臺灣博碩士論文加值系統

本研究之目的為利用血球凝集試驗與石英晶體微天秤(Quartz Crystal Microbalance, QCM)分析重組鳳梨莖部凝集素(rAcmJRL)與經由定點突變之重組鳳梨莖部凝集素(D136V)醣類結合部位的活性變化。本研究是將鳳梨莖部凝集素(AcmJRL)基因片段建構至含有穀胱甘肽S-轉移酶 (Glutathione S-transferase, GST)的pGEX-4T-1 GST Expression Vector，並命名為 pGEX-lec，之後將其轉型至表達菌株Competent Cell BL21(DE3)。利用大腸桿菌(Escherichia coli, E. coli)大量表現rAcmJRL蛋白質後，使用Glutathione Sepharose®4B膠體將其純化，再進行濃縮，即得到具有凝集血球功能的rAcmJRL。Azarkan M.等人在 2018年文獻中指出，將AcmJRL與香蕉凝集素(Banlec)和其他三種植物凝集素進行多序列比對，結果發現和其他三種植物凝集素進行多序列比對，結果發現 和其他三種植物凝集素進行多序列比對，結果發現 和其他三種植物凝集素進行多序列比對，結果發現 AcmJRL與 Banlec具有部分非常相似的胺基酸序列，而這段胺基酸序列是屬於保守胺基酸序列位置，另外還發現AcmJRL與Banlec一樣具有兩個相似的醣結合位點。根據 Meagher J. L.等人在 2005年文獻中提到，兩個醣結合位點都含有GG環(GG loop)以及配體結合環(ligand binding loop)，而這兩個環都會與醣類做結合。因此綜合上述文獻結果，本研究將rAcmJRL中的ligand binding loop醣類結合位置進行突變。為了探討rAcmJRL之活性變化，本研究利用不同醣類進行專一性血球凝集試驗以及QCM進行分析。本研究經由實驗證明rAcmJRL與D136V蛋白質濃度為1.8g/ml，而比活性為1.11x103 HAU/mg。經由醣類專一性血球凝集試驗之結果證明rAcmJRL對Mannose、Glucose等醣類具有專一性結合，D136V也一樣對Mannose、Glucose等醣類具有專一性結合。QCM分析結果發現D136V皆對Glucose與Galatose結合能力降低。

The purpose of this study was to analyze the activity changes of the rAcmJRL and site-directed mutagenesis rAcmJRL (D136V) carbohydrate binding sites using a hemagglutination test and a quartz crystal microbalance (QCM). In this study, the AcmJRL gene fragment was constructed into pGEX-4T-1 GST Expression Vector containing glutathione S-transferase (GST), named pGEX-lec, and then transformed into Competent Cell BL21 (DE3). The rAcmJRL protein was expressed in large amounts using Escherichia coli, purified using Glutathione Sepharose® 4B colloid, and concentrated to obtain rAcmJRL having a Agglutination blood cell function. Azarkan M. et al. pointed out in the literature in 2018 that AcmJRL get on multiple sequence alignment with Banlec and three other plant lectins, and it was result found that AcmJRL and Banlec have partially very much similar amino acid sequences, and this amino acid sequence belongs to a conservaed amino acid sequence position, and AcmJRL has been in addition found to have two similar sugar binding sites as Banlec. According to Meagher J. L. et al., in the 2005 literature mentioned, both sugar binding sites contain a GG loop and a ligand binding loop, both of which bind to the sugar. Therefore, based on the results of the above literature, this study mutated the sugar binding position of the ligand binding loop in rAcmJRL. In order to investigate the changes in the activity of rAcmJRL, this study used different saccharides for specific hemagglutination test and QCM analysis. This experiment proves through experiments, the rAcmJRL and D136V protein concentrations were 1.8 g/ml, and the specific activity was 1.11g/ml, and the specific activity was 1.11g/ml, and the specific activity was 1.11g/ml, and the specific activity was 1.11g/ml, and the specific activity was 1.11 g/ml, and the specific activity was 1.11 g/ml, and the specific activity was 1.11g/ml, and the specific activity was 1.11 g/ml, and the specific activity was 1.11 g/ml, and the specific activity was 1.11g/ml, and the specific activity was 1.11g/ml, and the specific activity was 1.11 g/ml, and the specific activity was 1.11 g/ml, and the specific activity was 1.11 g/ml, and the specific activity was 1.11 g/ml, and the specific activity was 1.11g/ml, and the specific activity was 1.11g/ml, and the specific activity was 1.11g/ml, and the specific activity was 1.11g/ml, and the specific activity was 1.11g/ml, and the specific activity was 1.11g/ml, and the specific activity was 1.11g/ml, and the specific activity was 1.11 g/ml, and the specific activity was 1.11 g/ml, and the specific activity was 1.11g/ml, and the specific activity was 1.11×103 HAU/mg. Through the results of the saccharide-specific hemagglutination test, it was proved that rAcmJRL has specific binding to Mannose, Glucose and other sugars, and D136V has the same specific binding to Mannose, Glucose and the like. QCM analysis showed that D136V had a reduced ability to bind glucose to galantamine.

中文摘要…………………………………………………………………………………………………………i
英文摘要……………………………………………………………………………………………………iii
目錄……………………………………………………………………………………………………………………v
圖目錄…………………………………………………………………………………………………………vii
第一章 前言…………………………………………………………………………………………………1
第二章 方法………………………………………………………………………………………………19
2.1 AcmJRL基因提取…………………………………………………………………………19
2.1.1 PCR………………………………………………………………………………………………19
2.1.2 DNA電泳………………………………………………………………………………………20
2.1.3 PCR產物純化……………………………………………………………………………20
2.2 載體建構…………………………………………………………………………………………21
2.2.1 PCR產物與T vector黏合…………………………………………………21
2.2.2 轉型………………………………………………………………………………………………21
2.2.3 pGEX-4T-1表達載體製備…………………………………………………22
2.3 重組AcmJRL…………………………………………………………………………………22
2.3.1 菌種培養…………………………………………………………………………………22
2.3.2 質體提取…………………………………………………………………………………22
2.3.3 限制酶剪切限制酶剪切……………………………………………………23
2.3.4 膠體純化…………………………………………………………………………………23
2.4 表現載體建構……………………………………………………………………………24
2.4.1 AcmJRL與pGEX-4T-1載體黏合……………………………………24
2.4.2 轉型……………………………………………………………………………………………25
2.4.3 定序與比對定序與比對……………………………………………………25
2.4.4 菌種保存…………………………………………………………………………………26
2.5 大量表現GST融合蛋白…………………………………………………………26
2.6 蛋白質純化與濃縮…………………………………………………………………27
2.7 蛋白質電泳分析………………………………………………………………………28
2.8 蛋白質濃度測定………………………………………………………………………29
2.9 血球凝集試驗……………………………………………………………………………30
2.9.1 紅血球懸浮液製備……………………………………………………………30
2.9.2 比活性分析……………………………………………………………………………30
2.9.3 醣類專一性分析…………………………………………………………………31
2.10 QCM分析……………………………………………………………………………………32
2.10.1 醣類晶片固定化製備……………………………………………………32
2.10.2 測定………………………………………………………………………………………32
2.11 定點突變…………………………………………………………………………………33
2.11.1 設計引子……………………………………………………………………………33
2.11.2 PCR…………………………………………………………………………………………33
第三章 結果…………………………………………………………………………………………35
3.1 AcmJRL基因片段製備 基因片段製備……………………………35
3.2 重組AcmJRL製備………………………………………………………………………35
3.3 定點突變………………………………………………………………………………………36
3.4 表現GST融合蛋白……………………………………………………………………37
3.5 蛋白質濃度與活性…………………………………………………………………38
3.6 醣類專一性分析………………………………………………………………………38
3.7 QCM分析………………………………………………………………………………………38
第四章 討論……………………………………………………………………………………………39
第五章 結論……………………………………………………………………………………………41
參考文獻……………………………………………………………………………………………………42
附錄一…………………………………………………………………………………………………………67
附錄二…………………………………………………………………………………………………………69
附錄三…………………………………………………………………………………………………………72
附錄四…………………………………………………………………………………………………………74
附錄五…………………………………………………………………………………………………………77
附錄六…………………………………………………………………………………………………………78
附錄七…………………………………………………………………………………………………………79
Extended Abstract……………………………………………………………………………80

林志偉 (2009)。鳳梨莖部凝集素純化與特性分析 (碩士論文)。國立虎尾科技大學生物科研究所。
施雯齡 (2011)。利用石英晶體微天平建立蕙蘭嵌紋病毒和齒舌輪斑病毒快速檢測系統 (碩士論文)。國立虎尾科技大學生物科技研究所。
鄒啟煇 (2010)。以石英晶體微天平探討重組鳳梨莖部凝集素醣類結合活性 (碩士論文)。國立虎尾科技大學生物科技研究所。
廖秋雯 (2013)。鳳梨莖部凝集素融合蛋白功能性表現與活分析 (碩士論文)。國立虎尾科技大學生物科技研究所。
蕭文宏 (2004)。利用半導體製程製作微小化之QCM陣列生物感測器 (碩士論文)。國立交通大學奈米科技研究所。
Azarkan M., Feller G., Vandenameele J., Herman R., El Mahyaoui R., Sauvage E., Vanden Broeck A., Matagne A., Charlier P., and Kerff F., (2018) Biochemical and structural characterization of a mannose binding jacalin-related lectin with two-sugar binding sites from pineapple (Ananas comosus) stem. Sci Rep., 8, 11508.
Alencar NM., Cavalcante CF., Vasconcelos MP., Leite KB., Aragão KS., Assreuy AM., Nogueira NA., Cavada BS., and Vale MR., (2005) Anti-inflammatory and antimicrobial effect of lectin from Lonchocarpus sericeus seeds in an experimental rat model of infectious peritonitis. J Pharm Pharmacol., 57, 919-22.
Atillasoy EO., Kapetanakis A., Itzkowitz SH., and Holt PR., (1998) Amaranthin lectin binding in the rat colon: response to dietary manipulation. Mt Sinai J Med., 65, 146-53.
Abebe T., Skadsen RW., and Kaeppler HF., (2005) A proximal upstream sequence controls tissue-specific expression of Lem2, a salicylate-inducible barley lectin-like gene. Planta., 221, 170-83.
BOYD WC., and SHAPLEIGH E., (1954) Separation of individuals of any blood group into secretors and non-secretors by use of a plant agglutinin (lectin). Blood., 9, 1194-8.
Barre A., Peumans WJ., Rossignol M., Borderies G., Culerrier R., Van Damme EJ., and Rougé P., (2004) Artocarpin is a polyspecific jacalin-related lectin with a monosaccharide preference for mannose. Biochimie., 86, 685-91.
Bourne Y., Zamboni V., Barre A., Peumans WJ., Van Damme EJ., and Rougé P., (1999) Helianthus tuberosus lectin reveals a widespread scaffold for mannose-binding lectins. Structure., 7, 1473-82.
Becker B., and Cooper MA., (2011) A survey of the 2006-2009 quartz crystal microbalance biosensor literature. J Mol Recognit., 24, 754-87.
Ciopraga J., Gozia O., Tudor R., Brezuica L., and Doyle RJ., (1999) Fusarium sp. growth inhibition by wheat germ agglutinin. 1428, 424-32.
Corbeau P., Haran M., Binz H., and Devaux C., (1994) Jacalin, a lectin with anti-HIV-1 properties, and HIV-1 gp120 envelope protein interact with distinct regions of the CD4 molecule. Mol Immunol., 31, 569-75.
Corbeau P., Pasquali JL., and Devaux C., (1995) Jacalin, a lectin interacting with O-linked sugars and mediating protection of CD4+ cells against HIV-1, binds to the external envelope glycoprotein gp120. Immunol Lett., 47, 141-3.
Chisholm ST., Parra MA., Anderberg RJ., and Carrington JC., (2001) Arabidopsis RTM1 and RTM2 genes function in phloem to restrict long-distance movement of tobacco etch virus. Plant Physiol., 127, 1667-75.
Cho NJ., Frank CW., Kasemo B., and Höök F., (2010) Quartz crystal microbalance with dissipation monitoring of supported lipid bilayers on various substrates. Nat Protoc., 5, 1096-106.
Dias Rde O., Machado Ldos S., Migliolo L., and Franco OL., (2015) Insights into animal and plant lectins with antimicrobial activities. Molecules., 20, 519-41.
Dixon MC., (2008) Quartz crystal microbalance with dissipation monitoring: enabling real-time characterization of biological materials and their interactions. J Biomol Tech., 19, 151-8.
Dirri F., Palomba E., Longobardo A., Zampetti E., (2017) Measuring Enthalpy of Sublimation of Volatiles by Means of Piezoelectric Crystal Microbalances. Orig Life Evol Biosph., 47, 533-544.
Favero J., Corbeau P., Nicolas M., Benkirane M., Travé G., Dixon JF., Aucouturier P., Rasheed S., Parker JW., and Liautard JP., (1993) Inhibition of human immunodeficiency virus infection by the lectin jacalin and by a derived peptide showing a sequence similarity with gp120. Eur J Immunol., 23, 179-85.
Gardères J., Bourguet-Kondracki ML., Hamer B., Batel R., Schröder HC., and Müller WE., (2015) Porifera Lectins: Diversity, Physiological Roles and Biotechnological Potential. Mar Drugs., 13, 5059-101.
Guillot J., Guerry M., Konska G., Caldefie-Chezet F., De Latour M., and Penault-Llorca F., (2004) Modification of glycoconjugates during the carcinogenesis: the case of mammary carcinomas. Bull Cancer., 91, 141-58.
Görlach J., Volrath S., Knauf-Beiter G., Hengy G., Beckhove U., Kogel KH., Oostendorp M., Staub T., Ward E., Kessmann H., and Ryals J., (1996) Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat. Plant Cell., 8, 629-43.
Hirabayashi J., Yamada M., Kuno A., and Tateno H., (2013) Lectin microarrays: concept, principle and applications. Chem Soc Rev., 42, 4443-58.
Hirabayashi J., (2004) Lectin-based structural glycomics: glycoproteomics and glycan profiling. Glycoconj J., 21, 35-40.
Hester G., Kaku H., Goldstein IJ., and Wright CS., (1995) Structure of mannose-specific snowdrop (Galanthus nivalis) lectin is representative of a new plant lectin family. Nat Struct Biol., 2, 472-9.
Hassan MA., Rouf R., Tiralongo E., May TW., and Tiralongo J., (2015) Mushroom lectins: specificity, structure and bioactivity relevant to human disease. Int J Mol Sci., 16, 7802-38.
Huang X., Bai Q., Hu J., and Hou D., (2017) A Practical Model of Quartz Crystal Microbalance in Actual Applications. Sensors (Basel)., 17.
Kabir S., (1998) Jacalin: a jackfruit (Artocarpus heterophyllus) seed-derived lectin of versatile applications in immunobiological research. J Immunol Methods., 212, 193-211.
Koo JC., Lee SY., Chun HJ., Cheong YH., Choi JS., Kawabata S., Miyagi M., Tsunasawa S., Ha KS., Bae DW., Han CD., Lee BL., and Cho MJ., (1998) Two hevein homologs isolated from the seed of Pharbitis nil L. exhibit potent antifungal activity. Biochim Biophys Acta., 1382, 80-90.
Lannoo N., and Van Damme EJ., (2014) Lectin domains at the frontiers of plant defense. Front Plant Sci., 5, 397.
Lannoo N., Peumans WJ., and Van Damme EJ., (2008) Do F-box proteins with a C-terminal domain homologous with the tobacco lectin play a role in protein degradation in plants? Biochem Soc Trans., 36, 843-7.
Mitchell CA., Ramessar K., and O'Keefe BR., (2017) Antiviral lectins: Selective inhibitors of viral entry. Antiviral Res., 142, 37-54.
Meagher JL., Winter HC., Ezell P., Goldstein IJ., and Stuckey JA., (2005) Crystal structure of banana lectin reveals a novel second sugar binding site. Glycobiology., 15, 1033-42.
Ma QH., Tian B., and Li YL., (2010) Overexpression of a wheat jasmonate-regulated lectin increases pathogen resistance. Biochimie., 92, 187-93.
Mirmohseni A., Razzaghi MA., Pourata R., Rastgouye-Hojagan M., and Zavareh S., (2009) Selective determination of ethyl acetate, acetone, ethanol, and methyl ethyl ketone using quartz crystal nanobalance combined with principle component analysis. J Environ Sci Health A Tox Hazard Subst Environ Eng., 44, 847-53.
Marx KA., (2003) Quartz crystal microbalance: a useful tool for studying thin polymer films and complex biomolecular systems at the solution-surface interface. Biomacromolecules., 4, 1099-120.
Maglio O., Costanzo S., Cercola R., Zambrano G., Mauro M., Battaglia R., Ferrini G., Nastri F., Pavone V., and Lombardi A., (2017) A Quartz Crystal Microbalance Immunosensor for Stem Cell Selection and Extraction. Sensors (Basel)., 17.
Nikitina VE., Loshchinina EA., and Vetchinkina EP., (2017) Lectins from Mycelia of Basidiomycetes. Int J Mol Sci., 18.
Peumans WJ., and Van Damme EJ., (1995) Lectins as plant defense proteins. Plant Physiol., 109, 347-52.
Peumans WJ., and Van Damme EJ., (1998) Plant lectins: specific tools for the identification, isolation, and characterization of O-linked glycans. Crit Rev Biochem Mol Biol., 33, 209-58.
Peumans WJ., Zhang W., Barre A., Houlès Astoul C., Balint-Kurti PJ., Rovira P., Rougé P., May GD., Van Leuven F., Truffa-Bachi P., and Van Damme EJ., (2000) Fruit-specific lectins from banana and plantain. Planta., 211, 546-54.
Procek M., Stolarczyk A., Pustelny T., and Maciak E., (2015) A Study of a QCM Sensor Based on TiO₂ Nanostructures for the Detection of NO₂ and Explosives Vapours in Air. Sensors (Basel)., 15, 9563-81.
Phongphanphanee S., Yoshida N., and Hirata F., (2011) Molecular recognition explored by a statistical-mechanics theory of liquids. Curr Pharm Des., 17, 1740-57.
Qian K., Deng Q., Fang G., Wang J., Pan M., Wang S., and Pu Y., (2016) Metal-organic frameworks supported surface-imprinted nanoparticles for the sensitive detection of metolcarb. Biosens Bioelectron., 79, 359-63.
Rinderle SJ., Goldstein IJ., and Remsen EE., (1990) Physicochemical properties of amaranthin, the lectin from Amaranthus caudatus seeds. Biochemistry., 29, 10555-61.
Rinderle SJ., Goldstein IJ., Matta KL., and Ratcliffe RM., (1989) Isolation and characterization of amaranthin, a lectin present in the seeds of Amaranthus caudatus, that recognizes the T- (or cryptic T)-antigen. J Biol Chem., 264, 16123-31.
Sharma V., and Surolia A., (1997) Analyses of carbohydrate recognition by legume lectins: size of the combining site loops and their primary specificity. J Mol Biol., 267, 433-45.
Swanson MD., Winter HC., Goldstein IJ., and Markovitz DM., (2010) A lectin isolated from bananas is a potent inhibitor of HIV replication. J Biol Chem., 285, 8646-55.
Swanson MD., Winter HC., Goldstein IJ., and Markovitz DM., (2010) A lectin isolated from bananas is a potent inhibitor of HIV replication. J Biol Chem., 285, 8646-55.
Swanson MD., Boudreaux DM., Salmon L., Chugh J., Winter HC., Meagher JL., André S., Murphy PV., Oscarson S., Roy R., King S., Kaplan MH., Goldstein IJ., Tarbet EB., Hurst BL., Smee DF., de la Fuente C., Hoffmann HH., Xue Y., Rice CM., Schols D., Garcia JV., Stuckey JA., Gabius HJ., Al-Hashimi HM., and Markovitz DM., (2015) Engineering a therapeutic lectin by uncoupling mitogenicity from antiviral activity. Cell., 163, 746-58.
Subramanyam S., Smith DF., Clemens JC., Webb MA., Sardesai N., and Williams CE., (2008) Functional characterization of HFR1, a high-mannose N-glycan-specific wheat lectin induced by Hessian fly larvae. Plant Physiol., 147, 1412-26.
Shimokawa M., Haraguchi T., Minami Y., Yagi F., Hiemori K., Tateno H., and Hirabayashi J., (2016) Two carbohydrate recognizing domains from Cycas revoluta leaf lectin show the distinct sugar-binding specificity-A unique mannooligosaccharide recognition by N-terminal domain. J Biochem., 160, 27-35.
Selyanchyn R., Korposh S., Wakamatsu S., and Lee SW., (2011) Respiratory monitoring by porphyrin modified quartz crystal microbalance sensors. Sensors (Basel)., 11, 1177-91.
Speight RE., and Cooper MA., (2012) A survey of the 2010 quartz crystal microbalance literature. J Mol Recognit., 25, 451-73.
Sauerbrey G., (1959) Verwendung von Schwingquarzen zur Wägung dünner Schichten und zur Mikrowägung. Z. Phys., 155, 206-222.
Tseng MC., Chang YP., and Chu YH., (2007) Quantitative measurements of vancomycin binding to self-assembled peptide monolayers on chips by quartz crystal microbalance. Anal Biochem., 371, 1-9.
Van Holle S., De Schutter K., Eggermont L., Tsaneva M., Dang L., and Van Damme EJM., (2017) Comparative Study of Lectin Domains in Model Species: New Insights into Evolutionary Dynamics. Int J Mol Sci., 18.
Van Parijs J., Broekaert WF., Goldstein IJ., and Peumans WJ., (1991) Hevein: an antifungal protein from rubber-tree (Hevea brasiliensis) latex. Planta., 183, 258-64.
Van Damme EJ., Hause B., Hu J., Barre A., Rougé P., Proost P., and Peumans WJ., (2002) Two distinct jacalin-related lectins with a different specificity and subcellular location are major vegetative storage proteins in the bark of the black mulberry tree. Plant Physiol., 130, 757-69.
Vashist S.K., and Vashist P., (2011) Recent Advances in Quartz Crystal Microbalance-Based Sensors. J Sens., 13.
Wright CS., (1997) New folds of plant lectins. Curr Opin Struct Biol., 7, 631-6.
Yang H., and Czapla TH., Isolation and characterization of cDNA clones encoding jacalin isolectins. J Biol Chem., 268, 5905-10.
Yang Y., Tu Y., Wang X., Pan J., and Ding Y., (2015) A label-free immunosensor for ultrasensitive detection of ketamine based on quartz crystal microbalance. Sensors (Basel)., 15, 8540-9.
Yang ZP., and Zhang CJ., (2009) Mechanism and kinetics of Pb(II) adsorption on ultrathin nanocrystalline titania coatings. J Hazard Mater., 172, 1082-6.