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標題: | 新型H7N9流感病毒蛋白質PB1-F2與蛋白酶體之關聯性研究 Study of the Relationship between the Novel Avian Influenza A(H7N9) Virus PB1-F2 Protein and Proteasome |
作者: | Kuo-Che Hung 洪國哲 |
指導教授: | 張世宗 |
關鍵字: | 新型H7N9流感病毒,PB1-F2,蛋白?體, H7N9 influenza virus,PB1-F2,proteasome, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 新型H7N9禽流感病毒的特色為高重症率與高死亡率,而流感病毒蛋白質PB1-F2是造成高重症率的因子之一。PB1-F2為病毒聚合酶PB1基因根據另外一個轉譯閱讀框架而轉譯出的蛋白質。研究指出PB1-F2可增進PB1聚合酶的活性,亦可藉由改變粒線體之膜電位而造成細胞凋亡,或者破壞宿主抵禦病毒入侵的機制,進而造成肺部的二次性細菌感染。本研究發現將新型H7N9流感病毒之PB1-F2基因於HEK293細胞表現時,其表現量可因加入蛋白酶體之抑制劑MG132,而有顯著上升的現象,顯示半衰期短暫的H7N9 PB1-F2蛋白質於細胞內的穩定性可能是由蛋白酶體所調控。進一步將PB1-F2分子上可能會被泛素化的所有離胺酸突皆變為精胺酸時,此PB1-F2的突變株於細胞中仍會被快速的降解,除非同時以MG132來處理細胞,才能偵測到PB1-F2的表現,顯示其降解可能並不需要經過泛素化。本研究亦於大腸桿菌中,成功表現及純化出N端為麥芽糖結合蛋白 (Maltose-binding protein, MBP) 與C端為PB1-F2的融合重組蛋白質 (MBP-F2)。將MBP-F2或MBP分別與20S蛋白酶體進行反應後,實驗結果顯示MBP-F2受到蛋白酶體的降解速率比MBP降解速率為慢,此結果與細胞實驗結果不同,詳細的反應機制尚須進一步探討。 The novel avian influenza virus H7N9 is known to have high morbidity and mortality. Influenza protein PB1-F2 is one of the factor that contributes to sever symptoms. PB1-F2 is translated from the alternative open reading frame of the viral PB1 polymerase gene segment. Previous studies showed that PB1-F2 could enhance the activity of PB1 polymerase, and could enter mitochondria intermembrane space to alter the membrane potential which results in cell apoptosis. It could also interact with mitochondrial antiviral signaling protein, damage the immunity of host cells, and cause secondary bacterial lung infection. This study found that the H7N9 PB1-F2 protein expression in HEK293 cell line was elevated by treated with proteasome inhibitor MG132, indicating that the stability of short-lived H7N9 PB1-F2 protein in cells might be regulated by proteasome. Furthermore, the PB1-F2 mutant without containing any potential ubiquitination lysine residues, which were replaced by arginines, was still degraded rapidly unless treated cells with MG132, demonstrating that the degradation of PB1-F2 might be ubiquitination-independent. In addition, the maltose binding protein (MBP) conjugated with a C-terminal PB1-F2 fusion protein (MBP-F2) was successfully expressed and purified by using Escherichia coli expression system. The purified MBP or MBP-F2 was then incubated with 20S proteasome respectively. The data showed that the degradation of MBP-F2 catalyzed by 20S proteasome in vitro was slower than that of MBP, revealing a different result observed in cellular experiments. More detailed investigation about the relationship between PB1-F2 and proteasome need to be further explored. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77917 |
DOI: | 10.6342/NTU201702950 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科技學系 |
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