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李琴, 王小引, 赵春澎等. 不同启动子和MAR组合对重组CHO细胞转基因表达的影响[J]. koko体育app 学报（医学版）, 2018, 49(1): 18-23.
引用本文: 李琴, 王小引, 赵春澎等. 有所不同加载子和MAR组合名字对协同CHO人体细胞转DNA表达出来的影响到[J]. 四川省读书学报（中医药学版）, 2018, 49(1): 18-23.
LI Qin, WANG Xiao-yin, ZHAO Chun-peng. et al. Effects of Different Promoters and MAR Combinations on Transgene Expression of Recombinant CHO Cells[J]. Journal of Sichuan University (Medical Sciences), 2018, 49(1): 18-23.
Citation: LI Qin, WANG Xiao-yin, ZHAO Chun-peng. et al. Effects of Different Promoters and MAR Combinations on Transgene Expression of Recombinant CHO Cells[J]. Journal of Sichuan University (Medical Sciences), 2018, 49(1): 18-23.

不同启动子和MAR组合对重组CHO细胞转基因表达的影响

Effects of Different Promoters and MAR Combinations on Transgene Expression of Recombinant CHO Cells

  • 摘要: 目的 分析不同启动子与核基质结合区（MAR）组合对中国仓鼠卵巢细胞（CHO）中转基因表达的影响。方法 将β-珠蛋白MAR(gMAR)与人巨细胞病毒早期启动子(CMV-IE)、猿猴空泡病毒40（SV40）启动子组合，构建两种不同启动子与gMAR组合的表达载体。转染CHO细胞，48 h后观察增强型绿色荧光蛋白（eGFP）的瞬时表达情况；G418筛选稳定转化的细胞株，流式细胞术分析稳定CHO细胞eGFP基因的表达水平，实时荧光定量PCR（qPCR）分析eGFP基因的相对拷贝数。结果 不含gMAR的表达载体，CMV-IE启动子eGFP表达明显强于SV40启动子；gMAR能提高CMV-IE启动子eGFP表达水平，但在SV40启动子中并未表现出提高作用。两侧含gMAR的表达载体比一侧含有gMAR的表达载体CMV-IE启动子eGFP表达荧光强；G418加压筛选后，含SV40启动子的gMAR表达载体eGFP基因表达不稳定，荧光逐渐减弱，因此，后期只对稳定表达eGFP基因的CMV-IE启动子表达载体进行流式细胞术和qPCR分析。流式细胞术检测结果显示，一侧含有与两侧含有gMAR的CMV-IE启动子表达载体eGFP基因表达量比不含gMAR的表达载体分别提高9.85倍和12.94倍；qPCR结果显示，以不含gMAR的载体的eGFP基因拷贝数为1，一侧含有与两侧含有gMAR的CMV-IE启动子表达载体eGFP基因相对拷贝数为3.68和9.25。结论 CMV-IE启动子活性强于SV40启动子；gMAR能提高CMV-IE启动子外源基因的表达水平，其机制可能与增加基因拷贝数相关。  
    Abstract: Objective To analyze the effects of different promoters and matrix attachment region (MAR) on the expression of transgene in Chinese hamster ovary (CHO) cells.Methods The expression vector was constructed by the combination of beta globin MAR (gMAR) with the human cytomegalovirus immediate-early promoter (CMV-IE) and simian virus 40 (SV40) promoter. These vectors were transfected into CHO cells, after 48 h, the transient expression of enhanced green fluorescent protein (eGFP) was observed; G418 was used to screen stably transformed cell lines, and the expression level of eGFP in CHO cells was analyzed by flow cytometry. The relative copy numbers of eGFP were analyzed by qPCR. Results Without gMAR expression vector, the expression of eGFP which was driven by CMV-IE promoter was stronger than that of SV40 promoter; gMAR could increase the expression level of eGFP driven by CMV-IE promoter, but did not show any enhancement in SV40 promoter. The expression level of eGFP which containing gMAR on both sides was stronger than that of gMAR on one side driven by CMV-IE promoter; After G418 screening, the expression level of eGFP containing gMAR driven by SV40 promoter wasunstable, the fluorescence gradually weakened, therefore, we only analyzed the expression vector stably expressing the eGFP gene driven by CMV-IE promoter by flow cytometry and qPCR. Compared with the expression vector without gMAR containing CMV-IE promoter, flow cytometry showed that the expression levels of eGFP on one and both sides with gMAR were increased by 9.85-fold and 12.94-fold, respectivley; The result of qPCR showed that the copy number of the eGFP gene without gMAR was set to 1, the copy number of the eGFP gene in the expression vector driven by CMV-IE with gMAR on one side and both sides were 3.68-fold and 9.25-fold, respectively. Conclusion The activity of CMV-IE promoter is stronger than that of SV40 promoter. gMAR can enhance the expression levels of transgene, which may be related to the increase of gene copy number.  
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