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系統識別號 U0026-2207201009545800
論文名稱(中文) SP-D基因變異性對於塵蟎過敏原結合能力的影響
論文名稱(英文) The effect of polymorphism of Pulmonary Surfactant Protein D on the binding ability to Dermatophagoides pteronyassinus
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 98
學期 2
出版年 99
研究生(中文) 羅文昇
研究生(英文) Wun-Sheng Luo
電子信箱 s1696413@ncku.edu.tw
學號 s1696413
學位類別 碩士
語文別 中文
論文頁數 77頁
口試委員 指導教授-王志堯
口試委員-張文粲
口試委員-葉才明
中文關鍵字 肺泡表面蛋白D  基因多型性  過敏氣喘  塵蟎過敏原 
英文關鍵字 Surfactant protein D  polymorphism  airway inflammation  allergen 
學科別分類
中文摘要 肺泡表面蛋白D (Surfactant protein D, SP-D),是由肺部中的肺泡第二型細胞(type II alveolar cells) 所分泌的介面活性蛋白,在調節肺部免疫防禦及發炎反應上扮演重要的角色。SP-D 藉由藉由其COOH-端 C類型 lectin 碳水化合物辨識區域 (Carbohydrate recognition domain , CRD) ,能夠與環境中吸入的致病菌結合並清除之,並能夠活化巨噬細胞 (Macrophage) 及先天性免疫反應。因此,SP-D與過敏原的結合能力可能將影響致敏反應的產生及調控免疫反應。而SP-D基因特定位置的單一核苷酸多型性(Single nucleotide polymorphism)會影響蛋白質的寡聚(Oligomerization)行為及其功能性。研究指出在SFTPD基因的三個位置中核苷酸改變會分別造成蛋白質胺基酸序列改變,分別是第11個胺基酸從甲硫胺酸(Methionine)轉變為穌胺酸(Threonine)(Met 11Thr),第160個胺基酸的位置由穌胺酸(Threonine) 轉變成丙胺酸(Alanine)(Thr160Ala),及第270個胺基酸的位置由絲胺酸(Serine)轉變為穌胺酸(Threonine) (Ser270Thr)。為了更進一步了解SP-D基因多型性對塵蟎過敏原結合能力造成的影響,我們以分子生物學方式構築了帶有四種不同單一核苷酸多型性的SP-D 質體,並在哺乳類動物細胞中成功表現出四種SP-D 重組蛋白,在胺基酸第11及160的位置,分別是Met11/Thr160, Met11/Ala160, Thr11/Thr160 及 Thr11/Ala160。使用脢聯免疫吸附測(ELISA)分析不同基因多型性之重組蛋白在與塵蟎萃取物結合能力,發現Met11基因型之結合能力較Thr11基因型為佳,而Thr160基因型之結合能力亦優於Ala160。另外,我們也使用凝集實驗分析四種基因多型性之重組蛋白在凝集大腸桿菌的能力是否有所差異,發現四種重組蛋白並沒有顯著的差異。因此,我們認為重組SP-D蛋白質具有凝集能力,且在SP-D蛋白質中第11或是第160個胺基酸因為基因多型性造成的改變會影響SP-D蛋白質的結合能力。
英文摘要 Pulmonary surfactant protein D (SP-D), as a calcium-dependent lectin secreted from alveolar type II cells, plays important roles in the regulation of pulmonary host defense and airway inflammation. SP-D interacts with a wide range of aero-born pathogens and allergens via its C-terminal carbohydrate recognition domains (CRD), activates alveolar macrophage and innate immune response. Therefore, the ability of SP-D in the binding of allergens may affect the allergen sensitization and allergen-induced immune response in the lung. Genetic variants of SP-D may influence its oligomerization and function in the binding of bacterial ligands. There are three single-nucleotide variations altering amino acids sequences of the mature form of SP-D protein in codon 11 (Met11Thr), 160 (Ala160Thr), and 270 (Ser270Thr) of the SFTPD gene. To study further on the mite allergen binding affinity in wild type and mutant type of SP-D, we constructed four single-nucleotide genetic variants of SFTPD gene in pcDNA3.1 vector and expressed the full-length recombinant SP-D protein, Met11/Thr160, Met11/Ala160, Thr11/Thr160, and Thr11/Ala160, with altered amino acid on the residue 11 or 160 from mammalian cells. On the other hand, we also purified native SP-D protein from amniotic fluid and recombinant neck/CRD SP-D from E. coli as the control proteins for the full length recombinant SP-D proteins. The binding ability to mite allergen of four genetic variants of SP-D was determined by ELISA, respectively. We found that the recombinant protein with Met11 had better binding ability to Dermatophagoides pteronyassinus (Der p) extract than Thr11, and with Thr160 had better ability than Ala160. Putting together, Met11/Thr160 haplotype recombinant SP-D had the best binding ability to mite allergen. Besides, in the assay of bacterial aggregation activity, we found that native SP-D needs both N-terminal and C-terminal function to achieve aggregation. Four genetic variants of SP-D had no significant different in aggregation ability. In summary, we constructed and expressed various mutants form of recombinant SP-D and suggested that SP-D genetic variants in amino acid residue 11 or 160 affect its binding ability.
論文目次 摘要 I
Abstract III
致謝 V
目錄 VI
圖目錄 VIII
緒論 1
過敏性氣喘與塵蟎 1
肺部先天性免疫防禦機制 2
SP- D的結構與先天性免疫功能 2
SP-D蛋白基因突變位置所造成之基因多型性 5
SP-D基因多型性與氣喘相關性 6
研究目標與策略 7
材料方法 8
一、細胞培養 8
二、 質體製備 (Plasmid preparation) 10
三、構築選殖基因質體 14
四、直接定點突變 (Site direct mutagenesis) 18
五、 基因殖入轉染 (Transfection) 20
六、西方墨點法轉漬 22
七、重組肺部表面蛋白D醣類辨識部位(Recombinant Neck/CRD SP-D )表現與純化 24
八、由羊水中純化SP-D 27
九、SP-D 定量 29
十、過敏原脢聯吸附實驗 (ELISA) 30
十一、凝集實驗 32
十二、SP-D預培養(pre-incubation) 與IL8 偵測 33
結果 34
一 、SP-D 基因表現質體建構與定序 34
二、SP-D 基因轉染 HEK293細胞株後之表現 35
三、大腸桿菌誘導生產之SP-D羧基醣類辨識部位 (N/CRD SP-D) 純化以及膠體電泳和西方墨點法分析 35
四、羊水中天然SP-D蛋白 (Native SP-D) 純化以及膠體電泳和西方墨點法分析 36
五、收取及分析不含血清的細胞培養液 (conditional medium) 36
六、細胞培養液 (conditional medium)中重組蛋白之功能性分析 37
七、基因型影響SP-D重組蛋白對塵蟎過敏原結合能力 37
八、基因型對於SP-D重組蛋白凝集大腸桿菌功能之分析 38
結論 39
討論 40
參考文獻 45
附錄 67
Appendix A. SP-D基因質體定序結果表 67
Appendix B. 藥品列表 75

參考文獻 Adam E., Hansen K.K., Astudillo O.F., Coulon L., Bex F., Duhant X., Jaumotte E., Hollenberg M.D., Jacquet A. (2006) The House Dust Mite Allergen Der p 1, Unlike Der p 3, Stimulates the Expression of Interleukin-8 in Human Airway Epithelial Cells via a Proteinase-activated Receptor-2-independent Mechanism. J Biol Chem 281:6910-6923. DOI: 10.1074/jbc.M507140200.
Akiyama J., Hoffman A., Brown C., Allen L., Edmondson J., Poulain F., Hawgood S. (2002) Tissue Distribution of Surfactant Proteins A and D in the Mouse. J Histochem Cytochem 50:993-996.
Brandt E.B., Mingler M.K., Stevenson M.D., Wang N., Khurana Hershey G.K., Whitsett J.A., Rothenberg M.E. (2008) Surfactant protein D alters allergic lung responses in mice and human subjects. J Allergy Clin Immunol 121:1140-1147.
Brinker K., Martin E., Borron P., Mostaghel E., Doyle C., Harding C., Wright J. (2001) Surfactant protein D enhances bacterial antigen presentation by bone-marrow derived dendritic cells. Am J Physiol 281:L1453 - 1463.
Crouch E., Persson A., Chang D., Heuser J. (1994) Molecular structure of pulmonary surfactant protein D (SP-D). J Biol Chem 269:17311-17319.
Crouch E., Rust K., Veile R., Donis-Keller H., Grosso L. (1993) Genomic organization of human surfactant protein D (SP-D). SP-D is encoded on chromosome 10q22.2-23.1. J Biol Chem 268:2976-2983.
Crouch E., Tu Y., Briner D., McDonald B., Smith K., Holmskov U., Hartshorn K. (2005) Ligand Specificity of Human Surfactant Protein D. J Biol Chem 280:17046-17056. DOI: 10.1074/jbc.M413932200.
Deb R., Shakib F., Reid K., Clark H. (2007) Major House Dust Mite Allergens Dermatophagoides pteronyssinus 1 and Dermatophagoides farinae 1 Degrade and Inactivate Lung Surfactant Proteins A and D. J Biol Chem 282:36808-36819. DOI: 10.1074/jbc.M702336200.
Floros J., Steinbrink R., Jacobs K., Phelps D., Kriz R., Recny M., Sultzman L., Jones S., Taeusch H.W., Frank H.A. (1986) Isolation and characterization of cDNA clones for the 35-kDa pulmonary surfactant-associated protein. J Biol Chem 261:9029-9033.
Floros J., Lin H., Garcia A., Salazar M., Guo X., DiAngelo S., Montano M., Luo J., Pardo A., Selman M. (2000) Surfactant protein marker alleles identify a subgroup of tuberculosis patients in a Mexican population. J Infect Dis 182:1473 - 1478.
Forbes L.R., Haczku A. (2010) SP-D and regulation of the pulmonary innate immune system in allergic airway changes. Clin Exp Allergy 40:547-562.
Foreman M.G., Kong X., DeMeo D.L., Pillai S.G., Hersh C.P., Bakke P., Gulsvik A., Lomas D.A., Litonjua A.A., Shapiro S.D., Tal-Singer R., Silverman E.K. (2010) Polymorphisms in Surfactant Protein D are Associated with COPD. Am J Respir Cell Mol Biol:2009-0360OC. DOI: 10.1165/rcmb.2009-0360OC.
Gardai S., Xiao Y., Dickinson M., Nick J., Voelker D., Green K., Henson P. (2003) By binding SIRP or calreticulin/CD91, lung collectins act as dual function surveillance molecules to suppress or enhance inflammation. Cell 115:13 - 23.
Guo X., Lin H.-M., Lin Z., Montano M., Sansores R., Wang G., DiAngelo S., Pardo A., Selman M., Floros J. (2001) Surfactant protein gene A, B, and D marker alleles in chronic obstructive pulmonary disease of a Mexican population. Eur Respir J 18:482-490.
Hansen S., Lo B., Evans K., Neophytou P., Holmskov U., Wright J.R. (2007) Surfactant Protein D Augments Bacterial Association but Attenuates Major Histocompatibility Complex Class II Presentation of Bacterial Antigens. Am J Respir Cell Mol Biol 36:94-102. DOI: 10.1165/rcmb.2006-0195OC.
Hartshorn K., White M., Tecle T., Tornoe I., Sorensen G., Crouch E., Holmskov U. (2007) Reduced influenza viral neutralizing activity of natural human trimers of surfactant protein D. Respir Res 8:9.
Hartshorn K.L., Crouch E., White M.R., Colamussi M.L., Kakkanatt A., Tauber B., Shepherd V., Sastry K.N. (1998) Pulmonary surfactant proteins A and D enhance neutrophil uptake of bacteria. Am J Physiol Lung Cell Mol Physiol 274:L958-969.
Heidinger K., König I.R., Bohnert A., Kleinsteiber A., Hilgendorff A., Gortner L., Ziegler A., Chakraborty T., Bein G. (2005) Polymorphisms in the human surfactant protein-D (SFTPD) gene: strong evidence that serum levels of surfactant protein-D (SP-D) are genetically influenced. Immunogenetics 57:1-7.
Husby S., Herskind A.M., Jensenius J.C., Holmskov U. (2002) Heritability estimates for the constitutional levels of the collectins mannan-binding lectin and lung surfactant protein D. A study of unselected like-sexed mono- and dizygotic twins at the age of 6–9 years. Immunology 106:389-394.
Janssen W.J., McPhillips K.A., Dickinson M.G., Linderman D.J., Morimoto K., Xiao Y.Q., Oldham K.M., Vandivier R.W., Henson P.M., Gardai S.J. (2008) Surfactant Proteins A and D Suppress Alveolar Macrophage Phagocytosis via Interaction with SIRP{alpha}. Am J Respir Crit Care Med 178:158-167. DOI: 10.1164/rccm.200711-1661OC.
Kölble K., Lu J., Mole S.E., Kaluz S., Reid K.B.M. (1993) Assignment of the Human Pulmonary Surfactant Protein D Gene (SFTP4) to 10q22-q23 Close to the Surfactant Protein A Gene Cluster. Genomics 17:294-298.
Kauffman H., Tamm M., Timmerman J.A., Borger P. (2006) House dust mite major allergens Der p 1 and Der p 5 activate human airway-derived epithelial cells by protease-dependent and protease-independent mechanisms. Clin Mol Allergy 4:5.
Kishore U., Wang J.Y., Hoppe H.J., Reid K.B. (1996) The alpha-helical neck region of human lung surfactant protein D is essential for the binding of the carbohydrate recognition domains to lipopolysaccharides and phospholipids. Biochem J 318:505-511.
Krueger M., Puthothu B., Gropp E., Heinze J., Braun S., Heinzmann A. (2006) Amino acid variants in Surfactant protein D are not associated with bronchial asthma. Pediatr Allergy Immunol 17:77-81.
Kuan S.F., Rust K., Crouch E. (1992) Interactions of surfactant protein D with bacterial lipopolysaccharides. Surfactant protein D is an Escherichia coli-binding protein in bronchoalveolar lavage. J Clin Invest 90:97-106.
Lahti M., Lofgren J., Marttila R., Renko M., Klaavuniemi T., Haataja R., Ramet A.M., Hallman M. (2002) Surfactant Protein D Gene Polymorphism Associated with Severe Respiratory Syncytial Virus Infection. [Article]. Pediatric Research June 51:696-699.
Leth-Larsen R., Nordenbaek C., Tornoe I., Moeller V., Schlosser A., Koch C., Teisner B., Junker P., Holmskov U. (2003) Surfactant protein D (SP-D) serum levels in patients with community-acquired pneumonia. Clin Immunol 108:29-37.
Leth-Larsen R., Garred P., Jensenius H., Meschi J., Hartshorn K., Madsen J., Tornoe I., Madsen H.O., Sorensen G., Crouch E., Holmskov U. (2005) A Common Polymorphism in the SFTPD Gene Influences Assembly, Function, and Concentration of Surfactant Protein D. J Immunol 174:1532-1538.
Lilly C.M. (2005) Diversity of asthma: Evolving concepts of pathophysiology and lessons from genetics. J Allergy Clin Immunol 115:S526-S531.
Lim B.L., Wang J.Y., Holmskov U., Hoppe H.J., Reid K.B.M. (1994a) Expression of the Carbohydrate Recognition Domain of Lung Surfactant Protein D and Demonstration of Its Binding to Lipopolysaccharides of Gram-Negative Bacteria. Biochemical and Biophysical Research Communications 202:1674-1680.
Lim B.L., Wang J.Y., Holmskov U., Hoppe H.J., Reid K.B.M. (1994b) Expression of the Carbohydrate Recognition Domain of Lung Surfactant Protein D and Demonstration of Its Binding to Lipopolysaccharides of Gram-Negative Bacteria. Biochem Biophys Res Commun 202:1674-1680.
Lin K.-W., Jen K.Y., Suarez C.J., Crouch E.C., Perkins D.L., Finn P.W. (2010) Surfactant Protein D-Mediated Decrease of Allergen-Induced Inflammation Is Dependent upon CTLA4. J Immunol 184:6343-6349. DOI: 10.4049/jimmunol.0901947.
Liu C.-F., Rivere M., Huang H.-J., Puzo G., Wang J.-Y. (2010) Surfactant protein D inhibits mite-induced alveolar macrophage and dendritic cell activations through TLR signalling and DC-SIGN expression. Clin Exp Allergy 40:111-122.
Madsen J., Kliem A., Tornoe I., Skjodt K., Koch C., Holmskov U. (2000) Localization of Lung Surfactant Protein D on Mucosal Surfaces in Human Tissues. J Immunol 164:5866-5870.
Österlund C., Grönlund H., Polovic N., Sundström S., Gafvelin G., Bucht A. (2009) The non-proteolytic house dust mite allergen Der p 2 induce NF-kB and MAPK dependent activation of bronchial epithelial cells. Clin Exp Allergy 39:1199-1208.
Palaniyar N., Nadesalingam J., Reid K. (2003) Innate immune collectins bind nucleic acids and enhance DNA clearance in vitro. Ann N Y Acad Sci 1010:467 - 470.
Palaniyar N., Nadesalingam J., Clark H., Shih M., Dodds A., Reid K.B.M. (2004) Nucleic Acid Is a Novel Ligand for Innate, Immune Pattern Recognition Collectins Surfactant Proteins A and D and Mannose-binding Lectin. J Biol Chem 279:32728-32736. DOI: 10.1074/jbc.M403763200.
Restrepo C.I., Dong Q., Savov J., Mariencheck W.I., Wright J.R. (1999) Surfactant Protein D Stimulates Phagocytosis of Pseudomonas aeruginosa by Alveolar Macrophages. Am J Respir Cell Mol Biol 21:576-585.
Sahly H., Ofek I., Podschun R., Brade H., He Y., Ullmann U., Crouch E. (2002) Surfactant Protein D Binds Selectively to Klebsiella pneumoniae Lipopolysaccharides Containing Mannose-Rich O-Antigens. J Immunol 169:3267-3274.
Singh M., Madan T., Waters P., Parida S.K., Sarma P.U., Kishore U. (2003) Protective effects of a recombinant fragment of human surfactant protein D in a murine model of pulmonary hypersensitivity induced by dust mite allergens. Immunology Letters 86:299-307.
Sorensen G.L., Hoegh S.V., Leth-Larsen R., Thomsen T.H., Floridon C., Smith K., Kejling K., Tornoe I., Crouch E.C., Holmskov U. (2009) Multimeric and trimeric subunit SP-D are interconvertible structures with distinct ligand interaction. Mol Immunol 46:3060-3069.
Stahlman M.T., Gray M.E., Hull W.M., Whitsett J.A. (2002) Immunolocalization of Surfactant Protein-D (SP-D) in Human Fetal, Newborn, and Adult Tissues. J Histochem Cytochem 50:651-660.
Strong P., Reid K.B.M., Clark H. (2002) Intranasal delivery of a truncated recombinant human SP-D is effective at down-regulating allergic hypersensitivity in mice sensitized to allergens of Aspergillus fumigatus. Clin Exp Allergy 130:19-24.
Strong P., Kishore U., Morgan C., Bernal A., Singh M., Reid K. (1998a) A novel method of purifying lung surfactant proteins A and D from the lung lavage of alveolar proteinosis patients and from pooled amniotic fluid. J Immunol Methods 220:139-149.
Strong P., Kishore U., Morgan C., Bernal A.L., Singh M., Reid K.B.M. (1998b) A novel method of purifying lung surfactant proteins A and D from the lung lavage of alveolar proteinosis patients and from pooled amniotic fluid. J Immunol Methods 220:139-149.
Takeda K., Miyahara N., Rha Y.-H., Taube C., Yang E.-S., Joetham A., Kodama T., Balhorn A.M., Dakhama A., Duez C., Evans A.J., Voelker D.R., Gelfand E.W. (2003) Surfactant Protein D Regulates Airway Function and Allergic Inflammation through Modulation of Macrophage Function. Am J Respir Crit Care Med 168:783-789. DOI: 10.1164/rccm.200304-548OC.
Van Rozendaal B., Van de Lest C., Van Eijk M., Van Helden H., Haagsman H. (1997) Pulmonary surfactant proteins A and D are involved in the early response to intratracheally aerosolized lipopolysaccharide. Biochem Soc Trans 25:S656.
Vandivier R.W., Ogden C.A., Fadok V.A., Hoffmann P.R., Brown K.K., Botto M., Walport M.J., Fisher J.H., Henson P.M., Greene K.E. (2002) Role of Surfactant Proteins A, D, and C1q in the Clearance of Apoptotic Cells In Vivo and In Vitro: Calreticulin and CD91 as a Common Collectin Receptor Complex. J Immunol 169:3978-3986.
Vercelli D. (2008) Discovering susceptibility genes for asthma and allergy. Nat Rev Immunol 8:169-182.
Wang J.-Y., Reid K.B.M. (2007) The immunoregulatory roles of lung surfactant collectins SP-A, and SP-D, in allergen-induced airway inflammation. Immunobiology 212:417-425.
Wang J.-Y., Kishore U., Reid K.B.M. (1995) A recombinant polypeptide, composed of the [alpha]-helical neck region and the carbohydrate recogniton domain of conglutinin, self-associates to give a functionally intact homotrimer. FEBS Letters 376:6-10.
Wang J.-Y., Kishore U., Lim B., Strong P., Reid K. (1996) Interaction of human lung surfactant proteins A and D with mite (Dermatophagoides pteronyssinus) allergens. Clin Exp Allergy 106:367-73.
Wang J.-Y., Shieh C.-C., You P.-F., Lei H.-Y., Reid Kenneth B.M. (1998) Inhibitory Effect of Pulmonary Surfactant Proteins A and D on Allergen-induced Lymphocyte Proliferation and Histamine Release in Children with Asthma. Am J Respir Crit Care Med 158:510-518.
White R., Damm D., Miller J., Spratt K., Schilling J., Hawgood S., Benson B., Cordell B. (1985) Isolation and characterization of the human pulmonary surfactant apoprotein gene. Nature 317:361-3.
William J.S., Pitud A.R., Robert A.W., Don R., Sasawan C., Matthew S.P., Ginger L.C., James M.S., Elizabeth C.M., Wanda P. (2010) Pest and allergen exposure and abatement in inner-city asthma: A Work Group Report of the American Academy of Allergy, Asthma & Immunology Indoor Allergy/Air Pollution Committee. J Allergy Clin Immunol 125:575-581.
Wright J.R. (2005) Immunoregulatory functions of surfactant proteins. Nat Rev Immunol 5:58-68.
Yamazoe M., Nishitani C., Takahashi M., Katoh T., Ariki S., Shimizu T., Mitsuzawa H., Sawada K., Voelker D.R., Takahashi H., Kuroki Y. (2008) Pulmonary Surfactant Protein D Inhibits Lipopolysaccharide (LPS)-induced Inflammatory Cell Responses by Altering LPS Binding to Its Receptors. J Biol Chem 283:35878-35888. DOI: 10.1074/jbc.M807268200.
Yong S.-J., Vuk-Pavlovic Z., Standing J.E., Crouch E.C., Limper A.H. (2003) Surfactant Protein D-Mediated Aggregation of Pneumocystis carinii Impairs Phagocytosis by Alveolar Macrophages. Infect. Immun. 71:1662-1671. DOI: 10.1128/iai.71.4.1662-1671.2003.
賴梅玉(2007) SP-D基因變異性與氣喘關係。國立成功大學分子醫學研究所碩士
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