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

Influence of different promoters on expression of transgene containing MAR expression vector in recombinant CHO cells

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(P<0.05);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(P<0.05)。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。

Objective To investigate the effect of different promoters on the expression level of transgene containing MAR expression vector in recombinant CHO cells. Methods The CMV promoter and β-globin MAR were amplified by PCR,then CMV promoter was replaced the SV40 promoter in pCAT1 for constructing the expression vector droved by CMV promoter. The control vectors of pCAT1 and pCAT2 without containing MAR were simultaneously transfected into the CHO cells. Then the stably trans- fected cell line was screened by G418. The CAT gene expression level was analyzed by ELISA. Results The expression level of CAT enzyme in the cells transfected with MAR-containing vectors was increased compared with the ceils transfected by pCATG and pCAT3 vectors without containing MAR,which were increased by 1.75 and 1.25 times respectively（P〈0.05） ;but CAT en- zyme expression level in the pCAT1 transfected ceils droved by SV40 promotor with the MAR-containing expression vectors was 1.4 times higher than that in the pCAT2 vector droved by the CMV promoter（ P〈0. 05）. Conclusion MAR can enhance the transgene expression level in stably recombinant CHO cells,and the promoting efficiency of SV40 promoter and MAR combination is superior to that of CMV promoter and MAR combination.