臺灣博碩士論文加值系統

摘 要
凝集素廣泛地存在於自然界不同生物體內，具有凝集紅血球及其他正常或變形細胞的能力，常被應用於細胞表面癌症辨識物及其他生物醫學的研究。本論文研究是從台灣沿岸採集之海洋大型海藻與分離培養的海洋微細藻類共三十種進行凝集活性檢測，並對具高活者藻類進行分離純化。以磷酸鹽緩衝液及20 ％乙醇分別萃取出海藻並以粗萃取液對人類A、B、O、AB血型、凝集缺陷患者以及大白鼠、綿羊及猴子三種動物的紅血球進行凝集活性檢測。結果發現在大型海藻中紅藻類之麒麟菜、綠藻類之裂片石蓴及褐藻類的重緣馬尾藻，具有較高之凝集力價達212 titer，而紅藻的龍鬚菜凝集力價較低，但具抗B型紅血球活性。微細藻類則普遍有較高凝集力價。以鏈蛋白酵素( pronase )或胰蛋白酵素( trypsin )處理人類及動物紅血球表面。大多提高粗萃取液的凝集活性8~64倍以上。在幾種測試的微細藻類其萃取液不需2價陽離子才具活性。海藻萃取液在4℃ 和 -20 ℃中經貯存6~12個月後，活性明顯降低。以鋸齒麒麟菜為例，酵素作用適合時間為2~6小時，在45 ℃下維持原有活性，但為不耐高溫種類。以DEAE Column分離純化出的鋸齒麒麟菜凝集素（ESA）蛋白質含量為每1g乾重藻體有10.2 mg。以SDS- PAGE凝膠電泳法得到該藻凝集素之分子量為29,000。醣抑制試驗結果顯示該凝集素對許多單醣及寡糖均有親和性。篩選過程中亦分離純化扁乳節藻的凝集素，具毒蛋白之溶血作用特性，分子量為26,000 Kda，該凝集素醣抑制試驗結果顯示該凝集素，對許多單醣及寡糖均有親和性。本論文亦利用西方點墨法初步篩選並發現藻類不具有抗癌功能的植物凝集素中。基於高凝集活性，海洋大型及微細藻類可做為研究親醣蛋白的生化及醫藥用途的材料。此外凝集素可能在藻類生物及生態上扮演重要角色。

Abstract
Agglutinins are widely distributed in nature. They are capable of agglutinating erythrocytes and other normal or transformal cells and often used in the studies for biomedical purposes. In this study, thirty species of marine macroalgae, collected from the northeastern coast of Taiwan, and marine unicellular algae were assayed for hemagglutination, followed by isolation and purification of agglutinins in the selected algae. Algal extracts in both phosphate buffer saline (PBS) and aqueous ethanol were assayed for agglutination against erythrocytes of both human normal blood groups, i.e. A,B,O,AB, and blood with coagulation disorder as well as against rate, sheep and monkey erythrocytes. The results indicate that all of the marine algae assayed can agglutinate human and animal erythrocytes. Of the macroalgae, Eucheuma serra (red alga), Ulva fasciata (green alga) and Sargassum duplicatum (brown alga) are most prominent for such agglutination, reaching 212 titers. Extract of Gracilaria lemaneiformis (red alga) is less active in hemagglutination, but it has strong afinity for B erythrocytes. Marine microalgae also contain agglutinins, with activity generally higher activity than that for macroalage.
Tretreatment of erythrocytes with pronase or trypsin for 2-6 hr improves markedly the detection of algal activity in general. Moreover, algal extracts remain active in agglutination at 45 ℃. The assayed microalgae have no requirement for divalent cations in hemagglutination. Agglutinating activity of crude algal extracts decreased markedly during the storage at 4 ℃ and —20 ℃ for 6-12 months.
The agglutinin of Eucheuma serra (ESA) was isolated by DEAE Column, containing 10.2 mg protein/g dry wt of algal powder, with a band of 29,000 on SDS-PAGE. ESA has the specific binding affinity for mono- and oligosaccharides. Moreover, the isolated protein from the red alga Galaxaura marginata demonstrates hemolysis activity, with molecular weight of 26,000 Kda. The protein also binds mono- and oligosaccharides. Further assay was employed for the detection of plant glycoprotein in algal extracts using antibodies of wheatgerm agglutinin (WGA) and concanavalin A (Con A) in ELISIA plate and western blotting.
In light of the generally strong activities, both marine macroalgae and microalgae are a potent source of lectins for study in biochemistry and medical applications. Some of the algal agglutinins probably have played important roles in algal biology and ecology.

封面
中文摘要
英文摘要
壹、縮寫
貳、緒論
參、實驗材料與儀器
肆、實驗方法
伍、結果
陸、討論
柒、圖表及圖表說明
捌、參考文獻
其他

五、參 考 文 獻
Akihiro, K. ,Hiroyuki M. and Jun-ichi O. and Hideo H. and Kanji H. 1999.
Occurrence of highly yielded lectins homologlus wihtin the genus
Eucheuma.Journal of Applied phycology.11:149-156.
Akihiro, K. ,Hiroyuki M. and Jun-ichi O. and Hideo H. and Kanji H. 1997. The
Marine red alga Eucheuma serra J. Agardh, a high yielding source of two
isolectins. Journal of Applied phycology. 9:331-338.
Bird, K.T., Chiles, T.C., Longley, R.E., Kendrick, A.F. and M.D. Kinkema (1993).
Agglutinins from marine macroalgae of the southeastern United States. J. Appl.
Phycol. 5: 2134-218.
Berheimer, A. (1952) Hemagglutinin in caterpillar bloods. Science 115:150-152.
Blunden, G. Farnham, W., Livett, B., Richardson, G. & Rogers, D. (1972) Br.
Phycol. J. 7: 279-285.
Blunden, G. and Rogers, D.J. (1975). Lloydia 38: 162-168.
Blunden,G., D.J. Roger and W. F. Farnham.1975.Survery of British seaweeds for
haemagglutinins. Lloydia 38: 162-168.
Boyd, W. C. (1950) Hemagglutinating substances of human cells in various Equptians
plants. J. mmunol. 65:281.
Boyd, W. C. and Reguera, R. M.(1949) Hemmglutinating substances for human cells
in various plants. J. Immunol. 62:333.
Boyd,W., and Brown, R. (1965) A specific agglutinin in the snail, Otala lactea. Nature
280:584-593.
Boyd, W.C., L R. Almodovar and L. G. Boyd.1966. Agglutinins in marine algae
for human erythrocytes. Transfusion 6:82-83.
Bolwell, G.P., Callow, J.A., Callow, M.E. and Evans, L.V. (1977) Cross-fertilisation in
fucoid seaweeds. Nature 268: 626-627.
Brain KR, Chalopin MC, Tumer TD, Blunden G, Wildgoose PB.(1973) Cytokinin
Activity of commercial aqueous extract. Plant Sci. Lett. 1:241-245.
Cushing, J. E.(1953) Individual variation in the hemagglutinin conten of yellowfin
tuna and bloods skipjack. J. Immunol. 68:543.
Chile, T.C. and K.T. Bird (1989) A comparative study of animal erythrocyte
agglutinins from marine algae. Com. Biochem. Physiol. 94B: 107-111.
Coats D.W., Tyler M.A. & Anderson D.M. 1984. Sexual processes in the life cycle
of Gyrodinium uncatenum (Dinophyceae). Journal of Phycology 20: 351-361.
Crouch IJ, Van Staden J. (1993) Evidence for the presence of plant Growth Regul.
13:21-29.
Deasi, P., and Springer, G. (1972) Eel serum anti-human blood group H(O) protein.
Methods Enzymol. 28:383-388.
Den, H., and Malinzak, D. (1977) Isolation and properties of ß-galactoside specific
lectin from chicle embryo thigh muscle. J. Biol. Chem. 252:5444-5448.
Etzler, M.E. (1986) Distribution and function of plant lectins. In: The Lectins (I.E.
Liener, N. Sharon and I.J. Goldstein, eds.). pp.371-435. Academic Press, Inc.
New York.
Estola, E. and Ovatia, O. (1955) Phytohemagglutinins in lichens. Ann. Med. Exp.
Biol. Fenn. 33:392-395.
Fabregas, J., A. Munoz, J. Llovo and J. Abalde.(1984) Agglutinins in marine red
algae. IRCS Med. Sci.12:298-299.
Ferreiros, C. M. and M. T. Criado.(1983) Purification and partial characterization of a
Fucus vesiculosus agglutinin. Res. Esp. Fisiol. 39:51-60.
Fabregas, J., Liovo, J. and A. Munoz (1983) Hemagglutinins in red seaweeds. Bot.
Mar. 28: 517-520.
Ford, W. W.(1911) The distribution of haemolysins, agglutinins,and poisons in fungi,
especially the Amanitas, the Entomas, the Lactarius, and the Inocybes. J.
Pharmcol. Exp. Ther. 2: 285-318.
Featonby-smith BC, Van Staden J. (1984) Identification and seasonal variation of
endogenous cytokinins in Ecklonia maxima ( Osbeck ) Papenf. Bot. Mar. 27:527-
531.
Ferreiros, C. M. and M. T. Criado.1983. Purification and partial
characterization Of a Fucus vesiculosus agglutinin. Rev. Esp. Fisiol. 39:51-60.
Frankel, A. E. 1997. Lectin-deficient ricin toxin intoxicates cells bearing the D-
mannose receptor. Carbohydrate Research.300(3): 251-258.
Fish, B.C., Rogers, D.J. and C.J. Barwell (1987) Agglutinins in red marine algae for
native and enzyme-modified erythrocytes. Br. Phycol. J. 22: 3030304.
Fugita, Y., Oishi, K., and Imahori, K. (1975) Purfifcation and properties of an anti-B
hemagglutinin produced by Streptomyces. Sp. Biochemistry 14:4465-4470.
Giloboa-Garber, N. (1972)Purification and properties of hemagglutinin from
Pseudomonas aeruginosa and its reaction with human blood cells. Biochim.
Biophys. Acta. 273:165-173.
Goto-Nance, R., Muramoto, K., Zenpo, Y. and H. Kamiya (1996) Purification and
characterization of the lectins of the soft coral Lobophytum variatum. Fish. Sci.
62: 297-301.
Grenenr Wc,Parker CM. Parker CW. (1976) Opposing effects of mitogenic and non-
mitogenic lectins on lymphocyte activation: evidence that wheat germ
agglutinin produces a negative signal. J. biol. Chem. 254:4017-4025.
Saze, H., and Conmernell, L. (1969) Studies on a hemagglutinin from the meadow
mushrooms II purification, composition and structure of Agraricus compestris
hemagglutinin. J. Biol. Chem. 244:4713-4719.
Shiomi, K., Yamanaka, H. and T. Kikuchi (1981) Purification and physiochemical
properties of a hemagglutinin (GVA-1) in the red alga Gracilaria verrucosa.
Bull. Jap. Soc. Sci. Fish. 47: 1079-1084.
Hammerstorm, S. and Kabat, E. (1969) Purification and characterization of a blood
group A reactive hemagglutinin from the snial, Helix pomatia, and a study of its
combining site. Biochemistry 8:2696-2705.
Hatakeyama, T., Himeshima, T., Komatsu, A. and Yamasaki, N. (1993) Purification
and characterization of two lectins from the sea cucumber Stichopus japonicus.
Biosci. Biotech. Biochem. 57: 1736-1739.
Hori, K., Ikegami, S., Miyazawa, K. and K. Ito (1988) Mitogenic and antineoplastic
isoagglutinis from the red alga Solieria robusta. Phytochemistry 27: 2063-2067.
Hori, K., Matsuda, H., Miyazawa, K. (1987). A mitogenic agglutinin from the red
alga Carpopeltis flabellata. Phytochemistry 26: 1335-1338.
Hori, K., Miyazawa, K., Fusetani, N., Hashimoto, K. and K. Ito (1986). Hypnins, low-
molecular weight peptidic agglutinins isolated from a marine red alga, Hypnea
japonica. Biochim. Biophys. Acta 873: 228-236.
Hori, K., Miyazawa, K. and K. Ito (1986). Preliminary characterization of agglutinins
from seven marine algal species. Bull. Jap. Soci. Fish. 52: 323-331.
Hori, K., Shimada, Y., Oiwa, C., Miyazawa, K. and Ito, K. (1993) Occurrence of a
novel group of hemagglutinins extractable by Pronase treatment in marine
algae. J. Appl. Phycol. 5: 219-223.
Hudgin, R., Pricer, W., Ashwell, G., and Stockat, R. (1974) The isolation and
properties of a rabbit liver binding protein specific for asilo-glycoproteins. J.
Biol. Chem. 249:5536-5543.
Johnsons, H. (1964) Human blood group A specific agglutinin of butter clam,
Saxidomus gigantens. Science 146:584-549.
Johnsons, B. (1941) Acta. Path. et Microbiol. Scandinv. Suppl. 54:456.
J. Sambrook., E. F. Fritsch and T. Maniatis.(1989) Molecular cloning : a labobatory
manual second edition.By cold spring Harbor Laboratory press printed in the
United States of America.B12.
Kamiya, H., K. Shiomi an Y. Shimizu. (1980) Marine biopolymers with cell
specificity III. Agglutinins in red alga cystoclonium purpureum:isolation and
characterization. J. Nat. Prod. 4.:136-139.
Kamiya, H., K. Ogata and K. Hori. (1982) Isolation and charaterization of a new
agglutinin in the red alga palmaria palmata(L.) O. Kuntze. Bot.Mar. 25:537-
540.
Kawagishi, H., mori, H. and kimura, A.(1994). A sialic acid-binding lectin from
the mushroom, Hericuim eriuaceum. FEBS Lett. 304:56-58.
Kaasaki, T., and Ashwell, G. (1976) Chemical and physical properties of a hepatic
membrane protein that specifically binds asilo-glycoproteins. J. Biol. Chem.
251:1296-1301.
Kawakubo, A., Makino, H., Ohnishi, J.-i., Hirohara, H. and Hori, K. (1997). The
marine red alga Eucheuma serra J. Agardh, a high yielding source of two
isolectins. J. Appl. Phycol. 9: 331-338.
Kobori, M. and H. Nojima. 1993. A simple treatment of DNA in a ligation
mixture prior to electroporation improves transformation frequency. Nucleic
acids research. 21:2728.
Kilpatrick DC. (1988) Accessory cell paradox: monocytes enhance or inhibit lectin-
mediated hyman T lymphocyte proliferation depending on the choice of
mitogen. Scand. J. Immunol. 24:11-19.
Kilpartick DC, Graham C, Urbaniak SJ. (1986) Inhibition of human lymphocyte
transformation by tomato lectin. Scand. J. Immunol. 24:11-19.
Kilpartick DC, McCurrach PN. (1987) Wheat germ agglutinin is mitogenic, non-
mitogenic and anti-mitogenic for hyman lymphoctyes. Scand. J.
Immunol.25:343-348.
Liao,Woan-Ru、Huang, Rang 2000. Agglutination of human and animal
erythrocytes in marine unicellular algae. Journal of Industrial Microbiology &
Biotechnology. 24：262-266. (SCI)
Liao,Woan-Ru、Huang, Rang and Su, Heui-Mei. 1999.Hemagglutinating activity of
lectins from marine microalgae. Proceedings of the Fifth International
Chrysophyte Symposium; Nova Hedwigia Beiheft.( in press )
Lis, H. and N. Sharon (1986). Applications of lectins. In: The Lectinss: Properties,
Functions and Applications in Biology and Medicine (Liener I.E., Sharon, N.
Goldstein, I.J., eds.) Academic Press, New York, 293-357.
Lima, H.C., Costa, F.H., Sampaio, A.H., Neves, S.A., Benevides, M.B., Teixeira,
D.I.A., Rogers, D.J. and Freitas, A.L.P. (1998) Induction and inhibition of
human lymphocyte trasformation by the lectin from the red marine alga
Amansia multifida. J. Appl. Phycol. 10: 153-162.
Marinkovich, V. A. (1964) Purification and charcaterization of the hemagglutinin
present in potatows.J. Immunol. 93:732.
Matsuda, T., Kabat, E. A. and Surolia, A. 1989. Carbohydrate binding
specificity of the basic lectin from winged bean (Psophocarpus tetragonolobus)
Mol. Immunology. 26:189-195.
Miller CO.(1965) Evidence for the natural occurrence of zeatin and dericatives:
Compounds from maize which promote cell division. Proc. Natl. Acad.
Sci.,USA.54:1052-1058.
Mooney PA, Van Staden J. (1986) Algae and cytokinins. J. Plant Physiol. 123:1-21.
Moreira RA,Cavada BS. (1984) Lectin from Canavalia brasillensis (Mart). Isolation,
characterization and behaviour during germination. Biologia plantarum 26:113-
120.
Bradford M. (1976). A rapid method for the quantitation fof microgram quantities of
proteins utilizing the principle of protein-dye binding. Anal. Biochem.,72:248-
254.
Nanskl, H. (1987) Lectin agglutination of thermophilic Campylobactor species. J.
Microbiol. 15:163-173.
Nathan, S. 1993. Lectin-carbohydrate complexes of plants and animals :an
atomic view. Trends Boichem Sci. 18:221-226.
Piller, V., Piller, F., and Cartron, J. -P. 1990. Comparison of the carbohydrate-
binding speificities of seven N-acetyl-d-galactosamine-recognizing lectins. Eur.
J. Biochem. 191:461-466.
Pustai A. (1991) Plant Lectins. Cambridge U. P. , Cambridge. 263pp.
Goto, R. Muranmoto, K. Yamazaki, M. and Kamiya H. ( 1992 ) Purification and
characterization of an agglutinin of the soft coral Sinularia species. Dec. Comp.
Immunol., 16:9-17.
Reed JC, McCurrach PM, Kilpatrick DC. (1985) Effect of wheat-germ agglutinin on
the interleukin pathway of human T lymphocyte activation. J. Immunol.
134:314-323.
Rogers, D.J. (1977). In Marine Nature Products Chemistry (Faulkner, D.J. & Fenical,
W.H. eds.), pp. 311-327, Plenum Press, N.Y.
Rogers, D.J. and K. Hori (1993) Marine algal lectins: new developments.
Hydrobiologia 260/261: 589-593.
Rogers, D.J. and G. Blunden (1980) Structural properties of the anti-B lectin from the
red alga Ptilota plumosa (Huds.) C. Ag. Bot. Mar. 23: 459-462.
Rogers, D.J. and B.C. Fish (1991) Marine algal lectins. In: Lectin Reviews (D.C.
Kilpatrick, E. Van Driessche and T.C. Bog-Hansen, eds.), Vol.1, pp.129-142.
Sigma Chemical Company, St. Loouis, MO.,U.S.A.
Rogers, D.J., Fish, B.C., Blunden, G., Farnjham, W.F., Carabvot, C., Morales, A.,
Rosquette, C.P., & Fredj, M. (1988). Br. Phycol. J. 23: 296.
Rogers, D.J., Fish, B.C. & Barwell, C.J. (1990) In Lectins: Biology,Biochemistry,
Clinical Bioxhemistry (Kocourek, J., & Freed, D.L.J.,eds.) 7: 49-52. Sigma
Chemical Company, St. Louis, MO, USA.
Rogers, D.J. & Blunden, G. (191980) Bot. Mar. 23: 459-462.
Rogers, D.J. & Topliss, J.A. (1983) Bot. Mar. 26: 301-305.
Rogers, D.J. & Topliss, J.A. (1979) J. Pharm. Pharmacol. 31: 11p.
Rogers, D.J., Topliss, J.A. & Blunden, G. (1979) Med. Lab. 36: 79-84.
Rogers, D.J., Blunden, G. & Evans, P.R. (1977) Med. Labv. Sci. 34: 193-200.
Rogers, D.J., Blunden, G., Guiry, M.D. & Northcott, M.J. (1982) Bot. Mar. 25: 399-
400.
Rogers, D.J., Blunden, G., Topliss, J.A. and M.D. Guiry (1980) A survey of some
marine organisms for haemagglutinins. Bot. Mar. 23: 569-577.
Rogers, D.J., Barwell, C.J., Fish, B.C. & Munoz, A. (1989) Br. Phycol. J. 24: 311
-312.
Rogers, D.J., & Galander, M.M. (1986) J. Pharm. Pharmacol. 38: 323-331.
Roger, D.J. & Galander, M.M. (1988) in Lctins: Biology, Biochemistry, Clinical
Biochemistry (Bog-Hanse, T.C. & Freed, D.L.J. eds) ^: 281-284. Sigma
Chemcial Company, St. Louis, MO. USA.
Rogers, D.J. and R.W. Loveless (1991) Electron microscopy of human erythrocytes
agglutinated by lectin from Codium fragile spp. tomentosoides and
pseudohaemagglutinin from Ascophyllum nodusum. J. Appl. Phycol.3: 83-86.
Rogers, D.J. & Loveless, R.W.(1985) Bota. Mar. 28: 133-137.
Vincent, J. P. , M. lazdunski, and H. Delaage, on the use of tetranitromethane as a
reagent for nitration.Eur. J. Biochem. 12:250.
Vehmeyer, K. et al.1998. Lectin-induced increase in clonogenic growth of
haematopoietic progenitor cells. European Journalo of Haematology 60(1):16-
20.
Tay SAB, Macleod JK, Palni LMS, Letham DS. (1985) Detction of cytokinins in a
seaweed extract.Phytochemistry. 24:2611-2614.
Tay SAB, Palni LMS, Macleod JK. (1987) Identification of cytokinins glucosides in a
seaweed extract. J. Plant Growth Regul. 5:133-138.
Tollefsen, S. E., and Korndekd, R. 1984. The B4 lectin from Vicia villosa Seeds
Interacts with N-acetylgalactisamine residues on erythrocytes with blood group
Cad specificity. Biochem. Biophys. Res. Commun. 123:1099-1106.
Tennigkeit, J. and H. Matzura. 1997. Nucleotide sequence analysis of a
choloramphenicol-resistance determinant from Argobacterium tumefacien
and identication of gene product. Genes. 113-116.
Waard, A. And Kornfeld, S. (1976) Isolation and properties of ß-galactoside binding
lectins of calf heart and lung. J. Bio. Chem. 251:7581-7587.
Wu, A. M. et al. 1988. Structural concepts of the human blood group A, B, H,
Le(a)(b), I, and I active glycoprotein purified from human ovarian cyst fluid.
Adv. Exp. Med. Biol. 228:351-394.
Wu, A. M. et al. 1991. Coding and classification of D-glactose,N-acetyl-D-
galactosamine and β-D-Galp-(1-3(4))-β-D-GacpNAC, specificities of
applied lectins. Carbohydr. Res. 213:127-143.
Wetprasit, N. Chulavatnatol,M. 1997. Determination of sugar specificity of
jackfruit lectin by a simple sugar-lectin binding assay using microtiter plate.
Biochemistry and Molecular Biology International. 42(2) :399-408.
Weiss, A. (1993). T Lymphocyte activation. In: Fundamental Immunology (Paul,
W.E., ed.). Raven Press, New York, 467-504.
Saito, T., and Matsuda, Y. (1984 ). Sexual agglutinin of mating- type minus gametes
in Chlamydomonas reinhardtii.II. Purification and characterization of minus
agglutinin and comparison with plus agglutinin. Arch. Microbiol. 139:95-9.
Shiomi, K., H. Kamiya and Y. Shimizu. (1979) Purification and characterization of an
agglutinin in the red alga Agardhiella tenera. Biochim. Biophys. Acta. 576:118-
127.
Shell, W. J., and Moore, W. S. (1980) Aggregation-dependent turnover of flagellar
Adhesion molecules in Chlamydomonas gametes.J. Cell Biol.84:203-10.
Shiomi, K., H. Yamanaka and T. Kikuchi. (1980) Biochemical properties of
hemagglutinins in the red alga Serraticardia maxima. Bull. Jap. Soc. Fish.
46:1369-1373.
Stockert, T., Morell, A., and Schemberg, I. (1974) Mammalian hepatic lectin. Science
186:365-366.