研究表明只有靈長類動物才存在的免疫球蛋白A1，其具有對抗微生物和其他的病毒感染的功能。IgA1存在於分泌物和細胞的表面，特別是黏膜細胞表面。但是有一些致病性細菌會分泌一種專一性很高的蛋白酶造成IgA1 重鏈與輕鏈連結區的水解而破壞人類 IgA1 的免疫功能，進而降低體內的防衛功\\\能，因此IgA1蛋白酶被認為具有致病的角色。雖然已有基因選殖等間接的方式推測了 IgA1 蛋白酶區域及反應中心位置，但是目前尚缺乏對此酵素的實際研究資料。為了進一步了解及證明蛋白酶的活性位點，本研究以流行性感冒嗜血桿菌(H. influenza)IgA1蛋白酶的基因為模板，利用聚合酶反應分別放大活性區域（相當於基因的1-885號核苷酸) 和蛋白酶區域 （相當於基因的1-2373號核苷酸)，並將放大的片段轉入中間載體pGEM-T進行DNA序列分析之後分別轉入表達載體pTrcHisA進行重組蛋白的表達。 將重組蛋白與人類免疫球蛋白A1進行活性測試，藉此進一步了解 IgA1 蛋白酶區域及水解活性的關係。本研究結果表明，蛋白酶結構區重組蛋白對人類免疫球蛋白A1重鏈具有水解活性，但是普遍被認為是具有反應中心重組蛋白並無法水解人類免疫球蛋白A1，同時證實蛋白酶結構區末端的高度保留區C-Box對IgA1的辨識及水解功能上是不需要的。

IgA1 (immunoglobulin A1) functions as an immune molecule, against microbial invaders and other pathogens, on the surface of animal and human cells, especially on the mucosal membrane. To impair the function of immunoglobulin the human immune system some bacteria secrete site-specific IgA1 proteases to cleave IgA1 at hinge region. The protease has therefore been implicated as a putative virulence factor. Although the alignment of the DNA for the proteases predicted the IgA1 protease domain and catalytic site, lack of experimental evidence hindered in both structural and functional enzymic studies. In order to elucidate and understand the exact activity, this study aims at to analyze catalytic site of IgA1 protease from H. influenza, and thus PCR was used to amplify the DNA for putative reactive site domain (1-885) and protease domain (1-2373), respectively, were cloned in pGEM-T and transferred to the expression vector, pTrcHis-A for production of the recombinant proteins. Analysis of the recombinant proteins shows that the Human IgA1 heavy chain hydrolyzed by the protease domain recombinant protein but putative reactive site. This demonstrates that protease domain is enough for protease activity. The putative reactive site domain alone is incapable in hydrolyzing Human IgA1.

目錄:
英文摘要
中文摘要
第一章 前言
1.1 IgA1蛋白酶的角色-----------------------------------------------8
1.2 IgA1蛋白酶結構------------------------------------------------10
1.3 IgA1蛋白酶前體結構--------------------------------------------12
1.4 IgA1蛋白酶分裂位置--------------------------------------------13
1.5 非分型流行性感冒嗜血桿菌---------------------------------------14
1.6 研究目的-------------------------------------------------------17
第二章 材料及方法
2.1 使用菌株、質體及生長條件 --------------------------------------19
2.2 製備質體去氧核醣核酸plasmid DNA -------------------------------20
2.3選殖NTHi500中885及2373片段 ----------------------------------21
2.4 製備勝任細胞並轉置---------------------------------------------23
2.5酶切作用及膠上純化 ---------------------------------------------25
2.5.1酶切確認--------------------------------------------------25
2.5.2酶切T-A cloning並純化------------------------------------25
2.5.3酶切TrcHisA載體及純化------------------------------------26
2.6 蛋白質表達及純化重組IgA1蛋白酶--------------------------------27
2.7 IgA1蛋白酶活性測試 --------------------------------------------28
第三章 結果
3.1 聚合酶連鎖反應放大非分型流行性感冒嗜血桿菌人類免疫球蛋白A1蛋白酶基因片段-------------------------------------------------------30
3.2 選殖非分型流行性感冒嗜血桿菌人類免疫球蛋白A1蛋白酶基因片段----31
3.3 基因定序 ------------------------------------------------------38
3.4 蛋白質表達及IgA1蛋白酶活性測試--------------------------------41
第四章 結論與討論-------------------------------------------------46
参考文獻-----------------------------------------------------------51

参考文獻:
Bachovchin, W. W., Plaut, A. G., Flentke, G. R., Lynch, M. and Kettner, C. A. (1990) Inhibition of IgAl proteinases from Neisseria gonorrhoeae and Hemophilus influenzae by peptide prolyl boronic acids. J. Bio. Chem. 265:3738-3743.

Barenkamp, S. J., and St Geme, J. W. 3rd. (1996) Identification of a second family of high-molecular-weight adhesion protein expressed by non-typable Haemophilus influenzae. Mol. Microbiol. 19:1215-1223.

Frandsen, E. V. G., Reinholdt, J., Kjeldsen, M. and Kilian, M. (1997) Inhibition of Prevotella and Capnocytophaga Immunoglobulin A1 proteases by human serum. Clin. Diagn. Lab. Immunol. 4:458-464.

Hui-Hsuan Chang (2006) Molecular Characterization of IgA1 protease from Non-typable Haemophilus influenza, Master thesis, Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan, R.O.C.

Halter, R., Pohlner, J., & Meyer, T. F. (1984).IgA protease of Neisseria gonorrhoeae: isolation and characterization of the gene and its extracellular product. The EMBO Journal, 3,1595-1601.

Kett, K., Brandtzaeg, P., Radl, J., and Haaijman, J. J. (1986) Different subclass distribution of IgA-producing cells in human lymphoid organs and various secretory tissues. J. Immunol. 136:3631-3635.

Kilian, M., Reinholdt, j., Lomholt, H.,Poulsen, K., & Frandsen, E.V. (1996). Biologiacl significance of IgA1 proteases in bacterial colonization and pathogenesis: critical evaluation of experimental evidence. APMIS - acta pathologica, mocrobiologica, et immunologica Scandinavica, 104, 321-338

Lorenzen, D. R., Düx, F., Wölk, U., Tsirpouchtsidis, A., Hass, G., and Meyer, T. F. (1999) Immunoglobulin A1 protease, an exoenzyme of pathogenic Neisseriae , is a potent inducer of proinflammatory cytokines. J. Exp. Med. 190:1049-1058.

Lomholt, H., Poulsen, K., and Kilian, M. (1995) Comparative characterization of the iga gene encoding IgA1 protease in Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae. Mol. Microbiol. 15: 495-506.

Lomoholt,H.,Van Alphen, L.,& Kilian, M.(1993). Antigenic variation of immunoglobulin A1 proteases amoung sequential isolates of Haemophilus influenza from healthy children and patients with chronic obstructive pulmonary disease. Infection and Immunity, 61, 4575-4581.

Mansa, B. and Kilian, M. (1986) Retained antigen-binding activity of Fab, fragments of human monoclonal immunoglobulin Al (IgAl) cleaved by IgAl Protease. Infect. Immun. 52:171-174


Mistry, D., and Stockley, R. A. (2005) Molecules in focus IgA1 protease. Int. J. Biochem. Cell. Biol. 38:1244-1248.

Mulks, M. H., Kornfeld, S. J., Frangione, B., and Plaut, A. G. (1982) Relationship between the specificity of IgA proteases and serotypes in Haemophilus influenzae. J. Infect. Dis. 146:266-274.


Murphy, T. F., Bernstein, J. M., Dryja, D. M., Campagnari, A. A., and Apicella, M. A. (1987) Outer membrane protein and lipooligosaccharide analysis of paired nasopharyngeal and middle ear isolates in otitis media due to nontypable Haemophilus influenzae: pathogenetic and epidemiological observations. J. Infect. Dis. 156: 723-731

Plaut, A. G., Genco, R. J., and Tomasi, T. B., Jr. (1974a) Isolation of an enzyme from Streptococcus sanguis, which specifically cleaves IgA. J. Immunol. 113:289-291.

Plaut, A. G., Wistar Jr., R. and Capra, J. D. (1974b) Differential susceptibility of human IgA immunoglobulins to streptococcal IgA protease. J. Clin. Invest. 54:1295-1300

Plaut, A. G. (1978) Microbial IgA proteases. N. Engl. J. Med. 298:1459-1463

Plaut, A.G., Gilbert, J. V., Artenstein, M. S., and Capra, J. D. (1975) Neisseria gonorrhoeae and Neisseria meningitidis: extracellular enzyme cleaves human immunoglobulin A. Science 190:1103-1105

Pohlner, J., Halter, R., Beyreuther, K., and Meyer, T. F. (1987) Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease. Nature (London) 325:458-462

Poulsen, K., Reinholdt, J.,jespersgaard, C.,Boye, K.,Brown, T. A.,Hauge, M.,& Kilian, M.(1998) A comprehensive genetic study of streptococcal immunoglobulin A1 protease: evidence for recombination within and between species. Infection and Immunity, 66, 181-190

Poulsen, K., Reinholdt, J., and Kilian, M. (1992) A comparative genetic study of serologically distinct Haemophilus influenzae type 1 immunoglobulin A1 proteases. J. Bacteriol. 174:2913-2921.

Qiu, J., Brackee, G. P., and Plaut, A. G. (1996) Analysis of the specificity of bacterial immunoglobulin A (IgA) proteases by a comparative study of ape serum IgAs as substrates. Infect. Immun. 64: 933-937.
Rao, V. K., Krasan, G. P., Hendrixson, D. R., and St Geme, J. W. 3rd. (1999) Molecular determinants of the pathogenesis of disease due to non-typable Haemophilus influenzae. FEMS Microbiol. Rev. 23:99-129.

Reinholdt, J. and Kilian, M. (1991) Lack of cleavage of immunoglobulin A (IgA) from rhesus monkeys by bacterial IgA1 proteases. Infect. Immun. 59: 2219-2221.

St Geme, J. W. 3rd., de al Morena, M. L., and Flkow, S. (1994) A Haemophilus influenzae IgA protease-like protein promotes intimate interaction with human epithelial cells. Mol. Microbiol. 14:217-223

St Geme, J. W. 3rd. (2002) Molecular and cellular determinants of non-typeable Haemophilus influenzae adherence and invasion. Cell. Microbiol. 4:191-200

Stull, T. L., Mendelman, P. M. Haas J. E., Schoenbron, M. A., Mack, K. D., and Smith, A. L.(1984). Characterization of Haemophilus Influenzae type b pill, Infect Immun, 46,: 787-796

Tsirpouchtsidis, A., Hurwitz, R., Brinkmann, V., Meyer, T. F. and Hass, G. (2002) Neisserial immunoglobulin A1 protease induces specific T-cell responses in humans. Infect. Immun. 70:335-344

Vitovski, S., Dunkin, K. T., Howard, A. J. and Sayers, J. R. (2002) Nontypeable Haemophilus influenzae in carriage and disease: a difference in IgA1 protease activity levels. J. Am. Med. Assoc. 287:1699-705.

V.Romanello, M.Marcacci, F.Dal Molin, M. Moschioni, S.Censini, A.Covacci, A.G.Baritussio, C.Montecucco, F.Tonello (2005) Cloning, expression, purification, and characterization of Streptococcus pneumonia IgA1 protease.

Vitovski, S., Read, R. C., and Sayers, J. R. (1999) Invasive isolates of Neisseria meningitidis possess enhanced immunoglobulin A1 protease activity compared to colonizing strains. FASEB J. 13:331-337

Weiser, J. N., Bae, D., Fasching, C., Scamurra, R. W., Ratner, A. J., and Janoff, E. N. (2003) Antibody-enhanced pneumococcal adherence requires IgA1 protease. Proc. Natl. Acad. Sci. USA 100:4215-4220.