Molecular cloning and the allergenic characterization of tropomyosin from forcipomyia taiwana
江啟峰、江主惠、李美芳
E-mail: [email protected]
ABSTRACT
Background：Forcipomyia taiwana is a tiny blood-sucking midge widely distributed in Taiwan. Like mosquito bites, midge bites can cause allergic reactions. Invertebrate tropomyosins are the panallergens responsible for cross-reactions between crustacea, insects, mites, nematodes, and different classes of mollusks. There are two purposes in this study. Firstly, we aimed to produce recombinant F. taiwana tropomyosin and investigate its allergenicity. Secondly, we aimed to investigate the inflammatory reaction of recombinant F. taiwana tropomyosin in human skin cell line, Hs68. Materials and Methods：We isolated total RNA of F. taiwana and
synthesized cDNA by RT-PCR using degenerate primers designed according to tropomyosin sequences of silverfish (Lepisma saccharina). The PCR product was ligated into TA cloning vector and sequenced to confirm the identity of the insert, and then subcloned into pET30a expression system. Recombinant protein was over-expressed in E.coli BL21 and purified using the His-Tag affinity column chromatography. The IgE-binding reactivity of purified recombinant protein was evaluated by immunoblot and ELISA. In addition, the skin fibroblast Hs68 cells were stimulated with rFor t 4 to evaluate the immunogenicity. Results：The cloned F. taiwana tropomyosin, named For t 4, comprised an 855-bp open reading frame and encoded a 32 kDa protein. The deduced amino acid sequence shared 58~67% identity with previously known allergenic tropomyosins. ELISA analysis revealed that rFor t 4 reacted with 24% (6/25) of F. taiwana-sensitized subjects. In addition, rFor t 4 showed 20~50% inhibition of IgE binding to F. taiwana crude extract by inhibition ELISA. It also stimulates IL-8 proteins secretion and up-regulates mRNA expression of IL-8, MCP-1, eotaxin and GM-CSF from human skin fibroblasts. Conclusions：Tropomyosin represents a minor allergen in F. taiwana extract. It is hoped that rFor t 4 will be useful for developing specific in vitro and in vivo diagnostics tools and immunotherapy of F.
taiwana allergy.
Keywords : Forcipomyia taiwana、allergy、For t 4、tropomyosin、Hs68 cells Table of Contents
目錄 封面內頁 簽名頁 授權書iii 中文摘要iv 英文摘要vi 致謝viii 目錄x 圖目錄xv 表目錄xvi 1. 前言 1.1 過敏疾病的介紹 1 1.2 過敏原的命名 2 1.3 過敏反應的致病機轉4 1.4 過敏疾病的診斷6 1.4.1 皮膚穿刺試驗(Skin Prick Test) 7 1.4.2 血清測試 7 1.5 過 敏疾病的預防及治療8 1.6 台灣鋏蠓9 1.6.1 台灣鋏蠓之分類9 1.6.2 台灣鋏蠓之形態10 1.6.3 台灣鋏蠓之分佈與生態11 1.6.4 台 灣鋏蠓之為害11 1.7 台灣鋏蠓過敏症12 1.7.1 昆蟲叮咬方式之分類12 1.7.1.1 叮螫昆蟲(Sting insects)12 1.7.1.2 叮咬昆蟲(Biting insects)12 1.7.2 昆蟲叮咬之不良反應13 1.8 原肌凝蛋白質(tropomyosin)過敏原的特性介紹14 1.9 趨化細胞激素15 1.10 研究目 的17 2. 實驗材料與方法19 2.1 實驗材料19 2.1.1 採集台灣鋏蠓雌蟲19 2.1.2 病患血清檢體之採集19 2.1.3 製備台灣鋏蠓全蟲 粗萃取物20 2.1.4 人類皮膚纖維母細胞Hs6820 2.2 化學藥品與器材20 2.2.1 一般化學試劑20 2.2.2 限制?﹞峸硐蘊袡Ⅲ簿?1 2.2.3 DNA電泳相關藥品21 2.2.4 蛋白質電泳相關藥品21 2.2.5 細胞趨化激素引子22 2.3 重要器材及儀器22 2.4 實驗方法22 2.4.1 台灣鋏蠓過敏原之分子選殖23 2.4.1.1 台灣鋏蠓RNA 之製備23 2.4.1.2 RNA 濃度之測定23 2.4.1.3 1st-strand cDNA synthesis23 2.4.1.4 設計tropomyosin degenerated 引子24 2.4.1.5 聚合? 嚍磥狨 (Polymerase Chain Reaction；PCR)24 2.4.1.6 DNA 電泳25 2.4.1.7 DNA 分子回收25 2.4.1.8 接合作用26 2.4.1.9 製備勝任細胞26 2.4.1.10 轉型作用27 2.4.1.11 微量質 體DNA 的萃取 (Miniprep)27 2.4.1.12 過敏原基因定序與同源性序列分析28 2.4.1.13 構築表現載體pET30a 載體29 2.4.1.14 表 現重組蛋白質及純化29 2.4.1.14.1 IPTG 誘導之劑量測定29 2.4.1.14.2 誘導蛋白質之時間測定30 2.4.1.14.3 蛋白質之純化30 2.4.1.15 重組蛋白質LPS 汙染之移除31 2.4.1.16 蛋白質濃度定量32 2.4.1.17 蛋白質電泳分析33 2.4.1.18 CBR 染色法
(Coomassie brilliant blue stain)35 2.4.2 重組蛋白質免疫特性分析35 2.4.2.1 西方墨漬法 (Western blotting)35 2.4.2.2 蛋白質二維 電泳分析37 2.4.2.3 銀染色法 (Silver stain)37 2.4.2.4 質譜儀分析與胜?汗頞q指紋鑑定38 2.4.2.5 以酵素聯結免疫吸附法 (ELISA)測定台灣鋏蠓專一型 免疫球蛋白E39 2.4.2.6 抑制型酵素聯結免疫吸附法40 2.4.3 細胞培養41 2.4.3.1 Hs68 細胞株之 培養41 2.4.3.2 細胞計數計算41 2.4.3.3 繼代培養42 2.4.3.4 Hs68 細胞之RNA 萃取 (RNA extraction)42 2.4.3.5 RNA 濃度測定 及1st-strand cDNA synthesis43 2.4.3.6 趨化激素基因之聚合? 嚍磥狨?3 2.4.3.7 IL-8濃度之測定43 2.4.3.8 統計分析44 3. 結 果46 3.1 台灣鋏蠓過敏原tropomyosin 基因之選殖與序列比對分析46 3.2 蛋白質之表現與分析46 3.2.1 表現載體pET30a 之構 築47 3.2.2 重組蛋白質之表現47 3.3 蛋白質體學分析48 3.4-重組過敏原與脊椎、非脊椎動物不同物種間tropomyosin .高保留 性序列比較分析49 3.5..重組過敏原之免疫活性分析49 3.5.1 酵素聯結免疫吸附法分析49 3.5.2 蛋白質電泳與免疫轉漬分析49
3.5.3 Inhibition ELISA50 3.6 台灣鋏蠓重組過敏原tropomyosin 刺激人類皮膚纖維母細胞 .株51 3.6.1 Dose response51 3.6.2 Time course51 3.7 討論52 4. 結論55 參考文獻77 附錄84
REFERENCES
5. 參考文獻 1.陳怡行。 台灣鋏蠓(小黑蚊)過敏臨床表徵、免疫機轉、過敏原分子選殖與鑑定之研究.私立東海大學/生命科學研究所
，2009。 2.陳錦生。 花蓮地區台灣鋏蠓之形態及族群動態研究.國立臺灣大學/植物病蟲害研究所，1980。 3.連日清。 小黑蚊(鋏蠓屬蠛 蠓亞屬)之分類、生態及防治.小黑蚊之發生、生態及防治研討會專刊。1-7. 2008。 4.江伯倫。 臺灣大學醫學院臨床醫學研究所，科學發 展， 2002。 5.李學進。 科學發展413期， 2007年5月。 6.Allergen nomenclature. WHO/IUS Allergen Nomenclature Subcommittee. World Health Organization, Geneva, Switzerland Bull World Health Organ 72, 797-806 (1994). 7.Aceituno, E. et al. Molecular cloning of major allergen from Cupressus arizonica pollen: Cup a 1. Clin Exp Allergy 30, 1750-1758 (2000). 8.Asturias, J. A. et al. Sequencing and high level expression in Escherichia coli of the tropomyosin allergen (Der p 10) from Dermatophagoides pteronyssinus. Biochim Biophys Acta 1397, 27-30 (1998).
9.Asturias, J. A. et al. Molecular characterization of American cockroach tropomyosin (Periplaneta americana allergen 7), a cross-reactive allergen.
J Immunol 162, 4342-4348 (1999).10. 10.Asturias, J. A., Eraso, E. and Martinez, A. Cloning and high level expression in Escherichia coli of an Anisakis simplex tropomyosin isoform. Mol Biochem Parasitol 108, 263-267 (2000). 11.Ayuso, R., Reese, G., Leong-Kee, S., Plante, M. and Lehrer, S. B. Molecular basis of hropod cross-reactivity: IgE-binding cross-reactive epitopes of shrimp, house dust mite and cockroach
tropomyosins. Int Arch Allergy Immunol 129, 38-48 (2002). 12.Ballmer-Weber, B. K. et al. Component-resolved in vitro diagnosis in carrot allergy:
does the use of recombinant carrot allergens improve the reliability of the diagnostic procedure. Clin Exp Allergy 35, 970-978 (2005). 13.Barletta, B. et al. Immunological characterization of a recombinant tropomyosin from a new indoor source, Lepisma saccharina. Clin Exp Allergy 35, 483-489 (2005). 14.Becker, W. M. and Reese, G. Immunological identification and characterization of i ndividual food allergens. J Chromatogr B Biomed Sci Appl 756, 131-140 (2001). 15.Bernardini, R. et al. Cross-reactivity between IgE-binding proteins from Anisakis simplex and
Dermatophagoides pteronyssinus. Int J Immunopathol Pharmacol 18, 671-675 (2005). 16.Chapman, M. D. Allergen nomenclature. Clin Allergy Immunol 21, 47-58 (2008). 17.Chuang, Y. Y., Lin, C. S., Wang, C. H. Yeh, C. C. Distribution and seasonal occurrence of Forcipomyia taiwana (Diptera: Ceratopogonidae) in the Nantou area in Taiwan. J Med Entomol 37, 205-209 (2000). 18.Chen, Y. H. et al. Hypersensitivity to
Forcipomyia taiwana (biting midge): clinical analysis and identification of major For t 1, For t 2 and For t 3 allergens. Allergy 60, 1518-1523 (2005).
19.Chen, Y. H., Lee, M. F., Tsai, J. J., Wu, H. J. and Hwang, G. Y. Specific IgE and IgG responses and cytokine profile in subjects with allergic reactions to biting midge Forcipomyia taiwana. Int Arch Allergy Immunol 150, 66-74 (2009). 20.Costongs, G. M. Bas, B. M. The first fully automated allergy analyser UniCAP: comparison with IMMULITE for allergy panel testing. Eur J Clin Chem Clin Biochem 35, 885-888 (1997).
21.Cox, L. Sublingual immunotherapy in pediatric allergic rhinitis and asthma: efficacy, safety, and practical considerations. Curr Allergy Asthma Rep 7, 410-420 (2007). 22.Cromwell, O. et al. Transition of recombinant allergens from bench to clinical application. Methods 32, 300-312 (2004).
23.Deinhofer, K. et al. Microarrayed allergens for IgE profiling. Methods 32, 249-254 (2004). 24.Descotes, J. Choquet-Kastylevsky, G. Gell and Coombs's classification: is it still valid Toxicology 158, 43-49 (2001). 25.Diaz-Perales, A. et al. Recombinant Pru p 3 and natural Pru p 3, a major peach allergen, show equivalent immunologic reactivity: a new tool for the diagnosis of fruit allergy. J Allergy Clin Immunol 111, 628-633 (2003).
26.Dreborg, S. The skin prick test in the diagnosis of atopic allergy. J Am Acad Dermatol 21, 820-821 (1989). 27.Foster, S., Roger Caras, Norman Arlott, and Amy Eisenberg. A Field Guide to Venomous Animals and Poisonous Plants (1998). 28.Frazier, C. A. Allergic reactions to biting insects.
Lancet 1, 911 (1972). 29.Garcia-Zepeda, E. et al. Human eotaxin is a specific chemoattractant foreosinophil cells and provides a new mechanism to explain tissue eosinophilia. Nat. Med 2, 449-456 (1996). 30.Golden, D. B. Insect sting allergy and venom immunotherapy. Ann Allergy Asthma Immunol 96, S16-21 (2006). 31.Golden, D. B., Kagey-Sobotka, A., Norman, P. S., Hamilton, R. G. and Lichtenstein, L. M. Outcomes of allergy to insect stings in children, with and without venom immunotherapy. N Engl J Med 351, 668-674 (2004). 32.Hashimoto, S. et al. p38
Mitogen-activated protein kinase regulates IL-8 expression in human pulmonary vascular endothelial cells. Eur Respir J 13, 1357-1364 (1999).
33.Henklova, P. et al. SB203580, a pharmacological inhibitor of p38 MAP kinase transduction pathway activates ERK and JNK MAP kinases in primary cultures of human hepatocytes. Eur J Pharmacol 593, 16-23 (2008). 34.Hoffman, D. Allergic Reactions to Biting Insects. American Academy of Allergy Asthma and Immunology, 161-174 (2003). 35.Ishizaka, K. Ishizaka, T. Identification of gamma-E-antibodies as a carrier of reaginic activity. J Immunol 99, 1187-1198 (1967). 36.Ishizaka, K., Ishizaka, T. Hornbrook, M. M. Physicochemical properties of reaginic antibody. V. Correlation of reaginic activity wth gamma-E-globulin antibody. J Immunol 97, 840-853 (1966). 37.Jansen, A., de Lijster de Raadt, J., van Toorenenbergen, A. W. and van Wijk, R. G. Allergy to pistachio nuts. Allergy Proc 13, 255-258 (1992). 38.Jeong, K. Y. et al. Allergenic characterization of tropomyosin from the dusky brown cockroach, Periplaneta fuliginosa. Clin Diagn Lab Immunol 11, 680-685 (2004). 39.Jeong, K. Y. et al. Molecular cloning and characterization of tropomyosin, a major allergen of Chironomus kiiensis, a dominant species of nonbiting midges in Korea. Clin Diagn Lab Immunol 11, 320-324 (2004). 40.Joenvaara, S. et al. Caveolar transport through nasal epithelium of birch pollen allergen Bet v 1 in allergic patients. J Allergy Clin Immunol 124, 135-142 (2009). 41.Jeoung, B. J. et al. Quantification of the major brown shrimp allergen Pen a 1 (tropomyosin) by a monoclonal antibody-based sandwich ELISA. J Allergy Clin Immunol 100, 229-234 (1997). 42.Kay, A. B.
Overview of 'allergy and allergic diseases: with a view to the future'. Br Med Bull 56, 843-864 (2000). 43.Kay, A. B. Allergy and Hypersensitivity:
History and Concepts. Allergy and Allergic Diseases 1, 19 (2008). 44.Larsen, J. N. and Lowenstein, H. Allergen nomenclature. J Allergy Clin
Immunol 97,577-578 (1996). 45.Lee, M. F. et al. Identification of hevamine and hev B 1 as major latex allergens in Taiwan. Int Arch Allergy Immunol 139, 38-44 (2006). 46.Leonard, E. J. and Yoshimura, T. Human monocyte chemoattractant protein-1(MCP-1). Immunol Today 11, 97-101 (1990). 47.Leung, P. S. et al. Identification and molecular characterization of Charybdis feriatus tropomyosin, the major crab allergen. J Allergy Clin Immunol 102, 847-852 (1998). 48.Levine, M. and Lockey, R. Monograph on insect allergy. American Academy of Allergy Asthma and Immunology (2003). 49.Liu, G. M. et al. Effects of boiling on the IgE-binding properties of tropomyosin of shrimp (Litopenaeus vannamei). J Food Sci 75 (2010). 50.Luscinskas, F. W. et al. C-C and C-X-C chemokines trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Ann N Y Acad Sci 902, 288-293 (2000). 51.Marampon, F. et al. MEK/ERK inhibitor U0126 affects in vitro and in vivo growth of embryonal rhabdomyosarcoma. Mol Cancer Ther 8, 543-551, doi:1535-7163 (2009). 52.Matsuda, T., Matsubara, T. and Hino, S. Immunogenic and allergenic potentials of natural and recombinant innocuous proteins. J Biosci Bioeng 101, 203-211 (2006). 53.Matsumae, H. et al. Specific removal of endotoxin from protein solutions by immobilized histidine. Biotechnol Appl Biochem 12, 129-140 (1990). 54.Metcalf, D. et al. In vitro action on hemopoietic cells of recombinant murine GM-CSF purified after production in Escherichia coli: comparison with purified native GM-CSF. Cellular Physiology 128, 421-431 (1986). 55.Mothes, N., Valenta, R. and Spitzauer, S. Allergy testing: the role of recombinant allergens.
Clin Chem Lab Med 44, 125-132 (2006). 56.Muller, U., Berchtold, E. and Helbling, A. Honeybee venom allergy: results of a sting challenge 1 year after stopping successful venom immunotherapy in 86 patients. J Allergy Clin Immunol 87, 702-709 (1991). 57.Nichol, J. Bites and Stings: The World of Venomous Animals. New York: Facts on File (1990). 58.Niederberger, V. Valenta, R. Recombinant allergens for immunotherapy. Where do we stand Curr Opin Allergy Clin Immunol 4, 549-554 (2004). 59.Ohsugi, T. Koito, A. Current topics in prevention of human T-cell leukemia virus type i infection: NF-kappa B inhibitors and APOBEC3. Int Rev Immunol 27, 225-253 (2008). 60.Osterlund, C. et al. The non-proteolytic house dust mite allergen Der p 2 induce NF-kappaB and MAPK dependent activation of bronchial epithelial cells. Clin Exp Allergy 39, 1199-1208 (2009). 61.Oude Elberink, J. N., De Monchy, J. G., Van Der Heide, S., Guyatt, G. H. and Dubois, A. E. Venom immunotherapy improves health-related quality of life in patients allergic to yellow jacket venom. J Allergy Clin Immunol 110, 174-182 (2002). 62.Ponath, P. D. et al. Cloning of the human eosinophil chemoattractant, eotaxin. Expression, receptor binding, and functional properties suggest a mechanism for the selective recruitment of eosinophils. J Clin Invest 97, 604-612 (1996). 63.Reisman, R. E. Stinging insect allergy. J Allergy Clin Immunol 64, 3-4 (1979).
64.Reese, G., Ayuso, R. and Lehrer, S. B. Tropomyosin: an invertebrate pan-allergen. Int Arch Allergy Immunol 119, 247-258 (1999). 65.Rhen, T.
Cidlowski, J. A. Antiinflammatory action of glucocorticoids--new mechanisms for old drugs. N Engl J Med 353, 1711-1723 (2005). 66.Santos, A. B.
et al. Cross-reactive IgE antibody responses to tropomyosins from Ascaris lumbricoides and cockroach. J Allergy Clin Immunol 121, 1040-1046 (2008). 67.Scharenberg, A. M. and Kinet, J. P. Allergy. Is localized immunoglobulin E synthesis the problem? Curr Biol 4, 140-142 (1994).
68.Shannon, M. F., Gamble, J. R. and Vadas, M. A. Nuclear proteins interacting with the promoter region of the human granulocyte/macrophage colony-stimulating factor gene. Proc Natl Acad Sci U S A 85, 674-678 (1988). 69.Shiraki. Investigation on general injurious insect. Taiwan Sotokufu Noji Shikenjo Tokubetsu Hokodu 8, 286-297 (1913). 70.Simons FER, e. Middleton's Allergy - Principles and Practice. Mosby Elsevier, 569-608 (2009). 71.Solaro, R. J. and Rarick, H. M. Troponin and tropomyosin: proteins that switch on and tune in the activity of cardiac myofilaments. Circ Res 83, 471-480 (1998). 72.Sturm, E. et al. Endotoxin-stimulated macrophages decrease bile acid uptake in WIF-B cells, a rat hepatoma hybrid cell line. Hepatology 31, 124-130 (2000). 73.Valenta, R. Recombinant allergen-based concepts for diagnosis and therapy oftype I allergy. Allergy 57 Suppl 71, 66-67 (2002). 74.Valenta, R. and Kraft, D. Recombinant allergen molecules: tools to study effector cell activation.
Immunol Rev 179, 119-127 (2001). 75.Wynn, T. A. IL-13 effector functions. Annu Rev Immunol 21, 425-456 (2003). 76.Xu, L. L., Warren, M. K., Rose, W. L., Gong, W. and Wang, J. M. Human recombinant monocyte chemotactic protein and other C-C chemokines bind and induce
directional migration of dendritic cells in vitro. J Leukoc Biol 60, 365-371 (1996). 77.Yeh, C. C. and Chuang, Y. Y. Colonization and bionomics of Forcipomyia taiwana (Diptera:Ceratopogonidae) in the laboratory. J Med Entomol 33, 445-448 (1996). 78.Yi, F. C. et al. Identification of shared and unique immunoglobulin E epitopes of the highly conserved tropomyosins in Blomia tropicalis and Dermatophagoides pteronyssinus. Clin Exp Allergy 32, 1203-1210 (2002).
