Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24025
標題: 以噬菌體表現尋找與流感病毒非結構蛋白NS1作用的結合胜肽
The use of phage display to screen the aptamer of influenza virus NS1 protein.
作者: 謝宗翰
Hsieh, Tsung-Han
關鍵字: phage display
噬菌體篩選
NS1
influenza virus
流感A型病毒非結構蛋白
出版社: 生物化學研究所
引用: 1. Cheng, A., S. Wong, and Y. Yuan, Structural basis for dsRNA recognition by NS1 protein of influenza A virus. Cell Res, 2009. 19(2): p. 187-195. 2. Garcia-Sastre, A., et al., Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology, 1998. 252(2): p. 324-30. 3. Kochs, G., A. Garcia-Sastre, and L. Martinez-Sobrido, Multiple Anti-Interferon Actions of the Influenza A Virus NS1 Protein. Journal of Virology, 2007. 81(13): p. 7011-7021. 4. Wang, T. and P. Palese, Unraveling the Mystery of Swine Influenza Virus. Cell, 2009. 137(6): p. 983-985. 5. Heikkinen, L., et al., Avian and 1918 Spanish influenza a virus NS1 proteins bind to Crk/CrkL Src homology 3 domains to activate host cell signaling. Journal of Biological Chemistry, 2008. 283(9): p. 5719. 6. Inglis, S., et al., The smallest genome RNA segment of influenza virus contains two genes that may overlap. Proceedings of the National Academy of Sciences, 1979. 76(8): p. 3790-3794. 7. Lamb, R. and P. Choppin, Segment 8 of the influenza virus genome is unique in coding for two polypeptides. Proceedings of the National Academy of Sciences, 1979. 76(10): p. 4908-4912. 8. Weber, F. and O. Haller, Viral suppression of the interferon system. Biochimie, 2007. 89(6-7): p. 836-842. 9. Hale, B., et al., The multifunctional NS1 protein of influenza A viruses. Journal of General Virology, 2008. 89(10): p. 2359. 10. Krug, R., et al., Intracellular warfare between human influenza viruses and human cells: the roles of the viral NS1 protein. Virology, 2003. 309(2): p. 181-189. 11. LU, Y., et al., Binding of the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the eIF-2 translation initiation factor. Virology, 1995. 214(1): p. 222-228. 12. Talon, J., et al., Activation of interferon regulatory factor 3 is inhibited by the influenza A virus NS1 protein. Journal of Virology, 2000. 74(17): p. 7989-7996. 13. Garcia, M., E. Meurs, and M. Esteban, The dsRNA protein kinase PKR: virus and cell control. Biochimie, 2007. 89(6-7): p. 799-811. 14. Min, J., et al., A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis. Virology, 2007. 363(1): p. 236-243. 15. Langland, J., et al., Inhibition of PKR by RNA and DNA viruses. Virus Research, 2006. 119(1): p. 100-110. 16. Falcon, A., et al., Interaction of influenza virus NS1 protein and the human homologue of Staufen in vivo and in vitro. Nucleic acids research, 1999. 27(11): p. 2241. 17. Bornholdt, Z.A. and B.V.V. Prasad, X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus. Nature, 2008. 456(7224): p. 985-988. 18. Bradley, D., Ultrastructure of bacteriophage and bacteriocins. Microbiology and Molecular Biology Reviews, 1967. 31(4): p. 230-314. 19. Smith, G. and V. Petrenko, Phage display. Chemical reviews, 1997. 97(2): p. 391-410. 20. Noren, K. and C. Noren, Construction of high-complexity combinatorial phage display peptide libraries. Methods, 2001. 23(2): p. 169-178. 21. Danner, S. and J. Belasco, T7 phage display: a novel genetic selection system for cloning RNA-binding proteins from cDNA libraries. Proceedings of the National Academy of Sciences, 2001. 98(23): p. 12954. 22. Cao, Z., et al., A novel functional motif of osteopontin for human lymphocyte migration and survival. Molecular Immunology, 2008. 45(14): p. 3683-3692.
摘要: 流感A型病毒的遺傳物質為單股負鏈RNA,由八個片段(segments)組成,第八個片段編碼出的蛋白之一為流感A型病毒的非結構蛋白1(Non-Structure protein 1)。NS1蛋白由230個胺基酸組成(視不同的流感病毒株而異)。分為N端的雙股核醣核酸結合區域與C端的效應功能區域。 由於病毒入侵宿主細胞後會引發宿主細胞的初級免疫系統來對抗病毒的入侵,而病毒NS1蛋白為多功能蛋白,可以與許多分子結合。包括NS1蛋白結構的N端 dsRNA binding domain 能與 dsRNA 、 PABP1(poly(A) binding protein 1)、RIG-1(Retinoic Acid-inducible Gene I)等結合,C端的 Effector domain 能與 eIF4G1(eukaryotic translation initiation factor 4G1)、CPSF(Cleavage and polyadenylation specificity factor)30kDa、hStaufen(human homologue of Drosophila melanogaster Staufen protein)、PKR(protein kinase R)、PABPII等等結合,目的是為了抑制宿主細胞產生干擾素(interferons),降低宿主細胞的抗病毒機制。舉例來說,目前已知病毒NS1蛋白會與病毒的 dsRNA 結合,避免宿主細胞內的 2’-5’ OAS(2''-5'' oligoadenylate synthetase)RNase L system活化,病毒RNA也就避免被宿主細胞的免疫系統辨識進而分解。NS1蛋白與宿主細胞內的 dsRNA 與RIG-1蛋白結合,能夠壓抑宿主的干擾素調節因子(IRF3, IRF7, IFN regulatory factors)的活化,進而抑制宿主細胞核內干擾素DNA轉錄為mRNA,使得宿主細胞無法正常的製造Interferon α/β蛋白。 由於NS1影響宿主細胞的抗病毒機制目前還沒有全盤的了解,如何利用NS1與細胞內分子的作用來控制病毒對宿主細胞的抗病毒機制是我們想要問的問題。因此我們希望能夠找到一個能夠與NS1蛋白結合的小胜肽片段,期許這個小胜肽片段能夠調控流感病毒感染宿主細胞的機制是我們的目標。 噬菌體表現(phage display)可以用來作為篩選廣大資料庫的工具,對於蛋白與蛋白之間,蛋白與DNA之間或是蛋白與RNA之間的結合作用分析提供快速方便的篩選方法。我們使用在 M13 噬箘體 pIII 殼蛋白表面表現12個胺基酸長度並且序列為隨機的噬菌體資料庫,與NS1蛋白進行噬菌體篩選實驗,經過淘選(panning)並以ELISA檢查篩選出的噬菌體與NS1蛋白的結合力,而後將感興趣的噬菌體定出基因序列,並且進一步以pulldown實驗檢驗我們篩選出的小胜肽片段與NS1蛋白的結合力,期許找出能夠與NS1蛋白有結合力的 aptamer。
The influenza A virus belongs to the Orthomyxoviridae family. Genome of influenza A virus contains 8 pieces of segmented single-stranded negative-sense RNAs and encodes 11 different proteins and 2 non-structural proteins including NS1 protein. Influenza A virus NS1 protein is a multifunctional protein. NS1 protein has 230 amino acids with 2 domains: N-terminal dsRNA binding domain and C-terminal effector domain. N-terminal domain can interact with dsRNA, poly A binding protein 1, and RIG-I (retinoic acid-induced gene I). C-terminal domain can interact with 30kDa submit of CPSF (cleavage and polyadenylation specificity factor), eIF4G1 (eukaryotic translation initiation factor 1), PKR (protein kinase R), PABPII (poly A binding protein 2), hStaufen (human homologue of Drosophila melanogaster Staufen protein). In most case, virus infection activates innate immune system of cell. NS1 protein can suppress IFN's induction of host cell. N-terminal dsRNA binding domain of NS1 protein binds dsRNA and prevents RNA of virus from being degraded by 2'-5' OAS (2'-5' oligoadenylate synthetase) RNase L pathway. NS1 protein can suppress activating transcription factors of host cell, include IFN regulatory factor 3 and 7, and inhibit host cell mRNA processing by binding dsRNA and RIG-1 protein in host cell to suppress host cell producing Interferon α/β protein. We hope to find a small aptamer that can inhibit the anti-viral pathway of influenza A virus by interacting with NS1 protein. Phage display provides a powerful method to screen a peptide library. We use NS1-S103F/I106M protein as a bait and screen by an M13 phage library containing random 12-mer peptides that are fused to the N-terminal domain of pIII coat protein. After several rounds of panning, we check the NS1-peptide interaction by ELISA and sequence the phage's DNA for the encoded peptide sequence. The interaction of phage-selected aptamer with NS1 will be further confirmed by GST-pull down assay.
URI: http://hdl.handle.net/11455/24025
其他識別: U0005-3107200913140500
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-3107200913140500
Appears in Collections:生物化學研究所

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