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  2. 農業暨自然資源學院
  3. 食品暨應用生物科技學系
Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90253
標題: 環境友善型食品級乳球菌口服疫苗系統之研發
Development of environment-friendly food-grade Lactococcus lactis systems as oral vaccine
作者: 李珮瑄
Pei-Hsuan Lee
關鍵字: Enterovirus 71;non-genetically modified environmental- friendly cell surface display system;recombinant VP1e-anchor motif fusion protein;live L. lactis vector;gram-positive enhancer matrix(GEM);腸病毒 71 型;非基改環境友善型表層展示系統 重組VP1e-anchor motif;融合蛋白質;乳球菌活載體;革蘭氏陽性菌加強基質
引用: 台灣乳酸菌協會 http://www.talab.org.tw/ 行政院衛生署疾病管制局(Centers for Disease Control, R.O.C., Taiwan) http://www.cdc.gov.tw/ 林志侯。乳酸菌載體表現系統之研究現況與趨勢。農業生技產業季刊。 周怡廷。2004。以菌體表層展示技術表現重組之表層蛋白及融合蛋白於大腸桿菌、乳酸桿菌及乳酸練球菌。國立中興大學食品暨應用生物科技學系碩士論文。 蘇政蕙。2005 年。挑選持續型強力啟動子並表現重組抗凍蛋白類似物於乳酸鏈球菌與乳酸桿菌中。國立中興大學食品暨應用生物科技學系碩士論文。 彭宣融。2006 年。開發重組乳酸鏈球菌口服疫苗。國立中興大學食品暨應用生物科技學系碩士論文。 黃馨慧。2007 年。增進第一型重組抗凍蛋白質類似物於乳酸鏈球菌之分泌表現。國立中興大學食品暨應用生物科技學系碩士論文。 羅詩晴。2009 年。以枯草桿菌分泌生產腸病毒 71 型 VP1 蛋白之部份表位片段及評估此表位片段對 BALB/c 小鼠之免疫效果。國立中興大學食品暨應用生物科技學系碩士論文。 吳柏彥。2010 年。研究枯草桿菌 xylose 誘導型系統與以枯草桿菌生產融合廣效性流感病毒 HA 抗原之疫苗。國立中興大學食品暨應用生物科技學系碩士論文。 劉衛綸。2013 年。利用乳酸鏈球菌表現純化重組人類第一型三葉因子及人類介白素-10。國立中興大學食品暨應用生物科技學系碩士論文。 Airaksinen, A. (2000). The VP1 Intracapsid Hook and Uncoating of Enteroviruses: National Public Health Institute. Audouy, S. A., van Roosmalen, M. L., Neef, J., Kanninga, R., Post, E., van Deemter, M., Metselaar, H., van Selm, S., Robillard, G. T., Leenhouts, K. J., & Hermans, P. W. (2006). Lactococcus lactis GEM particles displaying pneumococcal antigens induce local and systemic immune responses following intranasal immunization. Vaccine, 24(26), 5434-5441. Audouy, S. A., van Selm, S., van Roosmalen, M. L., Post, E., Kanninga, R., Neef, J., Estevao, S., Nieuwenhuis, E. E., Adrian, P. V., Leenhouts, K., & Hermans, P. W. (2007). Development of lactococcal GEM-based pneumococcal vaccines. Vaccine, 25(13), 2497-2506. Avall-Jaaskelainen, S., Kyla-Nikkila, K., Kahala, M., Miikkulainen-Lahti, T., & Palva, A. (2002). Surface display of foreign epitopes on the Lactobacillus brevis S-layer. Appl Environ Microbiol, 68(12), 5943-5951. Bahey-El-Din, M., Casey, P. G., Griffin, B. T., & Gahan, C. G. (2010). Efficacy of a Lactococcus lactis DeltapyrG vaccine delivery platform expressing chromosomally integrated hly from Listeria monocytogenes. Bioeng Bugs, 1(1), 66-74. Bahey-El-Din, M., & Gahan, C. G. (2010). Lactococcus lactis: from the dairy industry to antigen and therapeutic protein delivery. Discov Med, 9(48), 455-461. Bermudez-Humaran, L. G., Cortes-Perez, N. G., Le Loir, Y., Alcocer-Gonzalez, J. M., Tamez-Guerra, R. S., de Oca-Luna, R. M., & Langella, P. (2004). An inducible surface presentation system improves cellular immunity against human papillomavirus type 16 E7 antigen in mice after nasal administration with recombinant lactococci. J Med Microbiol, 53(Pt 5), 427-433. Brown, B. A., Oberste, M. S., Alexander, J. P., Jr., Kennett, M. L., & Pallansch, M. A. (1999). Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998. J Virol, 73(12), 9969-9975. Carr, F. J., Chill, D., & Maida, N. (2002). The lactic acid bacteria: a literature survey. Crit Rev Microbiol, 28(4), 281-370. Chart, H., Smith, H. R., La Ragione, R. M., & Woodward, M. J. (2000). An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5alpha and EQ1. J Appl Microbiol, 89(6), 1048-1058. Claus, H., Akca, E., Debaerdemaeker, T., Evrard, C., Declercq, J. P., Harris, J. R., Schlott, B., & Konig, H. (2005). Molecular organization of selected prokaryotic S-layer proteins. Can J Microbiol, 51(9), 731-743. de Vos, W. M., Kuipers, O. P., van der Meer, J. R., & Siezen, R. J. (1995). Maturation pathway of nisin and other lantibiotics: post-translationally modified antimicrobial peptides exported by gram-positive bacteria. Mol Microbiol, 17(3), 427-437. Engelhardt, H., & Peters, J. (1998). Structural Research on Surface Layers: A Focus on Stability, Surface Layer Homology Domains, and Surface Layer–Cell Wall Interactions. Journal of Structural Biology, 124(2–3), 276-302. Esteban, L. E., Temprana, C. F., Arguelles, M. H., Glikmann, G., & Castello, A. A. (2013). Antigenicity and immunogenicity of rotavirus VP6 protein expressed on the surface of Lactococcus lactis. Biomed Res Int, 2013, 298598. Faraldo, M. M., de Pedro, M. A., & Berenguer, J. (1992). Sequence of the S-layer gene of Thermus thermophilus HB8 and functionality of its promoter in Escherichia coli. J Bacteriol, 174(22), 7458-7462. Fernandez-Herrero, L. A., Olabarria, G., & Berenguer, J. (1997). Surface proteins and a novel transcription factor regulate the expression of the S-layer gene in Thermus thermophilus HB8. Mol Microbiol, 24(1), 61-72. Gilliland, S. E. (1990). Health and nutritional benefits from lactic acid bacteria. FEMS Microbiol Rev, 7(1-2), 175-188. Goh, Y. J., Azcarate-Peril, M. A., O'Flaherty, S., Durmaz, E., Valence, F., Jardin, J., Lortal, S., & Klaenhammer, T. R. (2009). Development and application of a upp-based counterselective gene replacement system for the study of the S-layer protein SlpX of Lactobacillus acidophilus NCFM. Appl Environ Microbiol, 75(10), 3093-3105. Holmgren, J., & Czerkinsky, C. (2005). Mucosal immunity and vaccines. Nat Med, 11(4 Suppl), S45-53. Kunji, E. R., Slotboom, D. J., & Poolman, B. (2003). Lactococcus lactis as host for overproduction of functional membrane proteins. Biochim Biophys Acta, 1610(1), 97-108. Le Loir, Y., Azevedo, V., Oliveira, S. C., Freitas, D. A., Miyoshi, A., Bermudez-Humaran, L. G., Nouaille, S., Ribeiro, L. A., Leclercq, S., Gabriel, J. E., Guimaraes, V. D., Oliveira, M. N., Charlier, C., Gautier, M., & Langella, P. (2005). Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production. Microb Cell Fact, 4(1), 2. Le Loir, Y., Gruss, A., Ehrlich, S. D., & Langella, P. (1998). A nine-residue synthetic propeptide enhances secretion efficiency of heterologous proteins in Lactococcus lactis. J Bacteriol, 180(7), 1895-1903. Lee, S. Y., Choi, J. H., & Xu, Z. (2003). Microbial cell-surface display. Trends Biotechnol, 21(1), 45-52. Leenhouts, K., Buist, G., & Kok, J. (1999). Anchoring of proteins to lactic acid bacteria. Antonie Van Leeuwenhoek, 76(1-4), 367-376. Madsen, S. M., Arnau, J., Vrang, A., Givskov, M., & Israelsen, H. (1999). Molecular characterization of the pH-inducible and growth phase-dependent promoter P170 of Lactococcus lactis. Mol Microbiol, 32(1), 75-87. Madsen, S. M., Hindre, T., Le Pennec, J. P., Israelsen, H., & Dufour, A. (2005). Two acid-inducible promoters from Lactococcus lactis require the cis-acting ACiD-box and the transcription regulator RcfB. Mol Microbiol, 56(3), 735-746. Medina, E., & Guzman, C. A. (2001). Use of live bacterial vaccine vectors for antigen delivery: potential and limitations. Vaccine, 19(13-14), 1573-1580. Melnick, J. L. (1984). Enterovirus type 71 infections: a varied clinical pattern sometimes mimicking paralytic poliomyelitis. Rev Infect Dis, 6 Suppl 2, S387-390. Melnick, J. L., Tagaya, I., & von Magnus, H. (1974). Enteroviruses 69, 70, and 71. Intervirology, 4(6), 369-370. Mierau, I., & Kleerebezem, M. (2005). 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Appl Microbiol Biotechnol, 68(6), 705-717. Mierau, I., Olieman, K., Mond, J., & Smid, E. J. (2005). Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications. Microb Cell Fact, 4, 16. Nouaille, S., Ribeiro, L. A., Miyoshi, A., Pontes, D., Le Loir, Y., Oliveira, S. C., Langella, P., & Azevedo, V. (2003). Heterologous protein production and delivery systems for Lactococcus lactis. Genet Mol Res, 2(1), 102-111. Ramasamy, R., Yasawardena, S., Zomer, A., Venema, G., Kok, J., & Leenhouts, K. (2006). Immunogenicity of a malaria parasite antigen displayed by Lactococcus lactis in oral immunisations. Vaccine, 24(18), 3900-3908. Ramirez, K., Ditamo, Y., Rodriguez, L., Picking, W. L., van Roosmalen, M. L., Leenhouts, K., & Pasetti, M. F. (2010). Neonatal mucosal immunization with a non-living, non-genetically modified Lactococcus lactis vaccine carrier induces systemic and local Th1-type immunity and protects against lethal bacterial infection. Mucosal Immunol, 3(2), 159-171. Renault, P. (2002). Genetically modified lactic acid bacteria: applications to food or health and risk assessment. Biochimie, 84(11), 1073-1087. Robinson, K., Chamberlain, L. M., Lopez, M. C., Rush, C. M., Marcotte, H., Le Page, R. W., & Wells, J. M. (2004). Mucosal and cellular immune responses elicited by recombinant Lactococcus lactis strains expressing tetanus toxin fragment C. Infect Immun, 72(5), 2753-2761. Rossi, F., Capodaglio, A., & Dellaglio, F. (2008). Genetic modification of Lactobacillus plantarum by heterologous gene integration in a not functional region of the chromosome. Appl Microbiol Biotechnol, 80(1), 79-86. Russell, W. M., & Klaenhammer, T. R. (2001). Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination. Appl Environ Microbiol, 67(9), 4361-4364. Sara, M., & Sleytr, U. B. (2000). S-Layer proteins. J Bacteriol, 182(4), 859-868. Shareck, J., Choi, Y., Lee, B., & Miguez, C. B. (2004). Cloning Vectors Based on Cryptic Plasmids Isolated from Lactic Acid Bacteria:Their Characteristics and Potential Applications in Biotechnology. Critical Reviews in Biotechnology, 24(4), 155-208. Sleytr, U. B. (1997). I. Basic and applied S-layer research: an overview. FEMS Microbiol Rev, 20(1–2), 5-12. Sleytr, U. B., Bayley, H., Sara, M., Breitwieser, A., Kupcu, S., Mader, C., Weigert, S., Unger, F. M., Messner, P., Jahn-Schmid, B., Schuster, B., Pum, D., Douglas, K., Clark, N. A., Moore, J. T., Winningham, T. A., Levy, S., Frithsen, I., Pankovc, J., Beale, P., Gillis, H. P., Choutov, D. A., & Martin, K. P. (1997). Applications of S-layers. FEMS Microbiol Rev, 20(1-2), 151-175. Slos, P., Dutot, P., Reymund, J., Kleinpeter, P., Prozzi, D., Kieny, M. P., Delcour, J., Mercenier, A., & Hols, P. (1998). Production of cholera toxin B subunit in Lactobacillus. FEMS Microbiol Lett, 169(1), 29-36. Smit, E., Oling, F., Demel, R., Martinez, B., & Pouwels, P. H. (2001). The S-layer protein of Lactobacillus acidophilus ATCC 4356: identification and characterisation of domains responsible for S-protein assembly and cell wall binding. J Mol Biol, 305(2), 245-257. Smit, E., & Pouwels, P. H. (2002). One repeat of the cell wall binding domain is sufficient for anchoring the Lactobacillus acidophilus surface layer protein. J Bacteriol, 184(16), 4617-4619. Smith, G. P. (1985). Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science, 228(4705), 1315-1317. Sorensen, H. P., & Mortensen, K. K. (2005). Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol, 115(2), 113-128. Studier, F. W. (1991). Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol, 219(1), 37-44. Studier, F. W., & Moffatt, B. A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol, 189(1), 113-130. Terpe, K. (2006). Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol, 72(2), 211-222. van Klompenburg, W., Paetzel, M., de Jong, J. M., Dalbey, R. E., Demel, R. A., von Heijne, G., & de Kruijff, B. (1998). Phosphatidylethanolamine mediates insertion of the catalytic domain of leader peptidase in membranes. FEBS Lett, 431(1), 75-79. van Roosmalen, M. L., Kanninga, R., El Khattabi, M., Neef, J., Audouy, S., Bosma, T., Kuipers, A., Post, E., Steen, A., Kok, J., Buist, G., Kuipers, O. P., Robillard, G., & Leenhouts, K. (2006). Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria. Methods, 38(2), 144-149. von Heijne, G. (1990). The signal peptide. J Membr Biol, 115(3), 195-201. Wadskov-Hansen, S. L., Willemoes, M., Martinussen, J., Hammer, K., Neuhard, J., & Larsen, S. (2001). Cloning and verification of the Lactococcus lactis pyrG gene and characterization of the gene product, CTP synthase. J Biol Chem,276(41), 38002-38009. Wells, J. M., & Mercenier, A. (2008). Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria. Nat Rev Microbiol, 6(5), 349-362. Wells, J. M., Wilson, P. W., Norton, P. M., Gasson, M. J., & Le Page, R. W. F. (1993). Lactococcus lactis: high-level expression of tetanus toxin fragment C and protection against lethal challenge. Mol Microbiol, 8(6), 1155-1162. Welman, A. D., & Maddox, I. S. (2003). Exopolysaccharides from lactic acid bacteria: perspectives and challenges. Trends Biotechnol, 21(6), 269-274.
摘要: 
Enterovirus is a general term for a group of viruses which contained many different types and has widely distributed all over the world. Enterovirus 71 (EV71) commonly causes Hand, Foot and Mouth Disease (HFMD) in children, in severe
cases even result in neurological and cardiorespiratory complications. EV71infection have been recently reported as Asian-Pacific regionale epidemics. Human beings are the only known hosts and source of infection. Currently, there are still no approved antiviral drugs or vaccines against EV71 infection yet. This study developed systems based on a concept of non-genetically modified environmental- friendly cell surface display. The Escherichia coli T7 expression
system express large amount of EV71 capsid protein VP1 epitopes(VP1e) fused with an anchor motif (designated as recombinant VP1e-anchor motif fusion protein). About 1.7mg/L of the purified recombinant VP1e-anchor motif fusion protein was obtained after purification. The purified fusion protein was anchored onto the outer surface of live L. lactis vector or gram-positive enhancer matrix(GEM). Results showed that the maximum binding capacity was about 105 molecules purified VP1e-anchor motif fusion protein /cell. Besides, the binding capacity of GEM was better than live L. lactis vector. The stabilities of bound fusion protein were evaluated by storage at three different temperatures (-80°C, 4 °C, and room temperature [20–30 °C]) during 6 months. The results showed that GEM-bound VP1e-anchor motif fusion protein was more stable than live L. lactis vector-bound. To confirm the stability, the microscopy analysis of the purified fusion protein was exmined and the fusion proteins definitely anchored on GEM. The non-genetically modified L. lactis (live or GEM) as vaccine carrier, can avoid the risk of genetically modified microorganisms (GMMs) to apply in human oral consumption. In another part of this study, the recombinant VP1e was expressed by L. lactis expression system. The constitutive expression vector (pNZSASVP1e) can
constitutively expressed recombinant VP1e(rVP1e) yielding 102.5μg/L. Another acid-inducible system (MpHI)( pNZAUS-SacBAVP1e) was constructed and which was expected to improve the production of rVP1e. Unfortunately, the secretion level
was not improved. Both expression levels were too low to examine the stability of rVP1e mixed with GEM. The VP1e was proved to be a good vaccine candidate, because anti-VP1
antiserum was successfully achieved previously. In the future, the improvement and evaluation of non-genetically modified cell surface display system as oral vaccine is
highly worthwhile to develope. To express the rVP1e by GRAS or food-grade system may provides safer and convenient tool to develop L. lactis-based food-grade oral vaccine.

腸病毒是一群病毒的總稱，型別繁多且廣泛分布於全球，其中以腸病毒 71型(human enterovirus 71)最容易引起手足口病及神經系統等相關之嚴重併發症，為亞太地區地方性的流行性傳染疾病，而人類是其已知的唯一宿主及感染源。目前並無任何有效的疫苗可供預防及治療。本論文利用非基改環境友善(environmental friendly)型表層展示(cell surface
display)系統之概念，以大腸桿菌表現系統大量表現融合有錨定功能性區塊(anchor motif)之腸病毒 71 型外鞘蛋白質 VP1 抗原決定部位VP1e，以最適純化條件純化重組融合蛋白質，經濃縮定量後可得到1.7mg/L 之重組 VP1e-anchor motif融合蛋白質，並將其分別與乳球菌活載體(live L. lactis vector)及革蘭氏陽性菌加強基質(gram-positive enhancer matrix, GEM)進行共置培養，結果證實重組VP1e-anchor motif 融合蛋白質能結合於細胞表面。並利用西方墨點法及免疫墨點法定量分析測得其最大結合能力(Maximum binding capacity)，結果顯示每個細胞約可與 105 分子之重組 VP1e-anchor motif 融合蛋白質結合。此外，GEM 的結合能力較乳球菌活菌載體好。一般疫苗之儲存溫度為 4°C，少數為-20°C 以下，若經長時間之存放且不失活，即為一高穩定性且具良好品質之疫苗。因此本論文另進行穩定性測定，將其置於-80°C、4°C 及室溫(20~30°C)三種不同溫度下存放數星期至數個月後，以蛋白質電泳及西方墨點法觀察重組 VP1e- anchor motif 融合蛋白質是否仍結合於細胞壁/GEM 上。結果顯示結合至 GEM 之重組VP1e- anchor motif 融合蛋白質較乳球菌活菌載體穩定。最後以掃描式電子顯微鏡(SEM)及免疫螢光顯微鏡(Immunofluorescence microscopy)分析照相確認。透過運用非基因工程改造乳酸菌株作為疫苗攜帶者(vaccine carrier)，可免除消費者對基因改造菌株於應用上之安全疑慮。此外，本論文亦利用乳酸菌表現系統分泌表現重組 VP1e 蛋白質。以本實驗室先前構築之持續型表現載體(pNZSASVP1e)表現重組 VP1e，但經純化後僅得到 102.5μg/L 之重組 VP1e，於是以 MpHI 酸誘導系統另構築一誘導型表現載體(pNZAUS-SacBAVP1e)，然而其胞外重組蛋白質之表現量甚低，因此皆無法由持續型及誘導型表現系統純化出足夠之重組 VP1e 蛋白質與 GEM 混合。本實驗室先前已經動物實驗成功製備出VP1特異性抗體，證實該蛋白質極具疫苗發展之潛力。未來可針對非基改表層展示系統進行動物試驗及疫苗效價評估。為避免全株基改微生物之諸多爭議，未來期待能將基改技術應用於安全級/食品級微生物系統，開發食品級口服乳酸菌疫苗，對人類有更多貢獻。
URI: http://hdl.handle.net/11455/90253
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