人apoE_7基因在NIH/3T3细胞中瞬时及稳定表达

Transient and Stable Expression of Human ApoE7 Gene in NIH/3T3 Cell Lines

目的为了探索人apoE_7基因受控于异源启动子MT-I时,能否在鼠类细胞中表达。方法自10.3 kb人apoE_7基因组DNA获得不带自身启动子的人apoE_7基因组DNA片段(60 kb),与小鼠金属硫蛋白启动子(MT-I)重组,构建pME_7真核表达质粒,用Lipofectamine转染NIH/3T3细胞。结果瞬时表达结果表明,重组pME_7质粒能够在NIH/3T3细胞中表达。稳定表达的结果则表明,人apoE_7基因的表达水平与其整合到宿主细胞基因组的拷贝数密切相关。ELISA测定结果显示,人apoE_7基因在NIH/3T3细胞中可以进行正确的转录剪切和翻译加工,合成有生物活性的人apoE_7蛋白;研究结果还表明:重金属可以诱导增强人apoE_7基因的高效表达,诱导后表达水平提高45%～60%。结论人apoE_7基因的表达不仅局限于人类的组织细胞,也可以在鼠类组织细胞中表达;而且MT-I启动子能够诱导表达人apoE_7基因,为人apoE_7转基因小鼠模型的建立奠定了基础。

To investigate the expressing profile of h-apoE_7 gene under the regulation of MT-I promoter in mouse cells. Methods The eukaryotic expressing plasmid pME_7 was constructed through cloning human apoE_7 genomic DNA(6.0 kb) into pMT at the site of Bgl Ⅱ/Hind Ⅲ just under the control of the mouse metallothionein-Ⅰ promoter. Transfection of pME_7 plasmid into NIH/3T3 cell lines by Lipo-fectamine was performed. Results The transient expression of recombinant pME_7 in NIH/3T3 cells showed that the expression was not restricted to human cells; The stable expression of pME_7 demonstrated that the levels of h-apoE_7 mRNA was highly related to the copies of integrated h-apoE_7 gene. Whereas the results of ELISA implicated that the exogenous human apoE_7 gene was transcribed, processed properly, and eventually translated into functional proteins. Futhermore, heavy metal (ZnSO_4) could enhance the expressing level of h-apoE_7 gene in transfected cell lines. The level of human apoE7 protein arised 45%～60% while induced. Conclusions It was suggested that MT-Ⅰ promoter could modulate the expression of recombinant h-apoE_7 gene correctly. Therefore, cellular studies provided the scientific basis for establishment of hapoE_7 transgenic mouse models.