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標題: 以螺旋胜肽連接子接合免疫調節蛋白
Conjugation of Immunoregulatory Proteins by Helix-Forming Linker
作者: 曾慈瀅
Tseng, Tzu-Ying
關鍵字: recombinant polymerase chain reaction Escherichia coli;重組聚合酶鏈反應 原核表現質體
出版社: 獸醫學系暨研究所
引用: 參考文獻 蔡淑婷。2003。C 蛋白之重組蛋白表現與應用。國立中興大學獸醫微生物學研究 所。台中。中華民國。 吳佩珊。2003。B 蛋白及趨化素之研究。國立中興大學獸醫微生物學研究所。 台中。中華民國。 Alfthan, K., Takkinen, K., Sizmann, D., Soderlund, H., and Teeri, T.T. (1995). Properties of a single-chain antibody containing different linker peptides. Protein Eng 8, 725-731. Arai, R., Ueda, H., Kitayama, A., Kamiya, N., and Nagamune, T. (2001). Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Eng 14, 529-532. Arai, R., Ueda, H., Tsumoto, K., Mahoney, W.C., Kumagai, I., and Nagamune, T. (2000). Fluorolabeling of antibody variable domains with green fluorescent protein variants: application to an energy transfer-based homogeneous immunoassay. Protein Eng 13, 369-376. Boulay, J.L., and Paul, W.E. (1993). Hematopoietin sub-family classification based on size, gene organization and sequence homology. Curr Biol 3, 573-581. Bulow, L. (1987). Characterization of an artificial bifunctional enzyme, beta-galactosidase/galactokinase, prepared by gene fusion. Eur J Biochem 163, 443-448. Chaudhary, V.K., Queen, C., Junghans, R.P., Waldmann, T.A., FitzGerald, D.J., and Pastan, I. (1989). A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin. Nature 339, 394-397. Haswell, L.E., Glennie, M.J., and Al-Shamkhani, A. (2001). Analysis of the oligomeric requirement for signaling by CD40 using soluble multimeric forms of its ligand, CD154. Eur J Immunol 31, 3094-3100. Hsu, Y.M., Lucci, J., Su, L., Ehrenfels, B., Garber, E., and Thomas, D. (1997). Heteromultimeric complexes of CD40 ligand are present on the cell surface of human T lymphocytes. J Biol Chem 272, 911-915. Hulse, D.J., and Romero, C.H. (2004). Partial protection against infectious bursal disease virus through DNA-mediated vaccination with the VP2 capsid protein and chicken IL-2 genes. Vaccine 22, 1249-1259. Huston, J.S., Levinson, D., Mudgett-Hunter, M., Tai, M.S., Novotny, J., Margolies, M.N., Ridge, R.J., Bruccoleri, R.E., Haber, E., Crea, R., et al. (1988). Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A 85, 5879-5883. Kaiser, P., Hughes, S., and Bumstead, N. (1999). The chicken 9E3/CEF4 CXC chemokine is the avian orthologue of IL8 and maps to chicken chromosome 4 syntenic with genes flanking the mammalian chemokine cluster. Immunogenetics 49, 673-684. Kaiser, P., and Mariani, P. (1999). Promoter sequence, exon:intron structure, and synteny of genetic location show that a chicken cytokine with T-cell proliferative activity is IL2 and not IL15. Immunogenetics 49, 26-35. Kaiser, P., Poh, T.Y., Rothwell, L., Avery, S., Balu, S., Pathania, U.S., Hughes, S., Goodchild, M., Morrell, S., Watson, M., et al. (2005). A genomic analysis of chicken cytokines and chemokines. J Interferon Cytokine Res 25, 467-484. Kogut, M., Rothwell, L., and Kaiser, P. (2002). Differential effects of age on chicken heterophil functional activation by recombinant chicken interleukin-2. Dev Comp Immunol 26, 817-830. Kothlow, S., Morgenroth, I., Tregaskes, C.A., Kaspers, B., and Young, J.R. (2008). CD40 ligand supports the long-term maintenance and differentiation of chicken B cells in culture. Dev Comp Immunol 32, 1015-1026. Kumar, S., Ahi, Y.S., Salunkhe, S.S., Koul, M., Tiwari, A.K., Gupta, P.K., and Rai, A. (2009). Effective protection by high efficiency bicistronic DNA vaccine against infectious bursal disease virus expressing VP2 protein and chicken IL-2. Vaccine 27, 864-869. Laing, K.J., and Secombes, C.J. (2004). Chemokines. Dev Comp Immunol 28, 443-460. Leonard, W.J. (2001). Cytokines and immunodeficiency diseases. Nat Rev Immunol 1, 200-208. Lillehoj, H.S., Choi, K.D., Jenkins, M.C., Vakharia, V.N., Song, K.D., Han, J.Y., and Lillehoj, E.P. (2000). A recombinant Eimeria protein inducing interferon-gamma production: comparison of different gene expression systems and immunization strategies for vaccination against coccidiosis. Avian Dis 44, 379-389. Ljungcrantz, P., Carlsson, H., Mansson, M.O., Buckel, P., Mosbach, K., and Bulow, L. (1989). Construction of an artificial bifunctional enzyme, beta-galactosidase/galactose dehydrogenase, exhibiting efficient galactose channeling. Biochemistry 28, 8786-8792. Maeda, Y., Ueda, H., Kazami, J., Kawano, G., Suzuki, E., and Nagamune, T. (1997). Engineering of functional chimeric protein G-Vargula luciferase. Anal Biochem 249, 147-152. Mallender, W.D., and Voss, E.W., Jr. (1994). Construction, expression, and activity of a bivalent bispecific single-chain antibody. J Biol Chem 269, 199-206. Marqusee, S., and Baldwin, R.L. (1987). Helix stabilization by Glu-...Lys+ salt bridges in short peptides of de novo design. Proc Natl Acad Sci U S A 84, 8898-8902. Morris, A.E., Remmele, R.L., Jr., Klinke, R., Macduff, B.M., Fanslow, W.C., and Armitage, R.J. (1999). Incorporation of an isoleucine zipper motif enhances the biological activity of soluble CD40L (CD154). J Biol Chem 274, 418-423. Nabeshima, S., and Ohkawa, H. (1990). [Inclusion body formation and renaturation of recombinant proteins]. Tanpakushitsu Kakusan Koso 35, 2688-2697. Nagasawa, T., Hirota, S., Tachibana, K., Takakura, N., Nishikawa, S., Kitamura, Y., Yoshida, N., Kikutani, H., and Kishimoto, T. (1996). Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 382, 635-638. Nagasawa, T., Tachibana, K., and Kishimoto, T. (1998). A novel CXC chemokine PBSF/SDF-1 and its receptor CXCR4: their functions in development, hematopoiesis and HIV infection. Semin Immunol 10, 179-185. Park, S.S., Lillehoj, H.S., Hong, Y.H., and Lee, S.H. (2007). Functional characterization of tumor necrosis factor superfamily 15 (TNFSF15) induced by lipopolysaccharides and Eimeria infection. Dev Comp Immunol 31, 934-944. Peacock, J.W., and Jirik, F.R. (1999). TCR activation inhibits chemotaxis toward stromal cell-derived factor-1: evidence for reciprocal regulation between CXCR4 and the TCR. J Immunol 162, 215-223. Pietravalle, F., Lecoanet-Henchoz, S., Blasey, H., Aubry, J.P., Elson, G., Edgerton, M.D., Bonnefoy, J.Y., and Gauchat, J.F. (1996). Human native soluble CD40L is a biologically active trimer, processed inside microsomes. J Biol Chem 271, 5965-5967. Read, L.R., Cumberbatch, J.A., Buhr, M.M., Bendall, A.J., and Sharif, S. (2005). Cloning and characterization of chicken stromal cell derived factor-1. Dev Comp Immunol 29, 143-152. Schat, K.A., Calnek, B.W., and Weinstock, D. (1986). Cultivation and characterisation of avian lymphocytes with natural killer cell activity. Avian Pathol 15, 539-556. Schnetzler, M., Oommen, A., Nowak, J.S., and Franklin, R.M. (1983). Characterization of chicken T cell growth factor. Eur J Immunol 13, 560-566. Shirozu, M., Nakano, T., Inazawa, J., Tashiro, K., Tada, H., Shinohara, T., and Honjo, T. (1995). Structure and chromosomal localization of the human stromal cell-derived factor 1 (SDF1) gene. Genomics 28, 495-500. Stepaniak, J.A., Shuster, J.E., Hu, W., and Sundick, R.S. (1999). Production and in vitro characterization of recombinant chicken interleukin-2. J Interferon Cytokine Res 19,515-526. Suzuki, Y., Rahman, M., and Mitsuya, H. (2001). Diverse transcriptional response of CD4(+) T cells to stromal cell-derived factor (SDF)-1: cell survival promotion and priming effects of SDF-1 on CD4(+) T cells. J Immunol 167, 3064-3073. Taga, T., and Kishimoto, T. (1992). Cytokine receptors and signal transduction. FASEB J 6, 3387-3396. Tan, K.B., Harrop, J., Reddy, M., Young, P., Terrett, J., Emery, J., Moore, G., and Truneh, A. (1997). Characterization of a novel TNF-like ligand and recently described TNF ligand and TNF receptor superfamily genes and their constitutive and inducible expression in hematopoietic and non-hematopoietic cells. Gene 204, 35-46. Tregaskes, C.A., Glansbeek, H.L., Gill, A.C., Hunt, L.G., Burnside, J., and Young, J.R. (2005). Conservation of biological properties of the CD40 ligand, CD154 in a non-mammalian vertebrate. Dev Comp Immunol 29, 361-374. Vainio, O., Ratcliffe, M.J., and Leanderson, T. (1986). Chicken T-cell growth factor: use in the generation of a long-term cultured T-cell line and biochemical characterization. Scand J Immunol 23, 135-142. Wels, W., Harwerth, I.M., Zwickl, M., Hardman, N., Groner, B., and Hynes, N.E. (1992). Construction, bacterial expression and characterization of a bifunctional single-chain antibody-phosphatase fusion protein targeted to the human erbB-2 receptor. Biotechnology (N Y) 10, 1128-1132. Wykes, M. (2003). Why do B cells produce CD40 ligand? Immunol Cell Biol 81, 328-331. Wykes, M., Poudrier, J., Lindstedt, R., and Gray, D. (1998). Regulation of cytoplasmic, surface and soluble forms of CD40 ligand in mouse B cells. Eur J Immunol 28, 548-559.
摘 要
在本研究中我們將A 蛋白以螺旋連接子分別接合B 蛋白或C 蛋白,本研
究所使用的螺旋連接子由胺基酸序列 EAAAK 重複三次組成 (EAAAK)3,共包
含15 個胺基酸。我們利用重組聚合酶鏈反應(recombinant polymerase chain
reaction, recombinant PCR)將螺旋連接子分別接合A 蛋白與B 蛋白或C 蛋白,
得到A-B 與A-C 兩個基因融合片段,將此二片段分別構築到原核表現質體
pET28 並將質體送入大腸桿菌誘導重組蛋白表現。將rA-B 與rA-C 純化後,經
MALDI-TOF 質譜儀分析鑑定並根據A、B 以及C 的功能進行蛋白的活性測試。
結果顯示,rA、rA-B 或rA-C 皆能趨化周邊血液單核球(peripheral blood
mononuclear cells, PBMCs),此外rA-B 能與表現B 蛋白受器之巨噬細胞或纖維
母細胞細胞株結合,另一方面,rA-C 能刺激周邊血液單核球的增生。整體而言,
(EAAAK)3 做為連接子能保有rA-B 中的A 與B 之活性或rA-C 中的A 與C 之

In this study, we linked A (A) with B (B) or with C (C) by a 15-amino acid helixforming
linker, (EAAAK)3. By performing recombinant polymerase chain reaction
(PCR), DNA sequence of (EAAAK)3 linker was introduced between that of A and B or
C to generate DNA fragments encoding A-B and A-C. Both DNA fragments were
cloned respectively into pET28 and transformed into Escherichia coli to induce
recombinant proteins expression. Purified recombinant A-B (rA-B) and A-C (rA-C)
proteins were further analyzed and by matrix-assisted laser desorption inoization-time
of flight mass spectrometry (MALDI-TOF MS) to verify their identities. Furthermore,
based on the functions of A, B, or C, we performed protein activity assays. Recombinant
A (rA), rA-B and rA-C all have chemotactic effect on peripheral blood
mononuclear cells (PBMCs). In addition, rA-B could bind to B receptor-expressing
cells, such as macrophages and fibroblast cell line. On the other hand, rA-C could
stimulate the proliferation of PBMCs. Taken altogether, our results indicated that
(EAAAK)3, as a peptide linker could retain the activities of A and B in rA-B or A and
C in rA-C.
其他識別: U0005-2008201015135400
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