Luận án Nghiên cứu biểu hiện kháng nguyên hemagglutinin (HA) tái tổ hợp của virus cúm A / H5N1 và đánh giá tính sinh miễn dịch trên gà

main of avian influenza H5N1 virus in E. coli BL21 in the fusion form with thioredoxin. 2) Expressing genes encoding HA1 globular and HA1-2 domain from avian influenza virus H5N1 in P. pastoris. 3) Selection suitable parameters for high HA expression 4) Evaluating the immunogenicity of recombinant HA protein in chicken trials. Results: The fusion protein of TrxHA1E and TrxHAl-2E with the molecular mass of 57 and 66 kDa were synthesized successfully in E. coli BL 21 cells in LB medium at 30°C under the regulation of T7 promoter which was induced by IPTG. The 108 fusion proteins were synthesized in inclusion body at 30 o C under induction of 0.5 mM IPTG. The effects of temperature (22, 25, 30 and 37 o C) and concentration of induction factor IPTG (0.1, 0.5, 1.0, 1.5 and 2.0 mM) were investigated. TrxHA1E was expressed at 30 o C accounts for 20% of total proteins, expression levels of TrxHA1E approximately 126 mg/l by ELISA. The solubility of TrxHA1-2E was over 70% as expressing at the temperature 25 o C and yield of TrxHA1-2E was 86 mg/l. Recombinant TrxHA1E protein then was separated from host soluble proteins by sonication combined with centrifugation, then solubilized in 2M urea had antigenicity to bind with antibody against HA of virus H5N1. TrxHA1E had capable of triggering the production of neutralizing antibodies in chicken when they were immunized with recombinant protein by subcutaneous injection or intranasal immunization. Hemagglutination inhibition (HI) titers of chickens sera after 2 weeks of booster immunization were 2.2-2.8 log2. The results showed that TrxHA1E had immunogenicity although the HI titers were at low levels. In P. pastoris, the fusion proteins were expressed in the two form: (1) containing protease sites thrombin (T) and enterokinase (E) that located on the linking sequence between trx and ha1 (Trx-TE-HA1), trx and ha1-2 (Trx-TE-HA1- 2); (2) removing the two protease sites on the linking sequence (TrxHA1, TrxHA1- 2). Both of Trx-TE-HA1 and Trx-TE-HA1-2 were expressed at low level and proteolyzed in the cultivation medium. In order to further improve the expression level of HA recombinant protein from A/H5N1 avian virus in P. pastoris we optimized the HA1 protein coding sequence and expressed it in P. pastoris X33, but foreign protein expression was also not stable. By gene construct removing the protease sites on the linking sequence reduced the proteolysis of the fusion protein and increased the expression proficiency in comparision with the prevous construct. In order to further improve the expression level of recombinant HA protein from A/H5N1 avian virus we optimized the HA5.1 protein coding sequence and expressed it in P. pastoris X33. After optimization, Codon Adaptation Index (CAI) value was improved from 0.69 to 0.98, without modifying the amino acid sequence 109 of the encoded protein. The synthetic mha1 with a length of 1 kb was inserted in to pPICZαmha1 then expressed in P. pastoris X33. MHA1 recombinant protein of 50 - 70 kDa was synthesized at 30 o C under induction of 1% methanol during 72 hours. Recombinant HA protein had biological function to agglutinate chicken’s blood cells at and had antigenicity to bind with antibody against HA of H5N1 virus. The structures of recombinant HA protein by removing the two protease sites on the linking sequences were also expressed in P. pastoris SMD1168. The recombinant proteins were produced with higher productivity and more stable. Different induction media including BMM, BMMY, BMMG, BMMGY and different induction ingredients were used to get higher fermentation yield of TrxHA1. Western blotting of fermentation culture supernatants with differ-ent media of the recombinant P. pastoris strain with anti - HA antibody revealed an obvious difference in the amount of the TrxHA1 protein produced between the different media, with a greater apparent yield being obtained from the suspension culture in modified BMMGY media with productivity estimated to be 3.4-fold higher than in standard media BMM and BMMY. Cultures in shake flasks provided expression levels of TrxHA1 approximately 14 mg/l. The recombinant protein was suffered from glycosylation and protease processing. The fed-batch fermentation at 10 L scale was developed for high-yield secretory expression of the recombinant Hemagglutinin protein. The TrxHA1 concentration of the clarified broth was found to be 84 mg/L. The TrxHA1 was partly purified by precipitation with 20 and 60% ammonium sulfate (mass/volumn) or purified using Co-NTA affinity chromatography. The doses of 100 μg TrxHA1 were immunized for two week-old chickens by subcutaneous injection or by intranasal administration for assessment of TrxHA1 immunogenicity. In subcutaneous injection chickens groups, haemagglutinin inhibition (HI) titer of chickens sera harvested after 2 weeks of booster immunization reached 7.0-7.2 log2. In intranasal immunization groups, HI titers were about 6.6-7.0 log2. 110 In conclusion, the study has demonstrated that P. pastoris can be used to express influenza TrxHA1 and TrxHA1-2 proteins with antigenicity and immunogenicity obtained. The expressed proteins were successfully purified from the expression culture without additional extraction steps, suggesting the feasibility of a cost-effective and large-scale production for vaccine purpose. Chickens vaccinated with TrxHA1 protein produced high neutralizing antibody titers, at rates comparable with the licensed inactivated H5N1 vaccine. Therefore, this system may act as an attractive and effective candidate vaccine to produce protein for avian influenza vaccines. 111 NHỮNG CÔNG TRÌNH CÔNG BỐ LIÊN QUAN ĐẾN LUẬN ÁN 1. Nguyễn Thị Thảo, Văn Thị Như Ngọc, Lê Thị Thu Hồng, Võ Viết Cường, Đỗ Thị Huyền, Trương Nam Hải (2010) Tối ưu hoá biểu hiện HA5.1 của virus cúm A/H5N1 trong nấm men P. pastoris SMD 1168. Hội nghị khoa học 35 năm thành lập Viện Khoa học & Công nghệ Việt Nam. Nxb. Khoa học tự nhiên và Công nghệ, 238-243. 2. Võ Viết Cường, Trần Thị Nhài, Đỗ Thị Huyền, Trương Văn Dung, Trương Nam Hải (2013) Nghiên cứu tinh chế và đánh giá khả năng sinh đáp ứng miễn dịch của protein HA5.1 từ virus cúm A/H5N1 biểu hiện trong Escherichia coli. Tạp chí Công nghệ sinh học, Số 3 (13):319-325. 3. Võ Viết Cường, Lê Thị Huệ, Đỗ Thị Huyền, Lê Quỳnh Giang, Nguyễn Thị Quý, Trương Nam Hải (2013) Biểu hiện gen HA5.1 được cải biến mã có hoạt tính sinh học trong nấm men Pichia pastoris X 33. Tạp chí Sinh học, Số 3: 255- 264. 4. Võ Viết Cường, Nguyễn Thị Quý, Nguyễn Thanh Ngọc, Lê Thị Thu Hồng, Đỗ Thị Huyền, Trương Văn Dung, Trương Nam Hải (2013) Đánh giá khả năng sinh đáp ứng miễn dịch trên gà của kháng nguyên HA tái tổ hợp biểu hiện trong nấm men Pichia pastoris. Tạp chí Sinh học, Số 4: 461-468. 5. Thi Quy Nguyen, Thi Thu Hao Van, Yu-Chen Lin , Thi Nhu Ngoc Van, Khanh Chi Bui, Quynh Giang Le, Thi Huyen Do, Thi Thu Hong Le, Viet Cuong Vo, Van Dung Truong, Peter M Smooker, Peter J Coloe, Nam Hai Truong (2014) A potential protein-based vaccine for influenza H5N1 from the recombinant HA1 domain of avian influenza A/H5N1 expressed in Pichia pastoris. Future Virology, 12(9): 1019-1031. 112 TÀI LIỆU THAM KHẢO 1. Aguilar-Yáñez JM, Portillo-Lara R, Mendoza-Ochoa GI, García-Echauri SA, López-Pacheco F, Bulnes-Abundis D, Salgado-Gallegos J, Lara-Mayorga IM, Webb-Vargas Y (2010) An Influenza A/H1N1/2009 hemagglutinin vaccine produced in Escherichia coli. PLoS One 5(7): 10.1371/journal.pone.0011694. 2. Ahmad M, Hirz M, Pichler H, Schwab H (2014) Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol 98(12): 5301-5317. 3. Akbarzadeh A, Dehnavi E, Aghaeepoor M, Amani J (2015) Optimization of Recombinant Expression of Synthetic Bacterial Phytase in Pichia pastoris Using Response Surface Methodology. Jundishapur J Microbiol 8(12): 10.5812/jjm.27553. 4. Amorij JP, Huckriede A, Wilschut J, Frijlink HW, Hinrichs WLJ (2008) Development of Stable Influenza Vaccine Powder Formulations: Challenges and Possibilities. Pharm. Res. 25 (6):10.1007/s11095-008-9559-6. 5. Anasontzis GE, Salazar Penã M, Spadiut O, Brumer H, Olsson L (2014) Effects of temperature and glycerol and methanol-feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris. Biotechnol Prog 30(3): 728-735. 6. Aoki FY, Boivin G, Roberts N (2007) Influenza virus susceptibility and resistance to oseltamivir. Antivir Ther 12(4B): 603-616. 7. Arinaminpathy N, McLean AR (2008) Antiviral treatment for the control of pandemic influenza: some logistical constraints. J R Soc Interface 5(22): 545-553. 8. Arjmand S, Lotfi AS, Shamsara M, Mowla SJ (2013) Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastoris. Electron J Biotechnol 16(1): 10.2225/vol16-issue1-fulltext-4. 9. Arnau C, Casas C, Valero F (2011) The effect of glycerol mixed substrate on the heterologous production of a Rhizopus oryzae lipase in Pichia pastoris system. Biochem Eng J 57: 30-37. 10. Athmaram TN, Saraswat S, Santhosh SR, Singh AK, Suryanarayana WS, Raj Priya, Gopalan N, Manmohan Parida, Lakshmana Rao PV, R Vijayaraghavan (2011) Yeast expressed recombinant Hemagglutinin protein of Novel H1N1 elicits neutralising antibodies in rabbits and mice. J Virol 8: 524-537. 113 11. Athmaram TN, Singh AK, Saraswat S, Srivastava S, Misra P, Rao MK, Gopalan N, Rao PL (2013) A simple Pichia pastoris fermentation and downstream processing strategy for making recombinant pandemic Swine origin influenza A virus hemagglutinin protein. J Ind Microbiol Biotechnol 40: 245-255. 12. Ausubel FM, Brent R, Kingston RE, Moore Đ, Seidman JG, Smith JA, Struhl K (2003) Current Protocols in Molecular Biology. John Wiley & Sons Inc. 13. Baigent SJ, McCauley JW (2001) Glycosylation of haemagglutinin and stalk- length of neuraminidase combine to regulate the growth of avian influenza viruses in tissue culture. Virus Res 79(1-2): 177-185. 14. Balamurugan V, Reddy GR, Suryanarayana VS (2007) Pichia pastoris: A notable heterologous expression system for the production of foreign proteins – Vaccines. Indian J Biotechnol 6: 175-186. 15. Bender C, Hall H, Huang J, Klimov A, Cox N, Hay A, Gregory V, Cameron K, Lim W and Subbarao K (1999) Characterization of the surface proteins of influenza A (H5N1) viruses isolated from humans in 1997 - 1998. Virology 254: 115-123. 16. Lê Trần Bình (2009) Đánh giá chất lượng vắc-xin cúm A/H5N1 cho gia cầm được sản xuất tại Việt Nam bằng chủng NIBRG-14. Báo cáo tổng kết đề tài cấp Nhà nước. Viện Công nghệ sinh học. Viện Hàn lâm khoa học và Công nghệ Việt Nam. 17. Blake TA, Williams TL, Pirkle JL, Barr JR (2009) Targeted N-linked glycosylation analysis of H5N1 influenza hemagglutinin by selective sample preparation and liquid chromatography/tandem mass spectrometry. Anal Chem 81(8): 3109-3118. 18. Bouvier NM, Palese P (2008) The biology of influenza viruses. Vaccine 26 (4): 49-53. 19. Boyle DB, Heine HG (1993) Recombinant fowlpox virus vaccines for poultry. Immunol. Cell Biol 71: 391-397. 20. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254. 21. Bright RA, Carter DM, Daniluk S, Toapanta FR, Ahmad A, Gavrilov V, Massare M, Pushko P, Mytle N, Rowe T, Smith G, Ross TM (2007) 114 Influenza virus-like particles elicit broader immune responses than whole virion inactivated influenza virus or recombinant hemagglutinin. Vaccine 25(19): 3871-3878. 22. Bright RA, Carter DM, Crevar CJ, Toapanta FR, Steckbeck JD (2008) Cross- Clade Protective Immune Responses to Influenza Viruses with H5N1 HA and NA Elicited by an Influenza Virus-Like Particle. PLoS One 3(1). doi:10.1371/journal.pone.0001501. 23. Bublot M, Pritchard N, Swayne DE, Selleck P, Karaca K, Suarez DL, Audonnet JC, Mickle TR (2006) Development and use of fowlpox vectored vaccines for avian influenza. Ann N Y Acad Sci 1081: 193-201. 24. Bublot M, Pritchard N, Cruz JS, Mickle TR, Selleck P, Swayne DE (2007) Efficacy of a fowlpox-vectored avian influenza H5 vaccine against Asian H5N1 highly pathogenic avian influenza virus challenge. Avian Dis 51(1): 498-500. 25. Bui C, Bethmont A, Chughtai AA, Gardner L, Sarkar S, Hassan S, Seale H, MacIntyre CR (2016) A Systematic Review of the Comparative Epidemiology of Avian and Human Influenza A H5N1and H7N9 - Lessons and Unanswered Questions. Transbound Emerg Dis 63(6): 602-620. 26. Nguyễn Văn Cảm, Tống Hữu Hiến, Nguyễn Tùng, Nguyễn Hoàng Đăng (2011) Kết quả công cường độc gà, vịt, ngan sau khi dùng vacxin cúm gia cầm tái tổ hợp H5N1 chủng Re-5 của Trung Quốc. Tạp chí Khoa học Kỹ thuật Thú y 2: 12-16. 27. Cardoso FM, Ibañez LI, Van den Hoecke S, De Baets S, Smet A, Roose K, Schepens B, Descamps FJ, Fiers W, Muyldermans S, Depicker A, Saelens X (2014) Single-domain antibodies targeting neuraminidase protect against an H5N1 influenza virus challenge. J Virol 88(15): 8278-8296. 28. Casali N, Preston A (2003) E. coli Plasmid Vectors: Methods and Applications Methods in Molecular Biology 35. Humana Press Inc. 29. Castrucci MR, Kawaoka Y (1993) Biologic importance of neuraminidase stalk length in influenza A virus. J Virol 67: 759-764. 30. Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev 24: 45-66. 115 31. Cereghino GP, Cereghino JL, Ilgen C, Cregg JM (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 13: 329-332. 32. Chen H, Smith GJ, Li KS, Wang J, Fan XH, Rayner JM, Vijaykrishna D, Zhang JX, Zhang LJ, Guo CT, Cheung CL, Xu KM, Duan L, Huang K, Qin K, Leung YH,Wu WL, Lu HR, Chen Y, Xia NS, Naipospos TS, Yuen KY, Hassan SS, Bahri S, Nguyen TD, Webster RG, Peiris JS, Guan Y (2006) Establishment of multiple sublineages of H5N1 influenza virus in Asia: implications for pandemic control. Proc Natl Acad Sci USA 103(8): 2845-2850. 33. Chen LM, Davis CT, Zhou H, Cox NJ, Donis RO (2008) Genetic compatibility and virulence of reassortants derived from contemporary avian H5N1 and human H3N2 influenza A viruses. PLoS Pathog 4(5): 10.1371/journal.ppat.1000072. 34. Chen H (2009) Avian influenza vaccination: the experience in China. Rev Sci Tech 28(1): 267-274. 35. Cheung C L, Rayner JM., Smith GJ, Wang P, Naipospos TS, Zhang J, Yuen KY, Webster RG, Peiris JS Guan Y, Chen H (2006) Distribution of amantadine-resistant H5N1 avian influenza variants in Asia. J Infect Dis 193: 1626-1629. 36. Chiu FF, Venkatesan N, Wu CR, Chou AH, Chen HW, Lian SP, Liu SJ, Huang CC, Lian WC, Chong P, Leng CH (2009) Immunological study of HA1 domain of hemagglutinin of influenza H5N1 virus. Biochem Biophys Res Commun 383(1): 27-31. 37. Ciarkowska A, Jakubowska A (2013) Pichia pastoris as an expression system for recombinant protein production. Postepy Biochem 59(3): 315-321. 38. Cornelissen LA, Vries RP, de Boer-Luijtze EA, Rigter A, Rottier PJ (2010) A Single Immunization with Soluble Recombinant Trimeric Hemagglutinin Protects Chickens against Highly Pathogenic Avian Influenza Virus H5N1. PLoS One 5(5):10.1371 /journal.pone.0010645. 39. Cos O, Ramón R, Montesinos JL, Valero F (2006) Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review. Microb Cell Fact 5(17): 10.1186/1475-2859-5-17. 116 40. Crevar CJ, Ross TM (2008) Elicitation of protective immune responses using a bivalent H5N1 VLP vaccine. Virol J 5(131): 10.1186/1743-422X-5-131. 41. Cuong NV, Truc VN, Nhung NT, Thanh TT, Chieu TT, Hieu TQ, Men NT, Mai HH, Chi HT, Boni MF, van Doorn HR, Thwaites GE, Carrique‐Mas JJ, Hoa NT (2016) Highly Pathogenic Avian Influenza Virus A/H5N1 Infection in Vaccinated Meat Duck Flocks in the Mekong Delta of Vietnam. Transbound Emerg Dis 63(2): 127–135. 42. Daly R, HeRNA TWM (2005) Expression of heterologous protein in Pichia pastoris: a useful experimental tool in protein engineering and production. J. Mol. Recognit 18: 119-138. 43. de Graaf M, Fouchier RA (2014) Role of receptor binding specificity in influenza A virus transmission and pathogenesis. EMBO J 33(8): 823-841. 44. de Jong JC, Palache AM, Beyer WE, Rimmelzwaan GF, Boon AC, Osterhaus AD (2003) Haemagglutination-inhibiting antibody to influenza virus. Dev. Biologicals 115: 63-73. 45. de Vries RP, de Vries E, Bosch BJ, de Groot RJ, Rottier PJ, de Haan CA (2010) The influenza A virus hemagglutinin glycosylation state affects receptor-binding specificity. Virology 403(1): 17-25. 46. Deshpande KL, Fried VA, Ando M, Webster RG (1987) Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence. Proc Natl Acad Sci USA 84(1): 36-40. 47. Doyle TM, Hashem AM, Li C, Van Domselaar G, Larocque L, Wang J, Smith D, Cyr T, FRNAsworth A, He R, Hurt AC, Brown EG, Li X (2013) Universal anti-neuraminidase antibody inhibiting all influenza A subtypes. Antiviral Res 100(2): 567-774. 48. Eichelberger MC, Wan H (2015) Influenza neuraminidase as a vaccine antigen. Curr Top Microbiol Immunol 386: 275-299. 49. Fan J, Liang X, Horton MS, Perry HC, Citron MP, Heidecker GJ (2004) Preclinical study of influenza virus A M2 peptide conjugate vaccines in mice, ferrets, and rhesus monkeys. Vaccine 22: 2993-3003. 50. Fukuyama S, Kawaoka Y (2011) The pathogenesis of influenza virus infections: the contributions of virus and host factors. Curr Opin Immunol 23(4): 481-486. 117 51. Galarza JM, Latham T, Cupo A (2005) Virus-like particle (VLP) vaccine conferred complete protection against a lethal influenza virus challenge. Viral Immunol 18(1): 244-251. 52. Gamblin SJ, Skehel JJ (2010) Influenza hemagglutinin and neuraminidase membrane glycoproteins. J Biol Chem 285(37): 28403-28409. 53. Gambotto A, Barratt-Boyes SM, de Jong MD, Neumann G, Kawaoka Y (2008) Human infection with highly pathogenic H5N1 influenza virus. Lancet 371(9622): 1464-1475. 54. Gao M, Soloff AC, Lu X, Montecalvo A, Nguyen DC, Matsuoka Y, Robbins PD, Swayne DE, Donis RO, Katz JM, Barratt-Boyes SM, Gambotto A (2006) Protection of mice and poultry from lethal H5N1 avian influenza virus through adenovirus-based mmmunization. J Virol 80(4):1959-1964. 55. Gao M, Shi Z (2013) Process Control and Optimization for Heterologous Protein Production by Methylotrophic Pichia pastoris. Chin. J. Chem. Eng 21(2) 216-226. 56. Ge J, Deng G, Wen Z, Tian G, Wang Y, Shi J, Wang X, Li Y, Hu S, Jiang Y, Yang C, Yu K, Bu Z, Chen H (2007) Newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and heterologous H5N1 avian influenza viruses. J Virol 81(1):150-158. 57. Gellissen G. (2000) Heterologous protein production inmethylotrophic yeasts. Appl. Microbiol. Biotechnol 54: 741-750. 58. Gleeson MA, White CE, Meininger DP, Komives EA (1998) Generation of protease-deficient strains and their use in heterologous protein expression. Methods Mol Biol 103: 81-94. 59. Gonçalves AM, Pedro AQ, Maia C, Sousa F, Queiroz JA, Passarinha LA (2013) Pichia pastoris: a recombinant microfactory for antibodies and human membrane proteins. J Microbiol Biotechnol 23(5): 587-601. 60. Gopal GJ, Kumar A (2013) Strategies for the production of recombinant protein in Escherichia coli. Protein J. 32(6): 419-425. 61. Grgacic EV, Anderson DA (2006) Virus-like particles: passport to immune recognition. Methods 40(1): 60-65. 62. Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and heterologous protein expression. Trends Biotechnol 22(7): 346-353. 118 63. Halbherr SJ, Ludersdorfer TH, Ricklin M, Locher S, Berger Rentsch M, Summerfield A, Zimmer G (2015) Biological and protective properties of immune sera directed to the influenza virus neuraminidase. J Virol 89(3): 1550-1563. 64. Lê Huy Hàm (2011) Nghiên cứu tạo giống bèo tấm tái tổ hợp mang kháng nguyên H5N1 phòng chống bệnh cúm gia cầm. Báo cáo tổng kết đề tài. 147- 149. Viện Di truyền nông nghiệp, Bộ NN và PTNT. 65. Trần Xuân Hạnh (2012) Sản xuất vaccine cúm gia cầm NAVET-VIFLUVAC, chủng NIBRG-14 ở Việt Nam. Tạp chí Khoa học Kỹ thuật Thú y 3: 89-93. 66. Trần Xuân Hạnh; Tô Thị Phấn; Đỗ Thanh Thủy; Phạm Sĩ Tú; Tô Long Thành; Tống Hữu Hiền; Nguyễn Hoàng Đăng; Đàm Thị Vui (2013) Thử hiệu lực của vacxin Navet-Vifluvac phòng bệnh cúm A/H5N1 Clade 1.1 và 2.3.2.1c bằng phương pháp công cường độc và tiếp xúc trực tiếp. Tạp chí Khoa học Kỹ thuật Thú y 5: 22-29. 67. Hanson A, Imai M, Hatta M, McBride R, Imai H, Taft A, Zhong G, Watanabe T, Suzuki Y, Neumann G, Paulson JC, Kawaoka Y (2016) Identification of Stabilizing Mutations in an H5 Hemagglutinin Influenza Virus Protein. J Virol 90(6): 2981-2992. 68. Nguyễn Lê Khánh Hằng, Lê Thị Quỳnh Mai (2016) Đặc điểm vi rút cúm A/H5N1 và A/H5N6 trên gia cầm tại Việt Nam, 2012-2015. Tạp chí Y học dự phòng 10(183): 126-133. 69. Nguyễn Lê Khánh Hằng, Ứng Thị Hồng Trang (2016) Tình trạng nhiễm vi rút cúm trên gia cầm khoẻ mạnh thu thập tại các chợ buôn bán, giết mổ gia cầm ở Hà Nội, 2012-2014. Tạp chí Y học dự phòng 10 (183): 119-125. 70. Hervé PL, Lorin V, Jouvion G, Da Costa B, Escriou N (2015) Addition of N- glycosylation sites on the globular head of the H5 hemagglutinin induces the escape of highly pathogenic avian influenza A H5N1 viruses from vaccine- induced immunity. Virology 486: 134-145. 71. Nguyễn Thị Minh Hiền, Lê Văn Hiệp, Lê Văn Bé, Nguyễn Thị Lan Phương, Trần Ngọc Nhơn, Đặng Thị Hồng Vân (2009) Nghiên cứu sản xuất vaccine cúm A/H5N1 cho người trên trứng gà có phôi từ chủng NIBRG-14. Tạp chí Y học dự phòng 2: 83-89. 72. Hilleman MR (2002) Realities and enigmas of human viral influenza: pathogenesis, epidemiology and control. Vaccine 20(25-26): 3068-2087. 119 73. Hirota K, Murata M, Sachi Y, Nakamura H, Takeuchi J, Mori K, Yodoi J (1999) Distinct roles of thioredoxin in the cytoplasm and in the nucleus. A two-step mechanism of redox regulation of transcription factor NF-kappaB. J Biol Chem 274(39): 27891-27897. 74. Nguyễn Thị Hoa, Đồng Văn Quyền (2015) Nghiên cứu tạo Baculovirus tái tổ hợp biểu hiện hemagglutinin (HA) của virus cúm A/H5N1 clade 2.3.2.1b. Tạp chí Công nghệ sinh học 37(1): 103-109. 75. Lê Thanh Hoà, Đoàn Thị Thanh Hương (2011) Nghiên cứu virus cúm A/H5N1 ở Việt Nam và Thái Lan: Dịch tễ phân tử, chẩn đoán và vaccine phòng chống. Báo cáo tổng hợp Kết quả khoa học và Công nghệ đề tài. Viện Công nghệ sinh học, Viện Hàn lâm khoa học và Công nghệ Việt Nam. 76. Holsinger LJ, Nichani D, Pinto LH, Lamb RA (1994) Influenza A virus M2 ion channel protein: a structure-function analysis. J Virol. 68(3):.1551-1563. 77. Horimoto T, Kawaoka Y (2006) Strategies for developing vaccines against H5N1 influenza A viruses. Trends Mol Med 12(11): 506-514. 78. Houser K, Subbarao K (2015) Influenza vaccines: challenges and solutions. Cell Host Microbe 17(3): 295-300. 79. Phạm Thanh Hồng, Nguyễn Văn Khoa, Cao Thị Bảo Vân (2015) Biểu hiện protein Hemagglutinin của vi rút cúm A/H5N1 trên hệ thống Baculovirus trong tế bào Sf9 (Spodoptera frugiperda) nuôi cấy huyền phù. Tạp chí Y học dự phòng 5 (165): 174-180. 80. Hu H, Gao J, He J, Yu B, Zheng P, Huang Z, et al. (2013) Codon Optimization Significantly Improves the Expression Level of a Keratinase Gene in Pichia pastoris. PLoS One 8(3): 10.1371/journal.pone.0058393. 81. Hu ZP, Yin J, Zhang YY, Jia SY, Chen ZJ, Zhong J (2012) Characterization of the immune responses elicited by baculovirus-based vector vaccines against influenza virus hemagglutinin. Acta Pharmacologica Sinica 33: 783-790. 82. Huang RB, Du QS, Wang CH, Chou KC (2008) An in-depth analysis of the biological functional studies based on the NMR M2 channel structure of influenza A virus. Biochem. Biophys. Res. Commun 377: 1243-1247. 83. Đỗ Thị Huyền, Bùi Hồng Vân, Văn Thị Như Ngọc, Trương Văn Dung, Trương Nam Hải (2009) Biểu hiện gen HA5 mã hoá kháng nguyên hemagglutinin (HA) của virus cúm A/H5N1 trong Escherichia coli. Tạp chí Công nghệ sinh học 7(2): 185-192. 120 84. Jegerlehner A, Schmitz N, Storni T, Bachmann MF (2004) Influenza A vaccine based on the extracellular domain of M2: weak protection mediated via antibody-dependent NK cell activity. J Immunol 172(9): 5598-5605. 85. Jeong H, Kim HJ, Lee SJ (2015) Complete Genome Sequence of Escherichia coli Strain BL21. Genome Announc 3(2): 10.1128/genomeA.00134-15. 86. Kang SM, Yoo DG, Lipatov AS, Song JM, Davis CT, Quan FS, Chen LM, Donis RO, Compans RW (2009) Induction of long-term protective immune responses by influenza H5N1 virus-like particles. PLoS One. 4(3): 10.1371/journal.pone.0004667. 87. Keawcharoen J, Amonsin A, Oraveerakul K, Wattanodorn S, Papravasit T, KRNAda S, Lekakul K, Pattanarangsan R, Noppornpanth S (2005) Characterization of the hemagglutinin and neuraminidase genes of recent influenza virus isolates from different avian species in Thailand. Acta Virol 49(4): 277-280. 88. Kelleher ZT, Sha Y, Foster MW, Foster WM, Forrester MT, Marshall HE (2014) Thioredoxin-mediated denitrosylation regulates cytokine-induced nuclear factor κB (NF-κB) activation. J Biol Chem 289(5): 3066-3072. 89. Khurana S, Verma S, Verma N, Crevar CJ, Carter DM (2010) Properly Folded Bacterially Expressed H1N1 Hemagglutinin Globular Head and Ectodomain Vaccines Protect Ferrets against H1N1 Pandemic Influenza Virus. PLoS One 5(7):10.1371/journal.pone.0011548. 90. Khurana S, Larkin C, Verma S, Joshi MB, Fontana J, Steven AC, King LR, Manischewitz J, McCormick W, Gupta RK, Golding H (2011) Recombinant HA1 produced in E. coli forms functional oligomers and generates strain-specific SRID potency antibodies for pandemic influenza vaccines. Vaccine 29(34): 5657-5665. 91. Kilbourne ED, Pokorny BA, Johansson B, Brett I, Milev Y, Matthews JT (2004) Protection of mice with recombinant influenza virus neuraminidase. J. Infect. Dis 189: 459-461. 92. Kopera E, Dwornyk A, Kosson P, Florys K, Sączyńska V, Dębski J, Adamczewska VC, Szewczyk B, Ostoja WZ, Grzelak K (2014) Expression, purification and characterization of glycosylated influenza H5N1 hemagglutinin produced in Pichia pastoris. Acta Biochim Pol 61(3): 597-602. 121 93. Krainer FW, Dietzsch C, Hajek T, Herwig C, Spadiut O, Glieder A (2012) Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway. Microb Cell Fact 11(12): 10.1186/1475-2859- 11-22. 94. Lambert LC, Fauci AS. (2010) Influenza Vaccines for the Future. N Engl J Med 363(21): 2036-2044. 95. Lardinois A, Steensels M, Lambrecht B, Desloges N, Rahaus M, Rebeski D, Berg T (2012) Potency of a recombinant NDV-H5 vaccine against various HPAI H5N1 virus challenges in SPF chickens. Avian Dis 56(4): 928-936. 96. Le TH, Nguyen NT (2014) Evolutionary dynamics of highly pathogenic avian influenza A/H5N1 HA clades and vaccine implementation in Vietnam. Clin Exp Vaccine Res 3: 117-127. 97. Lee EK, Kang HM, Kim KI, Choi JG, To TL, Nguyen TD, Song BM, Jeong J, Choi KS, Kim JY, Lee HS, Lee YJ, Kim JH (2015) Genetic evolution of H5 highly pathogenic avian influenza virus in domestic poultry in Vietnam between 2011 and 2013. Poult Sci 94(4): 650-661. 98. Lee YT, Kim KH, Ko EJ, Lee YN, Kim MC, Kwon YM, Tang Y, Cho MK, Lee YJ, Kang SM (2014) New vaccines against influenza virus. Clin Exp Vaccine Res 3(1): 12-28. 99. Li F, Yang S, Zhao L, Li Q, Pei J (2012) Synonymous condon usage bias and overexpression of a synthetic xynB Gene from Aspergillus niger NL1 in Pichia pastoris. Bio. Res 7(2): 2330-2343. 100. Li P, Anumanthan A, Gao XG, Ilangovan K, Suzara VV, Düzgüneş N, Renugopalakrishnan V (2007) Expression of recombinant proteins in Pichia pastoris. Appl Biochem Biotechnol 142: 105-124. 101. Li XL, Yang Y, Sun Y, Chen WJ, Sun RX, Liu K, Ma MJ, Liang S, Yao HW, Gray GC, Fang LQ, Cao WC (2015) Risk Distribution of Human Infections with Avian Influenza H7N9 and H5N1 virus in China. Sci Rep. 5: 10.1038/srep18610. 102. Li Y, Zhang X, Xu Q, Fu Q, Zhu Y, Chen S, Peng D, Liu X (2013) Characterisation and haemagglutinin gene epitope mapping of a variant strain of H5N1 subtype avian influenza virus. Vet Microbiol 162(2-4): 614-622. 103. Lin YJ, Deng MC, Wu SH, Chen YL, Cheng HC, Chang CY, Lee MS, Chien MS, Huang CC (2008) Baculovirus-derived hemagglutinin vaccine protects 122 chickens from lethal homologous virus H5N1 challenge. J Vet Med Sci 70(11): 1147-1152. 104. Liu WC, Gong T, Wang QH, Liang X, Chen JJ, Zhu P (2016) Scaling-up Fermentation of Pichia pastoris to demonstration-scale using new methanol- feeding strategy and increased air pressure instead of pure oxygen supplement. Sci Rep 6: 10.1038/srep18439. 105. Luczo JM, Stambas J, Durr PA, Michalski WP, Bingham J (2015) Molecular pathogenesis of H5 highly pathogenic avian influenza: the role of the haemagglutinin cleavage site motif. Rev Med Virol 25(6): 406-430. 106. Macauley-Patrick S, Fazenda ML, McNeil B, Harvey, L.M (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22: 249-270. 107. Macken CA, Webby RJ, Bruno WJ (2006) Genotype turnover by reassortment of replication complex genes from avian influenza A virus. J Gen Virol 87(10): 2803-2815. 108. Mahmood K, Bright RA, Mytle N, Carter DM, Crevar CJ, Achenbach JEHeaton PM, Tumpey TM, Ross TM (2008) H5N1 VLP vaccine induced protection in ferrets against lethal challenge with highly pathogenic H5N1 influenza viruses. Vaccine 26 (42): 5393-5399. 109. Marcelin G, Sandbulte MR, Webby RJ (2012) Contribution of antibody production against neuraminidase to the protection afforded by influenza vaccines. Rev Med Virol 22(4): 267-279. 110. Milián E, Kamen AA (2015) Current and emerging cell culture manufacturing technologies for influenza vaccines. Biomed Res Int: 10.1155/2015/504831. 111. Morvarid AR, Zeenathul NA, Tam YJ, Zuridah H, Mohdazmi ML, Azizon BO (2012) Effect of glycerol feed in methanol induction phase for Hepatitis B surface sntigen expression in Pichia pastoris strain KM71. Pertanika J. Sci. & Technol. 20 (1): 31-42. 112. Nayak B, Rout SN, Kumar S, Khalil MS, Fouda MM, et al. (2009) Immunization of Chickens with Newcastle Disease Virus Expressing H5 Hemagglutinin Protects against Highly Pathogenic H5N1 Avian Influenza Viruses. PLoS One 4(8):10.1371/journal.pone.0006509. 113. Neumann G, Hatta M, Kawaoka Y (2003) Reverse genetics for the control of avian influenza. Avian disease 47: 882-887. 123 114. Neumann G, Noda T, Kawaoka Y (2009) Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature 459: 931-939. 115. Neumann G, Kawaoka Y (2015) Transmission of influenza A viruses. Virology 279-280: 234-246. 116. Nguyễn Thị Bích Nga, Lương Thị Hồng Vân, Lê Thanh Hòa (2011) Phân tích đặc điểm chuỗi Polypeptide NA(N1) của một số chủng virus cúm A/H5N1 gây bệnh ở gia cầm thu thập các năm 2004 - 2009 tại Việt Nam. Tạp chí Công nghệ Sinh học 1: 47-54. 117. Nguyễn Thị Bích Nga, Lương Thị Hồng Vân, Lê Thanh Hòa (2011) Biến đổi di truyền HA (H5) virus cúm A/H5N1 qua phân tích trình tự gen của một số chủng gây bệnh ở gia cầm tại Việt Nam giai đoạn 2004-2010. Tạp chí Y học Việt Nam 2: 134-139. 118. Văn Thị Như Ngọc, Đỗ Thị Huyền, Nguyễn Thanh Nhàn, Nguyễn Phước Hải, Trương Văn Dung, Trương Nam Hải (2007) Biểu hiện gen ha5-1 mã hoá tiểu phần kháng nguyên Hemagglutinin (HA) của virus cúm A/H5N1 trong Escherichia coli. Tạp chí Công nghệ sinh học 5(3): 185-192. 119. Nicholson KG, Wood JM, Zambon M (2003) Influenza. Lancet 362(93970): 1733-1745. 120. Nwe N, He Q, Damrongwatanapokin S, Du Q, Manopo I, Limlamthong Y, Fenner B.J, Spencer L, Kwang J (2006) Expression of hemagglutinin protein from the avian influenza virus H5N1 in a baculovirus/insect cell system significantly enhanced by suspension culturê. BMC Microbiology 24: 6-16. 121. Okamatsu M, Nishi T, Nomura N, Yamamoto N, Sakoda Y, Sakurai K, Chu HD, Thanh LP, Van Nguyen L, Van Hoang N, Tien TN, Yoshida R, TakadaA, Kida H (2013) The genetic and antigenic diversity of avian influenza viruses isolated from domestic ducks, muscovy ducks, and chickens in northern and southern Vietnam, 2010–2012. Virus Genes 47: 317-329. 122. Palese P, Shaw ML (2007) Orthomyxoviridae: the viruses and their replication. In: Knipe DM, Howley PM, editors. Fields virology. Philadelphia, Lippincott Williams & Wilkin. 123. Paoletti E (1996) Application of poxvirus vectors to vaccination: an update. Proc.Acad.Sci. USA 93: 11349-11353. 124. Perdue ML, Suarez DL (2000) Structural features of the avian influenza virus hemagglutinin that influence virulence. Vet Microbiol 74: 77-86. 124 125. Phue JN, Lee SJ, Trinh L, Shiloach J (2008) Modified Escherichia coli B (BL21), a superior producer of plasmid DNA compared with Escherichia coli K (DH5α). Biotechnol Bioeng 101: 831-836. 126. Nguyễn Thị Lan Phương, Lê Văn Bé, Dương Hữu Thái, Lê Kim Hoà, Trần Ngọc Nhơn, Vũ Thị Thu Hương, Nguyễn Hoàng Tùng, Nguyễn Thị Lý (2015) Chất lượng vắc xin cúm IVACFLU-A/H5N1 dự tuyển thử nghiệm lâm sàng. Tạp chí Y học dự phòng 9 (169): 46-51 127. Pietrzak M, Macioła A, Zdanowski K, Protas-Klukowska AM, Olszewska M, Śmietanka K, Minta Z, Szewczyk B, Kopera E (2016) An avian influenza H5N1 virus vaccine candidate based on the extracellular domain produced in yeast system as subviral particles protects chickens from lethal challenge. Antiviral Res 133: 242-249. 128. Ping J, Lopes TJ, Nidom CA, Ghedin E, Macken CA, Fitch A, Imai M, Maher EA, Neumann G, Kawaoka Y (2015) Development of high-yield influenza A virus vaccine viruses. Nat Commun 6(8148): 10.1038/ncomms9148. 129. Potvin G, Ahmad A, Zhang Z (2012) Bioprocess engineering aspects of heterologous protein production in Pichia pastoris. Biochem. Eng 64: 91-105. 130. Pushko P, Pujanauski LM, Sun X, Pearce M, Hidajat R, Kort T, Schwartzman LM, Tretyakova I, Chunqing L, Taubenberger JK, Tumpey TM (2015) Recombinant H7 hemagglutinin forms subviral particles that protect mice and ferrets from challenge with H7N9 influenza virus. Vaccine 33(38): 4975-4982. 131. Qiao C, Jiang YP, Jia YQ, Tian GB, Liu M, Deng GH, Wang XR, Meng QW, Tang XY (2003) Protection of chickens against highly lethal H5N1 and H7N1 avian influenza viruses with a recombinant fowlpox virus co-expressing H5 haemagglutinin and N1 neuraminidase genes. Avian Pathol 32: 25-31. 132. Qiao C, Tian G, Jiang YP, Li Y, Shi J, Yu K, Chen H (2006) Vaccines developed for H5 highly pathogenic avian influenza in China. Ann NY Acad Sci 1081: 182-192. 133. Qiao C, Jiang Y, Tian G, Wang X, Li C, Xin X, Chen H, Yu K (2009) Recombinant fowlpox virus vector-based vaccine completely protects chickens from H5N1 avian influenza virus. Antiviral Res 81(3): 234-238. 134. Quan FS, Yoo DG, Song JM, Clements JD, Compans RW, Kang SM (2009) Kinetics of immune responses to influenza virus-like particles and dose- dependence of protection with a single vaccination. J Virol 83(9): 4489-4497. 125 135. Redkiewicz P, Sirko A, Kamel KA, Góra-Sochacka A (2014) Plant expression systems for production of hemagglutinin as a vaccine against influenza virus. Acta Biochim Pol 61(3): 551-560. 136. Rockman S, Brown LE, Barr IG, Gilbertson B, Lowther S, Kachurin A, Kachurina O, Klippel J, Bodle J, Pearse M, Middleton D (2013) Neuraminidase-inhibiting antibody is a correlate of cross-protection against lethal H5N1 influenza virus in ferrets immunized with seasonal influenza vaccine. J Virol 87(6): 3053-3061. 137. Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5(172): 10.3389/fmicb.2014.00172. 138. Rybicki EP (2010) Plant-made vaccines for humans and animals. Plant Biotechnol J 8(5): 620-637. 139. Sączyńska V (2014) Influenza virus hemagglutinin as a vaccine antigen produced in Bacteria. Acta Biochim Pol 61(3): 561-572. 140. Saelens X, Vanlandschoot P, Martinet W, Maras M, Neirynck S, Contreras R, Fiers W, Jou WM (1999) Protection of mice against a lethal influenza virus challenge after immunization with yeast-derived secreted influenza virus hemagglutinin. Eur J Biochem 260 (1): 166-175. 141. Sahdev S, Khattar SK, Saini KS (2008) Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem 307: 247-264. 142. Sambrook J, Russell DW (2001) Molecular Cloning. A Laboratory Manual, 3rd ed. Cold. Spring Harbor Laboratory, Cold Spring Harbor, NY. 143. Sanders CJ, Doherty PC, Thomas PG (2011) Respiratory epithelial cells in innate immunity to influenza virus infection. Cell Tissue Res 343: 13-21. 144. Schotsaert M, de Filette M, Fiers W, Saelens X (2009) Universal M2 ectodomain-based influenza A vaccines: Preclinical and clinical developments. Expert Rev. Vaccine 8: 499-508. 145. Schrauwen EJ, Fouchier RA (2014) Host adaptation and transmission of influenza A viruses in mammals. Emerg Microbes Infect 3(2): 10.1038/emi.2014.9. 126 146. Sguazzaa GH, Fuentealbaa NA, Tizzanoa MA, Galosi CM, Pecoraroa MR (2013) Expression of the hemagglutinin HA1 subunit of the equine influenza virus using a baculovirus expression system. Rev Argent Microbiol 45(4): 222-228. 147. Shehata AA, Fiebig P, Sultan H, Hafez M, Liebert UG (2012) Expression of Truncated Sequences of Influenza A Virus Subtype H5 in Pichia pastoris. J Am Sci 8(8): 433-441. 148. Shimizu Y S, Myint CO, Okamatsu M, Sakoda Y (2013) Influenza Virus-like Particles Containing HA, NA, and M1 Induced Protection in Chickens against a Lethal Challenge with the Highly Pathogenic H5N1 Avian Influenza Virus J Vaccines Vaccin 4 (201): 10.4172/2157-7560.1000201. 149. Shtyrya YA, Mochalova LV, Bovin NV (2009) Influenza Virus Neuraminidase: Structure and Function. Acta Naturae 1(2): 26-32. 150. Smirnov YA, Gitelman AK, Govorkova EA, Lipatov AS, Kaverin NV (2004) Influenza H5 virus escape mutants: immune protection and antibody production in mice. Virus Res 99(2): 205-208. 151. Lê Văn Sơn, Chu Hoàng Hà, Lê Trần Bình (2009) Nghiên cứu phát triển vaccine thực vật dùng qua đường miệng cho gia cầm phòng chống bệnh H5N1 ở Việt Nam. Báo cáo kết quả nghiên cứu Khoa học và Công nghệ. Viện Công nghệ Sinh học, Viện Hàn lâm khoa học và Công nghệ Việt Nam. 152. Spitsin S, Andrianov V, Pogrebnyak N, Smirnov Y, Borisjuk N, Portocarrero C (2009) Immunological assessment of plant-derived avian flu H5/HA1 variants. Vaccine 27: 1289-1292. 153. Sriwilaijaroen N, Suzuki Y (2012) Molecular basis of the structure and function of H1 hemagglutinin of influenza virus. Proc Jpn Acad Ser B Phys Biol Sci 88(6): 226-249. 154. Steinhauer DA (1999) Role of hemagglutinin cleavage for the pathogenicity of influenza virus. Virology 258(1): 1-20. 155. Subathra M, Santhakumar P, Naidu SS, Narasu LM, Senthilkumar TA, Sunil KL (2014) Expression of Avian Influenza Virus (H5N1) Hemagglutinin and Matrix Protein 1 in Pichia pastoris and Evaluation of their Immunogenicity in Mice. Appl Biochem Biotechnol 172: 3635-3645. 156. Suguitan AL, Cheng X, Wang W, Wang S, Jin H, Lu S (2011) Influenza H5 hemagglutinin DNA primes the antibody response elicited by the live 127 attenuated influenza A/Vietnam/1203/2004 vaccine in ferrets. PLoS One 6(7): 10.1371/journal.pone.0021942. 157. Swayne DE, Garcia M, Beck JR, Kinney N, Suarez DL (2000a). Protection against diverse highly pathogenic H5 avian influenza viruses in chickens immunized with a recombinant fowlpox vaccine containing an H5 avian influenza hemagglutinin gene insert. Vaccine 18: 1088-1095. 158. Swayne DE, Beck JR, Kinney N (2000b) Failure of a recombinant fowlpox virus vaccine containing an avian influenza hemagglutinin gene to provide consistent protection against influenza in chickens preimmunizedwith a fowl pox vaccine. Avian Dis 44: 132-137. 159. Swayne DE, Lee CW, Spackman E (2006) Inactivated North American and European H5N2 avian influenza virus vaccines protect chickens from Asian H5N1 high pathogenicity avian influenza virus. Avian Pathol 35(2): 141-146. 160. Tate MD, Job ER, Deng YM, Gunalan V, Maurer-Stroh S, Reading PC (2014) Playing hide and seek: how glycosylation of the influenza virus hemagglutinin can modulate the immune response to infection. Viruses 6(3): 1294-1316. 161. Dương Hữu Thái, Lê Văn Bé, Nguyễn Thị Lan Phương, Lê Kim Hòa (2013) Ứng dụng quy trình lõi sản xuất vaccine cúm A/H5N1 theo công nghệ nuôi cấy trên trứng gà có phôi ở quy mô công nghiệp. Tạp chí Y học dự phòng 9(145): 13-18. 162. Nguyễn Nam Thắng (2012) Đặc điểm phân tử các gen HA, NA và M của virus cúm gia cầm H5N1 lưu hành ở miền Bắc Việt Nam 2008-2009. Luận án Tiến sĩ Sinh học. Viện Công nghệ sinh học. 163. Tian G, Zhang S, Li Y, Bu Z, Liu P, Zhou J, Li C, Shi J, Yu K, Chen H (2005) Protective efficacy in chickens, geese and ducks of an H5N1-inactivated vaccine developed by reverse genetics. Virology 341(1): 153-162. 164. Nguyễn Ngọc Tiến, Hoàng Văn Năm; Văn Đăng Kỳ; Nguyễn Tùng; Ken Inui (2011) Lưu hành của virut cúm gia cầm độc lực cao H5N1 tại Việt Nam và vacxin phòng bệnh cúm gia cầm. Tạp chí Khoa học Kỹ thuật Thú y 6: 76-78. 165. Nguyễn Ngọc Tiến (2013) Tình hình dịch cúm gia cầm giai đoạn 2009-2012 và các giải pháp phòng chống. Tạp chí Khoa học Kỹ thuật Thú y 1: 82-90. 166. Tolner B, Smith L, Begent RH, Chester KA (2006) Production of recombinant protein in Pichia pastoris by fermentation. Nature Protocols 1(2): 1006-1021. 128 167. Tompkins SM, Zhao ZS, Lo CY, Misplon JA, Liu T, Ye Z, Hogan RJ, Wu Z, Benton KA, Tumpey TM (2007) Matrix protein 2 vaccination and protection against influenza viruses, including subtype H5N1. Emerg. Infect. Dis 13: 426 -435. 168. Tong S, Zhu X, Li Y, Shi M, Zhang J, Bourgeois M, Yang H, Chen X, Recuenco S, Gomez J, Chen LM, Johnson A, Tao Y, Dreyfus C, Yu W, McBride R, CRNAey PJ, Gilbert AT, Chang J, Guo Z, Davis CT, Paulson JC, Stevens J, Rupprecht CE, Holmes EC, Wilson IA, Donis RO (2013) New world bats harbor diverse influenza A viruses. PLoS Pathog 9(10): 10.1371/journal.ppat.1003657. 169. Treanor JJ, Wilkinson BE, Masseoud F, Hu-Primmer J, Battaglia R, O'Brien D, Wolff M, Rabinovich G, Blackwelder W, Katz JM (2001) Safety and immunogenicity of a recombinant hemagglutinin vaccine for H5 influenza in humans. Vaccine 19(14): 1732-1737. 170. Đậu Huy Tùng, Đồng Văn Quyền, Nguyễn Tùng, Nguyễn Nam Thắng, Trần Xuân Hạnh, Kim Young Bong, Đinh Duy Kháng (2012) Đánh giá hiệu lực vacxin cúm gia cầm H5N1 (chủng Nibrg14) trên một số Clade lưu hành ở Việt Nam năm 2011 và phân tích đặc tính di truyền của virut H5N1 clade 2.3.2.1b. Tạp chí Khoa học Kỹ thuật thú y 6: 5-16. 171. Cao Thị Bảo Vân, Nguyễn Văn Khoa (2012) Biểu hiện protein hemagglutinin của virus cúm A/H5N1 trong hệ thống baculovirus/tế bào côn trùng. Tạp chí Y học dự phòng 4: 28-34. 172. Nguyễn Thị Thu Vân (2007) Nghiên cứu thành công quy trình công nghệ sản xuất vaccine cúm A/H5N1 trên tế bào thận khỉ tiên phát ở quy mô phòng thí nghiệm. Báo cáo tổng kết Khoa học và Kỹ thuật đề tài. Viện Vệ sinh dịch tễ Trung ương. 173. Veits J, Wiesner D, Fuchs W, Hoffmann B, Granzow H, Starick E, Mundt E, Schirrmeier H, Mebatsion T, Mettenleiter TC, Oberdorfer AR (2006) Newcastle disease virus expressing H5 hemagglutinin gene protects chickens against Newcastle disease and avian influenza. Proc. Natl. Acad. Sci. USA 103: 8197-8202. 174. Velkov T, Ong C, Baker MA, Kim H, Li J, Nation RL, Huang JX, Cooper MA, Rockman S (2013) The antigenic architecture of the hemagglutinin of influenza H5N1 viruses. Mol Immunol 56(4): 705-719. 129 175. Wagner R, Matrosovich M, Klenk H (2002) Functional balance between haemagglutinin and neuraminidase in influenza virus infections. Med Virol 12(3): 159-166. 176. Wagner S, Klepsch MM, Schlegel S, Appel A, Draheim R, Tarry M, Högbom M, van Wijk KJ, Slotboom DJ, Persson JO, de Gier JW (2008) Tuning Escherichia coli for membrane protein overexpression. Proc Natl Acad Sci USA. 105(38): 14371-14376. 177. Wang J, Qiu JX, Soto C, DeGrado WF (2011) Structural and dynamic mechanisms for the function and inhibition of the M2 proton channel from influenza A virus. Curr. Opin. Struct. Biol 21: 68-80. 178. Wang J, Ma C, Wang J, Jo H, Canturk B, Fiorin G, Pinto LH, Lamb RA, Klein ML, DeGrado WF (2013) Discovery of novel dual inhibitors of the wild-type and the most prevalent drug-resistant mutant, S31N, of the M2 proton channel from influenza A virus. J Med Chem 56(7): 2804-2812. 179. Wanga CC, Chena RJ, Tsenga YC, Hsua CH, Hunga YF, Chena SW, Chenf CM, Khoof, Chenga TJ, Chenga YS, Jana JT, Wua CY, Maa C, Wonga CH (2009) Glycans on influenza hemagglutinin affect receptor binding and immune response. Proc Natl Acad Sci USA 106 (43): 18137-18142. 180. Watanabe Y, Ibrahim MS, Suzuki Y, Ikuta K (2012) The changing nature of avian influenza A virus (H5N1). Trends Microbiol 20(1): 11-20. 181. Webster RG (1998) Influenza: an emerging disease. Emerg Infect Dis 4: 436- 441. 182. Webster RG, Guan Y, Peiris M, Walker D, Krauss S, Zhou NN, Govorkova EA, Ellis TM, Dyrting KC, Sit T, Perez DR, Shortridge KF (2002) Characterization of H5N1 influenza viruses that continue to circulate in geese in southeastern China. J Virol 76(1): 118-126. 183. Webster RG, Govorkova EA (2014) Continuing challenges in influenza. Ann N Y Acad Sci 1323: 115-139. 184. Weinacker D, Rabert C, Zepeda AB, Figueroa CA, Pessoa A, Farías JG (2014) Applications of recombinant Pichia pastoris in the healthcare industry. Braz J Microbiol 44(4): 1043-1048. 185. Weli SC, Tryland M (2011) Avipoxviruses: infection biology and their use as vaccine vectors. Virol J 8(49): 10.1186/1743-422X-8-49. 130 186. Werten MW, van den Bosch TJ, Wind RD, Mooibroek H, de Wolf FA (1999) High-yield secretion of recombinant gelatins by Pichia pastoris. Yeast 15(11): 1087-1096. 187. Wildt S, Gerngross TU (2005) The humanization of N-glycosylation pathways in yeast. Nat Rev Microbiol 3(2): 119-128. 188. Wilson I A, Skehel J J, Wiley D C (1981) Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 Å resolution. Nature 289: 366- 373. 189. Wong SS, Webby RJ (2013) Traditional and new influenza vaccines. Clin Microbiol Rev 26(3): 476-492. 190. Xie QM, Ji J, Du LQ, Cao YC, Wei L, Xue CY, Qin JP, Ma JY, Bi YZ (2009) Preparation and immune activity analysis of H5N1 subtype avian influenza virus recombinant protein-based vaccine. Poult Sci 88 (8): 1608-1615. 191. Xiong C, Liu Q, Chen Q, Chen J (2013) Genome Sequence of a Reassortant H5N1 Avian Influenza Virus Isolated from Domestic Green-Winged Teal. Genome Announc 1(4): 10.1128/genomeA.00639-13. 192. Yang JL, Wang HL, Wang SX, Yang P, Liu K and Jiang Cheng-yu (2010) High-level expression, purification and characterization of codon-optimized recombinant hemagglutinin 5 proteins in mammalian cells. Chinese Medical Journal 123(8): 1073-1077. 193. Yang P, Ma C, Cui S, Zhang D, Shi W, Pan Y, Sun Y, Lu G, Peng X, Zhao J, Liu Y, Wang Q (2016) Avian influenza A(H7N9) and (H5N1) infections among poultry and swine workers and the general population in Beijing, China, 2013-2015. Sci Rep. 6(33877): 10.1038/srep33877. 194. Zhang N, Zheng BJ, Lu L, Zhou Y, Jiang S, Du L (2015) Advancements in the development of subunit influenza vaccines. Microbes Infect 17(2): 123-134. 195. Zhang R, Wang X, Su J, Zhao J, Zhang G (2010) Isolation and analysis of two naturally-occurring multi-recombination Newcastle disease viruses in China. Virus Res 151(1): 45-53. 196. Zhang W, Inan M, Meagher MM (2000) Fermentation Strategies for Recombinant Protein Expression in the Methylotrophic Yeast Pichia pastoris. Biotechnol. Bioprocess Eng 5: 275-287. 197. Zhang WH, Potter KJH, Plantz BA, Schlegel VL, Smith LA, Meagher MM (2003) Pichia pastoris fermentation with mixed-feeds of glycerol and 131 methanol: growth kinetics and production improvement. J Ind Microbiol Biotechnol 30: 210-215. 198. Zou W, Ke J, Zhu J, Zhou H, Jin M (2012) The antigenic property of the H5N1 avian influenza viruses isolated in central China. Virol J 9(148): 10.1186/1743-422X-9-148. 199. Invitrogen (2010a) Pichia Expression Kit, Instruction Manual Catalog no. K1710-01. 200. Invitrogen (2010b) pPICZA, B, and C, Instruction Manual Catalog no. V195 -20. 201. Invitrogen (2010c) EasySelect Pichia Expression Kit For Expression of Recombinant Proteins Using pPICZ and pPICZα in Pichia pastoris. Cat. no. K1740-01. 202. OIE (2008) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 203. 204. www.genscript.com/tools/rare-codon-analysis 205. www.wpro.who.int/emerging_diseases/AvianInfluenza/e