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

【摘要】：目的分析在重組CHO細(xì)胞中不同啟動(dòng)子對(duì)含核基質(zhì)結(jié)合區(qū)(MAR)表達(dá)載體轉(zhuǎn)基因表達(dá)的影響。方法 PCR擴(kuò)增CMV啟動(dòng)子及β-珠蛋白MAR,構(gòu)建含β-珠蛋白MAR表達(dá)載體pCAT1,隨后將CMV啟動(dòng)子替代pCAT1上SV40啟動(dòng)子構(gòu)建CMV啟動(dòng)子驅(qū)動(dòng)的表達(dá)載體pCAT2。pCAT1、pCAT2不含MAR的對(duì)照載體同時(shí)轉(zhuǎn)染CHO細(xì)胞,G418篩選穩(wěn)定轉(zhuǎn)化的細(xì)胞株,酶聯(lián)免疫吸附試驗(yàn)(ELISA)分析氯霉素乙酰轉(zhuǎn)移酶(CAT)基因的表達(dá)水平。結(jié)果含MAR表達(dá)載體轉(zhuǎn)染的細(xì)胞CAT酶表達(dá)量比不含MAR的pCATG和pCAT3載體轉(zhuǎn)染的細(xì)胞高,分別提高2.14倍和1.25倍(P0.05);而由SV40啟動(dòng)子驅(qū)動(dòng)含MAR表達(dá)載體pCAT1轉(zhuǎn)染的細(xì)胞CAT酶表達(dá)水平明顯比由CMV啟動(dòng)子驅(qū)動(dòng)的pCAT2載體高3.26倍(P0.05)。結(jié)論在穩(wěn)定重組CHO細(xì)胞中MAR能夠提高轉(zhuǎn)基因的表達(dá)水平,SV40啟動(dòng)子與MAR組合其啟動(dòng)效率優(yōu)于CMV啟動(dòng)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(cè)試實(shí)驗(yàn)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(guó)家自然科學(xué)基金資助項(xiàng)目(31371332)
【分類號(hào)】：Q78

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本文編號(hào)：1952210