本文選題：MAR + 啟動子　； 參考：《重慶醫(yī)學(xué)》2017年17期

【摘要】：目的分析在重組CHO細胞中不同啟動子對含核基質(zhì)結(jié)合區(qū)(MAR)表達載體轉(zhuǎn)基因表達的影響。方法 PCR擴增CMV啟動子及β-珠蛋白MAR,構(gòu)建含β-珠蛋白MAR表達載體pCAT1,隨后將CMV啟動子替代pCAT1上SV40啟動子構(gòu)建CMV啟動子驅(qū)動的表達載體pCAT2。pCAT1、pCAT2不含MAR的對照載體同時轉(zhuǎn)染CHO細胞,G418篩選穩(wěn)定轉(zhuǎn)化的細胞株,酶聯(lián)免疫吸附試驗(ELISA)分析氯霉素乙酰轉(zhuǎn)移酶(CAT)基因的表達水平。結(jié)果含MAR表達載體轉(zhuǎn)染的細胞CAT酶表達量比不含MAR的pCATG和pCAT3載體轉(zhuǎn)染的細胞高,分別提高2.14倍和1.25倍(P0.05);而由SV40啟動子驅(qū)動含MAR表達載體pCAT1轉(zhuǎn)染的細胞CAT酶表達水平明顯比由CMV啟動子驅(qū)動的pCAT2載體高3.26倍(P0.05)。結(jié)論在穩(wěn)定重組CHO細胞中MAR能夠提高轉(zhuǎn)基因的表達水平,SV40啟動子與MAR組合其啟動效率優(yōu)于CMV啟動子與MAR組合。
[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.
【作者單位】： 新鄉(xiāng)醫(yī)學(xué)院分析測試實驗室;新鄉(xiāng)醫(yī)學(xué)院生物化學(xué)與分子生物學(xué)教研室;
【基金】：國家自然科學(xué)基金資助項目(31371332)
【分類號】：Q78

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