在生物體內，外生性抗原被降解並帶到抗原呈現細胞表面以誘導產生特異性抗體，在這過程中，抗原可能因結構變化而導致抗原決定部位受到遮蔽使抗體無法與原生性蛋白有良好反應。 先前關於重組臺灣眼鏡蛇毒蛋白Cobrotoxin與原生性Cobrotoxin之抗原性比較已有深入報導，本論文目標在於探討是否存在某些非原生性Cobrotoxin之結構，暴露原生性毒蛋白所隱蔽之抗原決定部位而提升對抗體反應性。 首先以圓二色偏極光譜及Acrylamide淬熄Trp螢光分析得知重組與原生性Cobrotoxin二級結構之差異。 在ELISA及dot blotting實驗中發現重組Cobrotoxin對抗體之反應性明顯優於原生性毒蛋白，以特異性認氮端及碳端之抗體分析也是同樣結果，推測因結構差異導致，而以Ammonium thiocyanate elution分析發現重組Cobrotoxin對抗體親和力也有所提升。 在Trypsin及Chymotrypsin digestion實驗中得知重組Cobrotoxin較原生性毒蛋白易受到酵素水解，推測其對抗體之表達位向有所差異。 當Cobrotoxin碳端兩對雙硫鍵Cys43-54及Cys55-60突變後發現其抗原性有顯著衰退現象，且當重組及原生性毒蛋白回到線性狀態時抗原性則沒有差異，顯示抗體主要辨認部位具結構依賴性。 綜合上述結果得知原生態Cobrotoxin之結構可能隱蔽了某些抗原決定部位而使其對抗體反應性並非最佳狀態。

To induce the production of antibodies, exogenous antigens are taken up and degraded in antigen presenting cells in vivo. Since this process inevitably lead to distort antigen’s structure, it is likely that some arising antibodies following immunization may not react appropriately with native protein. In the present study, comparative studies on the reactivity of cobrotoxin and recombinant cobrotoxin toward anti-cobrotoxin antibodies were carried out. CD spectra and acrylamide quenching of Trp fluorescence showed that global structure of recombinant cobrotoxin was different from that of native toxin. Results of ELISA and dot blotting assay revealed that recombinant cobrotoxin had a superior reactivity toward anti-cobrotoxin antibodies than native toxin did. Reactivity with antibody fractions specifically against N-terminal region or C-terminal region of cobrotoxin also showed the same results. The binding of recombinant cobrotoxin with antibodies was stronger than that of cobrotoxin as revealed by ammonium thiocyanate elution assay. Recombinant protein was susceptible to reduce its antigenicity after tryptic digestion compared to cobrotoxin. Distorting disulfide linkages at C-terminus caused a marked decrease in immunoreactivity of recombinant cobrotoxin, indicating that anti-cobrotoxin antibodies mostly recognized conformation-dependent epitopes. Moreover, cobrotoxin and recombinant cobrotoxin showed a similar immunoreactivity under denaturing condition. Taken together, these results suggest that native conformation with cobrotoxin may unfavorably impede the interaction of some epitope(s) with anti-cobrotoxin antibodies.

目錄------------------------------------------------------------------1
中文摘要------------------------------------------------------------2
英文摘要------------------------------------------------------------3
縮寫表---------------------------------------------------------------4
緒論------------------------------------------------------------------5
實驗材料-----------------------------------------------------------10
實驗方法-----------------------------------------------------------12
結果-----------------------------------------------------------------28
討論-----------------------------------------------------------------37
圖表-----------------------------------------------------------------43
參考文獻-----------------------------------------------------------56

Basus, V. J., Billeter, M., Love, R. A., Stroud, R. M. and Kuntz, I. D. (1988) Structural studies of alpha-bungarotoxin. 1. Sequence-specific 1H NMR resonance assignments. Biochemistry 27, 2763-2771.

Basus, V. J. and Scheek, R. M. (1988) Structural studies of alpha-bungarotoxin. 2. 1H NMR assignments via an improved relayed coherence transfer nuclear overhauser enhancement experiment. Biochemistry 27, 2772-2775.

Chang, C. C., Yang, C. C., Nakai, K. and Hayashi, K. (1971a) Studies on the status of free amino and carboxyl groups in cobrotoxin. Biochim. Biophys. Acta 251, 334-344.

Chang, C. C., Yang, C. C., Hamaguchi, K., Nakai, K. and Hayashi, K. (1971b) Studies on the status of tyrosyl residues in cobrotoxin. Biochim. Biophys. Acta 236, 164-173.

Chang, C. C. and Yang, C. C. (1973) Immunochemical studies on the tryptophan-modified cobrotoxin. Biochim. Biophys. Acta 295, 595-604.

Chang, L. S., Kuo, K. W. and Chang, C. C. (1993) Status of tryptophan residue in cobrotoxin and alpha-bungarotoxin. Biochem. Mol. Biol. Int. 29, 435-442.

Chang, L. S., Kuo, K. W., Lin, J., Lin, S. R. and Chang, C. C. (1995a) Analysis of a conformation-independent epitope and a conformational epitope in a protein: a study on cobrotoxin from Taiwan cobra venom. J. Biochem. 117, 863-868.

Chang, L. S., Lin, J., Kuo, K. W., Lin, S. R. and Chang, C. C. (1995b) Characterization of epitopes in native and unfolded cobrotoxin: evidence of an immunodominant C-terminal region related to the production of precipitating and non-precipitating antibodies against cobrotoxin. J. Biochem. 118, 686-692.

Chang, L. S., Lin, S. R. and Chang, C. C. (1998) Unfolding/folding studies on cobrotoxin from Taiwan cobra venom: pH and GSH/GSSG govern disulfide isomerization at the C-terminus. Arch. Biochem. Biophys. 354, 1-8.

Chang, L. S., Lin, S. R. and Yang, C. C. (2001) Glutaraldehyde cross-linking alters the environment around Trp29 of cobrotoxin and the pathway for regaining its fine structure during refolding. J. Pept. Res. 58, 173-179.

Chang, L. S., Lin, R., Chen, K. C. and Chang, C. C. (2003) Enrichment of the antibodies against the C-terminus of Taiwan cobra cobrotoxin using dimeric glutaraldehyde-modified toxin as an immunogen. Toxicon 41, 181-186.

Chant, A., Kraemer-Pecore, C. M., Watkin, R. and Kneale, G. G. (2005) Attachment of a histidine tag to the minimal zinc finger protein of the Aspergillus nidulans gene regulatory protein AreA causes a conformational change at the DNA-binding site. Protein Expression and Purification 39, 152-159.

Chen, J., Liu, Y., Li, X., Wang, Y., Ding, H., Ma, G. and Su, Z. (2009) Cooperative effects of urea and L-arginine on protein refolding. Protein Expression and Purification 66, 82-90.

Dong le, V., Selvanayagam, Z. E., Gopalakrishnakone, P. and Eng, K. H. (2002) A new avidin-biotin optical immunoassay for the detection of beta-bungarotoxin and application in diagnosis of experimental snake envenomation. J. Immunol. Methods 260, 125-136.

Hatanaka, H., Oka, M., Kohda, D., Tate, S., Suda, A., Tamiya, N. and Inagaki, F. (1994) Tertiary structure of erabutoxin b in aqueous solution as elucidated by two-dimensional nuclear magnetic resonance. J. Mol. Biol. 240, 155-166.

Kimball, J. W. (1990) Introduction to Immunology, pp. 48-56, Macmillan, New York.

Kuo, K. W. and Chang, C. C. (1991) High affinity antibody to cobrotoxin prepared from the derivatives of glutaraldehyde-detoxified cobrotoxin. J. Biochem. 110, 863-867.

Kuo, K. W., Chang, L. S. and Chang, C. C. (1995) The structural loop II of cobrotoxin is the main binding region for nAChR and epitope in the region is conformation-dependent. J. Biochem. 117, 438-442.

Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

Le Goas, R., LaPlante, S. R., Mikou, A., Delsuc, M. A., Guittet, E., Robin, M., Charpentier, I. and Lallemand, J. Y. (1992) Alpha-cobratoxin: proton NMR assignments and solution structure. Biochemistry 31, 4867-4875.

Li, J. J., Liu, Y. D., Wang, F. W. and Su, Z. G. (2004) Hydrophobic interaction chromatography correctly refolding proteins assisted by glycerol and urea gradients. Journal of Chromatography A 1061, 193-199.

Li, M. and Su, Z. (2003) Separation and identification of different refolding components. Journal of Biotechnology 103, 119-127.

Macdonald, R. A., Hosking, C. S. and Jones, C. L. (1988) The measurement of relative antibody affinity by ELISA using thiocyanate elution. J. Immunol. Methods 106, 191-194.

Moise, L., Piserchio, A., Basus, V. J. and Hawrot, E. (2002) NMR structural analysis of alpha-bungarotoxin and its complex with the principal alpha-neurotoxin-binding sequence on the alpha 7 subunit of a neuronal nicotinic acetylcholine receptor. J. Biol. Chem. 277, 12406-12417.

Onda, M. (2009) Reducing the immunogenicity of protein therapeutics. Curr. Drug Targets 10, 131-139.

Osipov, A. V., Kasheverov, I. E., Makarova, Y. V., Starkov, V. G., Vorontsova, O. V., Ziganshin, R. Kh., Andreeva, T. V., Serebryakova, M. V., Benoit, A., Hogg, R. C., Bertrand, D., Tsetlin, V. I. and Utkin, Y. N. (2008) Naturally occurring disulfide-bound dimers of three-fingered toxins: a paradigm for biological activity diversification. J. Biol. Chem. 283, 14571-14580.

Parker, D. C. (1993) T cell-dependent B cell activation. Annu. Rev. Immunol. 11, 331-360.

Pullen, G. R., Fitzgerald, M. G. and Hosking, C. S. (1986) Antibody avidity determination by ELISA using thiocyanate elution. J. Immunol. Methods 86, 83-87.

Sivaraman, T., Kumar, T. K., Hung, K. W. and Yu, C. (2000) Comparison of the structural stability of two homologous toxins isolated from the Taiwan cobra (Naja naja atra) venom. Biochemistry 39, 8705-8710.

Stanley, C., Lew, A. M. and Steward, M. W. (1983) The measurement of antibody affinity: a comparison of five techniques utilizing a panel of monoclonal anti-DNP antibodies and the effect of high affinity antibody on the measurement of low affinity antibody. J. Immunol. Methods 64, 119-132.

Tsetlin, V. (1999) Snake venom α-neurotoxins and other 'three-finger' proteins. Eur. J. Biochem. 264, 281-286.

Wang, J. H. and Reinherz, E. L. (2002) Structural basis of T cell recognition of peptides bound to MHC molecules. Mol. Immonol. 38, 1039-1049.

Yang, C. C. (1965) Crystallization and properties of cobrotoxin from Formosan cobra venom. J. Biol. Chem. 240, 1616-1618.

Yang, C. C. (1967) The disulfide bonds of cobrotoxin and their relationship to lethality. Biochim. Biophys. Acta 133, 346-355.

Yang, C. C., Yang, H. J. and Huang, J. S. (1969) The amino acid sequence of cobrotoxin. Biochim. Biophys. Acta 188, 65-77.

Yang, C. C., Yang, H. J. and Chiu, R. H. (1970) The position of disulfide bonds in cobrotoxin. Biochim. Biophys. Acta 214, 355-363.

Yang, C. C., Chang, C. C. and Liou, I. F. (1974) Studies on the status of arginine residues in cobrotoxin. Biochim. Biophys. Acta 365, 1-14.

Yang, C. C., Lin, M. F. and Chang, C. C. (1977) Purification of anti-cobrotoxin antibody by affinity chromatography. Toxicon 15, 51-62.

Yang, C. C., Chang, C. C., Lin, M. F., Lee, H. Y. and Chuang, L. Y. (1978) Purification and properties of non-precipitating antibodies to cobrotoxin. Int. J. Peptide Protein Res. 12, 303-310.

Yang, C. C., Chuang, L. Y. and Chang, C. C. (1981) The non-precipitability character of non-precipitating antibody to cobrotoxin. Snake 13, 32-37.

Yang, C. C. and Chang, L. S. (1999) Biochemistry and molecular biology of snake neurotoxin. J. Chin. Chem. Soc. 46, 319-332.

Yoshii, H., Furuta, T., Yonehara, T., Ito, D., Linko, Y. Y. and Linko, P. (2000) Refolding of denatured/reduced lysozyme at high concentration with diafiltration. Biosci. Biotechnol. Biochem. 64, 1159-1165.

Yu, C., Lee, C. S., Chuang, L. C., Shei, Y. R. and Wang, C. Y. (1990) Two-dimensional NMR studies and secondary structure of cobrotoxin in aqueous solution. Eur. J. Biochem. 193, 789-799.

Yu, C., Bhaskaran, R., Chuang, L. C. and Yang, C. C. (1993) Solution conformation of cobrotoxin: a nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing study. Biochemistry 32, 2131-2136.