重组CHO细胞中不同启动子对含MAR表达载体转基因表达的影响

发布时间：2018-05-29 20:03

本文选题：MAR + 启动子　；参考：《重庆医学》2017年17期

【摘要】：目的分析在重组CHO细胞中不同启动子对含核基质结合区(MAR)表达载体转基因表达的影响。方法 PCR扩增CMV启动子及β-珠蛋白MAR,构建含β-珠蛋白MAR表达载体pCAT1,随后将CMV启动子替代pCAT1上SV40启动子构建CMV启动子驱动的表达载体pCAT2。pCAT1、pCAT2不含MAR的对照载体同时转染CHO细胞,G418筛选稳定转化的细胞株,酶联免疫吸附试验(ELISA)分析氯霉素乙酰转移酶(CAT)基因的表达水平。结果含MAR表达载体转染的细胞CAT酶表达量比不含MAR的pCATG和pCAT3载体转染的细胞高,分别提高2.14倍和1.25倍(P0.05);而由SV40启动子驱动含MAR表达载体pCAT1转染的细胞CAT酶表达水平明显比由CMV启动子驱动的pCAT2载体高3.26倍(P0.05)。结论在稳定重组CHO细胞中MAR能够提高转基因的表达水平,SV40启动子与MAR组合其启动效率优于CMV启动子与MAR组合。
[Abstract]:Objective to investigate the effect of different promoters on the expression of nuclear matrix binding region (Mar) expression vector in recombinant CHO cells. Methods CMV promoter and 尾 -globin marker were amplified by PCR, and 尾 -globin MAR expression vector pCAT1 was constructed. Then the CMV promoter was replaced by SV40 promoter on pCAT1 to construct CMV promoter driven expression vector pCAT2.pCAT1pCAT2 without MAR was transfected simultaneously. CHO cell line G418 was used to screen stable transformed cell lines. Enzyme linked immunosorbent assay (Elisa) was used to analyze the expression level of chloramphenicol acetyltransferase (CAT) gene. Results the expression of CAT enzyme in the cells transfected with MAR expression vector was higher than that with pCATG and pCAT3 vector without MAR. The expression level of CAT enzyme in the cells transfected with SV40 promoter was 3.26 times higher than that of pCAT2 vector driven by CMV promoter. Conclusion in stable recombinant CHO cells, MAR can improve the expression level of transgenic cells. The promoter efficiency of SV40 promoter combined with MAR is better than that of CMV promoter and MAR combination.
【作者单位】：新乡医学院分析测试实验室;新乡医学院生物化学与分子生物学教研室;
【基金】：国家自然科学基金资助项目(31371332)
【分类号】：Q78

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