甲型和乙型流感病毒M2离子通道作为抗病毒靶点的研究
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摘要
为了制备和筛选针对甲型和乙型流感病毒具有广谱和强抗病毒活力的抗体和多肽,我们应用杂交瘤技术分别制备了针对甲型流感病毒M2和乙型流感病毒BM2离子通道的单克隆抗体,同时应用噬菌体展示技术筛选出与乙型流感病毒M2离子通道膜外区高特异结合的噬菌体多肽,并分别从细胞水平上研究了它们的抗病毒活性。结果分别筛选到针对甲型和乙型流感病毒M2膜外区N末端8个保守氨基酸序列的单克隆抗体M2e8-7和BM2e-2,其中M2e8-7抗体高特异性结合已知21种甲型流感病毒M2突变型中的19种,是已报道的M2单克隆抗体中识别病毒种类最多的一株抗体。病毒中和实验显示M2e8-7能有效抑制所识别的不同甲型流感病毒突变型在MDCK细胞里的复制。与此类似,BM2e-2抗体有效抑制了不同乙型流感病毒株在MDCK细胞里的复制。此外,还筛选到了能与乙型流感病毒M2膜外区8个保守氨基酸序列高度特异结合的短肽VSFTPSF,其抗病毒活性还有待进一步研究。因此,M2离子通道的独特结构对于抗病毒感染具有重要的作用,本研究所筛选到的针对甲型和乙型流感病毒特异的单克隆抗体和短肽为临床检测和抗病毒药物的开发提供了重要的理论依据。
To investigate the broad spectrum antiviral activities in vitro, we have prepared and characterized the monoclonal antibodies against M2 protein channel of influenza A and B viruses. Not only have we applied the hybridoma technique to get the monoclonal antibodies against M2 protein of influenza A and B viruses, but also screened the phage displayed peptide specifically binding to BM2. We characterized the M2e8-7and BM2e-2 recognizing the N terminus eight highly conserved epitomes respectively. The mAb M2e8-7 exhibited high affinity reacted with 19 of the current known 21 influenza A M2e variants. A cytopathic assay showed that the mAb M2e8-7 potently inhibited the replication of variety influenza A virus. Seemly, the monoclonal antibody BM2e-2 potently protected MDCK cells from homologous, but not heterologous, virus infections. Besides, specific peptide sequence VSFTPSF secreting anti-BM2 mAbs were obtained and the mechanism of the inhibition was further discussed. These results indicate that antibody targeting the M2 proton channel is a promising therapeutic candidate for treating influenza virus infections, and that brought new insight in developing vaccine.
To investigate the broad spectrum antiviral activities in vitro, we have prepared and characterized the monoclonal antibodies against M2 protein channel of influenza A and B viruses. Not only have we applied the hybridoma technique to get the monoclonal antibodies against M2 protein of influenza A and B viruses, but also screened the phage displayed peptide specifically binding to BM2. We characterized the M2e8-7and BM2e-2 recognizing the N terminus eight highly conserved epitomes respectively. The mAb M2e8-7 exhibited high affinity reacted with 19 of the current known 21 influenza A M2e variants. A cytopathic assay showed that the mAb M2e8-7 potently inhibited the replication of variety influenza A virus. Seemly, the monoclonal antibody BM2e-2 potently protected MDCK cells from homologous, but not heterologous, virus infections. Besides, specific peptide sequence VSFTPSF secreting anti-BM2 mAbs were obtained and the mechanism of the inhibition was further discussed. These results indicate that antibody targeting the M2 proton channel is a promising therapeutic candidate for treating influenza virus infections, and that brought new insight in developing vaccine.
引文
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2. Taubenberger JK, R.A., Lourens RM, Wang R, Jin G,Fanning TG, Characterization of the 1918 influenza virus polymerase genes. Nature,2005. 437:p.889-893.
3. AS, F., Emerging and re-emerging infectious diseases:influenza as a prototype of the host-pathogen balancing act. Cell,2006.124:p.665-670.
4. Peter P, A.G., Influenza vaccines:present and future. J Clin Invest,2002.110: p.9-13.
5. PINTO L.H., L.R.A., Influenza virus proton channels. Photochem. Photobiol. Photobiol. Sci.,2006.5:p.629-632.
6. Skarlas, T., et al., Influenza virus H5NI hemagglutinin (HA) T-cell epitope conjugates:design, synthesis and immunogenicity. J Pept Sci.17(3):p. 226-32.
7. Colman, P.M., et al., Three-dimensional structure of a complex of antibody with influenza virus neuraminidase. Nature,1987.326(6111):p.358-63.
8. J.P., T., Synthetic peptide vaccine design:synthesisi and properties of a high-density multiple antigenic peptide system. PNAS USA,1988.85:p. 5409-5413.
9. S. Wong, M.R.C.P., M. Kwok-yung Yuen, Avian influenza virus infections in humans. Chest,2006.129:p.156-168.
10. PINTO L.H., H.L.J., LAMB R.A., Influenza virus M2 protein has ion channel activity. Cell,1992.69:p.517-528.
11. Ito, T., et al., Evolutionary analysis of the influenza A virus M gene with comparison of the M1 and M2 proteins. J Virol,1991.65(10):p.5491-8.
12. PINTO L.H., L.R. A., The M2 Proton Channels of Influenza A and B Viruses. J. Biol. Chem.,2006.281:p.8997-9000.
13. Romvary, J., et al., The role of wild birds in the spread of influenza viruses. Acta Microbiol Acad Sci Hung,1980.27(4):p.269-77.
14. Pinto, L.H., Holsinger, L.J. and Lamber, R.A., Infuenza virus M2 protein has ion channel activity. Cell,1992.69:p.517-528.
15. Zebedee, S.L. and R.A. Lamb, Influenza A virus M2 protein:monoclonal antibody restriction of virus growth and detection of M2 in virions. J Virol, 1988.62(8):p.2762-72.
16. Neirynck, S., Deroo, T., Saelens, X., Vanlandschoot, P., Jou, W.M., A universal infuenza A vaccine based on the extracellular domain of the M2 protein. Nat. Med,1999.5:p.1157-1163.
17. Osterhaus, A.D., et al., Influenza B virus in seals. Science,2000.288(5468):p. 1051-3.
18. A.M. Frace, A.I.K., T. Rowe, R.A. Black, J.M. Katz, Modified M2 proteins produce heterotypic immunity against influenza A virus. Vaccine,1999. 17(2237-2244).
19. BALANNIK V., L.R.A., PINTO L.H., The oligomeric state of the active BM2 ion channel protein of influenza B virus. J. Biol. Chem,2008.283:p. 4895-4904.
20. Treanor, J.J., et al., Passively transferred monoclonal antibody to the M2 protein inhibits influenza A virus replication in mice. J Virol,1990.64(3):p. 1375-7.
21. Sesma, A.F., Schulman, J.L. and Moran, T.M., A bispecific antibody recognizing infuenza A virus M2 protein restricts ejector cells to inhibit virus replication. J. Virol,1996.70:p.4800-4804.
22. Anar, C., et al., Evaluation of clinical data and antibody response following influenza vaccination in patients with chronic obstructive pulmonary disease. New Microbiol.33(2):p.117-27.
23. Krystyna M, J.F., Mark E, Goran K, Mare C, Laszlo O, Walter G, Induction of influenza type A virus specific resistance by immunization of mice with a synthetic multiple antigenic peptide vaccine that contains ectodomains of matrix protein 2. Vaccine,2003.21:p.2616-26.
24. Bukrinskaya, A.G., et al., Influenza virus uncoating in infected cells and effect of rimantadine. J Gen Virol,1982.60(Pt 1):p.49-59.
25. Niqueux, E., et al., Presence of serum antibodies to influenza A subtypes H5 and Nl in swans and ibises in French wetlands, irrespective of highly pathogenic H5N1 natural infection. Avian Dis.54(1 Suppl):p.502-8.
26. MOULD J.A., P.R.G., TAKEDA M., OHIGASHI Y.,VENKATARAMAN P., LAMB R.A., PINTO L.H, Influenza B virus BM2 protein has ion channel activity that conducts protons across membranes. Dev. Cell,2003.5:p. 175-184.
27. M. Schmidtke, U.S., B. Jahn, H.M. Dahse, A. Stelzner, A rapid assay for evaluation of antiviral activity against coxsachie virus B3, influenza virus A,andh erpes simplex virus type 1. J. Virol. Methods,2001.95:p.133-143.
28. BUI M, W.K.G., HEL ENIU A, Effect of M1 protein and low p H on nuclear t ransport of influenza virus ribnucleoproteins. Virology,1996.70(12):p. 8391-8401.
29. Schnell, J.R. and J.J. Chou, Structure and mechanism of the M2 proton channel of influenza A virus. Nature,2008.451(7178):p.591-5.
30. HA Y A J, W.A.J., SKEHEL J J The molecular basis of the specific anti2influenza action of amantadine. EMBO J,1985.4(11):p.3021-3024.
31. LAWRENCE H P, L.R.A., The M2 proton channel s of influenza A and B viruses. J Biol Chem,2006.281(14):p.8997-9000.
32. Kohler, G. and C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity. Nature,1975.256(5517):p.495-7.
33. GP, S., Filamentous fusion phage:novel expression vectors that display co-loned antigens on the virion surface. Science,1985.228:p.1315-1317.
34. Chi X S, H., Bolar T V, Detect ion and char act erization of new influenza B virus variant s in 2002. J Cl in Microbiol,2005.43(5):p.2345.
35. Morrison, A., et al., Characterization of a monoclonal antibody to erythroid related factor. Hybridoma (Larchmt).30(2):p.175-9.
36. Shaw, P.G., et al., Monoclonal antibody cocktail as an enrichment tool for acetylome analysis. Anal Chem.83(10):p.3623-6.
37. Gift, S.K., et al., Monoclonal Antibody m18 Paratope Leading to Dual Receptor Antagonism of HIV-1 gp120. Biochemistry.50(14):p.2769-79.
38. Garcia-Jurado, G., et al., Monoclonal Antibody That Recognizes a Domain on Heterogeneous Nuclear Ribonucleoprotein K and PTB-Associated Splicing Factor. Hybridoma (Larchmt).30(1):p.53-9.
39. Dai, Y.C., et al., Epitope mapping and structural analysis of the anti-Der p1 monoclonal antibody:insight into therapeutic potential. J Mol Med.
40. Hecker, J., et al., Generation and epitope analysis of human monoclonal antibody isotypes with specificity for the timothy grass major allergen Phl p 5a. Mol Immunol.48(9-10):p.1236-44.
41. Brand, T.M., M. Iida, and D.L. Wheeler, Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab. Cancer Biol Ther.11(9):p. 777-92.
42. Miller, A.K., et al., Characterization of site-specific glycation during process development of a human therapeutic monoclonal antibody. J Pharm Sci. 100(7):p.2543-50.
43. Deng, Y.Q., et al., A broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of E protein. PLoS One. 6(1):p. e16059.
44. Ozdemir, O., Measuring human mast cell-mediated cytotoxicity against human tumour cells:three-colour flow cytometry using monoclonal antibody target staining with annexin Ⅴ/propidium iodide co-labelling of death. Allergol Immunopathol (Madr).
45. Ren, X., et al., Phage displayed peptides recognizing porcine aminopeptidase N inhibit transmissible gastroenteritis coronavirus infection in vitro. Virology. 410(2):p.299-306.
46. Hu, L., et al., Screening of T7 phage displayed Bactrocera dorsalis (Hendel) antenna cDNA library against chemosensory protein. Arch Insect Biochem Physiol.75(3):p.174-86.
47. Shukla, G.S. and D.N. Krag, Phage-displayed combinatorial peptide libraries in fusion to beta-lactamase as reporter for an accelerated clone screening: Potential uses of selected enzyme-linked affinity reagents in downstream applications. Comb Chem High Throughput Screen.13(1):p.75-87.
48. Song, S.Y., et al., Successful application of the dual-vector system Ⅱ in creating a reliable phage-displayed combinatorial Fab library. Mol Cells, 2009.27(3):p.313-9.
49. He, Y.Y., et al., [Screening of mimic epitopes of Toxoplasma gondii antigen by phage-displayed peptide library]. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi,2007.25(6):p.515-7.
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