FIP-fve 蛋白之 N 端序列對其免疫活性之必要性

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୯ҥѠ᡼εᏢғނၗྍᄤၭᏢଣ!

༜᛬ᄤඳᢀᏢࣴز܌!

ᅺγፕЎ!

Graduate Institute of Horticulture and Landscape Architecture College of Bio-Resources and Agriculture

National Taiwan University Master Thesis

FIP-fve ೈқϐ N ᆄׇӈჹځխࣝࢲ܄ϐѸा܄

N-terminal of FIP-fve is essential for its immune activity

݅ྼ࣑

Yu-Shan Lin

ࡰᏤ௲௤Ǻ೚ ᇶ റγ Advisor: Fuu Sheu, Ph.D.

ύ๮҇୯ 103 ԃ 7 Д July, 2014

ଣ

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(3)

II

ठᖴ

ҁፕЎૈ໩ճֹԋ२Ӄाགᖴৱৣ ೚ᇶ റγǴԴৣନΑӧࣴز΢ගٮΑ೚

ӭགྷݤϷБӛѦǴΨᡣᏢғԖԾךวචޑޜ໔Ǵ๏ΑᏢғؼӳޑᏢಞᆶჴᡍᕉნǴ ߚதགৱǶԜѦǴԴৣޑ၉ᇟЇᏤךॺӧ଺Γೀ٣Бय़׳ࣁԋߏǴЪாΨόבᜢ ᚶᏢғϐғࢲǴගٮ೚ӭғࢲ΢ϐڐշǴܭԜҗ૱གᖴԴৣǶ

གᖴα၂ہ঩ ڬד፵ റγǵዐ௵໢ റγǵᕯ׆╕ റγૈ୼ኘϧቩ᎙ҁፕ ЎǴ٠ܭᏢғα၂ਔ๏ϒ೚ӭᝊ຦ࡌ᝼ᆶႴᓰǴ٬ҁፕЎૈ୼׳уֹ๓ǴܭԜལ

΢၈ኑᖴཀǴགৱԴৣॺޑᔅշǶ

ᖴᖴჴᡍ࠻ޑεᏢߏדؼǴ຾ٰ΋ࣴޑѤԃਔӀόᆅࢂғࢲᗋࢂჴᡍ΢೿ڙ ډᏢߏ೚ӭྣ៝ǴЀځനࡕᇴჴᡍޑਔং੿ޑഞྠգΑࡐӭǴ׆ఈ۳ࡕԖᐒ཮ૈ

ӆکᏢߏസ΋݆ǹགᖴӭωӭ᛬λқᏢۆӧғࢲ΢೚ӭ࿶ᡍޑϩ٦аϷჴᡍ΢ޑ Ӛᅿڐշǹགᖴ߷ֻᏢߏӧ፦᛼၂ᡍޑڐշǹགᖴٌधޑҥܭᏢߏௗЋڐշჴᡍ

࠻ޑӚ໨٣୍ǴΨᖴᖴգӧჴᡍਔಠϺ࣬ՔǴઔᅽӚՏᏢߏۆ΋Ϫ໩ճǶ੝ձག ᖴगᛰৣഡӧךখ຾ჴᡍ࠻ޑਔংǴऐЈޑ௲Αך೚ӭჴᡍ٠ܫЋᡣךԾρᏹ բǴᔅշךૈזೲޑΑှჴᡍ࠻ǴԿԜך΋ޔࡐགᖴৣഡޑ௲ᏢБଞǶ΋ࣴޑᏢ ߏۆՙ௵ǵ໡൛ǵ஀ཁǵߓ۔ǵ᝵ཫǵᑗᑗکऍӵӕኬӧғࢲ΢аϷჴᡍޑᏢಞ

΢๏Α೚ӭڐշǴགᖴգॺޑбрǼᖴᖴӕۛޑᏯ϶࢙Գǵۑᓄک໡࣓Ǵࡐ໒Ј

ૈ୼Ԗգॺޑ࣬ՔᆶගᒬǴᡣךॺޑᅺγғఱ໩ճ่״ǴӚՏךॺ଺ډΑǼ੝ձ

ࢂதഉך΋ଆӧъڹಠϺ໩ߡᇴፕЎޑ໡࣓ǴߚதགᖴǶགᖴჴᡍ࠻ӳუՔػᡫǵ ᓉॕǵۗ੿Ϸ଻ሸǴԖգॺӧޑჴᡍ࠻੿ࢂзΓӼЈǴکգॺࡋၸޑҺՖᗺᅀ೿

ࢂךࣔ຦ޑӣᏫǴΨགᖴεৎޑᡣךޑғࢲԖཥޑᡏ཮Ǵᜫ౥཰ࡕ϶ፉ٩ᙑǶΠ

΋ۛёངޑࢀࢋᆶ٫ฑǴᖴᖴیॺᔅԆፕЎঅׯǴΨགᖴӧനࡕჴᡍፂڈਔයی ॺᆢ࡭ჴᡍ࠻ޑၮբ໩ճǴઔᅽیॺ (ک଻ሸ) ፕЎϷჴᡍ೿ε໩ճǼགᖴᏢ׌

ۂ╈ᑣǵຐ໩ǵৎॕǵमᇞǵ३๩ǵࡘఃǵҲૠک؊ᐇǴԖεৎӧޑჴᡍ࠻൩ࢂ

ӧࣴزࣴزࣴزࣴزࣴز΢ග΢ග΢ග΢ග΢ග΢ ٮΑٮΑٮΑٮΑΑ೚೚೚೚೚೚ ᏢಞᆶჴჴჴჴჴᡍᕉᡍᕉᡍᕉᡍᕉᡍᕉნნნნნǴ

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III

кᅈ៿ઢᖂǴᖴᖴգॺޑഉՔǼᖴᖴల۩ۆۆǵ඲ۆکᕋঢڐշεৎೀ౛ჴᡍ࠻

ޑελ٣Ǵதத٦ڙډۆۆک඲ۆޑӳЋ᛬Ǵ੿ޑߚதҗ૱གᖴǴઔᅽӚՏيᡏ

଼நǵ΋ϪѳӼǶགᖴ෌༾܌ഋ҅ৱᏢߏᆶؔ੝ථϦљഋ໩ಹλۆܭჴᡍ΢ޑࡌ

᝼ᆶڐշǴऩؒԖգॺ೭ҁፕЎёૈᗋӧьᜢ྽ύǶќѦǴΨགᖴჴᡍύ឴࣊ޑ λႵǴܭԜԿ΢നుޑལཀǶ

གᖴඁᝑǵECHOǵ൛ဖǵऔݿǵ࿧໋೭΋ၡ΢ٰޑഉՔǴܤᄹததᡣգॺ

᠋ךӗधНǴଽᅟޑ࣬ᆫᡣך׳кᅈ߿਻ޑᝩុوΠѐǴᜫ௃ፉߏӧǶ

གᖴݿǵ༰ǵঢঢکۂۂӧ೭൳ԃٰޑഉՔᆶх৒Ǵᡣךคࡕ៝ϐኁޑֹԋ ΑᅺγᏢᐕǴଽᅟჴᡍϼఁഞྠգॺௗଌǴ܈ࢂЈ௃ݪ഼ਔգॺޑངᆶᜢᚶǴ೿

ࢂᡣךᝩុոΚ߻຾ޑΚໆǴགᖴգॺǼᖴᖴᜢЈךޑߏ፸ॺаϷ݅ৎл׌ۊ ۂǴයఈૈόູॄգॺයࡑޑᝩុوӛΓғΠঁ໘ࢤǶ

നࡕǴགᖴዐৎѶ΋ၡ΢ޑЍ࡭ǴऩؒԖգךёૈ཮ᒱၸ຾Ε΋ࣴ೭ঁྕធ εৎ৥ޑᐒ཮ǴΨёૈӧъ೼ԶቲǵคݤڗளᅺγᏢᐕǶ೭٤ԃǴჴᡍ΢ᆶғࢲ

΢ޑྣ៝೿གᐟܭЈǴᜫ҂ٰךॺ೿ૈᝩុԋߏǴ΋ଆ΋ޔՉǵӛ߻ՉǼઔᅽऍ ӳޑ҂ٰǶ

ᙣܭԜϩ٦ҁፕЎ๏܌ԖޑৎΓᆶܻ϶Ǵ᝘΢ךന၈ኑޑઔᅽǶ

݅ྼ࣑ ᙣᇞܭ ୯ҥѠ᡼εᏢ༜᛬ᄤඳᢀᏢࣴز܌

ύ๮҇୯ 103 ԃ 7 Д շεৎεৎεৎεৎৎೀ౛ೀ౛ೀ౛ೀ౛ೀ౛౛ჴᡍჴᡍჴᡍჴᡍჴᡍჴᡍ࠻࠻࠻࠻࠻

ᖴ ᖴ ᖴ

ᖴǴઔǴઔǴઔǴઔǴઔઔᅽӚᅽӚᅽӚᅽӚᅽӚՏيՏيՏيՏيՏيᡏᡏᡏᡏᡏᡏ ۆܭ

ۆܭ ۆܭ ۆܭ

ۆܭܭჴᡍჴᡍჴᡍჴᡍჴᡍ΢ޑ΢ޑ΢ޑ΢ޑ΢ޑࡌࡌࡌࡌࡌࡌ ᖴჴჴჴჴჴᡍύᡍύᡍύᡍύᡍύ឴࣊឴࣊឴࣊឴࣊឴࣊ޑޑޑޑޑ

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IV

ᄔा

ߎଞ⏯խࣝፓ࿯ೈқ FIP-fve ๧ڗԾߎଞ⏯ηჴᡏǴЎ᝘ࡰрځڀԖխࣝፓ

࿯ǵלᕎǵלၸ௵Ϸ෧጗วݹϸᔈ฻фਏǶΒભ่ᄬϩ݋ᡉҢǴFIP-fve ೸ၸٿ

ঁൂϡᡏ N ᆄ Į-helix а౧Н܄ᗖ่׎ԋᚈϡᡏǶҁჴᡍ࠻߻Γࣴزࡰр FIP- fve όሡ࿶ၸלচև᝘ಒझঅႬǴԶࢂ೸ၸᆶ MHC Ϸ TCR ϩηᗖ่ࢲϯ T

ಒझǴӢԜ௢ෳFIP-fve բҔБԄᜪ՟ຬלচǶҁࣴزҞޑԖΒǴ΋ࣁ᛾ჴᚈϡ ᡏ่ᄬჹܭ FIP-fve ޑխࣝፓ࿯ࢲ܄ࢂѸाޑǴΒࣁפр FIP-fve ᆶ MHC ک TCR ڀҬϕբҔϐ᛾ᏵǶࣁ௖૸ᚈϡᡏ่ᄬჹ FIP-fve խࣝፓ࿯ࢲ܄ϐቹៜǴ ஒ 1-13 ữ୷ለલѨ܈ӄߏ FIP-fve cDNA ᄬᑐܭ pET-32a(+) ၩᡏǴ٠೸ၸεဉ ఎ๵س಍߄౜ளϩηໆࣣऊ 26 kDa ϐ His-FIP-fve 1-114 ک His-FIP-fve 14-114

ೈқǶа enterokinase Ϫନ His-tag ࡕǴ࿶፦᛼Кჹዴᇡԋфڗள rFIP-fve 1-114 аϷ rFIP-fve 14-114Ƕխࣝፓ࿯ࢲ܄၂ᡍБय़ǴТࢤલѨ rFIP-fve 14-114 ڈᐟλ Ⴕ๠᠌ಒझϩݜ IFN-Ȗ ૈΚၨ rFIP-fve 1-114 ১ǴЪคݤڈᐟλႵ๠᠌ಒझ IFN-Ȗ Ϸ IL-2 ୷Ӣ߄౜Ƕᆕӝа΢่݀Ǵ௢ෳѨѐᚈϡᡏ่ᄬ೷ԋ FIP-fve խ

ࣝፓ࿯ࢲ܄फ़եǶӧ FIP-fve ᜪຬלচ܄፦௖૸΢Ǵ೸ၸӅӕխࣝ؈ᐘݤёޕ FIP-fve ᆶ߄य़ϩη MHC class II ڀҬϕբҔǴќѦว౜ FIP-fve ё೏ MHC class I Ϸځ Isotype IgG2a לᡏਂਆǶฅԶǴҁࣴزۘ҂೸ၸӅӕխࣝ؈ᐘݤ࣮

ـ FIP-fve ᆶ TCR ೱ่ϐ᛾ᏵǴӢԜ໻ૈ᛾ჴ FIP-fve ૈೱ่ APCs ߄य़ϩ η MHC class IIǶ

ᜢᗖӷǺຬלচǵHis ᑼӝೈқǵӕࠠᚈϡᡏǵխࣝፓ࿯ೈқǵӅӕխࣝ؈ᐘ

ࡰррррځڀځڀځڀځڀځڀڀԖխԖխԖխԖխԖխԖխࣝፓࣝፓࣝፓࣝፓࣝፓࣝፓ

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V

Abstract

FIP-fve, an immunomodulatory protein isolated from Flammulina velutipes, has

been demonstrated to have activities of immunomodulatory, anti-cancer, anti-allergic

and suppression of inflammation. Based on a secondary structure analysis, we known

that two FIP-fve monomers form homodimer through the hydrophobic interaction

between N-terminal Į-helics. Previous studies suggested that FIP-fve can mediate T

cells activation through the linkage between T cell receptors (TCR) on T cells and MHC

molecules on antigen present cells (APCs) without processing procedure of APCs. This

process is similar to the mechanism of superantigen-mediated immune response. The

aim of this study was to demonstrate the necessity of dimer structure for FIP-fve

immunomodulatory activity and to prove the directly binding of FIP-fve with MHC and

TCR. To study the relationship between the dimer structure and immunomodulatory

activity of FIP-fve, we constructed the full length 1-114 FIP-fve cDNA and truncated

14-114 FIP-fve cDNA on pET-32a(+) vector and expressed the His-fusion protein His-

FIP-fve 1-114 and His-FIP-fve 14-114. After digested by enterokinase to remove the

His-tag residue, rFIP-fve 1-114 and rFIP-fve 14-114 were purified with FPLC system.

The ability of rFIP-fve 14-114 to stimulate IFN-Ȗ production on murine splenocytes was

lower than rFIP-fve 1-114. Incubating murine splenocytes with rFIP-fve 14-114 could ina vvvvvelelelelelututututuipipipippeseses,,,,,hahahahahasssss

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VI

not elevate the gene expression of IFN-Ȗ and IL-2. According to the above results, we

suggested that the dimerization might be critical for FIP-fve immunomodulatory

activity. On the other hand, FIP-fve and RAW 264.7 cell lysate were co-

immunoprecipitated to determine superatigen-like characterization of FIP-fve. Results

indicated that FIP-fve could interact with MHC class II molecule directly. Interestingly,

we also found that FIP-fve was pulled down by both anti-MHC class I antibody and

IgG2a which was isotype of anti-MHC class I antibody. However, no co-

immunoprecipitated evidence showed that FIP-fve bound with TCR in this study. In

conclusion, we proved that FIP-fve directly bind with MHC class II molecule.

Key words: superantigen, His-fusion protein, homodimer, immunomodulatory protein,

co-immunoprecipitation

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VII

Ҟᒵ

α၂ہ঩཮ቩۓਜ... I

ठᖴ... II

ᄔा... IV

Abstract ... V

Ҟᒵ... VII

კҞᒵ... IX

ᕭቪ߄... X

ಃ΋ക ߻Γࣴز... 1

ಃ΋࿯! ߎଞ⏯ᙁϟ ... 1

ಃΒ࿯! ځдߎଞ⏯ԋϩᆶфਏϟಏ ... 2

ಃΟ࿯! ߎଞ⏯խࣝፓ࿯ೈқ FIP-fve ᙁϟ ... 5

ಃѤ࿯! ຬלচᙁϟ ... 10

ಃΒക! ࣴز୏ᐒᆶҞޑ... 12

ಃΟക! ׷਑ᆶБݤ... 13

ಃ΋࿯! ߎଞ⏯խࣝፓ࿯ೈқ FIP-fve પϯ ... 14

ಃΒ࿯! ᡂ܄ጤᡏႝݚϩ݋ (SDS-PAGE) ... 17

ಃΟ࿯! ೈқ፦ᔸԄᙯӑ ... 19

ಃѤ࿯! ՋБᙯᅄݤ (western blotting) ... 20

ಃϖ࿯! ख़ಔೈқ rFIP-fve 1-114 Ϸ truncated rFIP-fve 14-114 ୷Ӣᒧ෗ᆶᄬᑐ ... 21

ಃϤ࿯! ख़ಔೈқ rFIP-fve 1-114 Ϸ truncated rFIP-fve 14-114 ߄౜ᆶપϯ ... 25

ಃΎ࿯! ጤᡏϣНှ (In gel digestion) ... 27

ಃΖ࿯! λႵ๠᠌ಒझڗள ... 28

ಃΐ࿯! ሇનೱ่խࣝ֎ߕϩ݋ (Enzyme-linked immunosorbent assay, ELISA) ... 30

ಃΜ࿯! ಒझᕴ RNA ܜڗᆶϸᙯᒵ ... 31

ಃΜ΋࿯! ջਔۓໆᆫӝ䁙᜘ᙹϸᔈ (Real-time PCR) ... 32 ...IIII

II

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VIII

ಃΜΒ࿯! ѮᏘಒझਲ਼ RAW 264.7 ಒझ୻Ꭶ ... 34

ಃΜΟ࿯! Ӆӕխࣝ؈ᐘݤ (Co-immunoprecipitation) ... 35

ಃΜѤ࿯! ಍ीϩ݋ ... 38

ಃѤക ࣴز่݀... 39

ಃ΋࿯! ख़ಔೈқ rFIP-fve 1-114 Ϸ truncated rFIP-fve 14-114 ᒧ෗ᆶપϯ ... 39

ಃΒ࿯! ख़ಔೈқ rFIP-fve 1-114 Ϸ truncated rFIP-fve 14-114 ܭλႵ๠᠌ಒझϐխ ࣝࢲ܄ ... 42

ಃΟ࿯! аӅӕխࣝ؈ᐘݤ௖૸ FIP-fve ϐᜪຬלচ੝܄... 43

ಃϖക! ૸ፕ... 46

ಃ΋࿯! ख़ಔೈқ rFIP-fve 1-114 Ϸ truncated rFIP-fve 14-114 ೈқપϯ௖૸ ... 46

ಃΒ࿯! ТࢤલѨ truncated rFIP-fve 14-114 ೈқфૈ௖૸ ... 47

ಃΟ࿯! FIP- fve ᆶ߄य़ϩηᒃک܄ϐ௖૸ ... 48

ಃϤക! ่ፕᆶ҂ٰ৖ఈ... 51

ୖԵЎ᝘... 52

... ... ... 3434343434 ... ...3535353555 ...33338888 ...33333399999

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IX

კҞᒵ

კ΋ǵߎଞ⏯խࣝፓ࿯ೈқ FIP-fve ϐપϯ ... 58

კΒǵᄬᑐ pET-32a(+)-fve 1-114 Ϸ pET-32a(+)-fve 14-114 ߄౜ၩᡏ ... 59

კΟǵfve 1-114 ᆶ fve 14-114 Їηᘉቚ DNA ౢނႝݚკ ... 60

კѤǵpGM-T-fve 1-114ǵpGM-T-fve 14-114 ᆶ pET-32a(+) ज़ڋ⅋ϸᔈౢނ .. 61

კϖǵғౢ His-FIP-fve 1-114 ന٫ᇨᏤਔ໔ᆶᐚࡋϐՋБᙯᅄ่݀ ... 62

კϤǵख़ಔೈқ His-FIP-fve 1-114 ϐપϯ ... 63

კΎǵख़ಔೈқ His-FIP-fve 14-114 ϐપϯ ... 64

კΖǵа enterokinase Ϫନ His-FIP-fve 1-114 Ϸ His-FIP-fve 14-114 ϐ His-tag ౢނϩ݋... 65

კΐǵrFIP-fve 1-114 аϷ rFIP-fve 14-114 ϐՋБᙯᅄϩ݋ ... 66

კΜǵrFIP-fve 1-114 аϷ rFIP-fve 14-114 ፦᛼ϩ݋ೈқׇӈКჹ่݀ ... 67

კΜ΋ǵrFIP-fve 1-114 аϷ rFIP-fve 14-114 ڈᐟλႵ๠᠌ಒझϩݜ IFN-Ȗ ϐਏ ݀... 68

კΜΒǵrFIP-fve 14-114 คݤڈᐟλႵ๠᠌ಒझ IFN-Ȗ ک IL-2 ୷Ӣ߄౜ ... 69

კΜΟǵFIP-fve ᆶ߄य़ϩη MHC class II ԖҬϕբҔ ... 70

კΜѤǵFIP-fve ᆶ߄य़ϩη TCR VĮ ҂ـԖҬϕբҔ ... 71

კΜϖǵFIP-fve ᆶ߄य़ϩη TCR Vȕ ҂ـԖҬϕբҔ ... 72

კΜϤǵλႵ๠᠌ಒझઇ࿗నᆶ FIP-fve Ӆӕխࣝ؈ᐘౢނ SDS-PAGE ϩ݋კ ... 73

კΜΎǵλႵ๠᠌ಒझઇ࿗నᆶ FIP-fve Ӆӕխࣝ؈ᐘౢނ፦᛼ϩ݋Кჹ่݀ ... 74

...555555888888

౜ၩᡏ 55559

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X

ᕭቪ߄

Abbreviations

APC antigen presenting cell Bcl-xL B-cell lymphoma-extra large BSA bovine serum albumin

DC dendritic cell

FIP fungal immunomodulatory proteins

hPBMC human peripheral blood mononuclear cells

IFN-Ȗ Interferon gamma

IL-2 Interleukin-2

LC-MS liquid chromatography–mass spectrometry MAPK mitogen-activated protein kinase

MHC major histocompatibility complex OVA ovalbumin TCR T cell receptor

T-bet T-box transcription factor

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1

ಃ΋ക ߻Γࣴز

ಃ΋࿯! ߎଞ⏯ᙁϟ

⏯ᜪӢᔼᎦሽॶཱུଯǴЪڀᐱ੝ᗲऍ॥ښکαགǴԾђаٰջբࣁख़ा१׷

٬ҔǴԶ߈ԃٰ१Ҕ⏯ᜪӢځΟεᐒૈǺᔼᎦ੝܄ǵ༐ӳ੝܄аϷғނࢲ܄Զڙ ᢋҞǶӧᔼᎦ੝܄Бय़Ǵ⏯ᜪύНϩ֖ໆऊ 80-90%Ǵೈқ፦ᆶᗐᜪ֖ໆৡ౦ၨ

εǴεठ΢ёϩࣁ൤ܭೈқ፦ (20-50%)ǵ൤ܭᗐᜪ (40-70%) аϷٿԋϩӚъޑ

⏯๵ΟᅿǶ⏯ᜪޑᗐ፦аੇᝯᑗ (trehalose)ǵҒ៛ᎇǵߓ܎դᗐᎇ฻Γᜪဉၰၨ

ᜤ֎ԏϐեϩηᗐǴԶќ֖Ԗ ȕ -ဟᆫᑗک൳΍፦฻ό੃ϯ܄ᑧ१ᠼᆢǴӢԜឦ ܭ዗ໆၨեϐ१ࠔǶԪҽ֖ໆऊӧ 10% аΠǴԶિ፦֖ໆཱུե (1-8%)Ǵӭࢂа όႫکિެለࣁЬǶନ΢ॊගډ֖ԖӭᗐǵΟ亻ᜪکೈқ฻ࢲ܄ԋϩѦǴ⏯ᜪύ

֖Ԗᙦ൤ޑᆢғનB1ǵᆢғન B2ǵ๨ᡵለǵғނનаϷ࿶ഝفڰᎇᙯඤϐᆢғ ન D2ǴёբࣁᔼᎦံкϐٰྍǶ൩༐ӳ੝܄ٰᇥǴ⏯ᜪޑ॥ښٰԾܭԋϩύޑ GMPǵAMP ฻ਡ㧿ለаϷᎽữለǵ᝴݀ለ฻ǴќΨԖឦܭ؂ᅿ⏯ᜪᐱԖޑ३ښ ӵ݊૙ޑ݊૙ᎇǵ३⏯ଳᔿࡕғԋޑ३⏯ᆒ฻Ƕ⏯ᜪޑαག߾ٰԾҗ൳΍፦کဟ ᆫᗐಔԋޑಒझᏛǴόӕ⏯ᜪಒझᏛಔԋόӕǴӢԶ೷൩Αᐱ੝ޑ೬ૄαགǶ߈ ԃٰǴ೚ӭࣴزࡰр⏯ᜪύӭᗐ (polysaccharide)ǵΟ亻ᜪ (triterpene) کೈқڀ ԖלဍዦǵխࣝቚமǵלวݹǵᗉխЈՈᆅ੯ੰ฻ࢲ܄ǴӢᙴᕍᆶᛰҔфਏ٬⏯

ᜪڀԖଯޑ࿶ᔮሽॶ (Нഁڑǵοӝ҅ϢǴ1997)Ƕ

ߎଞ⏯ (Flammulina velutipes) ΞӜߎ⏯ǵО⏯ǵо⏯ǵ้࢓ߎᒲ⏯ǴमЎ߫

ӜGolden needle mushroomǴ܈ᆀ Enoki mushroomǴࣁதـ१Ҕ⏯ϐ΋Ǵځϩᜪ

΢ ឦ ܭ ੿ ๵ ߐ (Eumyeota) ǵ ᏼ η ๵ ٥ ߐ (Basidiomycotina) ǵ ቫ ๵ ᆜ (Hymenomycetes)ǵӕᏼη๵٥ᆜ (Homobasidiomycetes)ǵദ๵Ҟ (Agaricales)ǵ

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α᝱ࣽ (Tricholomataceae)ǵλО⏯ឦ (Flammulina) (ယǴ2006)Ƕߎଞ⏯ηჴᡏύ

֖Ԗ 89.8% НϩǴԶଳख़ύ֖ 58.0% ᅹНϯӝނǵ27.5% ಉೈқ፦ǵ7.4% Ԫ ϩаϷ 7.0% િ፦ (Ko, et al., 2007)Ƕữ୷ለಔԋ΢Ǵ؂լߎଞ⏯а֖Ԗ 30 డ լ lysine ک 28 լ glycine ٿޣࣁനӭǹ᝜ނ፦֖ໆ߾ࢂаႇ֖ໆനଯǴځԛࢂ

ᕗǵᗔ (Smiderle, et al., 2008)Ƕ

ಃΒ࿯! ځдߎଞ⏯ԋϩᆶфਏϟಏ

߈ԃٰ೚ӭߎଞ⏯ࢲ܄ނ፦࣬ᝩ೏ว౜ǴڀԖלᕎǵխࣝፓ࿯฻ߥ଼фਏǴ хࡴо⏯ન (flammulin)ǵלੰࢥೈқǵਡᑗᡏѨࢲೈқکߎଞ⏯ӭᗐ฻ǶԜѦǴ ߎଞ⏯η१ᡏ๧ڗނО⏯ಒझࢥન (flammutoxin, FTX) ߾ࢂ΋ڀԖಒझྋှբ ҔϐࢥೈқǶ

2.1. о⏯ન

о⏯નࣁ΋Ծߎଞ⏯ηჴᡏаН๧БԄڗளϐೈқǴനԐࢂҗ Komatsu ฻ Γ ܭ 1963 ԃ а ౷ ለ ሓ ؈ ᐘ ǵ Ό ᎇ ϩ ϯ ࡕ ӆ ࿶ DEAE-Sephadex ک CM- Sephadex ฻഍໚ᚆηቫ݋ᆅࢊપϯԶள (Komatsu, et al., 1963)Ƕځ؈ᐘதኧࣁ 2.5 SǴ฻ႝᗺ 8.9Ǵҗ SDS-PAGE ϩ݋ϩηໆࣁ 23.7 kDa (Zhang, et al., 1999)Ǵ Զ 2003 ԃԖᏢޣճҔႝቔ᠀ᚆηϯ፦᛼ (ESIMS) ׳ᆒዴޑीᆉځϩηໆࣁ 19891.13 Da (ڬ฻ΓǴ2003)Ƕо⏯નޑữ୷ለಔԋςϩ݋ֹԋǴаЧữለ (alanine)

֖ໆനଯԶό֖Ҙ౷ữለ (methionine) (ڬ฻ΓǴ2003)Ƕо⏯ન೏ᇡࣁࢂڀԖל ᕎૈΚϐೈқǴځё׭ڋλႵԺዦ S180 (sarcoma 180) ϷՆМဎНዦ (Ehrlich ascites tumor) ϐғߏǴගϲᑡᕎλႵӸࢲ౗Ǵ٠ቚуᑡᕎλႵޑᏔ۔ख़ໆǴᡉҢ о⏯નڀԖלᕎޑወΚǶќԖࣴزࡰрǴо⏯નޑ N ᆄׇӈᆶ෌ނਡᑗᡏѨࢲ

ೈқ (ribosome-inactivating proteins, RIPs) ࣬՟ǴΨࢂ೏ຎࣁڀԖלᕎǵלੰࢥ ߎ

ߎଞ⏯ଞ⏯ଞ⏯ଞ⏯ଞ⏯ηჴηჴηჴηჴηჴηჴᡏύᡏύᡏύᡏύᡏύ

ೈ

ೈқ፦қ፦қ፦қ፦қ፦қ፦ǵǵǵǵǵ7.7.7.7.7.4%4%4%4%4% ԪԪԪԪԪԪ ଞ⏯а⏯а⏯а⏯а⏯аа֖Ԗ֖Ԗ֖Ԗ֖Ԗ֖Ԗ 3333300000 డడడడడడ

֖ໆനനനനനଯǴଯǴଯǴଯǴଯǴځԛځԛځԛځԛځԛࢂࢂࢂࢂࢂ

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3

ޑёૈ᛾Ᏽϐ΋ (Wang and Ng, 2000)Ƕ

2.2. О⏯ࢥન

О⏯ࢥન๧ڗԾߎଞ⏯ηჴᡏǴځϩηໆऊࣁ 31 kDaǴ฻ႝᗺࣁ 5.4Ǵӧữ

୷ለಔԋύԖεໆޑՅ਽ለǴํ਽ለکҒ਽ለ (Bernheimer and Oppenheim, 1987)Ƕ 1974 ԃǴ Lin ი໗а 60% Ⴋک౷ለሓܭߎଞ⏯ηჴᡏઇ࿗నύᡶ݋рО⏯ࢥ નǴԜი໗ӧ߻΋ԃܭ૛⏯ (Volvariella volvaceae) ύપϯр΋ࢥೈқ (Lin, et al., 1973)ǴӢԜӕኬܭ NMR 1 ࠔسλႵဎ๚ύݙ৔О⏯ࢥનᔠෳځࢥ܄Ǵ٠ளډ LD50 ࣁ 2.45 mg/kgǴόၸО⏯ࢥનϐࢥ܄ё࿶ 100ÛC у዗ೀ౛ࡕֹӄѐନǴ Ъ٠คαܺࢥ܄ǹќѦӧࡕុࣴزύว౜О⏯ࢥનځᎩ܄፦Ǵх֖Ǻ1. ܭᐚࡋ 25 ȝg/mL ਔёྋှΓᜪ O ࠠआՈౚǴԶ້ᚆηӧғ౛ᐚࡋਔёа೽ϩ଍ᙯྋ ՈբҔǶ2. ೷ԋλႵֽ೽Нဍ܈Ӣੵภౢғסᡏϸᔈ (writhing reaction)ǴฅԜϸ ᔈё೽ϩ೏לಔᙃữᛰ׭ڋǶ3. ׭ڋՆМဎНዦಒझޑڥ֎Ъ೷ԋಒझဍ๞ǴԶ

້ᚆηӕኬૈ׭ڋО⏯ࢥન೷ԋޑՆМဎНዦಒझڥ֎ೲ౗Πफ़ (Lin, et al., 1975; Lin, et al., 1974)Ƕ

1998 ԃǴВҁ Tomita ი໗ుΕ௖૸О⏯ࢥનྋՈᐒڋǴว౜О⏯ࢥન཮Ӄ ЇଆΓᜪआಒझ᏾๞ࡕԶྋՈǴ٬आՈౚႇᚆηѦࢬǺќѦΨว౜࿶О⏯ࢥનೀ

౛ϐआՈౚӧႝηᡉ༾᜔ᢀჸΠǴёܭಒझ߄य़р౜Ѧ৩ 10 nmǵϣ৩ 5 nm ϐ ᕉރ่ᄬǴӢԜᇡࣁО⏯ࢥન೸ၸܭಒझ߄य़׎ԋ΋ঁᕉރޑեᆫނǴ٠ౢғ 4

²5 nm ޑᒃН܄ϾࢰӢԶ٬आՈౚྋှ (Tomita, et al., 1998)Ƕ1999 ԃǴWatanabe

฻Γᒧ෗р΋ႣෳϩηໆελᆶО⏯ࢥન࣬ӕϐ cDNA Тࢤ (Watanabe, et al., 1999)ǴԶ Tomita ฻ΓஒϐᆶО⏯ࢥન N ᆄ୷Ӣۓׇ่݀КჹǴዴᇡᒧ෗рޑ

ࢂ֖Ԗ 272 ữ୷ለޑО⏯ࢥન cDNA ТࢤǴ٠ғౢख़ಔО⏯ࢥન (rFTX)ǶԜ ѦǴдॺว౜೸ၸ፦᛼ۓׇڗளϐữ୷ለׇӈ໻Ԗ 251 ঁǴᆶ୷Ӣᒧ෗่݀ள 272 ঁ ่ ݀ ό ӕ Ǵ Ӣ Ԝ ғ ౢ Α લ Ю N ᆄ Met ک C ᆄ 20 ঁ ữ ୷ ለ ϐ

ႝᗺᗺࣁࣁࣁࣁࣁ 55555.4.4444ǴӧǴӧǴӧǴӧǴӧữữữữữ

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4

rFTX251 аϷӄߏ rFTX271Ǵว౜ rFTX251 ٩ฅڀԖྋՈૈΚǹฅԶ rFTX271 Ӣ׎ԋᚈϡᡏԶคݤܭՈౚ΢׎ԋϾࢰǴऩሡࡠൺྋՈૈΚ߾ሡ٬ҔીೈқϪନ

ಃ 252 ঁữ୷ለаࡕޑТࢤǴӢԜᇡࣁО⏯ࢥનচࢂᚈϡᡏ่ᄬǴฅӢಃ 251 ঁ ữ୷ለ Lys ک 252 ঁữ୷ለ Met ϐೱ่ჹીೈқଯࡋ௵གǴܰ೏Нှԋѝ֖

251 ঁữ୷ለϐൂᡏО⏯ࢥનǴӢԜڀԖྋՈࢥ܄ (Tomita, et al., 2004)ǶҞ߻О

⏯ࢥનϐࣴزۘ҂ឍܴځ C ᆄ 20 ঁữ୷ለӵՖ٬ϐ׎ԋᚈϡᡏǴԜ೽ϩԖࡑ

׳຾΋؁௖૸ǹԶО⏯ࢥનૈගϲဉၰಒझ CaCo-2 ޑಒझጢ೯೸܄ǴڀԖ໒ว ԋᛰࠔ֎ԏᏊϐወΚ (Watanabe et al., 1999)Ƕ

2.3. ߎଞ⏯ӭᗐ

ߎଞ⏯ӭᗐЬाٰԾܭηჴᡏᆶ๵ํᡏޑН๧ނǴѳ֡ϩηໆऊӧ 50~200

࿤໔ǴЬԾߎଞ⏯ύ๧ڗϐӭᗐᅿᜪᕷӭǴаԋҽ΢ٰ࣮Ǵёϩࣁೈқ่ӝӭᗐǵ аȕ -(1ʈ3)-D ဟᆫᗐࣁ่ᄬϐӭᗐǾ฻Ƕ

1968 ԃ Kamasuka ฻Γଞჹӭᅿൂη๵ߐ੿๵๧ڗӭᗐǴԶߎଞ⏯ӭᗐ (F.

velutipes polysaccharide, FVP) ΨܭԜਔ२ࡋ೏પϯԶр (Kamasuka, et al., 1968)Ǵ

ഌុ೏પϯрٰޑߎଞ⏯ӭᗐ߾ᗋԖ EA3ǵEA5ǵEA6ǵFVP1ǵFVP2Ǿ฻Ƕӧ EA3

ޑࣴزύǴIkekawa ೸ၸለНှ (acid hydrolysis) کΌㄽНှ (acetolysis) ฻ϯ ᏢБݤϩ݋ EA3Ǵว౜ځࣁ ȕ -(1ʈ3)-D ဟᆫᗐǴЪᕴᡏ֖ 92.5% ဟ๻ᑗǴڀ Ԗؼӳޑלᕎࢲ܄ (Ikekawa, et al., 1969; Ikekawa, et al., 1973; Yoshioka, et al., 1973)ǹEA5 ࢂҗ 45% ဟ๻ᑗǵ32% ъ٢ᑗǵ13.4% Ғ៛ᑗаϷ 7.2% ޑߓ܎

դᑗ܌ಔԋǴ຾΋؁೸ၸ concanavalin A-Sepharose 4B ᒃک܄ᆅࢊჄϩ EA5Ǵё ளόӕಔԋϐ EA500ǵEA505 ک EA510Ǵว౜ဟ๻ᑗ֖ໆനଯϐ EA500 Ԗനӳޑ לᕎࢲ܄ǴӢԜӆ೸ၸ Sephadex G-200 ᆅࢊӆԛჄϩ EA500 ள EA501ǵEA502ǵ EA503 ΟᅿӭᗐǴԶځύϩηໆനεޑ EA501 Ԗനӳޑלᕎࢲ܄Ǵځಔԋࣁ 42.3% ဟ๻ᑗǵ17.3% ъ٢ᑗǵ12.2%Ғ៛ᑗǵ6.7% ЕᑗаϷ 14.7% ߓ܎դᑗ ΚǹฅฅฅฅฅԶԶԶԶԶ rrrrrrFTFTFTFTFTFTX2X2X2X2XX 7171717171

٬Ҕ

٬ҔҔીೈીೈીೈીೈીೈқϪқϪқϪқϪқϪନନନନନନ ᄬǴ

ᄬǴ ᄬǴ ᄬǴ

ᄬǴฅӢฅӢฅӢฅӢฅӢฅӢಃಃಃಃಃ22222511111ঁঁঁঁঁঁ

ܰ೏೏೏೏೏НှНှНှНှНှԋѝԋѝԋѝԋѝԋѝ֖֖֖֖֖

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(Ikekawa, et al., 1982)Ƕ

1983 ԃǴOtagiri வߎଞ⏯ηჴᡏН๧ளќ΋ೈқ่ӝӭᗐ٠ڮӜࣁ EA6Ǵ

೸ၸаݙ৔ࣝभࡕӆ๏ϒ EA6 ޑ၂ᡍǴว౜ځԖշܭۯߏ L1210 ՈᕎኳԄύ ᑡᕎλႵޑტڮǴЪ٠όቹៜλႵխࣝس಍ύჹL1210 ᕎಒझޑ੝౦܄ (Otagiri, et al., 1983)ǹќѦǴIkekawa Ψว౜ᓉેݙ৔ EA6 ᆶԲआՈౚಒझ (sheep red blood cell) ܭ ICR λႵǴڗр๠᠌ಒझ೸ၸྋՈჴᡍϩ݋ว౜ EA6 ёගϲלԲ

आՈౚ IgM לᡏౢғǴ٠׭ڋݙ৔ԲआՈౚಒझ܌ЇଆޑλႵىඓဍ๞

(Ohkuma, et al., 1982)Ƕᆕӝа΢่݀Ǵளޕ EA6 ࣁ΋ڀלᕎǵխࣝፓ࿯Ϸלว ݹϐೈқ่ӝӭᗐǶ

ಃΟ࿯! ߎଞ⏯խࣝፓ࿯ೈқ FIP-fve ᙁϟ

2.1. FIP ৎ௼ϟಏ

⏯ᜪύխࣝፓ࿯ೈқ (immunomodulatory protein) നԐว౜ܭ 1989 ԃǴࢂ

җВҁᏢޣԾᡫ޲ύપϯϐ LZ-8 (Lin Zhi-8)ǴځࡕԾߎଞ⏯ύ๧ڗϐ FIP-fveǵ

݊׼ᡫ޲ (Ganoderma tsugae) ύ๧ڗϐ FIP-gts аϷ૛⏯ύ๧ڗрϐ FIP-vvo (Volvariella volvacea)Ǵว౜ԜѤᅿೈқޑữ୷ለׇӈ࣬՟ࡋၲ 44%ǴЪڀԖ 42%

ϐ ӕ ྍ ܄ (Lin, et al., 1997) Ǵ Ӣ Ԝ ᘜ ᜪ ࣁ ੿ ๵ խ ࣝ ፓ ࿯ ೈ қ ፦ (fungal immunomodulatory proteins, FIP) (Ko, et al., 1997)ǶFIP ৎ௼೏ᇡࣁࢂ΋ᅿ BRMs (biological response modifiers)Ǵ೸ၸ่ӝಒझ߄य़஑΋܄ڙᏔޑБԄǴၲډፓ࿯

խࣝس಍ޑҞޑ (चǴ2006; ঞǴ1993)Ƕ

2.2. FIP-fve ϐϩηᆶғϯ੝܄

ߎଞ⏯խࣝፓ࿯ೈқ FIP-fve ๧ڗԾߎଞ⏯ηჴᡏǴځϩηໆࣁ 12.7 kDaǴ җ 114 ঁữ୷ለ܌ಔԋǶFIP-fve ೈқ፦ׇӈςှрǴN ᆄڀԖΌㄽϯঅႬǴữ

ᗐ ᗐ ᗐ

ᗐ٠ڮ٠ڮ٠ڮ٠ڮ٠ڮڮӜࣁӜࣁӜࣁӜࣁӜࣁ EEEEEAAAAA6Ǵ 2

2 2 2

211101010 ՈᕎՈᕎՈᕎՈᕎՈᕎኳԄኳԄኳԄኳԄኳԄύύύύύύ ޑ੝౦౦౦౦౦܄܄܄܄܄ (((((OtOtOtOtOtagagagagagiriririririiiii,

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୷ ለ ׇ ӈ ύ લ Ю Ҙ ౷ ữ ለ (methionine) ǵ ъ ા ữ ለ (cysteine) а Ϸ ಔ ữ ለ (histsdine)ǴӢԜό֖ᚈ౷ᗖ (Ko, et al., 1995)ǶΒભ่ᄬႣෳ΢ǴFIP-fve җ 6 ঁ ȕ-strandsǵ΋ঁ ȕ-turn аϷ 3 ঁ Į-helices ಔԋǴځύٿঁ Į-helices Տܭ N ᆄǴ

΋ঁӧ C ᆄǴFIP-fve ೈқ፦ N ᆄ࣬ჹ C ᆄၨ౧НǴԜ܄፦ᆶխౚೈқख़᜘

ёᡂ୔࣬՟ (Kino, et al., 1989; Ko et al., 1997)Ƕӧ 2003 ԃǴPaaventhan ฻Γ೸

ၸೈқ፦่඲ᆶ X-ray ᙅ৔ϩ݋מೌǴ຾΋؁ᕕှ FIP-fve ӧԾฅύаߚӅሽ ᗖ่ϐӕࠠᚈϡᡏ (homodimer) ӸӧǴځ่ᄬࢂаൂϡᡏύ N ᆄ Į-helix ౧Н

܄ූ୷ Ala2, Leu5, Leu9, Leu12 ک Val13 ᆶќ΋࣬ჹൂϡᡏϐ Į-helices ׎ԋǴ Զӧ Į-helix ࡕௗ๱җٿঁ ȕ-sheet ܌ಔԋޑಃΟࠠᠼᆢೱ่ೈқ (fibronectin III, FNIII)ǶܭԜǴFIP-fve ࣁ FIP ৎ௼ύ२Տςှ݋่ᄬϐೈқ (Paaventhan, et al., 2003; Seow, et al., 2003)Ƕ

߈ԃٰࣴزࡰрǴճҔᗺँᡂޑБԄઇᚯ FIP-fve ่ᄬύႣෳᆶথ٢ᜪխࣝ

ಒझᗐᜪϐ่ӝՏ W24, T28, D34, T90, I91, and W111Ǵ٬ள FIP-fve ڈᐟΓᜪ Ոڬᜐಒझϩݜ IFN-Ȗ ૈΚΠफ़Ǵ௢ෳ FIP-fve ่ᄬ΢ޑᗐᜪ่ӝኳಔکΓᜪڬ ᜐՈൂਡౚޑᗐϯڙᏔჹխࣝϸᔈڀᜢᖄ܄ (Liu, et al., 2012)Ƕ

2.3. FIP-fve ϐխࣝፓ࿯ࢲ܄

ӧᡏѦ၂ᡍύǴ٬Ҕ 2 ȝg/mL FIP-fve ૈ٬ΓᜪѤᅿՈࠠϐआՈౚᏉ่ǴЪ ځૈΚ٠ό཮೏ 0.1 M D-glucose, D-galactose, D-fucose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine Ϸ lactose ฻ᗐᜪ׭ڋǶќѦǴFIP-fve ёڈᐟΓᜪڬᜐ

ՈరЃಒझ (human peripheral blood mononuclear cells, hPBMCs) ϐቚғǴᇨᏤ hPBMC வ G1/G0 ය຾Ε S යǴߦ຾ᙯᒵӢη T-bet (T-box transcription factor) ޑ߄౜ (चǴ2006)Ǵҭёڈᐟ hPBMC ϩݜ IFN-Ȗ (Ko et al., 1995)ǶऩႣӃ٬Ҕ p38 mitogen-activated protein kinase (p38 MAPK) ׭ڋᏊೀ౛ hPBMCǴ߾ FIP-fve คݤߦ٬ځϩݜ IFN-ȖǴӢԜ௢ෳ FIP-fve ࢂ೸ၸ p38 MAPK ೼৩ፓ࿯ T ಒझ

ne) )) а Ϸа Ϸа Ϸа Ϸа ϷϷಔ ữಔ ữಔ ữಔ ữಔ ữಔ ữለለለለለ Ǵ

ǴǴFIFIFIIIIPP-P-P--fvfvfvfvfveeeeeҗҗҗҗҗ66666ঁঁঁঁ c

c c

ceeseseses ՏՏܭՏܭՏܭՏܭՏܭ NNNNN ᆄǴᆄǴᆄǴᆄǴᆄǴ ᆶխխխխխౚೈౚೈౚೈౚೈౚೈқख़қख़қख़қख़қख़᜘᜘᜘᜘᜘

(18)

7

ౢғIFN-Ȗ (Wang, et al., 2004)Ƕ೸ၸ GST pull-down assay ่ӝ LC-MS/MS ϩ

݋Ǵ่݀ᡉҢ FIP-fve ᆶ hPBMC ύޑ integrin beta-3 ԖёૈڀҬϕբҔǴЪᆶ FIP-fve Ӆ୻Ꭶϐ hPBMC ځೈқ፦ႀữለᕗለϯܴᡉޑቚуǴа΢่݀ᡉҢ FIP-fve ёૈբҔܭ CD4⁺ T ಒझǴ٠وӛ Th1 ፓ࿯ϐխࣝϸᔈ (ঞǴ2003)Ƕ

FIP-fve ૈ୼फ़ե IL-5 ܌ፓ࿯ϐ༐ለ܄қՈౚቚғޑ௃׎Ǵ٠෧ե IL-5RĮ

୷Ӣ߄౜ǶќѦǴ྽ FIP-fve ᆶ༐ለ܄қՈౚӅ୻ᎦਔǴૈගଯ CD95 ޑ߄౜ǵ फ़եBcl-xL Ϸ pro-caspase 3 ޑ߄౜ໆǴ٬ځวғಒझ঒Ϋ (apoptosis) ޑ౜ຝǴ ᡉҢFIP-fve ૈ୼ׯ๓ᆶᏘለ܄ౚ࣬ᜢޑၸ௵วݹϸᔈ (Hsieh, et al., 2007)Ƕќ ԖࣴزճҔ 250 mM glutaraldehyde ٬ FIP-fve ׎ԋӭϡᡏǴ่݀ࡰр FIP-fve

׎ԋΟϡᡏϷѤϡᡏޑૈΚၨ FIP-gts ১ǴЪ೭٤ӭϡᡏคݤڈᐟ hPBMCs ϐ ቚғ (Ou, et al., 2009)Ƕ

FIP-fve ёڈᐟλႵ๠ಒझϩݜ IFN-Ȗ ٠ගଯ IL-2 ک IFN-Ȗ ୷Ӣᙯᒵ߄

౜ (Ko et al., 1995)ǴฅԶ FIP-fve ൂᐱᆶλႵ T ಒझӅ୻Ꭶޑ௃ݩΠǴคݤڈ ᐟ T ಒझቚғǹՠऩӧ࿶ paraformaldehyde ڰۓǵа mitomycin-C ೀ౛ (ูǴ 2008) ܈ᒟྣၸ (Hsieh, et al., 2003) ϐӄ๠ಒझӸӧޑ௃׎ΠǴ߾ёߦ຾ T ಒ झޑቚғǴ߄Ң FIP-fve ڈᐟλႵ T ಒझቚғሡाלচև᝘ಒझ (antigen presenting cell, APC) ӸӧǶFIP-fve ૈ୼ߦ຾λႵ T ಒझڙᏔ (T-cell receptor , TCR) VĮ 19 mRNA ޑ߄౜ໆၲচҁϤ७ǴЪऩ٬ҔЬाಔᙃ࣬৒܄ፄӝᡏ (major histocompatibility complex, MHC) ϐύک܄לᡏᆶλႵဎ๚ѮᏘಒझаϷ λႵ๠᠌ಒझӅ୻ᎦǴ߾׭ڋ T ಒझڙ FIP-fve ߦ຾ޑቚғ௃׎Ǵҭफ़եচҁ ёҗ FIP-fve ڈᐟޑλႵဎ๚ѮᏘಒझ IL-12 p35, IL-12 p40 аϷ IL-18 ޑ୷Ӣ ߄౜ໆ (ูǴ2008)ǶќѦǴFIP-fve ΨёаڈᐟဉᛠጢరЃ่ (mesenteric lymph node, MLN) Ϸ୻ओМඬ (Peyer’s patches, PP) ಒझౢғ IFN-Ȗ (ቅǴ2013)Ƕ

ᡏϣ၂ᡍБय़Ǵ߻Γࣴزว౜ FIP-fve ڀԖלၸ௵ǵלᕎаϷफ़ՈᑗϐወΚǶ ӧלၸ௵фਏ΢Ǵࣴزࡰр೸ၸႣӃܭဎ๚ݙ৔ FIP-fve ё׭ڋҗ BSA Їଆޑ ӝ LLLLLC-C-C-C-C-MSMSMSMSMSS/M/M/M/M/M/MSSSSS ϩϩϩϩϩ ڀ

ڀ ڀ ڀ

ڀҬϕҬϕҬϕҬϕҬϕϕբҔբҔբҔբҔբҔǴЪǴЪǴЪǴЪǴЪᆶᆶᆶᆶᆶᆶ Ǵ

Ǵ Ǵ Ǵ

Ǵа΢а΢а΢а΢а΢а΢่่่่่݀݀݀݀݀ᡉҢᡉҢᡉҢᡉҢᡉҢҢ ᔈ (ঞǴঞǴঞǴঞǴঞǴ20202020200303030303)))))ǶǶǶ

(19)

8

BALB/c λႵس಍܄ၸ௵ϸᔈ (systemic anaphylaxis)ǹӧ compound 48/80 ᇨᏤ ޑλႵىඓဍ๞၂ᡍύǴӕኬႣӃဎ๚ݙ৔ FIP-fve ё෧Ͽىඓဍ๞ᡏᑈԿ 49.3 % (Ko et al., 1995)Ƕ࿶җဎ๚ݙ৔ᚊռқೈқ (ovalbumin, OVA) ӆаαܺ

OVA ठ௵ޑ୏ނኳԄύǴᗯ१ FIP-fve ϐλႵคܴᡉၸ௵ϸᔈޑ߄౜ǴЪՈమ ύ OVA ஑΋܄לᡏ IgE ҭคᡉ๱ቚуǴԶ IgG2a Ԗ΢ϲޑ௃׎ǹځ๠ಒझᆶ OVA Ӆӕ୻ᎦࡕǴ΢మύ IFN-Ȗ ֖ໆቚуǴԶ IL-5 ෧ϿǴᡉҢᗯ१ FIP-fve ё

೸ၸ٬խࣝϸᔈᙯࣁа Th1 ࣁЬǴԶ෧ե Th2 ࠠၸ௵ϸᔈੱރ (Hsieh et al., 2003)Ƕӧफ़եՈᑗࣴز΢Ǵҗ Streptozotocin ᇨวಃ΋ࠠᑗֿੰޑ ICR λႵύǴ

๏ϒ FIP-fve คݤफ़եՈᑗޑᐚࡋǴόၸӧಃΒࠠᑗֿੰޑ ICR λႵύǴ๏ϒ FIP-fve ૈफ़եՈᑗޑᐚࡋǴځਏ݀ၨӕឦܭ FIP ৎ௼ϐ FIP-gts ᡉ๱ (चǴ2006)Ƕ

೸ၸλႵطᕎኳԄࣴز FIP-fve לᕎወΚǴว౜ᗯ१ FIP-fve ૈ୼ۯߏᑡᕎ (BNL 1MEA.7R.1 cell) λႵޑტڮǵ׭ڋဍዦελ٠׭ڋطᕎಔᙃޑՈᆅཥғǴ ЪλႵڬᜐՈൂਡౚޑ CD80, MHC class I, MHC class II ޑ߄౜ໆࣣԖගଯޑ౜

ຝǴ௢ፕ FIP-fve ёࢲϯӃϺϷ፾ᔈࠠխࣝϸᔈၲډלᕎਏ݀ǴЪ IFN-Ȗ תᄽ

ཱུख़ाفՅ (Chang, et al., 2010)Ƕஒ FIP-fve բࣁ՘ᏊᆶΓᜪ٢ᓐރੰࢥ (HPV)- 16 E7 ᕎ୷Ӣೈқ፦ӕਔխࣝλႵǴёගଯᑡᕎλႵӸࢲ౗ၲ 60%Ǵ٠ගϲλႵ ᡏϣჹ HPV-16 E7 ڀԖ஑΋܄לᡏ֖ໆаϷჹ (HPV)-16 E7 ڀ஑΋܄ޑ CD4⁺ Ϸ CD8⁺ T ಒझ IFN-Ȗ ϩݜໆǹ೸ၸಒझ౽෗၂ᡍǴዴᇡלဍዦ੝܄ᆶ IFN-Ȗǵ CD4⁺ Ϸ CD8⁺ T ಒझ࣬ᜢǶӆޣǴFIP-fve ёܭᡏϣ၂ᡍύڈᐟᐋँಒझ (dendritic cells, DCs) ϐԋዕ٠ᇨᏤלচ஑΋܄ޑ CD8⁺ T ಒझխࣝϸᔈ (Ding, et al., 2009)Ƕ

2.4. FIP-fve ϐӼۓ܄

ӧуπၸำύǴ१ނচ਑όЮ࿶եྕհএǵଯྕొฆ܈ࢂமለமᡵ฻ཱུᆄచ ҹೀ౛ǴӢԜऩኬࠔӼۓόܰڙྕࡋǵለᡵॶ܈ᓯᙒਔ໔฻చҹቹៜځࢲ܄Ǵ߾

ounnnnnd d d d d 44844848/8/8/8/8/8/8000000 ᇨᏤᇨᏤᇨᏤᇨᏤᇨᏤ ى

ى ى ى

ىඓඓඓဍඓဍඓဍඓဍ๞ᡏ๞ᡏ๞ᡏ๞ᡏ๞ᡏᑈԿᑈԿᑈԿᑈԿᑈԿԿ O

O O O

OVAVAVAVA)AA))) ӆаӆаӆаӆаӆаαܺαܺαܺαܺαܺܺ ޑ߄߄߄߄߄౜Ǵ౜Ǵ౜Ǵ౜Ǵ౜ǴЪՈЪՈЪՈЪՈЪՈమమమమమ

(20)

9

ԖၨεወΚ଺ࣁВࡕߥ଼१ࠔϐᔈҔǶҞ߻၂ᡍεӭаಒझቚғ܈ IFN-Ȗ ϩݜ ໆղᘐ FIP-fve ࿶уπ܈੃ϯೀ౛ࡕϐӼۓ܄ǶӧऐྕБय़Ǵа 100ÛC ଯྕೀ

౛ FIP-fve 60 ϩដࡕǴᗋߥԖ΋ъڈᐟλႵ๠ಒझቚғૈΚǴฅ 90 ϩដࡕ߾ε

೽ϩࢲ܄ςᎁઇᚯǴԶೀ౛ 2 λਔࡕǴFIP-fve ΨѨѐΑڈᐟ hPBMC ϩݜ IFN-Ȗ ϐૈΚǶόၸ FIP-fve ߾ёהڙӧ 121ʚ ఠ๵ 15 ϩដаϷ -80ʚ հএ 12 λਔǶӧऐለᡵ೽ϩǴܭ pH 2 ޑᕉნٿλਔ܈ pH 4-10 ޑᕉნѤλਔ FIP- fve ࣣૈߥԖځࢲ܄Ǵՠ྽ pH 13 ਔ FIP-fve ڈᐟλႵ๠ಒझቚғૈΚ෧ࣁऊ

72%Ǵڈᐟ IFN-Ȗ ϩݜໆΨफ़ࣁ 67%Ƕऩࢂ๏ϒ੿ޜଳᔿೀ౛ǴFIP-fve ᗨёڈ ᐟλႵ๠ಒझቚғǴՠಒझϩݜ IFN-Ȗ ޑਏ݀ࠅफ़եࣁচҁޑ΋ъ (Ou et al., 2009; Tong, et al., 2008)ǶࣁΑຑ՗१ࠔύ܌బу FIP-fve ޑߥӸයज़Ǵஒ FIP-fve аόӕఠ๵చҹೀ౛٠ෳ၂ځߥӸයज़Ǵ೸ၸႝݚϩ݋ว౜ FIP-fve аύ܄ᕉნ 121ÛC ೀ౛ 20 ϩដ܈а 138ÛC ೀ౛ 18 ࣾࡕǴԿϿૈ୼ܭ࠻ྕΠߥӸъԃǴ ऩа 130ÛC ೀ౛ 12 ࣾ܈а 121ʚ ೀ౛ 20 ϩដǴ߾ԿϿёӧ 4ÛC հᙒΠߥ Ӹъԃ (஭Ǵ2011)Ƕ

FIP-fve ӧኳᔕगǵဉٿ໘ࢤӚձ੃ϯύǴ࿶ၸ֖गೈқ䁙ϐኳᔕग੃ϯన

ೀ౛ǴᡉҢFIP-fve ٠ό཮Ԗ܌फ़ှǴԶऩࢂа֖Ԗી䁙ϐ੃ϯనኳᔕဉၰ੃ϯǴ FIP-fve ᗨϿ೽ϩ೏੃ϯ٠ౢғٿТࢤǴՠε೽ϩϝ҂೏੃ϯǶԶ࿶ኳᔕဉगၰ

੃ϯࡕޑ FIP-fve ёڈᐟ୻ओМඬಒझϩݜ IFN-ȖǴӢԜ᛾ܴ੃ϯࡕϐ FIP-fve ϝڀԖፓ࿯ဉၰխࣝϐࢲ܄ (ቅǴ2013)Ƕ

2.5. ख़ಔ FIP-fve ϐ౦ᡏ߄౜

ӧࣴزೈқ፦੝܄ਔǴऩሡڗளεໆЪ፦પޑೈқǴ܈ளډόӕܭϺฅೈқ ϐТࢤਔǴ߾ёճҔ୷Ӣख़ಔמೌڗளೈқኬࠔǶӃஒట߄ၲ኱ϐೈқ୷Ӣࡌᄬ ܭၩᡏ΢ǴஒၩᡏᙯΕ஌ЬಒझࡕǴ࿶Ԝεໆ߄ၲрҞ኱ೈқǴӆપϯ߾ёளϐǶ

߻Γа pGEX-2T expression vector ᄬᑐ pGEX-FIP-fveǴஒϐᙯΕεဉఎ๵ TG1 ғ܈܈܈܈܈ IIIIIFNFNFNFNNN---ȖȖȖȖȖȖ ϩݜϩݜϩݜϩݜϩݜ а

а а а

а 111100000000ÛÛCCCCC ଯྕଯྕଯྕଯྕଯྕೀೀೀೀೀೀ 9

9 9 9

9000 ϩដϩដϩដϩដϩដࡕ߾ࡕ߾ࡕ߾ࡕ߾ࡕ߾εεεεεε ᐟ hhhhhPBPBPBPBPBMCMCMCMCMC ϩݜϩݜϩݜϩݜϩݜ

(21)

10

٠а IPTG ᇨᏤख़ಔೈқεໆ߄౜ࡕǴௗޣճҔ Glutathione-Sepharose 4B ᒃک

܄ᆅࢊપϯ֖ GST-FIP-fve ᑼӝೈқǶளډ GST ᑼӝೈқࡕǴаᒣᇡ஑΋܄Ϫ Տϐሇન thrombin ϪΠ GST-tagǴջёӆԛપϯளډ reFIP-fve (࢒Ǵ1995)Ƕࣴ

ز ่ ݀ ว ౜ reFIP-fve ჹ Γ ᜪ आ Ո ౚ Ꮙ ໣ ૈ Κ ᆶ native fve ࣬ ӕ Ǵ ฅ ڈ ᐟ hPBMC ቚ෗ޑૈΚၨ native fve եΟ७ǶќѦ࢒ΨᄬᑐΑѤᅿલѨँᡂᅿೈқ

፦ reFIP-fve 14-114ǵreFIP-fve 1-103ǵreFIP-fve 14-103ǵreFIP-fve 28-103Ǵว౜

reFIP-fve 14-114 ᆶ reFIP-fve 1-114 ჹܭ hPBMC ቚ෗ૈΚനௗ߈ǴԶ reFIP-fve 1-103 ڈᐟ hPBMC ቚғૈΚफ़ࣁ 75%ǴreFIP-fve 14-103 ߾फ़ࣁ 40%ǶreFIP-fve 14-114 ᆶ reFIP-fve 1-114 ჹܭΓᜪआՈౚᏉ໣ૈΚΨനௗ߈Ǵՠ reFIP-fve 14- 103 आՈౚᏉ໣ૈΚΠफ़Կ 25%ǴԶ FIP-fve 28-103 ڈᐟ hPBMC ቚғૈΚᆶ ᏉՈࢲ܄ֹӄ഼ѨǴᡉҢ FIP-fve ϐ N ᆄ 14-28 ϐữ୷ለᆶ C ᆄ 103-114 ϐ ữ୷ለჹځխࣝቚமբҔԖᜢ (࢒Ǵ1995)ǶҭԖ߻Γ٬Ҕ pGEX-4T-1 ၩᡏᄬᑐ ٠߄౜ GST-FIP-fveǴ่݀ᡉҢ GST-FIP-fve 1-114 Ϸ GST-FIP-fve 1-103 ёڈᐟ hPBMC ϩݜ IFN-ȖǴՠ୤Ԗ GST-FIP-fve 1-114 ڀआՈౚᏉ໣ૈΚ (चǴ2006)Ƕ ҁࣴز࠻මճҔ pGEX-4T-1 س಍પϯр reFIP-fve 28-103Ǵ่݀ᡉҢځᆶѮᏘಒ झޑ่ӝૈΚၨ native fve եǴЪόᆶ T ಒझ่ӝǴЪΨѨѐࢲϯλႵ๠᠌ಒ झౢғ IFN-Ȗ ϐૈΚǴ௢ෳ FIP-fve ऩคݤ׎ԋᚈϡᡏ่ᄬǴ߾ځխࣝፓ࿯ࢲ

܄फ़ե (஭Ǵ2011)Ƕ

ಃѤ࿯! ຬלচᙁϟ

ຬלচࣁࢂᅿ೸ၸ่ӝ MHC ᆶ TCR Զڈᐟεໆ T ಒझޑࢥೈқǶҞ߻

ςޕޑຬלচऊԖ 40 ᅿǴЬाࢂҗߎ໳Յဟ๻ౚ๵ (Staphylococcus aureus) ϩ ݜޑߎ໳Յဟ๻ౚ๵ဉࢥન (Staphylococcus enterotoxin, SE) A, B, C1-3, D, E , G- R, U کύࢥ܄Ҷլᆕӝੱࢥન (toxic shock syndrome toxin-1, TSST-1)Ǵϯᖋ܄᜘

ephahahahaarororororosesese444444BBBBBB ᒃکᒃکᒃکᒃکᒃک аᒣ

аᒣ аᒣ аᒣ

аᒣᒣᇡ஑ᇡ஑ᇡ஑ᇡ஑ᇡ஑΋܄΋܄΋܄΋܄΋܄ϪϪϪϪϪ e

e(((((࢒࢒࢒Ǵ࢒Ǵ࢒Ǵ࢒Ǵ111919199595959595))))ǶࣴǶࣴǶࣴǶࣴǶࣴࣴ

࣬ ӕ

࣬ ӕӕӕǴ ฅǴ ฅǴ ฅǴ ฅǴ ฅڈ ᐟڈ ᐟڈ ᐟڈ ᐟڈ ᐟ

(22)

11

ౚ ๵ (Streptococcus pyogenes) ϩ ݜ ޑ ᜘ ౚ ๵ ዗ চ ܄ Ѧ ࢥ ન (streptococcal pyrogenic exotoxin, SPE) A, C, G-M ک SMEZǵSSAǴҭԖϿኧϩᜪࣁᡖዀ๵຋

෗ન (Mycoplasma arthritidis mitogen, MAM) аϷଷ܄่ਡੰओМఎ๵຋෗ન (Yersinia pseudotuberculosis mitogen, YPM)ǶՈనύϿໆޑຬלচջёڈᐟ T ಒ झޑቚғǴځъቚғᏊໆ (P50) ӧ 0.02-50 pg/mLǴ٠ߦ٬ T ಒझϩݜ IL-2, IFN- Ȗ ک TNF-ĮǴ຾ԶЇଆխࣝੱރӵύࢥ܄Ҷլǵၸ௵܄ሷݹǵฎआ዗ǵวᐨࣗԿ

ࢂԝΫ (Baker and Acharya, 2004; Fraser and Proft, 2008; Proft and Fraser, 2003)Ƕ ຬלচޑϩηໆନΑ YPM ࣁ 14.5 kDa ѦǴځᎩऊӧ 20-30 kDaǶόႽ໺

಍לচѸ໪Ӄ೏ APC տᏘ٠অႬࡕǴӆ࿶җ MHC ϩηև᝘๏ T ಒझǴຬל চ٠όሡा೏ APC অႬǴջёޔௗ่ӝ APC ߄य़΢ MHC class II ᆶ T ಒझ

΢ޑ TCRǴ٠٬ T ಒझቚғǶӧ 10⁵-10⁶ ޑ naïve T ಒझύǴ໺಍לচև᝘Б Ԅѝૈࢲϯځ 1%ǴԶຬלচё٬ 20 % ޑ naïve T ಒझࢲϯǶ

߄य़ϩη MHC class II ΢Ԗٿঁᆶຬלচ่ӝՏ࿼Ǵಃ΋ঁ generic site Տ ܭ Į-chain ΢ǹќ΋ঁ zinc-dependent site ߾ӧ ȕ-chain ΢Ǵځᆶຬלচޑᒃک

܄ࣁ generic site ޑԭ७ǶҞ߻ࣴزύว౜Ǵຬלচύ SPE-A, SSA, SEB, SEC1- 3, SEG аϷ TTST-1 ฻ࢂа N ᆄਸН୔ᆶ Į-chain ϐ generic site ᗖ่ǴԶ SPE-C, SPE-G, SPE-H, SMEZ, SEH (Hakansson, et al., 2000) ฻߾ࢂᆶ ȕ-chain ΢ zinc-dependent site ่ӝǴՠΨԖӕਔᆶ Į, ȕ-chain ӕਔᗖ่ϐຬלচǴӵ SEA, SED, SEE, MAM ฻ (Proft and Fraser, 2003)Ƕ

ຬלচ२ा่ӝҞ኱ࣁ CD4⁺ T ಒझǴ٠٬ځࢲϯԋಃ΋ࠠᇶշࠠ T ಒझ (Bavari and Ulrich, 1995)Ƕεӭኧޑຬלচӵ SEB (Swaminathan, et al., 1992) ࢂ

೸ၸ TCRVȕ ᆶ T ಒझ่ӝǴՠ SHE ߾ࢂᆶ TCRVĮ ᗖ่Ǵ٠೸ၸ real-time ᛾ Ᏽ᛾ܴځᒣᇡՏࣁ VĮ 27 (Pumphrey, et al., 2007)Ƕ

ન (((((stststststreeeptptptptptptococococococooococoo cacacaal l l l l ኧϩ

ኧϩϩ

ኧϩϩᜪࣁᜪࣁᜪࣁᜪࣁᜪᜪࣁᡖዀᡖዀᡖዀᡖዀᡖዀ๵຋๵຋๵຋๵຋๵຋຋ ओ

ओ ओ ओ

ओМఎМఎМఎМఎМఎМఎ๵຋๵຋๵຋๵຋๵຋෗ન෗ન෗ન෗ન෗નન চջёёёёёڈᐟڈᐟڈᐟڈᐟڈᐟ TTTTT ಒಒಒಒಒ

(23)

12

ಃΒക! ࣴز୏ᐒᆶҞޑ

ӧჴᡍ࠻߻Γูୗ๮ࣴزύǴว౜ FIP-fve ࢲϯ T ಒझޑᐒڋёૈᜪ՟ຬ ל চ Ǵ চ Ӣ Ԗ Ο Ǻ1. FIP-fve ό ሡ ा ל চ և ᝘ ಒ झ অ Ⴌ Ǵ ѝ ሡ а ࿶ paraformaldehyde ೀ౛ၸϐ๠᠌ಒझ଺ࣁלচև᝘ಒझջёࢲϯ T ಒझǶ2. FIP- fve ёࢲϯ TCR VĮ 19 ୷Ӣ߄౜ၲ 6 ७ǴӢԜ௢ෳࢂᙖҗ TCR VĮ 19 բࣁࢲ

ϯ T ಒझϐڙᏔǶ3. ऩ٬Ҕ MHC class I ᆶ MHC class II ύک܄לᡏǴ߾ё

׭ڋҗ FIP- fve Їଆޑ T ಒझቚғᆶלচև᝘ಒझ୷Ӣ߄౜Ƕᆕӝ΢ॊ่݀Ǵ

௢ෳ FIP- fve ࢲϯᐒڋᆶ MHC class IǵMHC class II ԖᜢǴЪёૈࢂ೸ၸᆶ TCR ೱ่Զࢲϯ T ಒझǴԶӵԜբҔᐒڋཱུ՟ຬלচǴӢԜа FIP-fve ࣁᜪຬ לচ଺ࣁଷᇥ (ูǴ2008)ǶќԖ߻ΓаӅӕխࣝ؈ᐘݤǴམଛՋБᏀᗺ่݀ளޕ FIP-fve ᆶ MHC II ϐ໔ڀҬϕբҔǴᆶ MHC I ߾ցǹԶӧ TCR VĮ аϷ TCR Vȕ ၂ᡍύǴѝளډ೽ϩ᛾Ᏽዴᇡٿޣᆶ FIP-fve ڀҬϕբҔ (ዐǴ2012)Ƕ

ќѦǴ߻Γ჋၂પϯТࢤલѨख़ಔೈқǴ೸ၸѐନ FIP-fve ữ୷ለ N ᆄ 1- 13 ೽ϩǴ٬ϐѨѐ Į-helix ӢԶคݤ೸ၸ౧Н܄ᗖ่׎ԋᚈϡᡏ่ᄬǶќѦΨ ғౢΑલѨ 1-27ǵ104-114 аϷ 13-27 ೽ϩϐೈқӅ 8 ಔǴႣයૈـӚঁТࢤ ჹխࣝࢲ܄ϐቹៜǴฅа pGEX 4T-1 բࣁၩᡏ٠ᙯΕεဉఎ๵ύ߄౜ϐख़ಔೈ

қғౢόܰǴ୤ૈڗள reFIP-fve 28-103ǴӢԜࡌ᝼ࡕΓ׳ඤၩᡏӆԛપϯТࢤ લѨख़ಔೈқ (஭Ǵ2011)Ƕ

ҁፕЎϐಃ΋೽ϩࣁ೸ၸ pET-32a(+) ၩᡏख़ཥᙯ෗٠પϯ rFIP-fve 1-114 ϷТࢤલѨ rFIP-fve 14-114Ǵࡕុෳ၂ځխࣝࢲ܄Ǵаᕕှᚈϡᡏ่ᄬܭ FIP-fve ϐख़ा܄ǶಃΒ೽ϩǴࣁ؃ FIP-fve ᆶ MHC ҬϕբҔ׳ޔௗޑ᛾ᏵǴӃஒ RAW 264.7 ಒझ܈λႵӄ๠ಒझઇ࿗ࡕᆶ FIP-fve ຾ՉӅӕխࣝ؈ᐘݤǴPull-Down ϐౢނа LC-MS/MS ϩ݋ǴႣයૈа׳ޔௗޑ᛾Ᏽ࣮ـᆶ FIP-fve ڀҬϕբҔ ϐೈқǶ

ޑᐒᐒᐒᐒᐒڋёڋёڋёڋёڋёૈᜪёૈᜪૈૈᜪૈᜪ՟ຬ՟ຬ՟ຬ՟ຬ՟ຬ՟ຬ Ⴌ Ǵ ѝ ሡѝ ሡѝ ሡѝ ሡѝ ሡааааа ࿶

(24)

13

ಃΟക! ׷਑ᆶБݤ

Ȝᛰࠔȝ

z NaCl, KCl, NH4Cl, EDTA, KHCO3, Na2HPO4, ȕ-mercaptoethanol, acetic acid, HCl, SDS, APS, TEMED, glycine, coomassie brilliant blue R250, DTT(1,4- dithiothreitol), bromophenol blue, methanol, Lysozyme, Trypan blue, Albumin from bovine serum (BSA), concanavalin A (ConA) ᖼԾ Sigma-Aldrich (St.

Louis, MO, USA)

z ౷ለሓᖼԾѠ᡼ޥ਑ިҽԖज़Ϧљ (ཥԮѱǴѠ᡼)

z NaOAc(CH3COONa) ᖼԾ Mallinckrodt Baker Inc. (Phillipsburg, USA) z Tris-base, Glycerol, polysorbate 20 (Tween 20) ǵ H2SO4 ᖼ Ծ J.T.Baker

(Phillipsburg, NJ, USA)

z KH2PO4, Na2HPO4, NaN3 ᖼԾ Riedel-deHaën® (Seelze, Germany) z 40% Acrylamide/Gly ᖼԾ Bio Basic Inc. (Ontario, Canada)

z BlockPRO™ Blocking Buffer ᖼԾ Visual protein (ཥчѱǴѠ᡼) z IPTG ᖼԾ BioShop Canada Inc. (Ontario, Canada)

z FBS ᖼԾ GIBCO® Life Technologies (Carlsbad, CA, USA)

z RPMI-1640 medium, 89% High-glucose DMEM ᖼԾ Hyclone (Logan, UT, USA)

z Taq DNA Polymerase Master Mix Red ᖼ Ծ Ampliqon (Copenhagen, Denmark)

z HealthView Nucleic Acid Stain ᖼԾ Genomics BioSci & Tech Corp. (ཥч ѱǴѠ᡼)

z 95% ethanol ᖼԾ϶کຩܰިҽԖज़Ϧљ (ཥчѱǴѠ᡼)

(25)

14

Ȝ઻׷ȝ

z Vivaspin 6 (3 kDa MWCO), vivaspin 20 (5 kDa MWCO) ᖼԾ GE Healthcare (UK)

z 25 Tǵ75 Tǵ48 Ͼಒझ୻Ꭶዬǵ50 mL ᆶ 15 mL ᚆЈᆅᖼԾ Corning Life Science (Tewksbury, MA, USA)

ಃ΋࿯! ߎଞ⏯խࣝፓ࿯ೈқ FIP-fve પϯ

Ȝ׷਑ȝ (1) ኬࠔǺ

z ߎଞ⏯Ǻ2012 ԃ 5 Д 24 ᖼԾഗӳຬѱ (ᆵчѱǴѠ᡼) (2) ၂Ꮚ

z ๧ڗనǺڗ 90 g NaClǵ5 mL ȕ-mercaptoethanolǵ250 mL acetic acidǴྋှ

ܭѐᚆηНࡕۓໆԿ5 LǶ

z CM52 carboxymethyl cellulose ᖼԾ Whatman Inc. (New Jersey, USA) z 0.01 M NaOAc (pH 5.2)Ǻڗ 4.1 g NaOAc аѐᚆηНྋှࡕǴа CH3COOH

ፓ᏾pH ॶԿ 5.2ǴۓໆԿ 5 LǶ

z 0.5 M NaOAc (pH 5.2) Ǻ ڗ 10.25 g NaOAc а ѐ ᚆ η Н ྋ ှ ࡕ Ǵ а CH3COOH ፓ᏾ pH ॶԿ 5.2ǴۓໆԿ 250 mLǶ

z ֖ 0.5 M NaCl ϐ 0.01 M NaOAcǺڗ 4.4 g NaCl ྋܭ 0.01M NaOAc ྋనύǴፓ᏾ pH ॶԿ 5.2 ࡕǴۓໆԿ 150 mLǶ

z PBS ጗ፂనǺڗ 8.01 g NaClǵ0.2 g KH2PO4ǵ0.2 g KClǵ1.15 g Na2HPO4

аѐᚆηНӣྋࡕǴፓ᏾ pH ॶࣁ 7.2 ӆۓໆԿ 1 LǶ (3) Ꮤ׷

z ݀Ҋᐒ Water blender ᖼԾ Royal Dutch Philips Electronics Ltd. (Amsterdam, ᖼԾ

ᖼԾ ᖼԾ ᖼԾ

ᖼԾ GGGGEE E EEHeHeHeHeHealalalalalthththththcacacacarererereee

ᖼԾ

(26)

15

Holland)

z ຬॣݢઇ࿗ᐒ Heat system Sonicator XL2020 ᖼԾ Qsonica (Melville, NY, USA)

z եྕᚆЈᐒ Allegra^TM X-22R Centrifuge ᖼԾ Beckman Coulter, Inc. (CA, USA)

z ෧ᓸᔅ੅ DOA-P104-A4 ᖼԾ Gast Manutacturiag, Inc. ( Europe) z 1 ဦᘠરᖼԾ Toyo Roshi Kaisha, Ltd. (Japan)

z Glass-microfiber discs, PESU membrane (0.2 ȝm, 0.45 ȝm)ᖼԾ Sartorius Stedim Biotech. (Germany)

z Cellu Sep 3,500 MWCO ೸݋ጢᖼԾ Membrane Filtration Products, Inc.

(Seguin, TX, USA)

z 0.2 mm ࣒ዟ੧ᖼԾቼว࣒ዟሺᏔ୘Չ (ཥԮѱǴѠ᡼)

z Gradient Mixer ᖼԾ Amersham Pharmacia Biotech, Inc. (Piscataway, NJ, USA)

z HPLC fraction collector ᖼԾ Advantec MFS, Inc. (CA, USA)

z NanoDrop® ND-1000 spectrophotometer ᖼԾ NanoDrop Technologies, Inc.

(Wilmington, DE, USA)

z PB-10 pH ෳۓሺᖼԾ Sartorious (Goettingen, Germany)

ȜБݤȝ

(1) ߎଞ⏯ઇ࿗ᆶ౷ለሓ؈ᐘ

ஒཥᗲߎଞ⏯ѐᚇ፦ગख़ࡕǴࢱృϪ࿗٠ܭ 4ÛC ੆࿼๧ڗనύǶ႖Ϻа

݀ҊᐒϷຬॣݢ᎜ᕏᐒઇ࿗ೀ౛ (ઇ࿗ 10 ࣾଶ 10 ࣾǴᕴઇ࿗ਔ໔ 10 ϩ ដ)Ǵ٠ஒઇ࿗నեྕᚆЈ (4ÛC, 9,500 rpm, 20 ϩដ) ԏ໣΢మనǶ΢మన೯ ၸٿТ࣬᠄ϐ 1 ဦᘠરѐନᚇ፦ࡕǴуΕ౷ለሓၲ 90% ؈ᐘೈқǴܭ 4ÛC

ic ic ic ic

ica a(((M(M(M(Melelelelelvivivivivilllllllllle,e,eee NNNNNY,Y,Y,Y,

(27)

16

ܫ࿼႖ڹǶಃΒϺаեྕᚆЈ (4ÛC, 9500 rpm, 10 ϩដ) ԏ໣ೈқ؈ᐘࡕǴӣ ྋೈқԿ 0.01 M NaOAc (pH 5.2) ጗ፂనǴ٠းΕ೸݋ኳӧ 4ÛC Π೸݋ΟϺǴ

೼ύӭԛ׳ඤ጗ፂనǴᡣೈқֹӄ࿼ඤӧ 0.01 M NaOAc (pH 5.2) ጗ፂనύǶ ԏ໣೸݋ጢϣኬࠔǴа glass fibre ک 0.45 ȝm ᘠરၸᘠǴܭ 4ÛC ΠߥӸࡑࡕ

ុપϯǶ

(2) CM-52 ጤᡏᆅࢊቫ݋

ஒ 0.5 M NaOAc (pH 5.2) ྋనуΕ CM-52 ጤᡏύǴᠳ܏֡Ϭࡕᓉ࿼ 30 ϩដǶλЈॹ௞΢మࡕǴӆуΕ 0.01 M NaOAc (pH 5.2)Ǵӕኬᠳ܏֡Ϭᓉ࿼Ǵ ख़ፄа0.01 M NaOAc (pH 5.2) ѳᑽጤᡏࡕǴஒጤᡏॹΕࢎӳϐቫ݋ᆅࢊύǶ ᙯ໒ᆅࢊΠБࢲਵ٬ӭᎩనᡏࢬрǴࡑጤᡏᓸჴǴॹΕ࣒ዟ੧ܭځഗᆄǴа጗

ፂనᡏࢬࢱਔޑፂᔐǶаᔅ੅ݙΕ 0.01 M NaOAc (pH 5.2) ࢬࢱᆅࢊԿࢬрన pH 5.2 ਔջֹԋྗഢǶ

٬Ҕᔅ੅ܜڗς೸݋Ъၸᘠϐߎଞ⏯ಉ๧నԿᆅࢊǴፓ᏾ࢬೲऊ؂ϩដ 30 mLǴᆢ࡭నय़ଯࡋᗉխጤᡏଳ௞Ƕಉ๧నֹӄ೯ΕጤᡏࡕǴа 0.01 M NaOAc (pH 5.2) ࢱѐ҂ᗖ่ϐೈқǴޔډࢬрనϐೈқ፦ᐚࡋλܭ 0.1 mg/mL ଶЗǶ നࡕǴа0.01 M NaOAc (pH 5.2) Ϸ֖ 0.5 M NaCl ϐ 0.01 M NaOAc (pH 5.2)

຾ՉఊࡋࢬࢱǴаԏ໣Ꮤ؂ 4 mL ԏԋ΋ᆅǴԏ໣Կؑගనӄ೽೯Εᆅࢊࣁ ЗǶ೸ၸ SDS-PAGE ዴᇡҞ኱ೈқՏ࿼Ǵڗၨપϐᆅኧஒೈқᐚᕭ٠೸݋Կ PBS ྋనύǴߥӸӧ 4ÛC ࡑࡕុჴᡍ٬Ҕ܈ܫܭ -20ÛC ߥӸǶ

(3) ೈқ፦ᐚࡋෳۓ

٬ҔNanoDrop^® ND-100 ӄݢߏϩӀӀࡋीෳໆǶෳໆ߻Ӄڗ 2 ȝL ѐᚆ ηНమዅෳໆ୷০Ǵа࡬᜔ર࡬ѐН੧Ǵӆڗ 2 ȝL ྋᏊਠ҅ङඳॶǴӕኬа

࡬᜔રᔔ࡬୷০Ƕࡕڗ 2 ȝL ኬࠔܭ୷০Ǵаݢߏ 280 nm ୀෳځೈқ፦ᐚࡋǶ

໣

໣ೈқೈқೈқೈқೈққ؈؈؈؈ᐘ؈ᐘ؈ᐘࡕǴࡕǴࡕǴࡕǴӣӣӣӣӣӣ 4

4 4

4ÛÛÛC CC Π೸Π೸Π೸Π೸೸݋Ο݋Ο݋Ο݋Ο݋ΟϺǴϺǴϺǴϺǴ H 5.5.5.5.5.2)2)2)2)2) ጗ፂ጗ፂ጗ፂ጗ፂ጗ፂ጗ፂనύనύనύనύనύనύǶ ΠߥߥߥߥӸӸӸӸࡑࡕ

(28)

17

ಃΒ࿯! ᡂ܄ጤᡏႝݚϩ݋ (SDS-PAGE)

Ȝ׷਑ȝ (1) ၂Ꮚ

z Tri-base (pH 8.8)Ǻ18.17 g Tris-base уΕѐᚆηНࡕǴа HCl ፓԿ pH 8.8Ǵ ۓໆԿ100 mLǶ

z Tri-base (pH 6.8)Ǻ6.0 g Tris-base уΕѐᚆηНࡕǴа HCl ፓԿ pH 6.8Ǵ ۓໆԿ100 mLǶ

z 10% SDSǺڗ 10 g SDS ણ҃уΕѐᚆηНԿ 100 mLǶ z 10% APSǺڗ 0.1 g APS ણ҃уΕѐᚆηНԿ 1 mLǶ

z 5 X SDS sample dyeǺڗ 2.5 mL 0.5 M Tris-base (pH 6.8), 4 mL 10% SDS, 2 mL glycerol, 1 mL ȕ-mercaptoetnanol, 2 mL 1% bromophenol blue аϷ 2 mL 1 M DTT షӝ֡ϬǴ࿼ܭ -20ÛC ഢҔǶ

z Tank bufferǺڗ 3 g Tris-base, 14.4 g glycine ک 10 mL 10% SDS, аѐᚆ ηНӣྋ٠ۓໆԿ 1 LǶ

z CBR ࢉᏊ 1% stain stockǺڗ 2 g coomassie brilliant blue R250 уѐᚆη НԿ 200 mLǶ

z CBR ࢉᏊǺڗ 62.5 mL 1% stain stock, 250 mL methanol, 50 mL acetic acid уѐᚆНԿ 500 mLǶ

z ଏࢉᏊǺڗ 500 mL methanol, 100 mL acetic acid аϷ 400 mL ѐᚆηН షӝǶ

z ሌࢉڰۓనǺڗ 105 mL 95% Ethanol ک 25 mL Acetic acidǴаѐᚆηН ۓໆԿ 250 mLǶ

z ሌࢉ௵ϯనǺڗ 79 mL 95% Ethanolǵ0.78 g Sodium thiosulphate ک 17 g Sodium acetateǴаѐᚆηНۓໆԿ 250 mLǶ

(29)

18

z ሌࢉϸᔈనǺڗ 0.625 g Silver nitrate уΕѐᚆηНӣྋԿᡏᑈ 250 mLǴ ٠ܭ٬Ҕ߻уΕ 0.1 mL FormaldehydeǶ

z ሌࢉว৖నǺڗ 6.25 g Na2CO3 аѐᚆηНӣྋԿᡏᑈ 250 mLǴ٠ܭ٬

Ҕ߻уΕ 0.05 mL FormaldehydeǶ

z ሌࢉଶЗనǺڗ 12.5 mL Acetic acidǴуѐᚆηНۓໆԿ 250 mLǶ (2) Ꮤ׷

z Mini-protein ˩ ೈқ፦ႝݚس಍ᖼԾ Bio-Rad Laboratories (Hercules, CA, USA)

z Thermomixer comfort 1.5 mL ᖼԾ Eppendorf (Hamburg, Germany) z PXi 4 Image analysis system ᖼԾ SYNGENE (Cambridge, UK)

ȜБݤȝ

(1) ᡂ܄ጤᡏᇙഢ

᠗ጤ࣒ТӃа 70% ଚᆒమዅ٠ᔔ࡬ଳృǴ࿼ܭڰۓࢎ΢֨ᆙ٠ዴߥόᅅన ࡕǴଛӳ 15% running gel ݙΕ࣒Т໔ऊΖϩᅈǴܭ΢БуΕ methanol ஒྋనᓸ ѳǶऊ 30 ϩដࡕᏉጤǴॹѐ methanol ٠ஒଛӳϐ 4% stacking gel ݙΕ࣒Т໔Ǵ

ِೲකΕᏁ਩Ǵᓉ࿼ 30 ϩដࡕᏉጤǶஒᏁ਩ܨΠࡕǴጤТջёາጤϩ݋Ǵ܈੆ݰ ӧ tank buffer ύܭ 4ÛC ߥӸ΋ຼǶ

(2) ጤᡏႝݚᆶࢉՅ

టϩ݋ϐೈқ፦ኬࠔፓ᏾ԿҞ኱ᐚࡋࡕǴڗ 20 ȝL ᆶ 5ȝL ϐ 5X SDS sample dye షӝ֡ϬǴа 99ÛC у዗ 10 ϩដ٬ೈқᡂ܄Ǵу዗ࡕڗр࿼ӇǶஒ᠗ӳϐጤ ТࢎӧႝݚኲύǴॹΕىໆ tank bufferǴ٠ݙΕςफ़ྕޑኬࠔܭጤТпኲύǴᇂ΢

ႝݚኲ΢ᇂǶೱௗႝྍٮᔈᏔǴڰۓႝᓸ 60 ҷ੝຾ՉႝݚǴࡑ sample dye வ stacking gel າԿ running gelǴׯႝᓸࣁ 110 ҷ੝ᝩុႝݚǶऊ 2 λਔႝݚёֹ

ྋԿᡏԿᡏԿᡏԿᡏԿᡏᡏᑈᑈᑈᑈᑈᑈ 2250500mmmmLLLLLLǴ

2 2 2 2

25050505050mmmmmmLLLLLLǴ٠Ǵ٠Ǵ٠Ǵ٠٠٠ܭ٬ܭ٬ܭ٬ܭ٬ܭ٬ܭ٬

(30)

19

ԋǴλЈஒጤᡏԾ࣒ТڗΠࡕǴа CBR ࢉᏊࢉՅ 30 ϩដǴӣԏࢉᏊࡕӆаѐᚆ ηН܈ଏࢉᏊଏԿङඳ೸ܴǴջёаྣጤس಍ߥӸکϩ݋ቹႽǶ

(3) ሌࢉ

ሌࢉ΋ᅿཱུࡋᡫ௵ޑೈқ፦ࢉՅݤǴ೸ၸೈқ፦΢ޑለ୷ᆶሌሓᚆη่ӝǴሌ ᚆηӧለ܄ᕉნύ೏ᗋচࣁߎឦሌǴё٬ጤᡏ΢ޑೈқ፦և౜ుලՅǶஒາӳϐ SDS-PAGE Ծ᠗ጤ࣒ዟڗΠܫΕࢉՅ౯ࡕǴуΕሌࢉڰۓన (fixation) འਗϸᔈ 30 ϩដǶॹ௞ڰۓనࡕǴуΕሌࢉ௵ϯన (sensitizing) ӕኬའਗϸᔈ 30 ϩដǶ

௵ϯֹԋࡕǴॹ௞నᡏ٠аѐᚆηНࢱѐӭܭనᡏǴ؂ԛϖϩដǴᕴӅ຾ՉΟԛǶ ࢱֹࡕஒనᡏॹଳǴуΕሌࢉϸᔈనϸᔈ 20 ϩដǶӣԏሌࢉϸᔈనǴӆԛаѐᚆ ηНࢱѐӭܭሌᚆηǴ؂ԛ΋ϩដǴӅࢱٿԛǶॹ௞ഭᎩనᡏǴуΕሌࢉว৖న (developing) ฻ࡑుලՅచ஥р౜ǴջёуΕଶЗన่״ϸᔈǶ10 ϩដࡕǴጤᡏࢉ

ՅջֹԋǴё຾Չࡕុ၂ᡍ܈аѐᚆηНߥӸǶ

ಃΟ࿯! ೈқ፦ᔸԄᙯӑ

Ȝ׷਑ȝ (1) ၂ᏊǺ

z 10 ७ CAPSǺڗ 22.13 g CAPS аѐᚆηНӣྋ٠ፓ᏾Կ pH 11 ࡕǴۓ ໆԿ 1 L ࡑҔǶ

z Transfer bufferǺڗ 200 mL 10 ७ CAPS ک 500 mL methanolǴаѐᚆη НۓໆԿ 2500 mLǴ࿼ܭ 4 ÛC ႣհǶ

(2) Ꮤ׷Ǻ

z BioRed ᔸԄᙯӑᏔ׷ᖼԾ Bio-Rad Laboratories (Hercules, CA, USA) z Hybond ECL Nitrocellulose membrane (0.22 μm) ᖼԾ GE Healthcare (UK)

ԏࢉ

ԏࢉࢉᏊࡕᏊࡕᏊࡕᏊࡕᏊࡕᏊࡕӆаӆаааѐᚆѐᚆѐᚆѐᚆѐᚆѐᚆ

(31)

20

z 3MM Chr Chromatography Paper ᖼԾ Whatman Inc. (New Jersey, USA)

ȜБݤȝ

ڗٿТᘠરǵາֹႝݚϐጤТаϷ NC ጢ੆ݰܭ transfer buffer ѳᑽ 10 ϩ ដǴஒ΢ॊނҹаᘠરǵጤТǵNC ጢǵᘠર฻໩ׇಔӝǴ٬ႝࢬБӛࣁ NC ጢ ԿጤТǴаᙯӑպ֨ӳࡕܫΕᙯӑኲ٠ॹΕtransfer buffer ܭځύǴӧ 4ÛC ᕉნΠ а 400 mA ᙯӑ 1.5 λਔǶ

ಃѤ࿯! ՋБᙯᅄݤ (western blotting)

Ȝ׷਑ȝ (1) לᡏǺ

z Anti-FIP-fve (D1 ਲ਼)Ǻҗჴᡍ࠻߻ΓᇙഢǴ5000 ७ีញ٬ҔǶ

z Anti-TCR alpha antibody (ab18861), Anti-TCR beta antibody (ab18862) ᖼԾ abcam plc. (Cambridge, UK)

z AP conjugated goat anti-mouse IgG antibody, AP conjugated goat anti-rabbit IgG antibody ᖼԾ Kirkegaard & Perry Laboratories (KPL), Inc. (MD, USA) (2) ၂ᏊǺ

z 10 ७ TBSǺڗ 2.42 g Tris-base ک 29.2 g NaCl аѐᚆηНۓໆԿ 1000 mLǶ

z TTBSǺڗ 100 mL 10 ७ TBS уΕ 1 mL Tween-20ǴаѐᚆηНۓໆԿ 1000 mLǶ

z TMB membrane peroxidase substrate ᖼԾ Kirkegaard & Perry Laboratories (KPL), Inc. (MD, USA)

z Immobilon^TMWestern Millipore Chemiluminescent HRP Substrate ᖼ Ծ w

w w w

wJJJJJerererrrrssseseses yyy,y, UUSASASASA))) ) ) )

(32)

21

Millipore corporation (MA, USA)

ȜБݤȝ

ᙯӑࡕޑ NC ጢа Blocking buffer ܭ 4ÛC ᕉნΠϸᔈ΋ఁǴ႖Ϻஒ΋לа blocking buffer ፾ໆีញࡕǴܭ࠻ྕΠའਗϸᔈ 1 λਔǶॹ௞΋לࡕǴॹΕ TTBS ࢱѐ҂ᗖ่ϐלᡏǴ؂ԛ 10 ϩដǴӅΟԛǶڗ TTBS ၂ໆีញΒלࡕǴᆶ NC ጢܭ࠻ྕΠའਗϸᔈ 1 λਔǶॹ௞ΒלࡕǴӕኬа TTBS ࢱѐ҂ᗖ่ϐלᡏǴ؂

ԛ 10 ϩដǴӅΟԛǶஒූᎩ TTBS ѐନࡕǴуΕ 1 mL TMB membrane peroxidase substrate ևՅǴࡑጢ΢р౜ᙔՅచ஥ࡕǴуΕѐᚆηНଶЗϸᔈǶऩట຾ՉհӀև ՅǴ߾ѐନ TTBS ࡕǴܭས࠻ΠуΕ 1 mL ฻ᡏᑈషӝϐ Chemiluminescent HRP SubstrateǴϸᔈऊ 30 ࣾࡕ౽ѐӭᎩనᡏǴஒ NC ጢܫΕྣጤس಍ύևՅǶևՅਔ аಕуБԄ؂ᚼӀ 30 ࣾᓯӸቹႽΌԛǴޔډհӀૻဦόӆቚமࣁЗǶ

ಃϖ࿯! ख़ಔೈқ rFIP-fve 1-114 Ϸ truncated rFIP-fve 14-114 ୷Ӣᒧ෗ᆶᄬᑐ

Ȝ׷਑ȝ

(1) cDNA ٰྍǺςஒ FIP-fve ୷Ӣӄߏᄬᑐܭ pGEX4T-1 vectorǴ٠ᙯࠠԿεဉ ఎ๵ XL-10 ύǴڗளԾύξᙴᏢεᏢ࢒ߪؼԴৣჴᡍ࠻Ƕ

(2) ፦ᡏٰྍǺpET-32a(+) ᙯࠠԿεဉఎ๵ DH-5Į ύǴჴᡍ࠻ߥӸ๵ᅿǶ (3) ၂ᏊǺ

z pGM-T PCR Cloning Kit ᖼԾ GMbiolab (ѠύѱǴѠ᡼)

z QIAquick Gel Extraction Kit ᖼԾ Qiagen Sciences (Maryland, USA) z Plasmid Miniprep Purification Kit ᖼԾ GMbiolab (ѠύѱǴѠ᡼)

z 5 X TBE BufferǺڗ 54 g Tris baseǵ27.5 g boric acid аϷ 20 mL 0.5 M EDTAǴፓ᏾ pH 8.0 ࡕǴۓໆԿ 1 LǶ

(33)

22

z LBAǺڗ 10 g Tryptoneǵ10 g NaClǵ5 g Yeast extract аϷ 15 g Bacto Agar аѐᚆηНۓ৒Կ 1 LǴྐ๵ࡕ٬ҔǶ

z LB ୻ᎦనǺڗ 10 g Tryptoneǵ10 g NaCl аϷ 5 g Yeast extract аѐᚆ ηНۓ৒Կ 1 LǴྐ๵ࡕ٬ҔǶ

(4) FIP-fve ߄౜୷ӢЇη೛ीǺ

z fve 1-114 forward primer (AAA + Kpn I + Enterokinase + S¹ A² T³ S⁴)Ǻ 5’- AAAGGTACCGACGACGACGACAAGTCCGCCACGTCG -3’

z fve 1-114 reverse primer (W K K T + stop codon + Xho I + AAA)Ǻ 5’- AAACTCGAGTCAAGTCTTCTTCCA -3’

z fve 14-114 forward primer (AAA + Kpn I + Enterokinase + K¹⁴ K¹⁵ I¹⁶ D¹⁷)Ǻ 5’- AAAGGTACCGACGACGACGACAAGAAGAAGATCGAT -3’

z fve 14-114 reverse primer (W K K T + stop codon + Xho I + AAA)Ǻ 5’- AAACTCGAGTCAAGTCTTCTTCCA -3’

(5) Ꮤ׷

z GeneAmp® PCR System 2700 ᖼԾ Applied Biosystems (CA, USA)

z Mupid2-plus DNA gel electrophoresis system ᖼԾ ADVANCE Co., Ltd.

(Japan)

ȜБݤȝ

(1) ᆫӝ䁙ೱᙹϸᔈ (polymerase chain reaction, PCR)

а֖Ԗ FIP-fve ӄߏ୷Ӣϐεဉఎ๵ XL-10 բࣁኳ݈ DNAǴڗ 4.6 ȝL

๵నуΕ 0.2 ȝL forward primerǵ0.2 ȝL reverse primer аϷ 5 ȝL Taq DNA Polymerase Master Mix RedǴషϬࡕஒᆅᏛనᡏᚆЈࡕǴа GeneAmp® PCR System ຾Չ colony PCRǴځచҹࣁ 94ÛC ϸᔈ 2 ϩដࡕǴௗޣ 94ÛC ϸᔈ 30 ࣾǵ55ÛC ϸᔈ 30 ࣾǵ72ÛC ϸᔈ 40 ࣾӅ 24 ঁൻᕉǴࡕа 72ÛC ϸᔈ 10

Ϸ Ϸ Ϸ

Ϸ 111155555ggg g g g BBBaBactcttooAgAgAgAgAgAgararar

astststtteeeeextxtxtxtrarararararactctctctctct аѐаѐаѐаѐаѐаѐᚆᚆᚆᚆᚆᚆ

(34)

23

ϩដࡕǴफ़ྕԿ 4ÛC ኩӸǶ

(2) DNA ᛏિጤᡏႝݚ (Agarose Gel Electrophoresis)

ܭጤᡏႝݚ຾Չъλਔ߻ǴӃ᠗ጤࡑҔǶаλТ DNA ጤᡏࣁٯǴڗ 20 mL 0.5 X TBE bufferǴ٩టϩ݋ϐ DNA ТࢤελόӕуΕ 1-3% ϐ agaroseǴ നࡕуΕ 0.75 ȝL HealthView Nucleic Acid Stain འϬܫΕ༾ݢ᝗у዗Ǵࡑݦ ប 2-3 ԛࡕ৾рǶஒฆӳጤᡏॹΕ᠗ጤး࿼Ǵක΢Ꮑ਩ࡕᗉӀࡑځᏉڰǶஒ ᏉጤֹԋϐጤТᏁ਩ܘଆǴܫΕႝݚኲύǴ٠ॹΕ 0.5 X TBE buffer ᇂၸጤ ᡏǶஒ PCR ౢނݙΕጤТǴ٠аӄႝᓸ຾ՉႝݚǶႝݚ่״ࡕǴа UV ᐩ ዴᇡచ஥Տ࿼Ǵӆа PXi 4 Image analysis system ૶ᒵቹႽǶ

(3) DNA ጤᡏપϯ

٬Ҕ QIAquick Gel Extraction Kit ຾ՉપϯǶ२ӃǴϪΠ֖ԖҞ኱ DNA

೽ϩϐጤᡏܫΕ 1.5 mL ࣁໆᚆЈᆅύǴуΕ 3 ७ᡏᑈޑ QG bufferǴܭ 50ÛC ϸᔈ 10 ϩដǶዴᇡጤᡏྋှࡕǴуΕ 1 ७ᡏᑈϐ isopropanol ࡕషϬǶڗ 700 ȝL ༾ໆᚆЈᆅύనᡏуΕ QIAquick colume ύǴаനଯᙯೲᚆЈ 1 ϩ ដǶᚆЈֹࡕନѐᘠనǴख़ፄ؁ᡯޔډ༾ໆᚆЈᆅύనᡏڗֹǶуΕ 500 ȝL QG buffer ܭ colume ύࢱѐූᎩጤᡏǴаനଯᙯೲᚆЈ 1 ϩដǶௗޣуΕ 750 ȝL PE bufferǴаനଯᙯೲᚆЈ 1 ϩដǶѐନᘠనࡕǴӆԛаനଯᙯೲᚆ Ј 3 ϩដǴаዴᇡֹӄѐନ PE buffer ύ ethanolǶᚆЈֹࡕǴஒ QIAquick colume ౽Կཥޑ 1.5 mL ༾ໆᚆЈᆅǴڗ 30 ȝL ѐᚆηНܭ colume ΢Ǵᓉ

࿼ 1 ϩដࡕǴӆԛаനଯᙯೲᚆЈ 1 ϩដջёܭΠనڗளપϯϐ DNA Т ࢤǶ

(4) T-A cloning کᙔқᑔ

ጤᡏࣁٯٯٯ ڗ

(35)

24

аၩᡏᆶකΕ DNA ڗໆࣁ 1: 3 ϐКٯǴ٩ pGM-T cloning kit ࡌ᝼ଛ БуΕ 1 ȝL T4 ligase ک 1 ȝL 10 X bufferǴуΕѐᚆηНፓ᏾ᡏᑈԿ 10 ȝL ࡕǴܭ 16ÛC Πϸᔈ 10 λਔ຾Չၩᡏᆶ DNA ϐௗӝ (ligation)Ƕ

ܭௗӝਔёྗഢᙔқᑔ୻ᎦҝǴ२Ӄڗྐ๵ࡕϐ LBA ࡑफ़ྕԿ 55ÛCǴ ܭค๵ᏹբѠύуΕ 100 ȝg/mL ampicillin ࡕॹܭ୻ᎦҝύǶफ़ྕࡕ LBA Ꮙ ڰǴӃஒ୻ᎦҝॹᇂᠴዬǴऊъλਔ୻ᎦҝН਻ԏଳࡕǴуΕ 50 ȝL X-gal (50 mg/mL) аϷ 20 ȝL 0.5 M IPTG ܭ୻ᎦҝύǴӧས࠻Πа 0.5 mm ࣒ዟ੧༡ ዬǴ٠࿼ܭ 37ÛC ୻ᎦъλਔǶ୻Ꭶҝྗഢӳࡕё຾Չ༡ዬǴڗ 10 ȝL ௗӝ

ֹԋϐ፦ᡏᆶ 50 ȝL DH-5Į ๵నܭ 1.5 mL ༾ໆᚆЈᆅύషӝǴа 45ÛCǵ45

ࣾࣁచҹ຾Չ heat shockǴheat shock ֹԋҥڅ࿼Ӈ 10 ϩដǶܭค๵ᏹբѠ ύуΕ 40 ȝL LB ୻ᎦనǴ٠ڗр๵న֡Ϭ༡Ѳܭྗഢӳϐᙔқᑔ୻ᎦҝǴа 0.5 mm ࣒ዟ੧༡ዬࡕǴ࿼ܭ୻Ꭶጃύа 37 ÛC ୻Ꭶ႖ڹǶಃΒϺǴࡷᒧқՅ

๵ပǴλໆ୻Ꭶࡕஒ๵నଌԿ୷ᓪԯլථғނࣽמϦљ຾Չਡለۓׇϩ݋Ƕ

(5) ፦ᡏ DNA ᇙഢ

ஒ֖Ԗ pET-32a(+) ޜ፦ᡏϐ DH-5Į εဉఎ๵λໆ୻Ꭶऊ 10 mLǴаന ଯᙯೲᚆЈڗள๵༧Կ༾ໆᚆЈᆅࡕǴуΕ 200 ȝL Solution I ܭᚆЈᆅύǴ а vortex БԄషӝઇ๵ࡕǴуΕ 200 ȝL Solution II ٠ᇸᇸٰӣᙌᙯ 10 ԛǴ Ԝਔಒझᝌੌన೴ᅌዂమǶௗޣуΕ 200 ȝL Solution IIIǴٰӣᙌᙯ 10 ԛషӝ

֡ϬǴԜਔ཮р౜қՅ؈ᐘނǴаനଯᙯೲᚆЈ 5 ϩដǶஒ Spin column း Ε Collection tube ύǴڗዂమϐ΢మనλЈ౽Կ spin columnǴаനଯᙯೲᚆ Ј 1 ϩដǶ౽ନ collection tube ύޑᘠనǴуΕ 700 ȝL Wash SolutionǴаന ଯᙯೲᚆЈ 1 ϩដǴख़ፄԜమࢱ؁ᡯ΋ԛǶ౽ନ collection tube ύޑᘠనǴ ӆԛаനଯᙯೲᚆЈ 3 ϩដǴаዴᇡֹӄѐନ PE buffer ύ ethanolǶஒ spin column ౽Կཥޑ 1.5 mL ༾ໆᚆЈᆅǴڗ 50 ȝL ѐᚆηНܭ colume ΢Ǵᓉ

looooonninniningngngngngngkkkkiititi ࡌ᝼ࡌ᝼ࡌ᝼ࡌ᝼᝼᝼ଛଛଛଛଛଛ ፓ

ፓ ፓ ፓ

ፓ᏾ᡏ᏾ᡏ᏾ᡏ᏾ᡏ᏾ᡏ᏾ᡏᑈԿᑈԿᑈԿᑈԿᑈԿ 11110 0 000 ȝȝȝȝLLLLLL gatititititiononononon)))ǶǶǶǶǶǶ

(36)

25

࿼ 1 ϩដࡕǴӆԛаനଯᙯೲᚆЈ 1 ϩដջёܭΠనڗளપϯϐ፦ᡏǴёߥ Ӹӧ -20ÛC ࡑҔǶ

(6) ज़ڋ䁙Ϫϸᔈ (restriction enzyme reaction) کௗӝϸᔈ (ligation)

а፦ᡏ DNA ᇙഢБݤڗளֹ֖᏾܈લѨϐ FIP-fve DNA Тࢤϐ pGM- T ፦ᡏࡕǴڗ 5 ȝg ፦ᡏуΕ 0.2 ȝL BSAǵ2 ȝL NEB buffer 1 ک 0.5 ȝL Xho IǴံѐᚆηНԿᕴᡏᑈ 20 ȝL ࡕǴܭ 37ÛC Πϸᔈ 1 λਔǴӆуΕ 0.15 ȝL Kpn IǴܭ 37ÛC Πϸᔈ 15 ϩដǴջֹԋज़ڋ䁙ϪϸᔈǶpET-32a(+) ޜ፦ᡏ

ज़ڋ䁙Ϫϸᔈ߾ࢂڗ 10ȝg ፦ᡏуΕ 0.5 ȝL BSAǵ5 ȝL NEB buffer 1ǵ 0.5 ȝL Xho I ک 0.5 ȝL Kpn IǴံѐᚆηНԿᕴᡏᑈ 50 ȝL ࡕǴܭ 37ÛC Πϸᔈ 75

ϩដǶஒज़ڋ䁙Ϫϸᔈֹϐ DNA аᛏિጤᡏႝݚϩ݋ࡕǴϪΠ֖Ҟ኱ DNA ϐጤᡏࡕǴа DNA ጤᡏપϯБݤջёڗளࣁ sticky end ϐ DNA ТࢤϷ linear ፦ᡏǶ

ஒપϯࡕϐ pET-32a(+) ፦ᡏᆶ DNA Тࢤа 1: 3 ϐКٯڗໆǴӆуΕ 1 ȝL T4 ligase ک 1 ȝL 10 X bufferǴуΕѐᚆηНፓ᏾ᡏᑈԿ 10 ȝL ࡕǴܭ 16ÛC Πϸᔈ 10 λਔ຾Չၩᡏᆶ DNA ϐௗӝǶ10 λਔࡕǴ٩߻ॊБݤஔ

ӝֹԋϐ፦ᡏᙯ׎ (transform) Կ DH-5Į εဉఎ๵Ǵ༡ዬࡷ๵λໆ୻Ꭶࡕஒ

๵నଌԿ୷ᓪԯլථғނࣽמϦљ຾Չਡለۓׇϩ݋ǶዴᇡਡለׇӈคᇤǴӆ ஒ፦ᡏԾ๵ύڗрǴᙯ׎Կ፾ӝೈқ߄౜ϐεဉఎ๵๵ਲ਼ BL-21Ǵ༡ዬࡷ๵ࡕ ӆԛ຾Չਡለۓׇϩ݋ǴКჹׇӈ҅ዴࡕǴջֹԋख़ಔೈқϐᒧ෗ᆶᄬᑐǶ

ಃϤ࿯! ख़ಔೈқ rFIP-fve 1-114 Ϸ truncated rFIP-fve 14-114 ߄౜ᆶપϯ

Ȝ׷਑ȝ (1) ၂ᏊǺ

z 0.5 M IPTGǺܭค๵ᕉნΠа 0.22 ȝm Ͼ৩ᘠጢၸᘠǴܭ 4ÛC ߥӸǶ પ

પ પ પ

પϯϐϯϐϯϐϯϐϯϐϐ፦ᡏ፦ᡏ፦፦ ǴёǴёǴёǴёёёߥߥߥߥߥߥ

(37)

26

z Lysozyme stockǺڗ 0.1 g Lysozyme ӣྋܭ 10 mL ѐᚆηНǴϩးࡕӇ -20ÛC ࡑҔǶ

z Binding bufferǺڗ 7.6 g Na3PO4•12H2Oǵ29.22 g NaCl ک 0.34 g imidazole уѐᚆηНӣྋࡕǴፓ᏾ pH ॶࣁ 7.4ǴӆۓໆԿ 1 LǶ

z Elution bufferǺڗ 7.6 g Na3PO4•12H2Oǵ29.22 g NaCl ک 34 g imidazole уѐᚆηНӣྋࡕǴፓ᏾ pH ॶࣁ 7.4ǴӆۓໆԿ 1 LǶ

z EKMax™ Enterokinase ᖼԾ Invitrogen (CA, USA) (2) Ꮤ׷Ǻ

z ÄKTA purifier FPLC system, fraction collector frac 900, HiTrap IMAC FF 5 mL ᖼԾ GE Healthcare (UK)

ȜБݤȝ

(1) ๵ਲ਼୻Ꭶᆶ His-tag ᑼӝೈқ߄౜

ڗ 50 ȝl ๵న୻Ꭶܭ֖ 100 ȝg/mL ampicillin ϐ 5 mL LB నᄊ୻Ꭶ୷ύ (37 ÛCǴ130 rpm)Ǵऊ 6 λਔࡕջֹԋλໆܫεǶڗࢲϯϐ๵న 1 mL уܭ֖

100 ȝg/mL ampicillin ϐ 1L LB నᄊ୻Ꭶ୷ύǴࡑ๵న୻ᎦԿ֎Ӏॶၲ

0.6~0.8 ࡕǴуΕ IPTG ᇨᏤ His-tag ᑼӝೈқ߄౜Ǵڗ๵నາጤࡕаՋБᏀ ᗺݤዴᇡ߄౜ਔ໔کᇨᏤᐚࡋࡕǴനಖа 0.4 mM IPTG ᇨᏤऊ 4 λਔǴа եྕଯೲᚆЈ 10 ϩដ (4ÛCǴ9,500 rpm) ѐନ୻Ꭶ୷Ǵԏ໣๵༧٠аѐᚆη НమࢱࡕǴᚆЈѐନූᎩ୻Ꭶ୷Ǵஒ๵༧Ӹܭ -80ÛC ࡑҔǶ

(2) ๵నኬࠔ΢ᐒ߻ೀ౛

๵༧ှএࡕǴஒ 1 L ๵నԏ໣ϐ๵༧а 40 mL Binding buffer ᝌੌࡕǴ уΕ 200 ȝL lysozyme stock ܭ 4ÛC ᠳ܏ 30 ϩដྋ๵Ǵӆаຬॣݢ᎜ᕏᐒઇ

࿗ೀ౛ (ઇ࿗ 10 ࣾଶ 10 ࣾǴᕴઇ࿗ਔ໔ 10 ϩដ)Ƕઇ࿗ࡕ๵నаեྕଯ ೲᚆЈ 10 ϩដ (4ÛCǴ9,500 rpm)Ǵԏ໣΢ቫዂమనǴஒϐа 0.45 ȝm ک 0.22

ᚆ ᚆ ᚆ

ᚆηНηНηНηНηННǴϩǴϩǴϩǴϩးࡕးࡕးࡕးࡕࡕࡕӇӇӇӇӇӇ

ک 00000.3.3.3.3. 4 4 4 4 4 4 gggg g g imimimimiimididididddazazazazazazooololololeeeee

(38)

27

ȝm Ͼ৩ᘠጢၸᘠǴᘠనӇ 4ÛC ࡑҔǶ

(3) а FPLC (Fast protein liquid chromatography) س಍પϯ His-tag ᑼӝೈқ ஒ FPLC س಍ᆅጕаѐᚆηНమࢱଳృࡕǴး΢ᆅࢊ٠ݙΕ 0.1 M NiSO4Ǵ٬ᙻᚆηᆶጤᡏ่ӝǴܭ 4ÛC ΠբҔǶ30 ϩដࡕǶӃаѐᚆηНమ ࢱᆅݙǴӆݙΕ binding buffer ѳᑽǴஒ࿶ၸ߻ೀ౛ϐ๵నа 3 mL/min ࢬೲ ݙΕᆅࢊǶࡑ๵నӄኧݙΕᆅࢊࡕǴа binding buffer ࢱѐ҂ᗖ่ೈқǴ೛ۓ Ο໘ࢤࢬࢱǺ1. ڰۓ 15% elution buffer ࢬࢱ 10 C.V.Ƕ2. ఊࡋ 15~60% Elution buffer ࢬࢱ 10 C.V.Ƕ3. ڰۓ 100% Elution buffer ࢬࢱ 4 C.V.Ƕԏ໣؂໘ࢤࢬ рనǴ೸ၸ SDS-PAGE ዴᇡ His-tag ᑼӝೈқࢬࢱрޑՏ࿼Ƕ

(4) His-tag Ϫନ

аᐚᕭᆅᐚᕭ࿶ FPLC પϯϐ His-tag ᑼӝೈқǴ٠ஒϐ೸݋Կ PBS

጗ፂྋనύǶෳ၂ EK ሇનϪନ His-tag ϐచҹǴനಖа 0.1 uL ሇનᆶ 20 ȝL ᑼӝೈқКٯܭ 4ÛC Πϸᔈ 2~4 ϺǶϪନֹ His-tag ϐኬࠔǴ࿶ᐚᕭ٠ а 0.22 ȝm Ͼ৩ᘠጢၸᘠࡕǴёҔܭࡕុಒझ၂ᡍύǶ

ಃΎ࿯! ጤᡏϣНှ (In gel digestion)

Ȝ׷਑ȝ (1) ၂ᏊǺ

z 0.1 M NH4HCO3Ǻڗ 7.9 g NH4HCO3 ӣྋܭѐᚆηНύǴፓ᏾ pH 8.0 ࡕ ۓໆԿ 1 LǶ

z 0.1 M DTT stockǺڗ 0.15 g DTT а 10 mL 0.1 M NH4HCO3 ӣྋǶ aggggg ᑼӝᑼӝᑼӝᑼӝᑼӝᑼӝೈқೈқೈқೈқೈқೈқ

ࢊ٠ݙΕ

(39)

28

ȜБݤȝ

ڗςֹԋ CBR ࢉՅ٠ଏࢉϐጤᡏǴҔЋೌΘϪΠҞ኱చ஥٠Ϫ࿗ࡕǴஒጤᡏ

းΕ 1.5 mL ༾ໆᚆЈᆅύǴуΕ 1 mL 40% ACN/ 0.1M NH4HCO3 ᎜ᕏ 10 ϩដǴ ᚆЈڗ΢మǴख़ፄԜ؁ᡯޔډጤᡏଏࣁคՅǶуΕ 1 mL ѐᚆηН᎜ᕏΟϩដࢱ ѐӭᎩ ACNǴᚆЈڗрనᡏǶख़ፄаѐᚆηНమࢱٿԛࡕǴܜрనᡏǴуΕ 100%

ACN ਁᕏಥНԿጤಈᡂқǴ٠а੿ޜᚆЈᐒܜѐྋᏊǶࡑጤᡏଳᔿࡕǴуΕ 50 ȝL 10 mM DTT/ 0.1M NH4HCO3 ᇂၸጤᡏǴਁᕏషϬԿጤ༧ݰ๞೸ܴࡕǴ࿼ܭ 56ÛC Πϸᔈ΋λਔǶڗр༾ໆᚆЈᆅǴᚆЈࡕѐନ΢మనǴуΕ 1 mL ѐᚆηН᎜ᕏΟ ϩដǴᚆЈڗрనᡏǶख़ፄаѐᚆηНమࢱٿԛࡕǴܜрనᡏ٠ܭ੿ޜΠѐྋᏊ30 ϩដǶࡑጤᡏଳᔿࡕǴזೲуΕ50 ȝL 55mM IAM/ 0.1M NH4HCO3 ᇂၸጤ༧Ǵਁ

ᕏషϬࡕܫ࿼སೀϸᔈ 45 ϩដǶѐନ΢మనǴуΕ 1 mL 40% ACN/ 0.1 M NH4HCO3 ࢱٿԛǴڗрనᡏࡕуΕ 1 mL ѐᚆηН᎜ᕏΟϩដǴᚆЈڗрనᡏǶ

ख़ፄаѐᚆηНమࢱٿԛࡕǴܜрనᡏ٠ܭ੿ޜΠѐྋᏊ 30 ϩដǶуΕӣྋܭ

0.1M NH4HCO3 ϐ trypsinǴፓ᏾ሇનᆶೈқ፦֖ໆКࣁ 1: 20 - 1: 100 ϐ໔Ǵ٠у Ε 0.1M NH4HCO3 ԿవၸጤᡏࡕǴܭ 37ÛC Πϸᔈ΋ఁǶಃΒϺǴஒ༾ໆᚆЈᆅ షϬ٠ЪᚆЈࡕǴԏ໣న࣬Կཥޑ༾ໆᚆЈᆅύǴ٠уΕ 50%ACN/ 0.1% FA ๧ڗ ጤᡏ 3 ԛǴ؂ԛ 30 ϩដǴ٠֎рన࣬ࡕᆶӃ߻న࣬ӝٳǶуΕ 80% ACN/ 0.1%

FA ຾Չനࡕ๧ڗǴ10 ϩដࡕ֎рన࣬ᆶӃ߻న࣬ӝٳǶஒ܌Ԗӝٳޑన࣬੿ޜᚆ ЈଳᔿǶࡑనᡏֹӄଳᔿࡕǴуΕ 10 ȝL 0.1% FA ྋှೈқТࢤǴ٠Կܭ -20ÛC ߥ Ӹࡑϩ݋Ƕ

ಃΖ࿯! λႵ๠᠌ಒझڗள

Ȝ׷਑ȝ

(1) ჴᡍ୏ނǺ

٠ ٠ ٠ ٠

٠ϪϪϪ࿗Ϫ࿗Ϫ࿗Ϫ࿗ࡕǴࡕǴࡕǴࡕǴࡕǴஒጤஒጤஒጤஒጤஒጤᡏᡏᡏᡏᡏᡏ O3 ᎜ᕏ᎜ᕏ᎜ᕏ᎜ᕏ᎜ᕏᕏ1010101011 ϩដϩដϩដϩដϩដϩដǴ ηН᎜ᕏΟϩΟϩΟϩΟϩដࢱ

(40)

29

z BALB/cByJNarl λႵᖼԾ୯ৎჴᡍ୏ނύЈ (ᆵчѱǴѠ᡼) (2) ၂ᏊǺ

z RBC lysis bufferǺ155 mM NH4Clǵ10 mM KHCO3ǵ0.1 mM EDTA аѐᚆ ηНྋှషӝࡕǴܭค๵ᕉნΠ٬Ҕ0.22 ȝm Minisart Ͼ৩ᘠጢၸᘠǴܭ 4±C ߥӸǶ

z ಒझ୻ᎦనǺ֖ 10 % FBS ϐ RPMI-1640 mediumǶ

z 0.4% (w/v) trypan blue ࢉᏊǺᆀڗ 2 g trypan blue ྋܭ 500 mL ѐᚆηН ύషϬǶ

(3) Ꮤ׷Ǻ

z ୊ΘǵᢍηᖼԾߞቺ (ᆵчѱǴѠ᡼) z ಒझीኧᏔᖼԾ Reichert Inc., (NY, USA)

z ಒझ୻Ꭶጃ HeraǵᚆЈᐒ Labofuge 400 ᖼԾ Heraeus (Hanau, Germany)

ȜБݤȝ

ஒλႵаΒ਼ϯᅹ឴࣊ࡕǴаଚᆒஒځӄيቔᔸ੃ࢥǴଌ຾ค๵ᏹբᆵǶԾλ ႵѰୁङ೽Տ࿼୊Πλ༧ѦҜǴኚ໒ѦҜࡕӆаќୋ୊Θ୊໒ϣҜǴஒ๠᠌ڗрܫ

࿼ PBS ύǶ٬Ҕᑃ࣒ࣳТӃаࡪᓸБԄᇸᇸڗрಒझ٠ӣྋܭ PBS ύǴஒಒझ న౽ΕᚆЈᆅǴа 1,500 rpm ᚆЈϖϩដѐନ΢మǶܡණಒझი༧ࡕǴуΕ 1 mL RBC lysis buffer ྋှआՈౚǴᇸܡᚆЈᆅ 1 ϩដࡕᓉ࿼ 2 ϩដ٬ځϸᔈֹӄǴ уΕ 9 mL PBS ଶЗϸᔈǶӆԛа 1,500 rpm ᚆЈϖϩដѐନ΢మǴуΕ୻Ꭶనᝌ

ੌಒझǴ٠а trypan blue ࢉᏊࢉՅीᆉಒझኧໆǴа୻Ꭶనፓ᏾࿼፾྽ᐚࡋǴջ ёᆶኬࠔӅ୻ᎦǶ

Ѡ᡼

Ѡ᡼Ѡ᡼))) ) ) )

mMMMMMEEEEEDTDTDTDTDTDTA A A A AA аѐаѐаѐаѐаѐаѐᚆᚆᚆᚆᚆᚆ Ͼ৩ᘠጢၸᘠၸᘠၸᘠၸᘠ ܭ

(41)

30

ಃΐ࿯! ሇનೱ่խࣝ֎ߕϩ݋ (Enzyme-linked immunosorbent assay, ELISA)

Ȝ׷਑ȝ (1) ၂ᏊǺ

z IFN-Ȗ ELISA DuoSet¯ ᖼԾ R&D Systems (Minneapolis, MN, USA) z Wash bufferǺܭ PBS ύуΕ 0.05% (v/v) ϐ Tween 20Ƕ

z Block buffer : 1% BSA ྋܭ pH 7.2 ϐ PBS ጗ፂనύǶ

z Tris-buffered Saline (TBS)Ǻڗ 3.02 g Tris ک 4.38 g NaCl ӣྋܭѐᚆη НύǴፓ᏾ pH ॶࣁ 7.2 ࡕǴۓໆԿ 500 mLǶ

z Reagent diluentǺڗ 0.1% BSA ک 0.05% Tween 20 ྋܭ Tris-buffered Saline ύǶ

z Stop solutionǺ1M H2SO4 ྋనǶ (2) Ꮤ׷Ǻ

z Costar 96-Well Half-Area Plates ᖼԾ Corning Life Science (Tewksbury, MA, USA)

z EnSpire® Multimode Plate Readers ᖼԾ PerkinElmer Inc. (MA, USA)

ȜБݤȝ

၂ ᡍ ߻ ΋ Ϻ ܭ 96 Ͼ ዬ ύ ؂ ਱ у Ε 50 ȝL а PBS ี ញ Կ ፾ ྽ ᐚ ࡋ ϐ capture antibodyǴܭ࠻ྕΠ coating ႖ڹǶಃΒϺஒనᡏॹନǴ؂਱уΕ 150 ȝL wash buffer ࢱѐ҂֎ߕܭዬ΢ϐ capture antibodyǴӅࢱΟԛǶஒዬύనᡏܡଳࡕǴ

؂਱уΕ 100 ȝL block bufferǴܭ࠻ྕ୻Ꭶ 1 λਔǶॹ௞ዬύనᡏǴӕኬа wash buffer మࢱΟԛࡕǴஒዬύనᡏܡଳǴа reagent diluent ஒ኱ྗࠔׇӈีញࡕǴ؂

ϾуΕ50 ȝL ኱ྗࠔ܈ಒझ୻Ꭶ΢మనǴܭ࠻ྕΠ୻Ꭶ 2 λਔǶॹ௞ዬύనᡏǴ Ҕwash buffer మࢱΟԛ٠ஒዬύనᡏܡଳǴ؂਱уΕ 50 ȝL а assay diluent ีញ

t

t t t

tasasas

ass

sssasas y,y,y,y,,EEEEELILILILILISASASASASA))

(42)

31

Կ፾྽ᐚࡋϐ capture antibodyǴܭ࠻ྕΠ୻Ꭶ 2 λਔǶॹ௞ዬύనᡏǴҔ wash buffer మࢱΟԛ٠ஒዬύనᡏܡଳǴ؂਱уΕ 50 ȝL а assay diluent ีញԿ፾྽

ᐚࡋϐ Streptavidin-HRPǴܭ࠻ྕΠᗉӀ୻Ꭶ 20 ϩដǶॹ௞ዬύనᡏǴҔ wash buffer మࢱϖԛ٠ஒዬύనᡏܡଳǴ؂਱уΕ 50 ȝL TMB substrateǴܭ࠻ྕΠᗉӀ

୻ᎦԿևՅֹӄਔǴ؂ϾуΕ25 ȝL stop solution аύЗևՅϸᔈǶа ELISA reader

ෳۓځӧ 450 nm ޑ֎ӀॶǴ٠а 540 nm ϐ֎ӀॶբࣁୖԵॶǴीᆉಒझ୻Ꭶ΢

మనύಒझᐟન֖ໆǶ

ಃΜ࿯! ಒझᕴ RNA ܜڗᆶϸᙯᒵ

Ȝ׷਑ȝ (1) ၂ᏊǺ

z Total RNA Miniprep kit ᖼԾ GMbiolab (ѠύѱǴѠ᡼)

z RNA to cDNA EcoDry™ Premix (Oligo dT) ᖼԾ Clontech Laboratories, Inc.

(CA, USA)

ȜБݤȝ

ԏڗ୻Ꭶࡕಒझܭ 1.5 mL ༾ໆᚆЈᆅύǴа PBS ጗ፂనࢱѐ୻ᎦనǴᚆЈ ѐନ܌ԖనᡏࡕǴуΕ 600 ȝL RNA lysis/2-ME solutionǴϸᙟؑණಒझࡕǴߥӸܭ -80ÛC ӇጃࡑҔǶஒ܌Ԗਔ໔ᗺϐಒझ຋ှన࿼ܭ࠻ྕΠှএǴܭಒझ຋ှనύу Ε 600 ȝL 70% ଚᆒ٠᎜ᕏ֡ϬࡕǴڗр 700 ȝL షӝన౽ډ RNA spin column ύǴ аനଯᙯೲᚆЈ΋ϩដǶᚆЈֹԋࡕǴѐନᘠనӆஒഭᎩషӝన౽ډ spin column ύǴӕኬᚆЈ΋ϩដࡕѐᘠనǶஒ RNA spin column ܫӣচҁޑᘠనԏ໣ᆅύǴ уΕ500 ȝL ϐ RNA wash solution IǴаനଯᙯೲᚆЈ΋ϩដࡕѐᘠనǶԜਔཥᗲ ଛ࿼ 80 ȝL DNase incubation buffer ᆶ 2 ȝL DNase I ϐషӝనǴ٠ڗ 80 ȝL షӝ

ύన ύన ύన ύన

ύనᡏᡏᡏǴᡏǴᡏǴᡏǴҔҔ wawawawaaashshshshss lu

lu lu lu

luenenennnnt ttt ีញีញีញีញีញԿ፾Կ፾Կ፾Կ፾Կ፾྽྽྽྽྽྽ ύన

ύన ύన ύన

ύనᡏǴᡏǴᡏǴᡏǴᡏǴᡏǴҔҔҔҔ wawawawawawashshshhh