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研究生:鍾文彬
研究生(外文):Wen-Pin Chung
論文名稱:海鱺免疫球蛋白基因選殖及特性研究
論文名稱(外文):The molecular cloning and characterization of cobia(Rachycentron canadum)immunoglobulin gene
指導教授:林正輝林正輝引用關係
指導教授(外文):Lin Cheng-Hui
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:79
中文關鍵詞:海鱺免疫球蛋白
外文關鍵詞:cobiaimmunoglobulin
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免疫球蛋白主要分布在血液、黏液等處,在生物體內主要可用來結合外來物質(抗原),進而引發後續的免疫反應,因此在體液性免疫反應中扮演重要的角色。其結構主要是由重鏈及輕鏈所組成,目前已知存在於硬骨魚類的免疫球蛋白主要以IgM為主。本實驗由海鱺頭腎選殖出2個Ig重鏈clones (RC8、RC10)全長為1926及1923 bp,皆演譯(deduced)出583個胺基酸,扣除signal peptide後,推測分子量約為64 kDa,輕鏈4個clones (RC12、RC17、RC21、RC31) 全長為926、966、1009及1006 bp,分別演譯(deduced)出236、244、241及241個胺基酸,扣除signal peptide後,推測分子量約為23 kDa。以此估算海鱺免疫球蛋白四體大小約為696 kDa。在蛋白質純化方面,以硫酸銨沉澱及膠體萃取純化約得到海鱺IgM,經由SDS-PAGE分析其重鏈與輕鏈推測分子量大小分別約為69 及30 kDa,以此推測出四體分子量大小為792kDa,經由免疫兔子產生抗體之抗血清,並利用西方點墨法檢測,結果所產生的兔子抗海鱺Ig之抗血清能偵測到海鱺免疫球蛋白。
The immunoglobulin (Ig) is distributed and located in blood, mucus, etc. when immunoglobulin combine to antigen can induce subsequence immune response. Therefore, it play an important role in homoral immunity. The immunoglobulin is mainly of IgM in the teleost. In this study, we have isolated two full length immunoglobulin heavy chain of cobia (RC8、RC10) from headkidney which containing 1926 and 1923 bp, both deduced 583 amino acid, and the molecular weight are about 65 kDa. Four full length of immunoglobulin light chain (RC12、RC17、RC21、RC31) is containing 926, 966, 1009 and 1006 bp, respectively. It’s can deduced 236, 244, 241 and 241 amino acid, and the molecular weight are about 23 kDa. The molecular weight of immunoglobulin tetramer nearly as 696 kDa. In protein purification, we utilize ammonium sulfate precipitation and gel extraction to isolate cobia IgM. Utilizating SDS-PAGE to estimate the molecular weight heavy chain is about 69 kDa and light chain is about 30 kDa, the molecular weight of immunoglobulin tetramer nearly as 792 kDa. The produce antiserum of antibody via immune rabbit. Analysis of Western blotting which antibody of rabbit, it can detected the immunoglobulin of cobia.
謝辭………………………………………………………………………....i
摘要………………………………………………………………………...ii
Abstrate……………………………………………………………………iii
目錄………………………………………………………………………..iv
圖表頁次………………………………………………………………….vii
壹、前言……………………………………………………………………1
一、海鱺之生態與養殖之簡介…………………………………........... 1
二、海鱺重要的疾病…………………………………………………… 2
三、魚類免疫系統……………………………………………………... 3
四、魚類免疫球蛋白之研究…………………………………………... 4
五、研究目的…………………………………………………………... 6
貳、材料方法………………………………………………………………7
一、海鱺免疫球蛋白之基因選殖……………………………………... 7
1-1分離海鱺免疫球蛋白重鏈及輕鏈基因之部分cDNA序列..7
1-1-1 海鱺total RNA分離……………………………………..7
1-1-2 RNA膠體電泳之分析……………………………………7
1-1-3 海鱺免疫球蛋白重鏈及輕鏈基因引子設計……………8
1-1-4 反轉錄酶反應……………………………………………8
1-1-5 聚合酶鏈鎖反應…………………………………………8
1-1-6 瓊脂膠體萃取……………………………………………9
1-1-7 定序載體的構築與製備…………………………………9
1-2 海鱺免疫球蛋白重鏈及輕鏈基因之3’ RACE..................12
1-2-1 海鱺免疫球蛋白重鏈及輕鏈基因引子設計…………..12
1-2-2 第一股cDNA之合成…………………………………..12
1-2-3 擴增3’ cDNA端………………………………………..12
1-3 海鱺免疫球蛋白重鏈及輕鏈基因之5’ RACE..................13
1-3-1 海鱺頭腎mRNA純化………………………………….13
1-3-2第一股cDNA之合成……………………………………13
1-3-3 擴增5’ cDNA端………………………………………..14
1-3-4 PCR產物之定序………………………………………...15
二、海鱺免疫球蛋白之純化………………………………………….15
2-1 海鱺血清分離……………………………………………..15
2-2 海鱺血清免疫球蛋白之純化……………………………..15
2-3 還原態之SDS-聚丙烯胺膠體電泳………………………16
2-4 非還原態之SDS-聚丙烯胺膠體電泳……………………17
2-5 銀染………………………………………………………..18
2-6 蛋白質濃度測定…………………………………………..18
三、製備酵素連結免疫吸附的二級抗體……………………………..19
3-1 兔子抗海鱺免疫球蛋白(rabbit anti-cobia antibody)之
製備……………………………………………………….19
3-2、西方點墨法之分析………………………………………19
參、實驗結果………………………………………………………….....21
一、海鱺免疫球蛋白重鏈基因序列結果…………………………….21
二、海鱺免疫球蛋白重鏈基因序列比較…………………………….21
三、海鱺免疫球蛋白輕鏈基因序列………………………………….22
四、海鱺免疫球蛋白輕鏈基因序列比較…………………………….23
五、海鱺免疫球蛋白純化…………………………………………….24
六、海鱺免疫球蛋白免疫轉漬分析………………………………….25
肆、討論……………………………………………………………….....26
一、海鱺免疫球蛋白重鏈基因序列與比對………………………….26
二、海鱺免疫球蛋白輕鏈基因序列與比對………………………….27
三、海鱺免疫球蛋白之純化結果…………………………………….28
四、海鱺免疫球蛋白免疫轉漬分析………………………………….29
伍、參考文獻……………………………………………………………..30
邵廣昭,1996。台灣常見魚介貝類圖說(下)。台灣省漁業局。
張賜玲、謝介士、周瑞良、蘇茂森,1999。海鱺繁養殖技術簡介。
養魚世界。第14-26頁。
Acton, R. T., Weinheimer, P. F., Hall, S. J., Niedermeier, W., Shelton, E.
and Bennett, J. C., 1971. Tetrameric immune macroglobulins in
three orders of bony fishes. Proc. Natl. Acad. Sci. 68, 107-111.
Anderson, D. R., Atkinson, P. H. and Grimes, W. J., 1985. Major
carbohydrate structures at five glycosylation sites on murine IgM
determined by high resolution 1H-NMR spectroscopy. Arch.
Biochem. Biophys. 243, 605-618.
Chen, S. L., Xu, M. Y., Hu, S. L. and Li, L., 2004. Analysis of immune-relevant genes expressed in red sea bream spleen. Aquaculture 240, 115-130.
Chou, R. L., Su, M. S. and Chen, H. Y., 2001. Optimal dietary protein
and lipid levels for juvenile cobia (Rachycentron canadum).
Aquaculture 193, 81–89.
Coscia, M. R., Morea, V., Tramontano, A. and Oreste, U., 2000.
Analysis of a cDNA sequence encoding the immunoglobulin heavy chain of the Antarctic teleost Trematomus bernacchii.Fish Shellfish Immunol. 10, 343-357.
Davie, J. M. and Paul, W. E., 1972. Receptors on immunocompetent cells.
IV. Direct measurement of avidity of cell receptors and cooperative
binding of multivalent ligands. J Exp Med. 135, 643-659.
Ellis, A. E., 1999. Immunity to bacteria in fish. Fish Shellfish Immunol.
9, 291-308.
Espelid, S., Halse, M., Solem, S. T. and Jorgensen, T. O., 2001. Immunoglobulin genes and antibody responses in the spotted wolfish (Anarhichas minor Olafsen). Fish Shellfish Immunol. 11, 399-413.
Ghaffari, S. H. and Lobb, C. J., 1989. Nucleotide sequence of channel catfish heavy chain cDNA and genomic blot analyses. Implications for the phylogeny of Ig heavy chains.J. Immunol. 143, 2730-2739.
Ghaffari, S. H. and Lobb, C. J., 1997. Structure and genomic organization of a second class of immunoglobulin light chain genes in the channel catfish. J. Immunol. 159, 250-258.
Hansen, J. D., Landis, E. D. and Phillips, R. B., 2005. Discovery of a unique Ig heavy-chain isotype (IgT) in rainbow trout: Implications for a distinctive B cell developmental pathway in teleost fish. Proc. Natl. Acad. Sci. U.S.A. 102, 6919-6924.
Hatten, F., Fredriksen, A., Hordvik, I. and Endresen, C., 2001. Presence
of IgM in cutaneous mucus, but not in gut mucus of Atlantic salmon,
Salmo salar. Serum IgM is rapidly degraded when added to gut
mucus. Fish Shellfish Immunol. 11, 257-268.
Hordvik, I., Thevarajan, J., Samdal, I., Bastani, N. and Krossøy, B.,
1999. Molecular cloning and phylogenetic analysis of the Atlantic
salmon immunoglobulin D gene. Scand. J. Immunol. 50, 202-210.
Hordvik, I., Berven, F. S., Solem, S. T., Hatten, F. and Endresen, C.,
2002. Analysis of two IgM isotypes in Atlantic salmon and brown
trout. Mol. Immunol. 39, 313-321.
Kobayashi, K., Hara, A., Takano, K. and Hirai, H., 1982. Studies on
subunit components of immunoglobulin M from a bony fish, the
chum salmon (Oncorhynchus keta). Mol. Immunol. 9, 95-103.
Mestecky, J., Kulhavy, R., Schrohenloher, R. E., Tomana, M. and
Wright, G. P., 1975. Identification and properties of J chain isolated
from catfish macroglobulin. J. Immunol. 115, 993-997.
Okamoto. K., Ikemura. H., Savan. R. and Sakai. M., 2003. Cloning,
sequence and variability analysis of expressed immunoglobulin light
chain genes from yellowtail Seriola quinqueradiata. Fish Shellfish
Immunol. 14, 55-70.
Ota, T., Nguyen,T. A., Huang, E., Detrich, H.W. and Amemiya, C. T., 2003a.Positive Darwinian selection operating on the immunoglobulin heavy chain of Antarctic fishes. J. Exp. Zool. 95B, 45-58.
Ota, T., Rast, J. P., Litman, G. W. and Amemiya, C. T., 2003b. Lineage-restricted retention of a primitive immunoglobulin heavy chain isotype within the Dipnoi reveals an evolutionary paradox.Proc. Natl. Acad. Sci. U.S.A. 100, 2501-2506.
Palenzuela. O., Sitja-Bobadilla, A. and Alvarez-pellitero, P., 1996.
Isolation and partial characterization of serum immunoglobulins
from sea bass (Dicentrarchus labrax L.) and gilthead sea bream
(Sparus aurata L.) Fish Shellfish Immunol. 6, 81–94.
Partula, S., Schwager, J., Timmusk, S., Pilstrom, L. and Charlemagne, J., 1996. A second immunoglobulin light chain isotype in the rainbow trout. Immunogenetics 45, 44-51.
Pastoret, P. P., Griebel, P., Bazin, H. and Govaerts, A., eds. 1998.
Immunology of fishes.Handbook of Vertebrate Immunology pp.
1–5 San Diego: Academic Press.
Picchietti. S., Scapigliati. G., Fanelli. M., Barbato. F., Canese. S.,
Mastrolia. L., Mazzini. M. and Abelli. L., 2001. Sex-related
variations of serum immunoglobulins during reproduction in gilthead
sea bream and evidence for a transfer from the female to the eggs.
J. Fish Biol. 59, 1503–1511.
Pilstrom, L. and Bengten, E., 1996. Immunoglobulin in fish—genes,
expression and structure. Fish Shellfish Immunol.6, 243–262.
Pucci, B., Coscia, M. R. and Oreste, U., 2003. Characterization of serum
immunoglobulin M of the Antarctic teleost Trematomus bernacchii.
Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 135, 349-357.
Roitt, I. M. and Delves, P. J., 2001. Essential immunology. pp. 37-45
Blackwell Science, Inc., Malden., USA
Ross, D. A., Wilson, M. R., Miller, N. W., Clem, L. W. and Warr, G. W.,
1998. Evolutionary variation of immunoglobulin mu heavy chain
RNA processing pathways: origins, effects, and implications.
Immunol. Rev. 166, 143-151.
Saha, N. R., Suetake, H. and Suzuki, Y., 2004. Characterization and expression of the immunoglobulin light chain in the fugu: evidence of a solitaire type. Immunogenetics 56, 47-55
Saha, N. R., Suetake, H. and Suzuki, Y., 2005. Analysis and
characterization of the expression of the secretory and membrane
forms of IgM heavy chains in the pufferfish, Takifugu rubripes. Mol.
Immunol. 42, 113-124.
Shakin-Eshleman, S. H., Spitalnik, S. L. and Kasturi, L., 1996 The
amino acid at the X position of an Asn-X-Ser sequon is an important
determinant of N-linked core-glycosylation efficiency. J. Biol. Chem.
271, 6363-6366.
Solem, S. T. and Jorgensen, T. O., 2002. Characterisation of
immunoglobulin light chain cDNAs of the Atlantic salmon, Salmo salar L.; evidence for three IgL isotypes. Dev. Comp. Immunol. 26, 635-647.
Srisapoome, P., Ohira, T., Hirono, I. and Aoki, T., 2004. Genes of the
constant regions of functional immunoglobulin heavy chain of
Japanese flounder, Paralichthys olivaceus. Immunogenetics 56,
292-300.
Stenvik, J. and Jorgensen, T. O., 2000. Immunoglobulin D (IgD) of
Atlantic cod has a unique structure. Immunogenetics. 51, 452-461.
Uchida, D., Hirose, H., Chang, P. K., Aranishi, F., Hirayabu, E., Mano,
N., Mitsuya, T., Prayitno, S. B. and Natori, M., 2000.
Characterization of Japanese eel immunoglobulin M and its level in
serum. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 127,
525-532.
Warr, G. W., 1995. The immunoglobulin genes of fish. Dev Comp.
Immunol. 19, 1-12.
Watts, M., Munday, B. L. and Burke, C. M., 2001a. Isolation and partial
characterisation of immunoglobulin from southern bluefin tuna
Thunnus maccoyii Castelnau. Fish Shellfish Immunol. 11, 491-503.
Watts, M., Munday, B. L. and Burke, C. M., 2001b. Production,
characterization and diagnostic use of antisera to southern bluefin
tuna (Thunnus maccoyii Castelnau) immunoglobulin. Aquaculture
199, 245–257.
Willett, C. E., Cortes, A., Zuasti, A. and Zapata, A. G., 1999. Early
hematopoiesis and developing lymphoid organs in the zebrafish. Dev
Dyn. 214, 323-336.
Wilson, M., Bengten, E., Miller, N. W., Clem, L. W., Du, P. L. and
Warr, G. W., 1997. A novel chimeric Ig heavy chain from a teleost
fish shares similarities to IgD. Proc. Natl. Acad. Sci. 94, 4593-4597.
Wilson, M. R., and Warr, G. W., 1992. Fish immunoglobulins and the
genes that encode them. Annu. Rev.Fish Dis. 2, 201-221.
Wright, J. F., Shulman, M. J., Isenman, D. E. and Painter, R. H., 1990.
C1 binding by mouse IgM. The effect of abnormal glycosylation at
position 402 resulting from a serine to asparagine exchange at
residue 406 of the mu-chain. J. Biol. Chem. 265, 10506-10513.
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