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標題: 以RGD-EGFP融合蛋白質作為細胞穿透現象之探討
Investigation of RGD-EGFP Fusion Proteins in RGD-Mediated Protein Internalization
作者: 江翰
Chiang, Han
關鍵字: Integrin;Integrin;EGFP;RGD;Internalization;fusion protein;增強型綠色螢光蛋白;RGD;穿透作用;融合蛋白
出版社: 獸醫微生物學研究所
RGD (arginine-glycine-aspartate)是許多胞外基質(extracellcular matrix;ECM)中與integrin的辨識特定序列(motif),與細胞結合後,發動胞內訊息傳導的連鎖調控,使細胞發生構形重組、形態改變及生理性質之變化。目前已知RGD為外界多數病原穿入胞內的辨識特定序列,而穿透作用(Internalization)係指外源性物質藉由胞膜上的接受體傳導而進入細胞內之過程,其涵蓋侵入(penetration)及內吞作用(endocytosis)。因此,本實驗構築具EGFP (enhanced green fluorescent protein)、口蹄疫病毒VP1蛋白上G-H loop的RGD序列及不同的RGD序列部份的融合蛋白載體,然後進行該載體的誘導表現和蛋白的純化,再測試其功能。在螢光倒立顯微鏡下觀察到細胞穿透現象,且在共軛焦顯微鏡分析中多次得到相同的結果。顯示RGD-EGFP融合蛋白對於PK-15或MDBK細胞具有穿透作用的能力。目前正針對穿透效益的提昇及功能表現作進一步的探討。

Many extracellular matrices recognizes integrin through their RGD motif, such binding cell mediates the signal transduction cascade that might result in rearrangment of cytoskeleton, and alteration in physical properties. Recently, RGD has been observed as the recognition motif for most invasive pathogens. These pathogens enter or being internalized into host cell with the help of specific receptors on the membrane. Either penetration or endocytosis considered as a way of internalization. In our experiment, we constructed a plasmid that overexpresses fusion protein vectors that contains the G-H loop of VP1 protein of foot-and-mouth disease virus (FMDV) and an enhanced green fluorescent protein (EGFP). In addition, RGD motif of in other contexts was also expresses as fusion protein with EGFP. The fusion proteins were overexpressed in E. coli and the proteins were purified and tested for their function in internalization. It was observed by inverted microscopy and confocal microscopy that RGD-EGFP fusion proteins could be internalized by the PK-15 and MDBK cell line. We are now improving efficiency of internalization.
Appears in Collections:微生物暨公共衛生學研究所

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