跳到主要內容

臺灣博碩士論文加值系統

(18.97.9.172) 您好!臺灣時間:2025/02/10 12:19
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:李陽春
研究生(外文):Yang-Chun Lee
論文名稱:以幽門螺旋桿菌(Helicobaterpylori)之尿素免疫處理雞所產雞蛋蛋黃微膠囊化後抗體的安定性研究
論文名稱(外文):Study on the Stability of Microencapsulated Antibody prepared from the Yolk of Eggs Laid by Hens Immunized with urease from Helicobater pylori
指導教授:張鴻民張鴻民引用關係
指導教授(外文):Prof. Hung-Min Chang , Ph. D.
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:102
中文關鍵詞:幽門螺旋桿菌胃蛋白胰蛋白凝乳胰蛋白尿素環狀糊精阿拉伯膠水解澱粉
外文關鍵詞:Helicobater pyloripepsintrypsinchymotrypsinliposomecyclodextringum arabicsoluble starch
相關次數:
  • 被引用被引用:4
  • 點閱點閱:312
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
中文摘要 在流行病學的研究上發現,十二指腸潰瘍及慢性胃炎的患者,皆可分離出幽門螺旋桿菌,且此菌可能會引起胃癌,故此菌在胃腸消化道方面,的確是一個可怕的殺手。近來對幽門螺旋桿菌的研究可說不勝枚舉,但探討其在被動免疫上的應用卻是非常缺乏,但被動免疫需要大量的抗體,故需尋找經濟且豐富的抗體來源。 本研究是以幽門螺旋桿菌(Helicobater pylori)的尿素為抗原對10隻雞隻肌肉注射後,將免疫雞隻所產雞蛋之蛋黃液經不同濃度(10%及20%)的包覆材質(環狀糊精、阿拉伯膠、水解澱粉及酪蛋白)經微膠囊化後,以胃腸道蛋白分解酵素如胃蛋白(pepsin)、胰蛋白(trypsin)及凝乳胰蛋白(chymotrypsin)(酵素/受質=1/40)在37℃進行水解,探討其抗體殘存活性與水解率,並比較與添加不同濃度(10%及20%)包覆材質的蛋黃粉末之抗體活性的殘存情形。最後探討以liposome包覆BSA及IgY的包覆效率,並觀察包覆IgY者以胃蛋白(pepsin,15μg/ml)及酸水解(pH 1.8及pH 2.8,37℃),探討抗體在liposome中對酵素及酸的安定性。 實驗結果發現:將幽門螺旋桿菌之尿素粗萃液以Sephacryl S-300膠體過濾層析後,對10隻來亨雞肌肉注射連續五週,發現蛋黃及血清中抗體之ELISA值在第四週有明顯上升,第五週達到最高點,但抗體量在血清中與在蛋黃中的差異頗大,顯示血液中之IgG轉移成IgY的量並不大。將蛋黃液包覆10%及20%環狀糊精、水解澱粉及酪蛋白組經微膠囊化後對抗體具有保護作用,與添加10%及20%環狀糊精、水解澱粉及酪蛋白組之噴霧乾燥蛋黃粉末比較,有較高之抗體活性及水解率,且包覆材質濃度10%比20%之抗體活性高;而包覆10%及20%阿拉伯膠經微膠囊化後對抗體具有保護作用,與添加10%及20%阿拉伯膠組之噴霧乾燥蛋黃粉末比較,有較高之抗體活性及水解率,且包覆材質濃度20%比10%之抗體活性高。以liposome包覆BSA,於lecithin/cholesterol比為1:0.25時有較高的包覆效率(約71%),在包覆IgY時也有相似的結果(約69%)。表示以脫水-復水的方法可有效的包覆IgY;將包覆IgY之liposome以胃蛋白在pH 1.8及pH 2.8下水解,隨lecithin/cholesterol比例增加,ELISA值也隨著增加,表示liposome對IgY有保護的效果;將包覆IgY之liposome在pH 1.8及pH 2.8下水解,隨lecithin/cholesterol比例增加,ELISA值也隨著增加,表示liposome在酸的環境下對IgY有保護的效果。
Numerous epidemiological studies indicate a causative relationship between the presence of Helicobater pylori and histologically confirmed gastritis,gastric ulcer and doudenal ulcers.The most notable feature of H. pylori is the production of extremely active urease that could be isolated from cells.Many basic researches on H. pylori were conducted in resent decade.However,applications of H. pylori as the antigen for passive immunization against gastric ulcers and duodenal ulcers have not been reported.Since passive immunization requires a large quanity of antibody,therefore the search for cheap and abundent source of antibody become the important issue.Besides,orally administrated antibodies are sensitive to protease and gastric acid and the consideration for protection of antibody against acid and proteases upon ingestion have to be improved.In this study,purified urease from H. pylori was used as antigen to intramuscularly immunize 10 hens on the thighs.The relative ELISA(enzyme-linked immunosorbent assay) value of antibody in serum and egg yolk,diluted 1,000 folds,against urease was determined during the immunization period.To improve the antibody stability against acid and protease,the collected egg yolks were microencapsulated with various concentration(10 and 15%)of materials(cyclodextrin,gum arabic,soluble starch and casein)by a laboratory-prepared spray-drier(dried at 58±3℃).For protease test,the microencapsulated egg yolk powder was hydrolyzed with protease(pepsin at pH2.0 or 4.0;trypsin,chymotrypsin at pH 7.6) and the relative ELISA value against urease as well as the degree of hydrolysis was determined using spray-dried yolk powder as control.Liposomes of IgY(immunoglobulin in yolk) were also prepared,by repeated dehydration and rehydration of lecithin and cholesterol mixture,to investigate the effect of liposome composition on IgY stability against acid (Ph 1.8 and 2.8)and pepsin(15μg/ml).Meanwhile,the encapsulation efficiency of IgY in liposomes was discussed and compared with that of BSA.Results showed that the relative ELISA value of IgY in both serum and yolk began to increase in the third weel of immunization.The mcroencapsulation of egg yolk with 10 and 20% cyclodextrin,soluble starch,gum arabic and casein showed effective protection effect on the stability of IgY against acid and proteases.The highest encapsulation efficiency of BSA and IgY in liposomes prepared with the ratio of lecithin to cholesterol 1/0.25 was found to be 71 and 69%,respectively.However,liposomes with higher cholesterol content (lecithin/cholesterol=1/1) showed more effective protection effect on IgY against acid (pH 1.8 and 2.8) and pepsin hydrolysis.
目錄頁碼中文摘要 Ⅰ英文摘要 Ⅲ前言1第Ⅰ章 文獻整理3一、幽門螺旋桿菌的發現3二、幽門螺旋桿菌的重要性3(一)、胃炎4 (1).急性胃炎4 (2).慢性胃炎4(二)、十二指腸潰瘍4(三)、胃癌5三、幽門螺旋桿菌的致病因子及致病機轉8(一)、運動性8(二)、尿素11四、幽門螺旋桿菌尿素(E.C.3.5.1.5)簡介11五、幽門螺旋桿菌的被動免疫15六、抗體在雞蛋蛋黃中的分佈16七、抗體及IgY的結構16八、被動免疫的應用22九、微膠囊化技術之應用23(1). 噴霧乾燥微膠囊化法23(2).Liposomes23第Ⅱ章 材料與方法25一、藥品與器材251.菌種252.藥品253.儀器29二、實驗方法301. 幽門螺旋桿菌的培養、分離純化與鑑定302.尿素活性測定303.抗原的製備314.肌肉注射355.抗體活性之測定356.粗IgY製備367.蛋白質的定量368.蛋黃液的微膠囊化409.蛋黃粉末之酵素水解40 (1)胃蛋白40 (2)胰蛋白、凝乳胰蛋白4310.水解率之測定43(1)部份水解43(2)完全水解4311.Liposome的製備及胃蛋白水解測定44(1)Liposome的製備44(2)胃蛋白水解測定4512.膠過濾層析46第Ⅲ章 結果與討論49一、幽門螺旋桿菌的培養、分離純化與鑑定49二、以幽門螺旋桿菌之尿素作為抗原對雞所引起的免疫反應53三、蛋黃中抗體微膠囊化後對胃腸道蛋白質分解酵素之耐受性 57(1).酵素水解之安定性 57(2).水解率之測定 78四、Liposome的包覆效率及對胃蛋白質之耐受性 81第Ⅳ章 結論86參考文獻88
參考文獻王瑞廷﹐1995。醫用微生物學與免疫學。初版﹐第十五章﹐合記圖書出版社﹐台北。呂姿靜,1995。利用多醣體由雞蛋蛋黃中分離純化IgY之研究。國立台灣大學食品科技研究所碩士論文。何敏夫﹐1990。臨床生化學﹐第五章﹐合記圖書出版社﹐台北。林慶文,1983。蛋之化學與利用,二版,第二章,華香園出版社, 台北。陳玉堂,1996。以Streptococcus mutans免疫處理雞蛋所產生抗體之性質及其安定性之研究。國立台灣大學食品科技研究所碩士論文。陳增興,1996。幽門螺旋桿菌(Helicobater pylori)。臨床醫學 38:83-90。陳昭誠,1998。牛乳免疫球蛋白G安定性及IgG強化乳製品之研究。國立台灣大學食品科技研究所博士論文。戴金華﹐1997。雞蛋蛋黃免疫球蛋白Y的簡介。食品工業月刊﹐第29卷第06期。 歐陽瑞芬,1997。以Streptococcus mutans(c)免疫處理雞所產雞蛋蛋黃中抗體之安定性研究。國立台灣大學食品科技研究所碩士論文。清水 誠,1993。雞卵抗體と哺乳動物抗體との比較。農化67: 1430-1432。Adler-Nissen, J. 1979. Determination of degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. J. Agric. Food Chem. 27:1256-1262.Bangham, A.D., Standish, M.M. and Watkins, J.C. 1965. Diffusion of univalent ions across the lamellae of swollen phospholipids. J. Mol. Biol. 13:238-252.Chen, X.G., Correa, P., Offerhaus, J., Rodriquez, E., Janney, R., Hoffman, E., Fox, J., Hunter, F. and Diavolitsis, S. 1986. Ultrastructure of the gastric mucosa harboring campylobacter-like organisms. Am. J. Clin. Pathol. 86:575-582.Cuenca, R., Blanchard, T.G., Czinn, S.J., Nedrud, J.G., Monath, T.P., Lee, C.K. and Redline, R.W. 1996. Therapeutic immunization against Helicobater mustelae in naturally infected ferrets. Gastroenterology 110:1770-1775.Czinn, S.J. and Nedrud, J.G. 1991. Oral immunization against Helicobater pylori. Infect. Immun. 59:2359-2363.Dixon, M.F. 1994. Pathophysiology of Helicobater pylori infection. Scand. J. Gastroenterol. 29 Suppl 201:7-10.Dixon, M.F. 1994. Pathophysiology of Helicobater pylori infection. Scand. J. Gastroenterol. 29 Suppl 201:7-10.Dunn, B.E., Campbell, G.P., Perez-Perez, G.I. and Blaser, M.J. 1990. Purification and characterization of urease from Helicobater pylori. The journal of biological chemistry. 265:9464-9469.Dunn, B.E., Vakil, N.B., Schneider, B.G., Miller, M.M., Zitzer, J.B., Peutz, T. and Phadnis, S.H. 1997. Localization of Helicobater pylori urease and heat shock protein in human gastric biopsies. Infec. Immun. 65:1181-1188.Eaton, K.A., Brooks, C.L., Morgan, D.R. and Krakowka, S. 1991. Essential role of urease in pathogenesis of gastritis induced by Helicobater pylori in Gnotobiotic Piglets. Infection and Immunity 59:2470-2475.Evans, D.J., Evans, D.G., Kirkpatrick, S.S. and Graham, D.Y. 1991. Characterization of the Helicobater pylori urease and purification of its subunits. Microbial Pathogenesis. 10:15-26.Ferrero, R.F., Kansau, I.N. and Labigne, A. 1997. Virulence factors produced by H.pylori. In “The immunobiology of H.pylori:from pathogenesis to prevention”, Ernst P.B. and Smith P.D.,pp29-45.Lippinccot-Raven, Philadelphia.Ferrero, R.L.,Thiberge, J.M., Huerre, M. and Labigne, A. 1994. Recombinant antigens prepared from the urease subunits of Helicobater spp.:evidence of protection in a mouse model of gastric infection. Infec. Immun. 62:4981-4989.Fitzgerald, O. and Murphy, P. 1950. Studies on the physiological chemistry and clinical significance of urease and urea with special reference to the human stomach. Ir. J. Med. Sci. 292:97-159.Forman, D. 1996. Helicobater pylori and gastric cancer. Scand. J. Gastroenterol 31 Suppl 215:48-51.Gear, M.W., Truelove, S.C. and Whitehead, R. 1971. Gastric ulcer and gastritis. Gut 12:639-645. Gilbert,A.B.1971. The egg : its physical and chemical aspects. In ” Physiology and Biochemistry of the Domestic Fowl”(Bell D. J. and Freeman B. M. eds.)Vol.3, Academic Press, New York.Gootz, T.D., Perez-Pertz, G.I., Clancy, J., Martin, B.A., Tait-Kamradt, A. and Blaser, M.J. 1994. Immunological and molecular characterization of Helicobater felisi urease. Infec. Immun. 62:793-798.Graham, D.Y. and Go, M.F. 1993. Helicobater pylori:current status. Gastroenterology 105:279-282.Harlow, E. and Lane, D. 1988. Antibodies. p103-113. Cold Spring Harbor Laboratory. Harris, P.R., Harry, L.T., Perez-Perez, G.I., Blaser, M.J. and Smith, P.D. 1996. Helicobater felisi urease is a potent stimulus of mononuclear phagocyte activation and inflammatory cytokine production. 111:419-425.Harry, L.T., Coresia, M.J., Rosenthal, L.E. and Jones, B.D. 1988. Characterization of urease from Campylobacter pylori. J. Clinical Microbiology. 26:831-836.Hatta, H., Kim, M. and Yamamoto, T. 1990. A novel isolation method for hen egg yolk antibody, 'IgY''. Agric. Biol. Chem. 54: 2531-2535.Hattori, T. and Sugihara, H. 1996. The pathological sequence in the development of gastric cancer:I. Scand. J. Gastroenterol. 31 Suppl 214:34-35. Hazell, S.L. and Lee, A. Campylobacter pyloridis,urease,hydrogen ion back diffusion and gastric ulcers. Lancet 2:15-17.Hu, L.T. and Harry, L.T. 1990. Purification and N-terminal analysis of urease from Helicobater pylor. Infec. Immun. 58:992-998.Hunt, R.H. 1996. The role of Helicobater pylori in pathogenesis:the spectrum of clinical outcomes. Scand. J. Gastroenterol. 31 Suppl 220:3-9.Kim, Y.D., Morr, C.V. and Schenz, T.W. 1996. Microencapsulation paoperties of gum arabic and several food protein:liquid orange oil emulsion particles. J. Agric. Food Chem. 44:1308-1313.Kim, Y.D. and Morr, C.V. 1996. Microencapsulation properties of gum arabic and several food protein:spray-dried orange oil emulsion particles. J. Agric. Food Chem. 44:1314-1320.Kirby, C. and Gregoriadis, G. 1984. Dehydration-rehydration vesicles:a simple method for high yield drug entrapment in liposomes. Bio/Technology 2:979-984.Kirby, C.J. and Gregoriadis, G. 1980. The effect of the cholesterol content of small unilamellar liposomes on the fate of their lipid components in vivo. Life Sci. 27:2223-2230. Kronvall, G., Seal, U. S., Finstad, J. and JR.R.C.W.1970.Phylogenetic insight into evolution of mammalian Fc fragment of G globulin using Staphylococcal protein A. J. Immunol.104:140-147.Kuby, J. 1994. Immunology 2nd edition. W. H. Freeman and company, New York. Lechago, J. and Correa, P. 1993. Prolonged achlorhydria and gastric neoplasia:is their a causal relationship? Gastroenterology 104(8):1554-6.Lee, A. 1993. Pathogenicity of Helicobater pylori:a perspective. Infect. Immun. 61:1601-10.Lee, A. 1994. The microbiology and epidemiology of Helicobater pylori infection. Scand. J. Gastroenterol.29 Suppl 201:2-6.Lee, A. 1996. The nature of Helicobater pylori. Scand. J. Gastroenterol. 31 Suppl 214:5-8.Lee, A. 1996. Prevention of Helicobater pylori infection. Scand. J. Gastroenterol. 31 Suppl 215:11-15.Lee, C., Weltzin, R. and Thomas, W.D. 1995. Oral immunization with recombinant Helicobater pylori urease induces secretory IgA antibodies and protects mice from challenge with Helicobater felis. J. Infect. Dis. 172:161-172.Lieber, C.S. and Lefevre, A. 1959. Ammonia as a source of hypoacidity in patients with uraemia. J. Clin. Invest. 38:1271-1277.Luck, J.M. and Seth, T.N. 1924.Gastric urease. Biochem. J. 18:1227-1231.Marshall, B.J. 1983. Unidentified curved bacillus on gastric epithelium in active chronic gastritis. Lancet I:1273-5. Marshall, B.J. and Warren, J.R. 1984. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet I:1311-5. Marshall, B.J., Mcgechie, D.B., Rogers, P.A. and Glancy, R.J. 1985. Pyloric Campylobater infection and gastroduodenal disease. Med. J. Aust. 142:439-44.Marshall, B.J. and Langton, S.R. 1986. Urea hydrolysis in patients with Campylobacter pylori infection. Lancet I:1965-1966. Marshall, B.J. 1989. History of the discovery of C. pylori. In “Campylobater pylori in gastritis and peptic ulcer disease. Blaser, M.J. New York. 7-23. Martin, W.G., ans Vandegaer, J.E. and Cook, W.H. 1957. Fraction of livetin and the molecular weights of the α- and β-components. Can. J. Biochem. Physiol. 35:241-250.Megraud, F., Neman-Simha, V. and Brugmann, D. 1992. Futher evidence of the toxic effect of ammonia produced by Helicobater pylori urease on human epithelial cells. Infect. Immun. 60:1858-63.Moran, A.P. 1996. Pathogenic properties of Helicobater pylori. Scand. J. Gastroenterol. 31 Suppl 215:22-31.Otake, S., Nishihara, Y. Makimura, M. Hatta, H., Kim, M. Yamamoto, T. and Hirasawa, M. 1991. Protection of rats against dental caries by passive immunization with hen-egg-yolk antibody (IgY). J. Dent. Res. 70:162-166.Otani, H., Matsumoto, K., Saeki, A. and Hosono, A. 1991. Comparative studies on properties of hen egg yolk IgY and rabbit serum IgG antibodies. Lebens. Wissen. Technol. 24: 152-158.Palley, L.S., Murphy, J., Yan, Y., Taylor, N.,Polidoro, D. and Fox, J. 1993. The effects of an oral immunization scheme using muramyl dipeptide as an adjuvant to prevent gastric Helicobater mustelae infection of ferrets(abstrate). Acta. Gastro-Enterol Belg. 56(suppl):54.Pappo, J., Thomas, W.D., Kabok, Z., Taylor, N.S., Murphy, J.C. and Fox, J.G. 1995. Effects of oral immunization with recombinant urease on murine Helicobater felis gastritis. Infect. Immun. 63:1246-1252.Parsonnet, J., Friedman, G.D., Friedman, G.D. and Sibley, R.K. 1991. Helicobater pylori infection and the risk of gastric carcinoma. N. Engl. J. Med. 325:1127-1131.Pick, U. 1981. Liposomes with a large trapping capacity prepared by freezing and thawing of sonicated phospholipid mixtures. Arch. Biochem. Biophys. 212:186-194.Riecken, E.O. and Zeitz, M. 1995. Helicobater pylori:mechanisms of pathogenicity. In”Malignancy and chronic inflammation in the gastrointestinal tract.”(Schulzke, J.D., Epple, H.J., Bojarski, C., Kirstein, F. and Riecken, E.O.Rokkas, T., Papatheodorou, G., Karameris, A., Mavrogreogis, A., Kalogeropoulos, N. and Giannikos, N. 1993. Can H. pylori eradication restore vitamin C levels in gastric juice?(abstr) Gastroenterology(in press). Scott, N., Lansdown, M. and Diament, R. 1990. Helicobater gastritis and intestinal metaplasia in a gastric cancer family. Lancet 335:728. Shimizu, M., Fitzsimmons, R.C. and Nakai, S. 1988. Anti-E. coli immunoglobulin Y isolated from egg yolk of immunized chickens as a potential food ingredient. J. Food Sci. 53: 1360-1366.Shimizu, M., Miwa, Y., Hashimoto, K. and Goto, A. 1993. Encapsulation of chicken egg yolk immunoglobulin G(IgY) by liposomes. Biosci. Biotech. Biochem. 57(9): 1445-1449.Shimizu, M., Fitzsimmons, R.C. and Nakai, S. 1988. Anti-E. coli immunoglobulin Y isolated from egg yolk of immunized chickens as a potential food ingredient. J. Food Sci. 53: 1360-1366.Sipponen, P., Kosunen, T.U., Valle, J., Riihla, M. and Seppala, K. 1992. Helicobater pylori infection and chronic gastritis in gastric cancer. J. Clin. Pathol. 45:319-23.Sipponen, P. 1994. Gastric cancer─a long-term consequence of Helicobater pylori infection? Scand. J. Gastroenterol 29 Suppl 201:24-27.Sobala, G.M., Crabtree, J.E., Dixon, M.F., Schorah, C.J., Taylor, J.D., Rathbone, B.J., Heatley, R.V. and Axon, A.T. 1991. Acute Helicobater pylori infection:clinical features,local and systemic immune response,gastric mucosal histology,and gastric juice ascorbic acid concentrations. Gut 32:1415-18. Stuart, C., Van Stratum, E. and Rustigian, R. 1945. Further studies on urease production by Proteus and related organisms. J. Bacteriol. 49:437-444.Warren, J.R. 1983. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet I:1273.Watanabe, H. 1996. The pathological sequence in the development of gastric cancer:Ⅱ. Scand. J. Gastroenterol 31 Suppl 214:36-39.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊