- 學位論文
建立可誘導性動物醣基化系統的水稻細胞以用於生產醫藥用醣蛋白
Establishment of an inducible mammalian glycosylation system in rice for application of pharmaceutical glycoproteins
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摘要
顆粒巨噬細胞集落刺激因子 (Granulocyte-macrophage colony-stimulating factor, GM-CSF) 是細胞激素的一種，目前主要使用基因重組方式生產老鼠的GM-CSF (mGMCSF) 做基礎醫學研究。mGMCSF可利用哺乳類動物細胞生產，但有成本高、產量與生物安全性低等缺點，因此我們期望利用水稻細胞生產mGMCSF。此重組蛋白可以在水稻細胞中表現，但是在蛋白質的醣基化後修飾中會加上植物特有的木醣 (xylose) 與岩藻醣 (fucose)，可能引發過敏反應，因此我們利用基因工程的方法，在水稻細胞內以RNA干擾 (RNA interference, RNAi) 的模式降低木醣轉移酶 (XT) 與岩藻糖轉移酶 (FT) 的表現量，並導入人類醣基化模式以生產mGMCSF。由於減少這兩個植物特有的醣基轉移酶，可能會影響植物細胞的生長與生理，因此我們將持續表現的啟動子置換為受糖所調控的α- AMY3啟動子，在細胞培養液中利用糖的有無來調控RNAi的表現，目前我們得到16株水稻轉植細胞株。 我對懸浮細胞做連續多天的缺糖處理，分析水稻轉植細胞株內XT與FT基因的表現量與醣蛋白質的修飾情況。利用Q-RT-PCR分析，缺糖6小時後，水稻轉植細胞株之XT與FT mRNA已經下降，而用西方墨點 (Western blot) 分析醣蛋白的醣化情況，發現缺糖處理12小時後，木醣與岩藻醣添加在醣蛋白的程度，已明顯下降。為了得知分泌至培養基中的蛋白質是否會加上木醣與岩藻醣，分別取細胞缺糖不同時間的培養基來分析醣蛋白質的醣化程度，缺糖24小時後，分泌至細胞外的醣蛋白質已難偵測到木醣與岩藻醣的存在。本研究的目標為利用可誘導性動物醣基化系統的水稻懸浮細胞來生產mGMCSF。
並列摘要
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein and a cytokine that stimulates stem cells to produce granulocytes and monocytes. Recombinant GM-CSF is widely applied in basic medical research. Recombinant mouse GM-CSG (mGMCSF) can be produced by mammalian cells, but the drawbacks of this expression system are high cost and biosafety risk. In our research we choose rice suspension cell system to produce mGMCSF. We have successfully expressed mGMCSF in rice cell. However, plant specific β-1,2-xylosyltransferase (XT) and α-1,3-fucosyltransferse (FT) add xylose and fucose to glycoproteins, respectively, and these two sugar residues may induce human allergic reactions. Based on our experiences with rice expression system, RNA interference (RNAi) was used to reduce XT and two FTs to humanize mGMCSF production in rice cell. In our original design, the triple RNAi knockdown system was controlled by a strong and constitutive promoter to generate hairpin loops of double strand RNA molecules that are able to continuously induce gene silence for three plant specific glycosyltransferases. However, these enzymes maybe essential for plant cells, and their absences may lead to abnormal physiological responses. To construct an inducible system, α-AMY3 promoter was chosen because of its tight regulation by sugar. Sofar, we have obtained 16 transgenic rice cell lines. At RNA level, FT and XT transcripts were reduced after 6 hours of sugar starvation. At protein level, the secreted glycoproteins contained low β-1,2-xylose and α-1,3-fucose after 12 hours of sugar starvation. After refreshing the sugar free medium and further starving the cells for another 12 hours, we were not able to detect any xylose and fucose in secreted glycoproteins with our inducible RNAi rice cell lines, suggesting that sugar free medium can turn on RNAi to block plant specific N-glycosylation pathways.
參考文獻
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