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研究生:江政儒
研究生(外文):Chiang Cheng-Ju
論文名稱:新穎性細胞貼附材質的開發:重組日本腦炎病毒蛋白
論文名稱(外文):Recombinant Glycosaminoglycan-binding proteins of Japanese Encephalitis Virus as a novel cell adhesive material
指導教授:吳夙欽
指導教授(外文):Wu Suh-Chin
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:64
中文關鍵詞:醣胺素細胞貼附套膜蛋白醣胺素結合區細胞外間質
外文關鍵詞:glycosaminoglycancell adhesionenvelopeGAG-binding siteextracellular matrix
相關次數:
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細胞外間質為細胞生長的環境,聚集細胞形成組織,藉由訊號傳遞調控細胞的生長、分裂與分化,並且調節組織內不同類型細胞的功能,另一方面也提供細胞遷移的路徑。許多細胞外間質蛋白具有多重結合區域,如醣胺素結合區和間質內多醣體的結合或是細胞貼附序列和細胞表面受器的結合,使整個細胞外間質與細胞緊密的結合在一起。研究指出在日本腦炎病毒套膜蛋白的兩個區域E286-312以及E391-416可能是為醣胺素結合區,此區域可以和細胞表面的醣胺素結合,被認為是病毒感染細胞時,進行細胞貼附的關鍵。因此,以帶有醣胺素結合區的重組日本腦炎病毒套膜片段蛋白分析細胞貼附的狀況,進一步評估此蛋白發展為人工細胞外間質材料的可能性為本研究的目的。實驗中日本腦炎病毒套膜蛋白主要分成兩個片段,其中一個片段涵蓋了被推測是為醣胺素結合區的區域 (JEV E277-420/32a),並且運用點突變的方式在同一片段引入一RGD細胞貼附序列(JEV RGD/32a),另外的一個片段則不涵蓋被推測是為醣胺素結合區的區域(JEV E292-402/32a)。利用大腸桿菌表現系統生產重組蛋白,接著以再次摺疊的方式進行內涵體蛋白的純化。最後以細胞貼附分析測試不同片段的重組日本腦炎病毒套膜蛋白對於BHK-21細胞是否有幫助貼附的效果。結果顯示JEV E292-402/32a、JEV E277-420/32a以及JEV RGD/32a能夠幫助BHK-21細胞的貼附,並在蛋白進行固定濃度為10μg/ml時細胞貼附達到飽和,而肝素與細胞混合後再加入蛋白處理過的96孔培養皿,能夠產生抑制貼附的效果。在有血清的條件下,不論肝素是先與細胞混合或先與重組蛋白混合,JEV E277-420/32a以及JEV RGD/32a幫助細胞貼附的程度會增加。實驗的結果顯示血清中可能有某些成分和肝素以及JEV E277-420/32a、JEV RGD/32a重組蛋白產生未知的作用增加BHK-21細胞貼附的程度。
The major function of extracellular matrix (ECM) is aggregating cells into tissues, signaling cells to grow, proliferate, and differentiate, coordinating the diverse function of cells of different types, and providing a path for cell migration. Multiadhesive matrix proteins, such as fibronectin and laminin, which contain several binding domains including GAG-binding regions and cell-binding sequences, attach cells to the matrix. It has been proposed that two putative GAG-binding regions, E286-312 and E391-416, capable of binding to the GAGs on the surface of cells reside in the envelope protein of Japanese encephalitis virus. Virus can attach to cells by binding to the GAGs on the surface of cells, which was considered as an indispensable step in the early stage of virus infection. Hence, by measuring the adhesion of cells to the immobilized fragments of E protein will we be able to understand the interaction between cells and protein and to estimate the potential of a cell-adhesive material out of this protein. Briefly, the gene of JEV E protein was constructed into pET32a vector as JEV E292-402/32a and JEV E277-420/32a, in addition, a single amino acid mutation was introduced to create an RGD-motif-carrying mutant, JEV RGD/32a. After expressed in an E.coli. system, pure proteins obtained by on-column refolding process and typical IMAC procedure were subsequently ready for cell adhesion assay. JEV E292-402/32a, JEV E277-420/32a, and JEV RGD/32a coated microplates promoted BHK-21 cell attachment in a dose-dependent manner while heparin inhibited BHK-21 cell attachment to JEV E292-402/32a, JEV E277-420/32a, and JEV RGD/32a coated microplates. Under serum condition, however, the results were quite different. When heparin added to cells, on the contrary, BHK-21 cell attachment to JEV E277-420/32a and JEV RGD/32a coated microplates was profoundly increased, but cell attachment to JEV E292-402/32a coated microplates remained inhibited. If heparin was mixed with proteins, an enhanced BHK-21 cell attachment to JEV E277-420/32a and JEV RGD/32a coated microplates was detected, and no prominent effect was found on JEV E292-402/32a coated microplates. JEV E277-420/32a and JEV RGD/32a coated microplates enhanced BHK-21 cell attachment whether we combine heparin with cells or proteins, which possibly indicated the involvement of certain serum factor in cell adhesion.
頁次
第一章 序論
1.1 細胞外間質…………………………………..………..………1
1.2 細胞表面蛋白多醣………………………...…….……………2
1.3 日本腦炎病毒與其套膜蛋白……………..….…….…………4
1.4 醣胺素結合區與細胞貼附序列………………………………6
1.5 幫助細胞貼附的蛋白…………………………………………8
1.6 細胞黏附性蛋白的應用………………………………………9
1.7 實驗目的……………………………………………………..10

第二章 材料與方法
2.1 病毒與菌株
2.1.1 日本腦炎病毒………………………………………12
2.1.2大腸桿菌菌株…………………………………….…12
2.2 細胞株………………………………………….……………12
2.3 醱酵槽……………………………………………..……...…13
2.4 日本腦炎病毒套膜蛋白基因片段之載體構築點突變…….13
2.5 日本腦炎病毒套膜重組蛋白之表現,製備與純化
2.5.1 重組蛋白之表現……………………………………15
2.5.2 重組蛋白溶液的製備………………………………16
2.5.3 重組蛋白之純化………………………....…………17
2.6 內涵體蛋白的純化………………………………….………19
2.7 細胞貼附能力分析…………………………...………..……20

第三章 結果
3.1日本腦炎病毒套膜蛋白基因片段之載體構築與點突變…...22
3.2日本腦炎病毒套膜重組蛋白之表現,製備與純化
3.2.1 細菌生長曲線與蛋白表現情形 …………………24
3.2.2 重組蛋白之純化………………………..…………25
3.3內涵體蛋白的純化……………………………………...……26
3.4細胞貼附能力分析
3.4.1細胞貼附能力分析…………………………...……27
3.4.2肝素對細胞貼附能力的影響……………...………27

第四章 討論
4.1日本腦炎病毒套膜重組蛋白在大腸桿菌之表現情形…...…30
4.2內涵體蛋白的純化………………………………….…..……32
4.3細胞貼附能力分析………………………………….…..……34

第五章 結論………………………………………..……………..…38
參考文獻………………………………………………………………39
圖表……………………………………………………………………44





圖目次
頁次
圖1.日本腦炎套膜蛋白片段序列以及實驗的目標蛋白…………….44
圖2.pET22b以及pET32a表現載體意示圖………………………..…46
圖3.表現載體構築流程……………………………………………….47
圖4.重組蛋白表現、純化流程……………………………………….48
圖5.內涵體蛋白的純化流程示意圖………………………………….49
圖6.細胞貼附分析流程示意圖……………………………………….49
圖7. JEV E261-420/22b重組蛋白以IPTG誘導表現之結果…….….….50
圖8. JEV E292-402/22b重組蛋白以IPTG誘導表現之結果………..….50
圖9. JEV E261-420/32a重組蛋白以IPTG誘導表現之結果………..….51
圖10. JEV E292-402/32a重組蛋白以IPTG誘導表現之結果………….51
圖11.分析JEV E261-420/32a重組蛋白溶解性之結果…………………52
圖12.分析JEV E292-402/32a重組蛋白溶解性之結果…………………52
圖13. JEV E277-420/32a細菌生長曲線和IPTG誘導蛋白表現情形….53
圖14. JEV RGD/32a細菌生長曲線和IPTG誘導蛋白表現情形……54
圖15. JEV E277-420/32a和JEV RGD/32a蛋白溶解性之分析結果…...55
圖16. JEV E277-420/32a重組蛋白的純化……………………….……..55
圖17. JEV RGD/32a重組蛋白的純化……………………………..…56
圖18. JEV E292-402/32a重組蛋白的純化……………………………...56
圖19. JEV E277-420/32a和JEV RGD/32a內涵體蛋白的純化……..…57
圖20.純化後的蛋白以及西方墨點…………………………………...58
圖21.重組蛋白對細胞貼附的關係…………………………………...59
圖22.細胞貼附一個小時後的結果…………………………………...60
圖23. Heparin對細胞進行貼附所造成的影響………………………61
圖24.在有血清的條件下Heparin和細胞混合後細胞貼附的結果.…63
圖25.在有血清的條件下Heparin和蛋白混合後細胞貼附的結果….64
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