| 摘要: | 中文摘要
粒細胞 - 巨噬細胞集落刺激因子（GM-CSF）是一種細胞因子，作用在白血球細胞生長增殖和分化中。 在先前的研究中，小鼠GM-CSF (mGM-CSF) 重 組蛋白（rmGM-CSF）已成功使用αAmy3 啟 動子表達在水稻懸浮液中，細 胞培養在2-L生物反應器中，mGM-CSF的最高產 量可達24.6 mg / L。糖訊息可通過三個cis-elements，GC box，G box和TATCCA element調控αAmy3啟動子。屬 於1R-MYB 家族中的 三 種MYB 轉 錄 因 子OsMYBS1，OsMYBS2 和OsMYBS3 與αAmy3啟 動 子 的TATCCA element 相互作 用, 並 在 糖 調 控 的αAmy3的 表 達。 OsMYBS2是repressor，它在含糖培養的水稻細胞中表達，並與TATCCA element結合，使得αAmy3表現降低。為了增加rmGM-CSF的表達，本論文使用RNAi 策略抑制OsMYBS2 的表達,藉由降低OsMYBS2在水稻懸浮培養細胞中表現量，來增加αAmy3 啟動子表達rmGM-CSF。我的研究結果顯示，OsMYBS2基因靜默細胞中，mGM-CSF基因的表達分別在含糖與缺糖情況下較野生型高出1.3〜5.25倍與1.13〜4.4 倍。檢查rmGM-CSF 蛋白質的表達顯示，OsMYBS2靜默細胞中的蛋白質遠高於野生型,在缺糖第5天的OsMYBS2 基因靜默細胞培養液中，rmGM-CSF 的 產量比野生型高 1.7-2.5 倍，重組rmGM-CSF的產量最高為29.90μg/ mL.重 組rmGM-CSF蛋白質在更長的缺糖處理時,直到第11 天,產量仍然多於野生型。這些研究顯示，降低轉錄因子OsMYBS2，可增加αAmy3所驅動的 mGM-CSF並產 生更多的rmGM-CSF。
;Abstract
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that functions in cell proliferation and differentiation, especially in the growth of white blood cells. In previous studies, the expression of recombinant protein of mGM-CSF (rmGM-CSF) was drove by the αAmy3 promoter in rice suspension cultured cells. The cells were scaled up successfully in a 2-L bioreactor and the highest yield of rmGM-CSF was 24.6 mg/L. Sugar regulates the rice αAmy3 promoter through cis-elements, the GC box, the G box, and the TATCCA element. Three MYB transcription factors OsMYBS1, OsMYBS2, and OsMYBS3, that belong to 1R-MYB, interact specifically with the TATCCA element of αAmy3 promoter and play roles in sugar-regulated αAmy3 expression. The OsMYBS2 is repressor, that is expressed in sugar fed cells, can bind to the TATCCA element, so only a low level of αAmy3 is expressed. To increase the production of rmGM-CSF, the RNAi strategy was used to repress expression of OsMYBS2. The production of the mGM-CSF by aAmy3 promoter-base rice recombinant protein expression system was examined in OsMYBS2 knockdown rice suspension cultured cells. Our results showed that the expression of mGM-CSF gene was higher 1.3 – 5.25 fold under sugar starvation and 1.13 – 4.4 fold in sugar available condition in the OsMYBS2 knockdown cells than wild type. The Western blotting analysis showed that rmGM-CSF protein in OsMYBS2 knockdown was much higher than wild type. In day 5, productioon of rmGM-CSF in OsMYBS2 knockdown cell lines were higher 1.7 – 2.5 fold than in wild-type cell line. The highest yield of recombinant rmGM-CSF was 29.90 µg/mL in 5 days. The high rmGM-CSF productions in OsMYBS2 knockdown cell lines were still able to detect in 11 days of sugar starvation, as compared to in wild type. These studies demonstrated that knockdown of a transcription repressor OsMYBS2 increase the αAmy3 drove mGM-CSF production in sugar starvation of suspension rice cells. |
