標題: 昆蟲細胞內N-醣化過程的修飾
Modification of N-Glycosylation Pathway in Insect Cell
作者: 林芷吟
Lin, Chih-Yin
林苕吟, 陳宏文
Tiao-Yin Lin, Hung-Wen Chen
生物科技學系
關鍵字: 昆蟲細胞;N-醣化過程;Insect Cell;N-Glycosylation
公開日期: 1997
摘要: 以桿狀病毒╱昆蟲細胞系統表現外來蛋白質的缺點是其無法有效製造出含 有sialic acid及galactose的complex type醣蛋白質,以致製造出的醣蛋 白質無法在血液中維持正常的半生期。為了使昆蟲細胞的醣化過程更接近 哺乳動物細胞的醣化過程,我們希望在昆蟲細胞內表現三種醣基轉移酉每 (GlcNAc-transferase II、galactosyltransferase、 sialyltransferase),以改進其醣化過程。而人類抗胰蛋白酉每 (ha1- AT)是一個含三個二叉或三叉complex type寡醣鏈的醣蛋白質。在此我們 設計一起表現抗胰蛋白酉每 及三種醣基轉移酉每 ,希望製造出更完全醣 化的人類抗胰蛋白在本篇文章中,我們成功地製造出幾組重組桿狀病毒: BacAT,只表現ha1-AT;BacATgn,表現ha1-AT及GnTII,以此類推,另有 BacATg、BacATgng、BacATgng23、BacATgng26,利用這些重組桿狀病毒分 別感染Sf9昆蟲細胞及High-Five昆蟲細胞以進行表現。我們發現帶有醣基 轉移酉每 基因的重組病毒,所表現出的ha1-AT,其分子量較大。而以 peptide:N-glycosidase F與endoglycosidase H進行切割反應分析,發現 不同重組病毒所表現的ha1-AT,其寡醣鏈有所差異。相同重組病毒在Sf9 、High-Five不同細胞中所表現的ha1-AT,其寡醣鏈結構可能也不同。又 與COS-1細胞中所表現的h Recombinant glycoproteins synthesized from the baculovirus- insect cell expression system lack of complex type N- oligosaccharides. These products will not maintain a reasonable half-life in the circulation for therapeutics. To overcome this problem, we proposed to enhance the capability of N- glycosylation machinery by co-expression of GlcNAc-transferase II (GnTII), galactosyltransferase, and sialyltransferase with the target protein. Human a1-antitrypsin (ha1-AT), protecting elastic fibers in the lung from excess elastase activities, is a plasma glycoprotein containing three complex type N- oligosaccharides. We have successfully established six recombinant baculovirus strains harboring ha1-AT and different combinations of transferases. Using peptide:N-glycosidase F and endoglycosidase H, we analyzed the glycan structures of the ha1- AT expressed from insect cells infected with the recombinant virus strains including BacAT, BacATgn, Ba
URI: http://140.113.39.130/cdrfb3/record/nctu/#NT860111010
http://hdl.handle.net/11536/62589
Appears in Collections:Thesis