two groups. The mice were all infected with tumor in two groups, and there was no difference in tumor size. Conclusion The eukaryotic expression vector pcDNA3-MAGE-1 is successfully constructed, and the Hepa1- 6 cell line which can stably express human MAGE-1 gene is established with good proliferation and tumorigenesis ability.