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ࣇ࢓࣮ࢪᥦ♧࢔ࣞ࢖ᆺࣉࣟࣇ࢓࢖ࣜࣥࢢࢩࢫࢸ࣒ࢆ

ᇶ㍈࡜ࡍࡿ㧗ᛶ⬟ኚ␗ᢠయࡢຠ⋡ⓗ๰〇

2019

⏕࿨ศᯒ໬Ꮫ

ᮌཱྀ

⿱㈗

i ┠ḟ ␎ㄒ⾲ --- vi ⥴ㄽ --- 1 ᮏㄽ --- 8 ➨1❶ 㧗ᛶ⬟ኚ␗య༢㞳ຠ⋡ࡢホ౯⣔࡜ࡋ࡚ࡢ ᢠయࣇࣛࢢ࣓ࣥࢺࣛ࢖ࣈ࣮ࣛࣜࡢᵓ⠏ --- 8 ➨1⠇ ᗎ --- 8 ➨2⠇ ᢠࢥࣝࢳࢰ࣮ࣝࣔࣀࢡ࣮ࣟࢼࣝᢠయࡢㄪ〇࡜ㅖᛶ㉁ --- 9 ➨3⠇ ᢠࢥࣝࢳࢰ࣮ࣝᢠయ୍ᮏ㙐Fvࣇࣛࢢ࣓ࣥࢺࡢㄪ〇࡜ㅖᛶ㉁ --- 11 ➨4⠇ ኚ␗ᢠయࣇࣛࢢ࣓ࣥࢺࣛ࢖ࣈ࣮ࣛࣜࡢᵓ⠏࡜ࣃࣥࢽࣥࢢ࡟ࡼࡿ 㧗ぶ࿴ຊኚ␗యࡢ᥈⣴ --- 16 ➨5⠇ ⪃ᐹ--- 18 ➨2❶ ⧄⥔≧ࣇ࢓࣮ࢪࡢ㧗ឤᗘ᳨ฟࢆ┠ⓗ࡜ࡍࡿᢠࣇ࢓࣮ࢪ ᢠయࣇࣛࢢ࣓ࣥࢺ-ࣝࢩࣇ࢙࣮ࣛࢮ⼥ྜࢱࣥࣃࢡ㉁ࡢ๰〇 --- 19 ➨1⠇ ᗎ --- 19 ➨2⠇ ᢠࣇ࢓࣮ࢪࣔࣀࢡ࣮ࣟࢼࣝᢠయࡢㄪ〇࡜ㅖᛶ㉁ --- 20 ➨3⠇ ᢠࣇ࢓࣮ࢪᢠయ୍ᮏ㙐Fvࣇࣛࢢ࣓ࣥࢺࡢㄪ〇࡜ㅖᛶ㉁ --- 22 ➨4⠇ ᢠࣇ࢓࣮ࢪscFv-ࣝࢩࣇ࢙࣮ࣛࢮ⼥ྜࢱࣥࣃࢡ㉁ࡢ๰〇࡜᭷⏝ᛶ --- 25 ➨5⠇ ⪃ᐹ--- 29 ➨3❶ 㧗ᛶ⬟ኚ␗యࡢຠ⋡ⓗ๰〇ࢆྍ⬟࡜ࡍࡿ᪂つ࢔ࣞ࢖ᆺ ࣉࣟࣇ࢓࢖ࣜࣥࢢࢩࢫࢸ࣒ࡢ㛤Ⓨ --- 30 ➨1⠇ ᗎ --- 30 ➨2⠇ ࢔ࣞ࢖ᆺࣉࣟࣇ࢓࢖ࣜࣥࢢࢩࢫࢸ࣒ࢆྍ⬟࡜ࡍࡿ ࣇ࢓࣮ࢪᥦ♧᮲௳ࡢ᳨ウ --- 33 ➨3⠇ ࢔ࣞ࢖ᆺࣉࣟࣇ࢓࢖ࣜࣥࢢࢩࢫࢸ࣒ࢆ⏝࠸ࡓ㧗ぶ࿴ຊኚ␗యࡢ᥈⣴ --- 36 ➨4⠇ పoff-rateᣦྥ࢔ࣞ࢖ᆺࣉࣟࣇ࢓࢖ࣜࣥࢢࢩࢫࢸ࣒࡟ࡼࡿ 㧗ぶ࿴ຊኚ␗యࡢ᥈⣴ --- 39 ➨5⠇ 㧗ぶ࿴ຊኚ␗యࡢච␿ ᐃἲ࡟࠾ࡅࡿ᭷⏝ᛶ --- 44 ➨6⠇ ⪃ᐹ--- 46

ii ⤖ㄽ --- 48 ㅰ㎡ --- 50 ᐇ㦂ࡢ㒊 --- 51 ◊✲඲⯡࡟㛵ࡍࡿ㡯┠ --- 51 i. ⿦⨨ ii. ჾᮦ iii. ࢯࣇࢺ࢙࢘࢔ iv. ⦆⾪ᾮ v. ࢫࢸࣟ࢖ࢻ㢮 vi. ࢺࣜࢳ࣒࢘ᶆ㆑໬ྜ≀ vii. ᢠయ㢮࠾ࡼࡧᢠయ㛵㐃ヨ⸆ viii. ᢠయ௨እࡢච␿໬Ꮫ㛵㐃ヨ⸆ ix. 㓝⣲㢮 x. ᇶ㉁⁐ᾮ xi. ࡑࡢ௚ࡢヨ⸆࣭ჾᮦ xii. ᇵᆅ㢮 xiii. ⣽⬊࡜ࣇ࢓࣮ࢪ xiv. ࣋ࢡࢱ࣮DNA xv. ࣉࣛ࢖࣐࣮ xvi. ࢟ࢵࢺ㢮 xvii. 㟁ẼὋື⏝ࢤࣝ࡜Ὃື᮲௳ xviii. ᐇ㦂ື≀ xix. ౫㢗ศᯒ ➨1❶௜ᒓᐇ㦂 --- 59 ➨2⠇௜ᒓᐇ㦂 --- 59 1.2.1㡯 CS-࢔ࣝࣈ࣑ࣥ⤖ྜయ (CS-BSAࡲࡓࡣCS-OVA) ࡢㄪ〇 1.2.2㡯 ᢠCSᢠయ⏘⏕ࣁ࢖ࣈࣜࢻ࣮࣐ᰴࡢᶞ❧࠾ࡼࡧ ᢠCSࣔࣀࢡ࣮ࣟࢼࣝᢠయࡢ⏘⏕ 1.2.3㡯 ELISA࡟ࡼࡿᇵ㣴ୖΎ୰ᢠCSᢠయࡢ᳨ฟ 1.2.4㡯 Scatchardἲ࡟ࡼࡿ⤖ྜᐃᩘࡢ⟬ฟ 1.2.5㡯 Ab-CS#3ࢆ⏝࠸ࡿELISA ➨3⠇௜ᒓᐇ㦂 --- 61

iii

1.3.1㡯 Ab-CS#3࠾ࡼࡧAb-CS#10ᢠయྍኚ㒊㑇ఏᏊࢆྵࡴcDNAࡢㄪ〇 1.3.2㡯 Ab-CS#3ᢠయH㙐ྍኚ㒊㑇ఏᏊ (#3V_H) ࢆྵࡴDNA᩿∦ࡢㄪ〇 1.3.3㡯 Ab-CS#3ᢠయL㙐ྍኚ㒊㑇ఏᏊ (#3V_L) ࢆྵࡴDNA᩿∦ࡢㄪ〇 1.3.4㡯 Ab-CS#10ᢠయྍኚ㒊㑇ఏᏊ (#10V_H࠾ࡼࡧ#10V_L) ࢆྵࡴDNA᩿∦ࡢㄪ〇 1.3.5㡯 Ab-CS#3࠾ࡼࡧAb-CS#10ᢠయࡢV_HࡲࡓࡣV_L㑇ఏᏊࢆྵࡴ DNA᩿∦ࡢࢧࣈࢡ࣮ࣟࢽࣥࢢ 1.3.6㡯 Ab-CS#3࠾ࡼࡧAb-CS#10ᢠయࡢV_H࠾ࡼࡧV_L㑇ఏᏊࡢሷᇶ㓄ิࡢỴᐃ 1.3.7㡯 scFv㑇ఏᏊᵓ⠏ࡢࡓࡵࡢV_H-DNA࠾ࡼࡧV_L-DNA᩿∦ࡢㄪ〇 1.3.8㡯 Ab-CS#3ᢠయscFv㑇ఏᏊࡢࢡ࣮ࣟࢽࣥࢢ 1.3.9㡯 CS#10-scFv㑇ఏᏊࡢస〇࡜኱⭠⳦࡬ࡢᑟධ 1.3.10㡯 ྍ⁐ᆺCS#3-scFv㑇ఏᏊࡢస〇࡜኱⭠⳦࡬ࡢᑟධ 1.3.11㡯 ྍ⁐ᆺCS#10-scFv㑇ఏᏊࡢస〇࡜኱⭠⳦࡬ࡢᑟධ 1.3.12㡯 ྍ⁐ᆺscFvࡢస〇࡜⢭〇 1.3.13㡯 Scatchardἲ࡟ࡼࡿ⤖ྜᐃᩘࡢ⟬ฟ 1.3.14㡯 ྍ⁐ᆺCS#3-scFvࢆ⏝࠸ࡿELISA ➨4⠇௜ᒓᐇ㦂 --- 66 1.4.1㡯 Error-prone PCR࡟ࡼࡿኚ␗scFv㑇ఏᏊࡢㄪ〇 1.4.2㡯 ኚ␗scFv㑇ఏᏊࡢࢡ࣮ࣟࢽࣥࢢ࡜DNAሷᇶ㓄ิࡢỴᐃ 1.4.3㡯 KM13࣊ࣝࣃ࣮ࣇ࢓࣮ࢪࡢㄪ〇 1.4.4㡯 KM13࣊ࣝࣃ࣮ࣇ࢓࣮ࢪࡢຊ౯⟬ᐃ 1.4.5㡯 ኚ␗scFvᥦ♧ࣇ࢓࣮ࢪࣛ࢖ࣈ࣮ࣛࣜࡢㄪ〇࡜ࣇ࢓࣮ࢪࡢຊ౯⟬ᐃ 1.4.6㡯 ᢠCSάᛶࢆᣢࡘኚ␗scFvᥦ♧ࣇ࢓࣮ࢪࡢࣃࣥࢽࣥࢢ࡟ࡼࡿ㑅ᢥ 1.4.7㡯 ࢫࢡ࣮ࣜࢽࣥࢢ࡟⏝࠸ࡿࣔࣀࢡ࣮ࣟࢼࣝኚ␗scFvᥦ♧ࣇ࢓࣮ࢪࡢㄪ〇 1.4.8㡯 ࣔࣀࢡ࣮ࣟࢼࣝኚ␗scFvᥦ♧ࣇ࢓࣮ࢪࡢࢫࢡ࣮ࣜࢽࣥࢢ 1.4.9㡯 ࣔࣀࢡ࣮ࣟࢼࣝኚ␗scFvᥦ♧ࣇ࢓࣮ࢪࡢㄪ〇 1.4.10㡯 ࣔࣀࢡ࣮ࣟࢼࣝኚ␗scFvᥦ♧ࣇ࢓࣮ࢪࡢᢠCSάᛶࡢホ౯ 1.4.11㡯 ྍ⁐ᆺኚ␗scFvࡢㄪ〇 1.4.12㡯 Scatchardἲ࡟ࡼࡿ⤖ྜᐃᩘࡢ⟬ฟ ➨2❶௜ᒓᐇ㦂 --- 71 ➨2⠇௜ᒓᐇ㦂 --- 71 2.2.1㡯 VCSM13࣊ࣝࣃ࣮ࣇ࢓࣮ࢪࡢㄪ〇 2.2.2㡯 VCSM13࣊ࣝࣃ࣮ࣇ࢓࣮ࢪࡢຊ౯⟬ᐃ 2.2.3㡯 ᢠVCSM13ᢠయ⏘⏕ࣁ࢖ࣈࣜࢻ࣮࣐ᰴࡢᶞ❧࠾ࡼࡧᢠVCSM13 ࣔࣀࢡ࣮ࣟࢼࣝᢠయࡢㄪ〇

iv 2.2.4㡯 ELISA࡟ࡼࡿᇵ㣴ୖΎ୰ᢠVCSM13ᢠయࡢ᳨ฟ 2.2.5㡯 Ab-M13#71ࢆ⏝࠸ࡿ➇ྜELISA 2.2.6㡯 ࢖࣒ࣀࣈࣟࢵࢺἲ࡟ࡼࡿAb-M13#71ࡢ࢚ࣆࢺ࣮ࣉࡢ᥎ᐃ ➨3⠇௜ᒓᐇ㦂 --- 72 2.3.1㡯 Ab-M13#71ᢠయྍኚ㒊㑇ఏᏊࢆྵࡴcDNAࡢㄪ〇 2.3.2㡯 Ab-M13#71ᢠయH㙐ྍኚ㒊㑇ఏᏊ (V_H) ࢆྵࡴDNA᩿∦ࡢㄪ〇 2.3.3㡯 Ab-M13#71ᢠయL㙐ྍኚ㒊㑇ఏᏊ (V_L) ࢆྵࡴDNA᩿∦ࡢㄪ〇 2.3.4㡯 Ab-M13#71ᢠయࡢV_HࡲࡓࡣV_L㑇ఏᏊࢆྵࡴDNA᩿∦ࡢ ࢧࣈࢡ࣮ࣟࢽࣥࢢ 2.3.5㡯 Ab-M13#71ᢠయࡢV_H࠾ࡼࡧV_L㑇ఏᏊࡢሷᇶ㓄ิࡢỴᐃ 2.3.6㡯 M13-scFv㑇ఏᏊᵓ⠏ࡢࡓࡵࡢV_H-DNA࠾ࡼࡧV_L-DNA᩿∦ࡢㄪ〇 2.3.7㡯 Overlap extension PCR࡟ࡼࡿM13-scFv㑇ఏᏊࡢᵓ⠏࡜ࢧࣈࢡ࣮ࣟࢽࣥࢢ 2.3.8㡯 ྍ⁐ᆺM13-scFvࡢⓎ⌧࡜㟁ẼὋື 2.3.9㡯 ྍ⁐ᆺM13-scFvࢆ⏝࠸ࡿ➇ྜELISA 2.3.10㡯 ࣅ࢜ࢳࣥ໬VCSM13ࣇ࢓࣮ࢪࡢస〇 2.3.11㡯 ࣂ࢖࢜ࣞ࢖࣮ࣖᖸ΅ἲ࡟ࡼࡿ⤖ྜᐃᩘࡢ⟬ฟ ➨4⠇௜ᒓᐇ㦂 --- 75 2.4.1㡯 CS#10-scFv-Gaussiaࣝࢩࣇ࢙࣮ࣛࢮ⼥ྜࢱࣥࣃࢡ㉁స〇ࡢࡓࡵࡢ GLuc㑇ఏᏊࢆྵࡴDNA᩿∦ࡢㄪ〇 2.4.2㡯 CS#10-scFv-Gaussiaࣝࢩࣇ࢙࣮ࣛࢮ⼥ྜࢱࣥࣃࢡ㉁స〇ࡢࡓࡵࡢ CS#10-scFv㑇ఏᏊࢆྵࡴDNA᩿∦ࡢㄪ〇

2.4.3㡯 Overlap extension PCR࡟ࡼࡿCS#10-scFv-GLuc㑇ఏᏊࡢᵓ⠏࡜ ࢧࣈࢡ࣮ࣟࢽࣥࢢ 2.4.4㡯 CS/ࣅ࢜ࢳࣥ2㔜ᶆ㆑࣏ࣜ-L-ࣜࢩࣥࡢస〇 2.4.5㡯 CS#10-scFv-Gaussiaࣝࢩࣇ࢙࣮ࣛࢮ⼥ྜࢱࣥࣃࢡ㉁ࡢⓎ⌧࡜㟁ẼὋື 2.4.6㡯 CS#10-scFv-Gaussiaࣝࢩࣇ࢙࣮ࣛࢮ⼥ྜࢱࣥࣃࢡ㉁ࡢⓎග ᐃ 2.4.7㡯 CS#10-scFv-Gaussiaࣝࢩࣇ࢙࣮ࣛࢮ⼥ྜࢱࣥࣃࢡ㉁ࡢ᳨ฟឤᗘࡢ᳨ウ 2.4.8㡯 M13-scFv-Gaussia ࣝࢩࣇ࢙࣮ࣛࢮ⼥ྜࢱࣥࣃࢡ㉁స〇ࡢࡓࡵࡢ M13-scFv㑇ఏᏊࢆྵࡴDNA᩿∦ࡢㄪ〇 2.4.9㡯 M13-scFv-Gaussia ࣝࢩࣇ࢙࣮ࣛࢮ⼥ྜࢱࣥࣃࢡ㉁స〇ࡢࡓࡵࡢ GLuc㑇ఏᏊࢆྵࡴDNA᩿∦ࡢㄪ〇

2.4.10㡯 Overlap extension PCR࡟ࡼࡿM13-scFv-GLuc㑇ఏᏊࡢᵓ⠏࡜ ࢧࣈࢡ࣮ࣟࢽࣥࢢ

2.4.11㡯 ྍ⁐ᆺM13-scFv-GLucࡢⓎ⌧࡜㟁ẼὋື 2.4.12㡯 ྍ⁐ᆺM13-scFv-GLucࡢ⤖ྜ⬟ホ౯࡟⏝࠸ࡿ

v 2.4.13㡯 ྍ⁐ᆺM13-scFv-GLucࢆ⏝࠸ࡿࣇ࢓࣮ࢪELISA ➨3❶௜ᒓᐇ㦂 --- 80 ➨2⠇௜ᒓᐇ㦂 --- 80 3.2.1㡯 ࣇ࢓࣮ࢪస〇᮲௳ࡢホ౯࡟⏝࠸ࡿscFvᥦ♧ࣇ࢓࣮ࢪࡢస〇 3.2.2㡯 ELISA࡟ࡼࡿࣇ࢓࣮ࢪస〇᮲௳ࡢホ౯ 3.2.3㡯 ࢔ࣞ࢖ᆺ㑅ᢥἲ࡟⏝࠸ࡿᇵᆅ⤌ᡂ࠾ࡼࡧᇵ㣴᫬㛫ࡢホ౯ ➨3⠇௜ᒓᐇ㦂 --- 81 3.3.1㡯 ኚ␗scFv㑇ఏᏊࣛ࢖ࣈ࣮ࣛࣜ࡟ࡼࡿ኱⭠⳦ࡢᙧ㉁㌿᥮࡜ࢥࣟࢽ࣮ศ㞳 3.3.2㡯 M13-scFv-GLucࢆ⏝࠸ࡓⓎගᙉᗘࡢࣉࣟࣇ࢓࢖ࣜࣥࢢ 3.3.3㡯 ࣇ࢓࣮ࢪࡢᅇ཰࡜ࢧࣈࢡ࣮ࣟࢽࣥࢢ 3.3.4㡯 ࣔࣀࢡ࣮ࣟࢼࣝࣇ࢓࣮ࢪࡢㄪ〇࡜ᢠCSάᛶࡢホ౯ 3.3.5㡯 ྍ⁐ᆺኚ␗scFvࡢㄪ〇 3.3.6㡯 Scatchardἲ࡟ࡼࡿ⤖ྜᐃᩘࡢ⟬ฟ 3.3.7㡯 ྍ⁐ᆺኚ␗scFvࡢ኱㔞ㄪ〇࡜⢭〇 3.3.8㡯 ⤊ṆࢥࢻࣥࡀᏑᅾࡍࡿྍ⁐ᆺኚ␗scFvࡢ࢔࣑ࣀ㓟ࡢྠᐃ ➨4⠇௜ᒓᐇ㦂 --- 83 3.4.1㡯 CS#10-scFv-GLuc⼥ྜࢱࣥࣃࢡ㉁ࢆ⏝࠸ࡓoff-rate㑅ᢥ᮲௳ࡢ᳨ウ 3.4.2㡯 M13-scFv-GLucࢆ⏝࠸ࡓⓎගᙉᗘࡢࣉࣟࣇ࢓࢖ࣜࣥࢢ 3.4.3㡯 ࣇ࢓࣮ࢪࡢ෌ቑᖜ࡜M13-scFv-GLucࢆ⏝࠸ࡓoff-rateࡢほ  3.4.4㡯 ࣔࣀࢡ࣮ࣟࢼࣝࣇ࢓࣮ࢪࡢㄪ〇࡜ELISA 3.4.5㡯 ྍ⁐ᆺኚ␗scFvࡢㄪ〇 3.4.6㡯 Scatchardἲ࡟ࡼࡿ⤖ྜᐃᩘࡢ⟬ฟ 3.4.7㡯 ྍ⁐ᆺኚ␗scFvࡢ኱㔞ㄪ〇࡜⢭〇 3.4.8㡯 ࣅ࢜ࢳࣥ໬CS-BSAࡢస〇 3.4.9㡯 ࣂ࢖࢜ࣞ࢖࣮ࣖᖸ΅ἲ࡟ࡼࡿゎ㞳㏿ᗘᐃᩘࡢ ᐃ ➨5⠇௜ᒓᐇ㦂 --- 84 3.5.1㡯 ྍ⁐ᆺኚ␗scFvࢆ⏝࠸ࡿELISA ᘬ⏝ᩥ⊩ --- 86

vi ␎ㄒ⾲

AAP: abridged anchor primer Amp: ampicillin

ATP: adenosine 5’-triphosphate

AUAP: abridged universal amplification primer BSA: bovine serum albumin

cDNA: complementary DNA

CDR: complementarity-determining region cfu: colony-forming unit

CH: heavy chain constant region

CL: light chain constant region

CTP: cytidine 5’-triphosphate CS: cortisol

CT: cotinine dATP: deoxy ATP dCTP: deoxy CTP dGTP: deoxy GTP

DMSO: dimethyl sulfoxide DNA: deoxyribonucleic acid

dNTP: deoxynucleoside triphosphate dTTP: deoxy TTP

E2: estradiol-17b

EDTA: ethylenediaminetetraacetic acid ELISA: enzyme-linked immunosorbent assay Fab: antigen-binding fragment

FLuc: firefly luciferase FR: framework region GLuc: Gaussia luciferase GSP: group specific primer GTP: guanosine 5’-triphosphate IgG: immunoglobulin G

IPTG: isopropyl 1-thio-b-D-galactopyranoside Kan: kanamycin

NHS: N-hydroxysuccinimide (N-hydroxysuccinimidyl) MOI: multiplicity of infection

vii mRNA: messenger RNA

OVA: ovalbumin

PAGE: polyacrylamide gel electrophoresis PCR: polymerase chain reaction

PEG: polyethylene glycol pfu: plaque-forming unit POD: peroxidase

PVDF: poly(vinylidene fluoride)

RACE: rapid amplification of cDNA ends RLuc: Renilla reniformis luciferase RNA: ribonucleic acid

RT-PCR: reverse transcriptase-PCR scFv: single-chain Fv fragment SDS: sodium dodecyl sulfate

TdT: terminal deoxynucleotidyl transferase TMB: 3,3’,5,5’-tetramethylbenzidine TTP: thymidine 5’-triphosphate

Tris: tris(hydroxymethyl)aminomethane VH: heavy chain variable region

VL: light chain variable region

௨ୗ࡟ሷᇶࡢ␎ㄒࢆ♧ࡍ. A: adenine C: cytosine G: guanine T: thymine ௨ୗ࡟࢔࣑ࣀ㓟ࡢ␎ㄒࢆ♧ࡍ. A: alanine C: cysteine D: aspartic acid E: glutamic acid F: phenylalanine G: glycine H: histidine I: isoleucine

viii K: lysine L: leucine M: methionine N: asparagine P: proline Q: glutamine R: arginine S: serine T: threonine V: valine W: tryptophan Y: tyrosine

1 ⥴ㄽ ᢠయࡣ, ື≀ࡢయෆ࡟ᢠཎࡀ౵ධࡋࡓ่⃭࡟ࡼࡾ⏘⏕ࡉࢀࡿ⢾ࢱࣥࣃࢡ㉁࡛࠶ࡾ, ≉ ᐃࡢศᏊᵓ㐀ࢆ⢭ᐦ࡟ㄆ㆑ࡋ࡚ᙉ࠸ぶ࿴ຊ࡛⤖ྜࡍࡿᛶ㉁ࢆᣢࡘࡓࡵ, ᖜᗈ࠸ศ㔝࡛ ά⏝ࡉࢀ࡚࠸ࡿ. 2018ᖺࡢࣀ࣮࣋ࣝ໬Ꮫ㈹࠾ࡼࡧ⏕⌮Ꮫ࣭་Ꮫ㈹ࡣ࠸ࡎࢀࡶᢠయ࡟㛵ಀ ࡍࡿᴗ⦼࡟ᑐࡋ࡚ᤵ୚ࡉࢀࡓ. 1,2) ᢠయࡢస〇ࡸᛂ⏝࡟㛵㐃ࡍࡿ㐍Ṍࡀே㢮࣭♫఍ࡢⓎᒎ ࡟኱ࡁࡃᐤ୚ࡍࡿࡇ࡜ࢆᨵࡵ࡚࿘▱ࡉࡏࡿᛌᣲ࡜ゝ࠼ࡼ࠺. ་⒪ศ㔝࡛ࡣ, ࿘▱ࡢࡼ࠺࡟, ᢠయࡑࡢࡶࡢࡀ⒴ࡸ⮬ᕫච␿ᛶ⑌ᝈࡢ἞⒪⸆࡜ࡋ࡚౑ ⏝ࡉࢀ࡚࠸ࡿ. ඃࢀࡓศᏊᶆⓗ⸆࡜࡞ࡾ࠺ࡿ㈨㉁ࢆഛ࠼, ᢎㄆ௳ᩘࡶᛴቑࡋ࡚࠸ࡿࡇ࡜ ࠿ࡽ, 3) ࡑࡢ㟂せࡢ㧗ࡉࡀ࠺࠿ࡀ࠼ࡿ. ୍᪉, ᢠయࡣ, デ᩿࣭ศᯒヨ⸆࡜ࡋ࡚ࡶ୙ྍḞ࡛ ࠶ࡿ. 4) ᢠయࢆ⏝࠸ࡿᚤ㔞ᐃ㔞ἲࡣࠕච␿ ᐃἲ (࢖࣒ࣀ࢔ࢵࢭ࢖) ࠖ࡜⥲⛠ࡉࢀ, ⏕య ヨᩱࡢࡼ࠺࡞」㞧࡞⤌ᡂࡢ࣐ࢺࣜࢵࢡࢫ࡟ྵࡲࢀࡿࡈࡃᚤ㔞ࡢᶆⓗศᏊࢆᐃ㔞ࡍࡿ࠺ ࠼࡛୙ྍḞࡢ᪉ἲㄽ࡛࠶ࡿ. యᾮ୰ࡢ࣍ࣝࣔࣥࡢືែゎᯒࡣ, ච␿ ᐃἲࡢ㛤Ⓨ࡟ࡼࡾ ྍ⬟࡟࡞ࡗࡓ࡜ゝࡗ࡚ࡶ㐣ゝ࡛ࡣ࡞࠸. ᮏἲࡣ᧯సࡀ⡆౽᳨࡛యฎ⌮⬟ຊࡶ኱ࡁࡃ, ࢜ ࣥࢧ࢖ࢺศᯒࡸ࣐ࢫࢫࢡ࣮ࣜࢽࣥࢢ࡟ࡶ᭱㐺࡛࠶ࡿ. ዷፎ᳨ᰝࡸ࢖ࣥࣇ࢚ࣝࣥࢨࡢデ ᩿࡟⏝࠸ࡽࢀ࡚࠸ࡿࠕ࢖࣒ࣀࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮ࠖࡣ, ࡑࡢ≉㛗ࢆά࠿ࡋࡓᨵⰋἲ࡛࠶ ࡿ. ච␿ ᐃἲࡢᵓ⠏࡟ࡣᢠయࡀᚲ㡲࡛࠶ࡿࡀ, ච␿᪉ἲࡢᕤኵ࡟ࡼࡾᵝࠎ࡞ศᏊ࡟ᑐ ࡍࡿᢠయࡀᚓࡽࢀࡿࡓࡵ, ⸆≀ࡸࢫࢸࣟ࢖ࢻࡢࡼ࠺࡞పศᏊ໬ྜ≀࠿ࡽࢱࣥࣃࢡ㉁࡞ ࡝ࡢ㧗ศᏊ໬ྜ≀ࡲ࡛, ከᵝ࡞ศᏊࡢ ᐃࡀྍ⬟࡛࠶ࡿ. ᢠయࢆᚓࡿ࡟ࡣ, ୍⯡࡟ᐇ㦂 ື≀ࢆᶆⓗ≀㉁ (ᢠཎ) ࡛㐣ච␿ࡍࡿ. ࡍ࡞ࢃࡕ, ᶆⓗ≀㉁ࢆ㠀⤒ཱྀⓗ࡟⧞ࡾ㏉ࡋᢞ୚ ࡍࡿࡀ, ⿕ච␿ື≀࡟ᑐࡋ࡚␗✀ࡢື≀࡟⏤᮶ࡍࡿ㧗ศᏊ ໬ྜ≀࡛࠶ࢀࡤ, ㏻ᖖ, ᢠయ ࡢ⏘⏕ࡀច㉳ࡉࢀࡿ. ௚᪉, పศᏊ໬ྜ≀ࡣ୍⯡࡟ච␿ཎᛶࢆ♧ࡉࡎ, ࡑࡢࡲࡲື≀࡟ ᢞ୚ࡋ࡚ࡶᢠయࡣ⏘⏕ࡉࢀ࡞࠸. ࡋ࠿ࡋ, 㐺ษ࡞࣮࢟ࣕࣜࣖศᏊ࡜ඹ᭷⤖ྜࡉࡏࡓࡢࡕ ࡟ᢞ୚ࡍࡿ࡜, పศᏊ໬ྜ≀ࡀᢠཎỴᐃᇶ (࢚ࣆࢺ࣮ࣉ) ࡜ࡋ࡚ാࡁ, ࡇࢀࢆㄆ㆑ࡍࡿ ᢠయࡀᚓࡽࢀࡿࡼ࠺࡟࡞ࡿ. ࡇࡢࡼ࠺࡞໬ྜ≀ࡣච␿໬Ꮫࡢ㡿ᇦ࡛ࠕࣁࣉࢸࣥࠖ࡜⥲⛠ ࡉࢀࡿ. ච␿ᚋࡢື≀࠿ࡽ᥇⾑ࡋ, ⾑ᾮจᅛᡂศࢆศ㞳ࡍࡿࡇ࡜࡛ᚓࡽࢀࡿᢠ⾑Ύࡣ, ࡑࡢࡲ ࡲศᯒヨ⸆࡜ࡋ࡚฼⏝ࡍࡿࡇ࡜ࡀ࡛ࡁࡿ. ࡋ࠿ࡋ, ྵࡲࢀࡿᢠయࡣ」ᩘࡢᢠయ⏘⏕⣽⬊ ࡟⏤᮶ࡍࡿ࣏ࣜࢡ࣮ࣟࢼࣝᢠయ࡛࠶ࡿࡓࡵ, ୍ᐃရ㉁ࡢࡶࡢࢆ౪⤥ࡋ⥆ࡅࡿࡇ࡜ࡣᅔ 㞴࡛࠶ࡗࡓ. ࡇࡢၥ㢟ࡣ, ࣁ࢖ࣈࣜࢻ࣮࣐ἲ࡟ࡼࡿࣔࣀࢡ࣮ࣟࢼࣝᢠయస〇ᢏ⾡ࡢ☜❧ ࡟ࡼࡾ, ᢤᮏⓗ࡟ඞ᭹ࡉࢀࡓ. 5) ⿕ච␿ື≀ࡢ⭁⣽⬊ࢆྲྀࡾฟࡋ, ࣑࢚࣮࣐ࣟ⣽⬊࡜⼥ ྜࡉࡏ࡚స〇ࡋࡓࣁ࢖ࣈࣜࢻ࣮࣐ࢆࢡ࣮ࣟࣥ໬ࡍࡿࡶࡢ࡛, ༢୍ࡢᢠయ⏘⏕⣽⬊࡟⏤ ᮶ࡍࡿᆒ㉁࡞ᢠయศᏊࡀᚓࡽࢀࡿ. ࣔࣀࢡ࣮ࣟࢼࣝᢠయࡣ ᐃࡢᶆ‽໬࡟᭷฼࡞ࡇ࡜ ࠿ࡽ࣏ࣜࢡ࣮ࣟࢼࣝᢠయࡼࡾ฼⏝౯್ࡀ㧗ࡃ, ᭱㏆ࡢච␿ ᐃἲ࢟ࢵࢺࡢከࡃࡀ᥇⏝ ࡍࡿ࡟⮳ࡗ࡚࠸ࡿ.

2 ච␿ ᐃἲࡣ, ࡑࡢ ᐃཎ⌮࠿ࡽ㠀➇ྜἲ࡜➇ྜἲ࡟኱ูࡉࢀࡿ. 㠀➇ྜἲࡣ, 㓝⣲ ࡸᨺᑕᛶྠ఩య࡛ᶆ㆑ࡋࡓᢠయࢆᢠཎ ( ᐃᑐ㇟≀) ࡟ᑐࡋ࡚㐣๫࡟཯ᛂࡉࡏ, ⏕ᡂࡋ ࡓᢠཎᢠయ」ྜయࡢ㔞ࢆᶆ㆑ࡢࢩࢢࢼࣝᙉᗘ࠿ࡽࣔࢽࢱ࣮ࡍࡿࡶࡢ࡛࠶ࡾ, ᢠཎࡢቑ ຍ࡟ᛂࡌ࡚ࢩࢢࢼࣝᙉᗘࡶቑຍࡍࡿ (ᅗ 1a). ࢖࣒ࣀ࣓ࢺࣜࢵࢡ࢔ࢵࢭ࢖࡜ࡶ࿧ࡤࢀ࡚ ࠸ࡿࡀ, 1 ศᏊ୰࡟ 2 ࡘ௨ୖࡢᢠཎỴᐃᇶ (࢚ࣆࢺ࣮ࣉ) ࢆ᭷ࡋ࡚࠸ࡿ㧗ศᏊᢠཎ࡛࠶ ࢀࡤ, 2✀㢮ࡢᢠయ࡛ᢠཎࢆᤕᤊࡍࡿtwo-site࢖࣒ࣀ࣓ࢺࣜࢵࢡ࢔ࢵࢭ࢖ (ࢧࣥࢻ࢖ࢵࢳ ࢔ࢵࢭ࢖) ࡢ㐺⏝ࡀྍ⬟࡛, 㧗ឤᗘ࡞࢔ࢵࢭ࢖⣔ࢆᵓ⠏ࡋࡸࡍ࠸. 6,7) ㏆ᖺ, ࢧࣥࢻ࢖ࢵ ࢳ࢔ࢵࢭ࢖ࢆfL (ࣇ࢙࣒ࢺࣜࢵࢺࣝ) ࣞ࣋ࣝࡢᚤᑠᐜჾࡢ࡞࠿࡛⾜࠺͆ࢹࢪࢱࣝELISA͇ ࡀ㛤Ⓨࡉࢀ, 8–10) _{femtomolar~attomolar} ࣞ࣋ࣝࡢឤᗘࡀ㐩ᡂࡉࢀ࡚࠸ࡿࡇ࡜ࡣ≉➹࡟್ࡍ ࡿ. ୍ ᪉, ➇ྜἲ ࡣ,  ᐃᑐ㇟ ࡢᢠཎ (㠀ᶆ㆑ᢠ ཎ) ࡜, ୍ᐃ㔞ࡢ ᶆ㆑ᢠཎ ࢆ, ୍ ᐃ㔞ࡢ ᢠయ࡟ᑐࡋ࡚➇ྜⓗ࡟཯ᛂࡉࡏࡿ. 㠀ᶆ㆑ᢠཎࢆຍ࠼࡞࠸࡜ࡁ, ᶆ㆑ᢠཎࡢ୍㒊ࡣᢠయ ࡜⤖ྜࡋ (B⏬ศ), ୍㒊ࡣ㐟㞳ࡢ≧ែ࡛ṧࡿ (F⏬ศ). ࡇࡢ཯ᛂ⣔࡟㠀ᶆ㆑ᢠཎࡢᶆ‽ ရࢆຍ࠼࡚࠸ࡃ࡜, ᶆ㆑ᢠཎ࡜➇ྜࡋ, ῧຍࡍࡿᢠཎࡢቑຍ࡟ᛂࡌ࡚B⏬ศࡢ๭ྜࡀῶ ᑡࡍࡿࡓࡵ, ⏝㔞స⏝᭤⥺ࡀᚓࡽࢀࡿ (ᅗ 1b). ୍⯡࡟ୖ㏙ࡢࢧࣥࢻ࢖ࢵࢳ࢔ࢵࢭ࢖࡯ ࡝ࡢ ᐃឤᗘࡣᚓ㞴࠸ࡶࡢࡢ, ࡑࡢศᏊ඲యࡀ༢⊂ࡢ࢚ࣆࢺ࣮ࣉ࡜ࡋ࡚ࡩࡿࡲ࠺ࣁࣉ ᅗ 1. 㠀➇ྜἲ (ࢧࣥࢻ࢖ࢵࢳ࢔ࢵࢭ࢖) ࠾ࡼࡧ➇ྜἲࡢཎ⌮ (a) 㠀➇ྜἲࡢ௦⾲౛࡜ࡋ࡚, two-site࢖࣒ࣀ࣓ࢺࣜࢵࢡ࢔ࢵࢭ࢖ (ࢧࣥࢻ࢖ࢵࢳ࢔ࢵࢭ࢖) ࡢཎ⌮ࢆ♧ ࡍ. ᅛᐃ໬ᢠయ࡟ᑐࡋ࡚ᢠཎࢆῧຍࡋ, ูࡢ࢚ࣆࢺ࣮ࣉࢆㄆ㆑ࡍࡿᶆ㆑ᢠయ᳨࡛ฟࡍࡿ. ຍ࠼ࡿᢠཎࡢ㔞ࢆ ቑຍࡉࡏࡿ࡜, ᚓࡽࢀࡿࢩࢢࢼࣝᙉᗘࡶቑຍࡍࡿ. (b) ➇ྜἲࡢཎ⌮ࢆ♧ࡍ. ୍ᐃ㔞ࡢᢠయ࡟୍ᐃ㔞ࡢᶆ㆑ ᢠཎࢆ㠀ᶆ㆑ᢠཎ࡜࡜ࡶ࡟ຍ࠼ࡿ. 㐺ษ࡞᪉ἲࢆ⏝࠸࡚B⏬ศ࡜F⏬ศࢆศ㞳ࡋ࡚ (ࡇࡢ౛࡛ࡣᢠయࢆᅛ ┦࡟ᅛᐃ໬ࡍࡿࡇ࡜࡛㐩ᡂࡉࢀࡿ), 㠀ᶆ㆑ᢠཎࢆῧຍࡋ࡞࠿ࡗࡓ࡜ࡁࡢB⏬ศࡢࢩࢢࢼࣝࢆB₀, ῧຍࡋࡓ ࡜ࡁࡢࡑࢀࢆB࡜ࡋ, B₀࡟ᑐࡍࡿBࡢⓒศ⋡ (B/B₀%) ࢆ, ῧຍࡋࡓᶆ‽ရࡢ㔞࡟ᑐࡋ࡚ࣉࣟࢵࢺࡍࢀࡤ, ྑୗࡀࡾࡢᶆ‽᭤⥺ࡀᚓࡽࢀࡿ.

3 ࢸࣥ࡟ᑐࡋ࡚ࡶ㐺ᛂࡀྍ⬟࡛࠶ࡿ. ච␿ ᐃἲࡢឤᗘ࡟ᙳ㡪ࡍࡿᅉᏊ࡜ࡋ࡚, ᢠཎࡸᢠయࡢᶆ㆑࡟⏝࠸ࡽࢀࡿ࣐࣮࣮࢝ ≀㉁ࡢ᳨ฟឤᗘࡶᣲࡆࡽࢀࡿࡀ, ⏝࠸ࡿᢠయࡢᶆⓗᢠཎ࡟ᑐࡍࡿ⤖ྜぶ࿴ຊࡀ࡜ࡾࢃ ࡅ㔜せ࡛࠶ࡿ. ୍⯡࡟, ⤖ྜᐃᩘK_aࡀ኱ࡁ࠸࡯࡝, ᥮ゝࡍࢀࡤゎ㞳ᐃᩘK_d (= 1/K_a) ࡀᑠ ࡉ࠸࡯࡝, 㧗ឤᗘ࡞ ᐃࡀྍ⬟࡟࡞ࡿ. ➇ྜἲ࡛ࡣ, ཯ᛂ⣔࡟౑⏝ࡍࡿᶆ㆑ᢠཎ㔞࡟ᑐ ࡋ࡚ ᢠయ㔞ࢆ ᢚ࠼ࡿᚲせࡀ ࠶ࡿࡓࡵ, ࡑࡢᙳ㡪ࡀ㢧 ⴭ࡟⾲ࢀ ࡿ. ࣁࣉࢸࣥᢠ ཎ (25-ࣄ ࢻࣟ࢟ࢩࣅࢱ࣑ࣥ D₃) ࡢࣛࢪ࢜࢖࣒ࣀ࢔ࢵࢭ࢖ (ᨺᑕᛶྠ఩యᶆ㆑ᢠཎࢆ⏝࠸ࡿ➇ྜ ᆺච␿ ᐃἲ) ࢆࣔࢹࣝ࡜ࡋ࡚ᶆ‽᭤⥺ࢆࢩ࣑࣮ࣗࣞࢩࣙࣥࡋࡓ౛࠿ࡽ, ➇ྜἲ࡛࠶ࡗ ࡚ࡶᢠయࡢK_aࡀ10 12 _M-1 ௨ୖ࡛࠶ࢀࡤ, subfemtomoleࣞ࣋ࣝࡢឤᗘࡀᮇᚅ࡛ࡁࡿࡇ࡜ࡀ ♧ࡉࢀ࡚࠸ࡿ. 11) ᨺᑕᛶྠ఩యࡼࡾࡶ㧗ឤᗘ࡟㏣㊧ࡀྍ⬟࡞㓝⣲࡞࡝ࢆᶆ㆑ࡋ, ᶆ㆑ᢠ ཎࡢῧຍ㔞ࢆῶࡌࡿࡇ࡜࡛, attomoleࣞ࣋ࣝࡢ᳨ฟࡶྍ⬟࡜᥎ᐃࡉࢀࡿ. ࡋ࠿ࡋ, ື≀ࢆ ච␿ࡍࡿᚑ᮶ࡢᢠయ⏘⏕ἲ࡟ࡼࡾ, K_aࡀ1™10 11 _M-1 ࢆୖᅇࡿࡼ࠺࡞ᢠయࡀᚓࡽࢀࡿࡇ࡜ ࡣ⛥࡛࠶ࡿ. ࣁ࢖ࣈࣜࢻ࣮࣐ἲ࡟ࡼࡾK_a = 1.7™10 12 _M-1 ࡢࣔࣀࢡ࣮ࣟࢼࣝᢠࢪࢦ࢟ࢩࣥ ᢠయࢆᚓࡓ࡜ࡍࡿ౛ࡶ࠶ࡿࡀ, 12) ࡇ࠺ࡋࡓ㧗ぶ࿴ຊᢠయࢆ☜ᐇ࡟ᚓࡿࡓࡵࡢ᫂☜࡞ᣦ㔪 ࡣぢᙜࡓࡽ࡞࠸. ௒᪥, ᢠయࡢศᏊᵓ㐀ࢆ㑇ఏᏊ࡛ࣞ࣋ࣝᨵኚࡍࡿࠕᢠయᕤᏛࠖࡀ㐍Ṍࡋ, ື≀࠿ࡽᚓ ࡽࢀࡿኳ↛ࡢᢠయࢆୖ ᅇࡿᶵ⬟ࢆഛ࠼ࡓேᕤ ᢠయ (ኚ␗ᢠయ) ࢆ๰〇ࡍࡿࡇ࡜ࡀྍ⬟ どࡉࢀ࡚࠸ࡿ. ື≀࡟㢗ࡽࡎ, in vitro ࡢᐇ㦂⣔࡛ᢠయࡢᶵ⬟ࢆᨵⰋࡋ࡚࠸ࡃࡓࡵ, ᢠయ ࡢࠕヨ㦂⟶ෆศᏊ㐍໬ࠖ࡜ࡶ࿧ࡤࢀࡿ◊✲㡿ᇦ࡛࠶ࡿ. ᮏἲ࡟ࡼࡾୖグࡢࡼ࠺࡞㧗ぶ࿴ ຊᢠయࡀᚓࡽࢀࡿ࡞ࡽࡤ, ච␿ ᐃἲࡢᢤᮏⓗ࡞㧗ឤᗘ໬ࡀྍ⬟࡜ᮇᚅࡉࢀࡿ. ᢠయᕤ Ꮫ ࡣ 1980 ᖺ ௦ᮎᮇ ࠿ࡽ 1990 ᖺ௦ ๓༙࡟ ࠿ࡅ ࡚ᛴ ㏿࡟㐍 ᒎࡋ, ᢏ⾡ᇶ ┙ࡀ ☜❧ࡉ ࢀ ࡓ. 13,14) ࡑࡢዎᶵࡣ, ᢠཎ࡜ࡢ⤖ྜ࡟㛵ࢃࡿᢠయྍኚ㒊ࡢ㑇ఏᏊࢡ࣮ࣟࢽࣥࢢ 15,16) ࡜, ኱⭠⳦ࢆᐟ୺࡜ࡋ࡚పศᏊࣇࣛࢢ࣓ࣥࢺ (ᚋ㏙) ࡢⓎ⌧ࡀྍ⬟࡟࡞ࡗࡓࡇ࡜ 17–19) ࡛࠶ ࡿ. ᢠయࡢᶵ⬟ᨵኚࡣ, ୍⯡࡟ḟࡢᡭ㡰࡟ࡼࡾ⾜ࢃࢀࡿ. ࡲࡎ, ࣁ࢖ࣈࣜࢻ࣮࣐ἲ࡞࡝࡛ ᪤࡟ᚓࡽࢀ࡚࠸ࡿࠕኳ ↛ࡢࠖᢠయ [ࡍ࡞ࢃࡕච ␿ࢢࣟࣈࣜࣥ; ୺࡟ immunoglobulin G (IgG)] ࢆపศᏊ໬ࡍࡿ. ከࡃࡢሙྜ, ࡑࡢH㙐࡜L㙐ࡢྍኚ㒊ࢻ࣓࢖ࣥ (ࡑࢀࡒࢀVH, VL) ࢆ ே ᕤ ࡢ ࣜ ࣥ ࢝ ࣮ ࣌ ࣉ ࢳ ࢻ ࡛ 㐃 ⤖ ࡋ ࡓ ୍ ᮏ 㙐 Fv ࣇ ࣛ ࢢ ࣓ ࣥ ࢺ (single-chain Fv fragment; scFv) ࡟ኚ᥮ࡍࡿ. IgG ࡢᢠཎ⤖ྜ㒊఩ (ࣃࣛࢺ࣮ࣉ) ࡣVH࡜ VLࡢ㛫࡟఩⨨ ࡍࡿࡓࡵ, scFvࡶྠᵝࡢࣃࣛࢺ࣮ࣉࢆᙧᡂࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡾ, ᑐᛂࡍࡿIgGࡢᢠཎ ⤖ྜ⬟ࢆಖᣢࡋ࡚࠸ࡿࡇ࡜ࡀከ࠸. ࡋ࠿ࡶscFvศᏊࡢ኱ࡁࡉࡣIgGࡢ⣙1/6࡛࠶ࡾ (ᅗ 2), ኱⭠⳦ෆ࡛Ᏻᐃ࡟Ⓨ⌧࡛ࡁࡿࡇ࡜࡟ຍ࠼, 20,21) ༢୍ࡢ࣮࢜ࣉ࣮ࣥࣜࢹ࢕ࣥࢢࣇ࣮ࣞ ࣒࡟ࡼࡾ㌿෗, ⩻ヂࡉࢀࡿࡓࡵ, 㑇ఏᏊ᧯సࡀᐜ࡛᫆࠶ࡿ. ḟ࡟, ࡇࢀࢆࣉࣟࢺࢱ࢖ࣉ ࡜ࡋ࡚ఱࡽ࠿ࡢ᪉ἲ࡛ࣛࣥࢲ࣒ኚ␗࠶ࡿ࠸ࡣ㒊఩≉␗ⓗ࡞ኚ␗ࢆᑟධࡋ, 㐺ษ࡞ᐟ୺ ࡟Ⓨ⌧ࡉࡏࡿࡇ࡜࡟ࡼࡾከᵝᛶ࡟ᐩࡴኚ␗ scFv ࡢࣛ࢖ࣈ࣮ࣛࣜࢆᚓࡿ. ࡇࡢ࡞࠿࠿ࡽ ࠕഅ↛࡟ࠖ⏕ᡂࡋࡓ⛥ᑡ࡞ᨵⰋᆺኚ␗ scFv ࢆ, ኱㔞࡟๪⏕ࡍࡿࠕᨵᝏᆺࠖscFv ࠿ࡽศ

4 㞳ࡋࡘࡘ⋓ᚓࡍࡿࡢ࡛࠶ࡿ. ࡇࡢᕤ⛬ࢆຠ⋡ࡼࡃ⾜࠺ࡓࡵ࡟, 㑇ఏᆺ-⾲⌧ᆺᑐᛂᆺᢏ ⾡ࡀ㛤Ⓨࡉࢀࡓ. ࡍ࡞ࢃࡕ, ⾲⌧ᆺ࡛࠶ࡿscFvࢱࣥࣃࢡ㉁࡜, ࡑࢀࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊ ࢆ, ྠ୍ࡢ⢏Ꮚࡢᵓᡂᡂศ࡜ࡋ୍࡚య໬ࡉࡏࡿᢏ⾡࡛࠶ࡿ. ࡇࢀ࡟ࡼࡾ, ᨵᝏᆺscFv࡟ ᇙἐࡋࡓᨵⰋᆺ scFv ࡢ᳨ฟ࣭༢㞳࡜, ࡑࡢ」〇࡟ࡼࡿᚲせ㔞ࡢ⏕⏘ࢆ㐃⥆ⓗ࡟⾜࠺ࡇ ࡜ࡀྍ⬟࡜࡞ࡗࡓ. scFv㑇ఏᏊࡢሷᇶ㓄ิ࠿ࡽ, ᨵⰋᆺscFvࡢ1ḟᵓ㐀ࢆ᥎ᐃࡍࡿࡇ࡜ ࡶᐜ࡛᫆࠶ࡿ. 㑇ఏᆺ-⾲⌧ᆺᑐᛂᆺᢏ⾡ࡢ௦⾲౛ࡣࣇ࢓࣮ࢪᥦ♧ἲ࡛࠶ࡿ. ࡇࡢ᪉ἲࢆ1985ᖺ࡟ሗ ࿌ࡋࡓSmithࡣ, ᮏἲࢆᢠయᶵ⬟ࡢᨵኚ࡟ᛂ⏝ࡋࡓWinter ࡜ඹ࡟2018ᖺࡢࣀ࣮࣋ࣝ໬ Ꮫ㈹ࢆཷ㈹ࡋࡓ. 1,22–24) ࣇ࢓࣮ࢪᥦ♧ἲ࡜ࡣ⧄⥔≧ࣂࢡࢸࣜ࢜ࣇ࢓࣮ࢪ⢏Ꮚࡢ⾲㠃࡟࣌ ࣉࢳࢻࡸࢱࣥࣃࢡ㉁࡞࡝ࡢᶆⓗศᏊࢆᥦ♧ࡍࡿᢏ⾡࡛࠶ࡾ, ⏕ᡂࡍࡿᶆⓗศᏊᥦ♧ࣇ ࢓࣮ࢪࡢࢤࣀ࣒ෆ࡟ࡑࡢศᏊࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡀ⤌ࡳ㎸ࡲࢀࡿࡓࡵ, 㑇ఏᆺ࡜⾲⌧ ᆺࡢᑐᛂࡀ㐩ᡂࡉࢀࡿ. ࣛࣥࢲ࣒ኚ␗ࢆᑟධࡋࡓ scFv 㑇ఏᏊࣛ࢖ࣈ࣮ࣛࣜࢆ୍ᣲ࡟ࣇ ࢓࣮ࢪᥦ♧ࡍࡿࡇ࡜࡟ࡼࡾ, ኚ␗ scFv ᥦ♧ࣇ࢓࣮ࢪࣛ࢖ࣈ࣮ࣛࣜࡀస〇ࡉࢀࡿ. ࡇࡢ ࡞࠿࠿ࡽᨵⰋᆺ scFv ࢆ㑅ᢥࡍࡿ᪉ἲ࡜ࡋ࡚, ࠕࣃࣥࢽࣥࢢࠖ࡜࠸࠺ᡭἲࡀ⏝࠸ࡽࢀࡿ (ᅗ 3). ࡍ࡞ࢃࡕ, ࣇ࢓࣮ࢪࣛ࢖ࣈ࣮ࣛࣜ඲యࢆᅛ┦࡟ᅛᐃ໬ࡋࡓᢠཎ࡟୍ᣲ࡟཯ᛂࡉ ࡏ, ⤖ྜࡋࡓࣇ࢓࣮ࢪࡢࡳࢆఱࡽ࠿ࡢ᪉ἲ࡛⁐ฟࡍࡿ. ࡇ࠺ࡋ࡚ᚓࡽࢀࡿᢠཎ≉␗ⓗ࡞ scFv ᥦ♧ࣇ࢓࣮ࢪࡣࡈࡃᚤ㔞࡛࠶ࡿࡀ, ࣇ࢓࣮ࢪࡀ኱⭠⳦࡟ឤᰁࡋ࡚ቑṪࡍࡿᛶ㉁ࢆ ฼⏝ࡋ࡚㎿㏿࡟ᚲせ㔞ࢆ」〇ࡍࡿࡇ࡜ࡀ࡛ࡁࡿ. ࡇࡢࢧ࢖ࢡࣝࢆ⧞ࡾ㏉ࡍࡇ࡜࡛, ┠ⓗ ࡢࣇ࢓࣮ࢪࢆ⃰⦰ࡋ, ᭱⤊ⓗ࡟༢㞳ࡍࡿ (ᅗ 3). ࡇࡢ᪉ἲ࡟ࡼࡾ, ᮏ᮶⏕యෆ࡛㉳ࡁ࡚ ࠸ࡿᢠయࡢぶ࿴ᛶᡂ⇍㐣⛬ࢆᶍೌࡋ࡚ᢠయࡢぶ࿴ຊࢆ㧗ࡵࡿࡇ࡜ࡀྍ⬟࡛࠶ࡾ,ࠕヨ㦂 ⟶ෆぶ࿴ᛶᡂ⇍ࠖ࡜࿧ࡤࢀ࡚࠸ࡿ. ࡑࡢᡂᯝ࡜ࡋ࡚, ࢱࣥࣃࢡ㉁࡞࡝ࡢ㧗ศᏊᢠཎ࡟ᑐ ࡋ࡚ࡣ, 1000ಸ௨ୖࡶࡢぶ࿴ຊࡢྥୖࢆ⤒࡚, ࡑࡢK_aࡀ10 10 ࢆୖᅇࡿᢠయࢆస〇ࡋࡓ౛ ࡀ࠸ࡃࡘ࠿ሗ࿌ࡉࢀ࡚࠸ࡿ. 13) ࡞࠾, 㑇ఏᆺ࡜⾲⌧ᆺࢆᑐᛂࡉࡏࡿᡭẁ࡜ࡋ࡚, 㓝ẕࡢ ᅗ 2. ᢠయ (IgG) ࠾ࡼࡧ୍ᮏ㙐Fvࣇࣛࢢ࣓ࣥࢺ (scFv) ࡢᵓ㐀  ච␿ ᐃἲ࡟୍⯡ⓗ࡟⏝࠸ࡽࢀࡿᢠయࡣIgG࡛࠶ࡿ. 2ᮏࡢH㙐࡜L㙐࠿ࡽ࡞ࡿࢱࣥࣃࢡ㉁࡛, ࡑࢀࡒ ࢀNᮎ➃࡟ྍኚ㒊 (VH, VL) ࢆᣢࡘ. ᢠཎ⤖ྜ㒊఩ (ࣃࣛࢺ࣮ࣉ) ࡣVH࡜VLࡢ㛫࡟఩⨨ࡍࡿ. ྍኚ㒊ෆ࡟ ࡣᢠཎࡢ⤖ྜ࡟኱ࡁࡃ㛵୚ࡍࡿ┦⿵ᛶỴᐃ㒊 (complementarity-determining region; CDR) ࡀ3࢝ᡤ, ࡑࡋ࡚, ࡑࡢᵓ㐀ࢆᨭᣢࡍࡿᯟ⤌ࡳ㓄ิ (framework region; FR) ࡀ4࢝ᡤᏑᅾࡍࡿ. scFvࡣVH࡜VLࢆྍືᛶ࡟ᐩ ࡴேᕤࡢ࣮ࣜࣥ࢝࣌ࣉࢳࢻ࡛㐃⤖ࡋࡓᢠయࣇࣛࢢ࣓ࣥࢺ࡛࠶ࡿ.

5 ⣽⬊⾲㠃࡟ᏑᅾࡍࡿศᏊࢆ฼⏝ࡍࡿ㓝ẕ⾲ᒙᥦ♧ἲ

25)

ࡸ, ↓⣽⬊ࢱࣥࣃࢡ㉁ྜᡂ⣔ࢆ ฼⏝ࡋ࡚ messenger ribonucleic acid (mRNA)-ࣜ࣎ࢯ࣮࣒-ᶆⓗࢱࣥࣃࢡ㉁ࡢ」ྜయࢆᙧ ᡂࡉࡏࡿࣜ࣎ࢯ࣮࣒ࢹ࢕ࢫࣉࣞ࢖ 26) ࡶ㛤Ⓨࡉࢀ࡚࠸ࡿࡀ, ྲྀࡾᢅ࠸ࡢ⡆౽ࡉࡸࢩࢫࢸ ࣒ࡢᏳᐃᛶ࡟ඃࢀࡿࡇ࡜࠿ࡽࣇ࢓࣮ࢪᥦ♧ἲࡀ᭱ࡶᬑཬࡋ࡚࠸ࡿ. ࡋ࠿ࡋ࡞ࡀࡽ, ࣇ࢓࣮ࢪᥦ♧ἲ࡟࠾ࡅࡿᨵⰋᆺศᏊ✀ࡢ㑅ᢥຠ⋡࡟ࡘ࠸࡚ࡣᨵၿࡍ ࡭ࡁၥ㢟ࡀṧࡉࢀ࡚࠸ࡿ. ࣃࣥࢽࣥࢢࢆ⾜ࡗ࡚ࡶ, ࡼࡾ㧗࠸ぶ࿴ຊࢆ♧ࡍኚ␗యࡀࡼࡾ ඃඛⓗ࡟⃰⦰ࡉࢀࡿ࡜ࡣ㝈ࡽࡎ, ୍⯡ⓗ࡟⁐ฟࡉࢀࡓከᩘࡢ scFv ᥦ♧ࣇ࢓࣮ࢪࢡ࣮ࣟ ࣥ࡟ࡘ࠸࡚ಶูࡢゎᯒ (ࢫࢡ࣮ࣜࢽࣥࢢ) ࡀᚲせ࡛࠶ࡿ. ࡑࡢ⤖ᯝ, 㧗ぶ࿴ຊኚ␗యࡢ ༢㞳࡟ከ኱࡞ປຊ࡜᫬㛫ࢆ㈝ࡸࡍࡇ࡜ࡀᑡ࡞ࡃ࡞࠸. ࡇࢀࡣ, ୺࡟ḟࡢ 2ࡘࡢྍ⬟ᛶ࡟ ㉳ᅉࡍࡿࡶࡢ࡜⪃࠼ࡽࢀࡿ. ࡲࡎ, ࣛ࢖ࣈ࣮ࣛࣜᵓᡂ࣓ࣥࣂ࣮ (scFv 㑇ఏᏊಖᣢ⳦࠶ࡿ ࠸ࡣ scFv ᥦ♧ࣇ࢓࣮ࢪࡢࢡ࣮ࣟࣥ) ࡢቑṪ⋡ࡢᕪࡀᣲࡆࡽࢀࡿ. ࣃࣥࢽࣥࢢࢆᐇ⾜ࡍ ࡿ࡟㝿ࡋ࡚ࡣ, ኚ␗scFv㑇ఏᏊࢆᑟධࡋࡓ┤ᚋ࡟ᚓࡽࢀࡿࠕ࢜ࣜࢪࢼࣝࣛ࢖ࣈ࣮ࣛࣜࠖ ࡢᙧ㉁㌿᥮኱⭠⳦ࢆ෌ᇵ㣴ࡋࡓࡢࡕ, ࣊ࣝࣃ࣮ࣇ࢓࣮ࢪࢆឤᰁࡉࡏ࡚༑ศ㔞ࡢࣇ࢓࣮ ࢪࢆ⏕⏘ࡍࡿᚲせࡀ࠶ࡿ. ࡑࡢ㝿, ኱⭠⳦ࡢቑṪ㏿ᗘࡸࣇ࢓࣮ࢪ⢏Ꮚࡢᙧᡂ⋡ (ࣃࢵࢣ ࣮ࢪࣥࢢ⋡) ࡟ࢡ࣮ࣟࣥ㛫࡛኱ࡁ࡞ᕪࡀ⏕ࡌ࠺ࡿ. ᕼᑡ࡞ᨵⰋᆺኚ␗యࡢࢡ࣮ࣟࣥࡀ㡰 ㄪ࡟ቑṪࡍࡿಖドࡣ࡞ࡃ, 㔝⏕ᆺࡸᨵᝏᆺࢡ࣮ࣟࣥ࡟Ὲửࡉࢀࡿྍ⬟ᛶࡶ࠶ࡾ࠺ࡿ (ᅗ 4). ᐇ㝿, Derdaࡽࡣ, ࣃࣥࢽࣥࢢ࡟࠾ࡅࡿྛ㐣⛬ࡢ๓ᚋ࡛ᚓࡽࢀࡿ࣌ࣉࢳࢻᥦ♧ࣇ࢓࣮ ࢪࢡ࣮ࣟࣥࢆ㏣㊧ࡋࡓ⤖ᯝ, ࣛ࢖ࣈ࣮ࣛࣜࡢከᵝᛶࡣᶆⓗ≀㉁࡟ᑐࡍࡿ㑅ᢥࡼࡾࡶࡴ ࡋࢁቑᖜࡢ㐣⛬࡛኱ࡁࡃῶᑡࡋ࡚࠸ࡃ, ࡘࡲࡾᑡᩘࡢኚ␗ࢡ࣮ࣟࣥ✀࠿ࡽ࡞ࡿ㞟ᅋ࡟ ཰᮰ࡋ࡚࠸ࡃࡇ࡜ࢆ♧ࡋࡓ. 27) ࡇࢀࡣ, ࣃࣥࢽࣥࢢ࡛᭱⤊ⓗ࡟ᚓࡽࢀࡿࣇ࢓࣮ࢪ㞟ᅋࡣ, ᅗ 3. ࣇ࢓࣮ࢪᥦ♧ἲࢆ฼⏝ࡋࡓヨ㦂⟶ෆศᏊ㐍໬ࡢཎ⌮ ࣇ࢓࣮ࢪᥦ♧ἲࢆ⏝࠸࡚స〇ࡋࡓኚ␗scFvᥦ♧ࣇ࢓࣮ࢪࣛ࢖ࣈ࣮ࣛࣜࢆᅛ┦࡟ᅛᐃ໬ࡋࡓᢠཎ࡟཯ᛂ ࡉࡏ, ᢠཎ≉␗ⓗ࡞ࣇ࢓࣮ࢪࢆᤕᤊࡍࡿ. ᮍ཯ᛂࡢࣇ࢓࣮ࢪࢆὙίࡋࡓࡢࡕ, ᅛ┦࡟ṧࡿࣇ࢓࣮ࢪࢆ㓟ࡸ ሷᇶ࡞࡝࡛⁐ฟࡋ, ኱⭠⳦࡟ឤᰁࡉࡏࡿࡇ࡜࡛, ᢠཎ≉␗ⓗ࡞ࣇ࢓࣮ࢪࡀ⃰⦰ࡉࢀࡓ2ḟࣛ࢖ࣈ࣮ࣛࣜࡀ స〇ࡉࢀࡿ. ࡇࡢࢧ࢖ࢡࣝ (ࣃࣥࢽࣥࢢ࡜࿧ࡤࢀࡿ) ࢆ⧞ࡾ㏉ࡋ࡚, ┠ⓗࡢᛶ㉁ࢆࡶࡘࢡ࣮ࣟࣥࢆ⋓ᚓࡍ ࡿ.

6 ᢠཎ⤖ྜ⬟ࡢ㧗ࡉࡼࡾ, ࡴࡋࢁ」〇⋡ࡢ㧗ࡉࢆ཯ᫎࡋࡓࡶࡢ࡛࠶ࡿࡇ࡜ࢆព࿡ࡍࡿ. ࡉࡽ࡟, ࣃࣥࢽࣥࢢ࡛ࡣ, 㝈ࡽࢀࡓ㔞ࡢᅛᐃ໬ᢠཎ࠶ࡿ࠸ࡣࣅ࢜ࢳࣥ࡞࡝ࡢ࢔ࣇ࢕ࢽ ࢸ࢕ᢳฟ⏝ࢱࢢࢆ௜ຍࡉࢀࡓᢠཎ࡟ᑐࡋ࡚, Ⳙ኱࡞✀㢮ࡢ scFv ᥦ♧ࣇ࢓࣮ࢪࢡ࣮ࣟࣥ ࡀྠ᫬࡟཯ᛂࡍࡿࡇ࡜࡟࡞ࡿ. 」〇⋡ࡀ㧗ࡃ኱㔞࡟ቑᖜࡉࢀࡓᨵᝏᆺ࠶ࡿ࠸ࡣ㔝⏕ᆺ ࡢscFvᥦ♧ࣇ࢓࣮ࢪࡀ➇ྜⓗ࡟཯ᛂࡍࡿࡇ࡜࡟ࡼࡾ, ࡈࡃᚤ㔞ࡢᕼᑡ࡞ᨵⰋᆺscFvᥦ ♧ࣇ࢓࣮ࢪࡢᤕᤊࡀጉࡆࡽࢀࡿࡇ࡜ࡣ᝿ീ࡟㞴ࡃ࡞࠸ (ᅗ 5). ᅗ 5. ࣃࣥࢽࣥࢢ࡟࠾ࡅࡿ➇ྜ཯ᛂ ぶ࿴ຊࡀ㧗ࡃ, 」〇⋡ࡢప࠸ࢡ࣮ࣟࣥ࡜, ぶ࿴ຊࡀపࡃ, 」〇⋡ࡢ㧗࠸ࢡ࣮ࣟࣥࡀඹᏑࡍࡿሙྜ, 」〇 ⋡ࡢ㧗࠸ࢡ࣮ࣟࣥࡀࣛ࢖ࣈ࣮ࣛࣜࡢ኱㒊ศࢆ༨ࡵࡿ. ࡇࡢࣛ࢖ࣈ࣮ࣛࣜࢆࡲ࡜ࡵ࡚ᢠཎ࡟཯ᛂࡉࡏࡓሙ ྜ, పぶ࿴ຊࢡ࣮ࣟࣥ࡟ᢠཎࡀ༨᭷ࡉࢀ࡚ࡋࡲ࠸, ⋓ᚓࡍ࡭ࡁ㧗ぶ࿴ຊࢡ࣮ࣟࣥࡀ⤖ྜࡏࡎ, ࣛ࢖ࣈࣛࣜ ࣮࠿ࡽ⬺ⴠࡋ࡚ࡋࡲ࠺ࡇ࡜ࡶ࠶ࡾ࠺ࡿࡔࢁ࠺. ᅗ 4. ࣇ࢓࣮ࢪࣛ࢖ࣈ࣮ࣛࣜస〇࡟࠾ࡅࡿ኱⭠⳦ࡢቑṪ㏿ᗘࡸࣃࢵࢣ࣮ࢪࣥࢢ⋡ࡢᕪࡢᙳ㡪  scFvᥦ♧ࣇ࢓࣮ࢪឤᰁᚋࡢ኱⭠⳦ࡢቑṪ㏿ᗘࡸࣇ࢓࣮ࢪ⢏Ꮚࡢࣃࢵࢣ࣮ࢪࣥࢢ⋡ࡀ␗࡞ࡿሙྜ࡟స〇 ࡉࢀࡿࣇ࢓࣮ࢪࣛ࢖ࣈ࣮ࣛࣜࡢ≧ែࢆ♧ࡍ. ࢡ࣮ࣟࣥ (a) ࡢቑṪ㏿ᗘࡸࣃࢵࢣ࣮ࢪࣥࢢ⋡ࢆᶆ‽࡜ࡍࡿ ࡜, ࢡ࣮ࣟࣥ (b) ࡣࣃࢵࢣ࣮ࢪࣥࢢ⋡ࡣᶆ‽࡛ࣞ࣋ࣝ࠶ࡿࡀቑṪ㏿ᗘࡀ㏿ࡃ, ࢡ࣮ࣟࣥ (c) ࡣቑṪ㏿ᗘࡣ ᶆ‽࡛ࣞ࣋ࣝ࠶ࡿࡀࣃࢵࢣ࣮ࢪࣥࢢ⋡ࡀ㧗࠸. ࢡ࣮ࣟࣥ (a) ࡀ┠ⓗࡢᨵⰋᆺ࡛࠶ࡗࡓ࡜ࡋ࡚ࡶ, ࣃࣥࢽࣥ ࢢࢆ⧞ࡾ㏉ࡍࡇ࡜࡟ࡼࡾ, (b) ࡸ (c) ࡟Ὲửࡉࢀࡿࡇ࡜ࡀ࠶ࡾ࠺ࡿ.

7 ௨ୖࡢࣃࣥࢽࣥࢢ࡟㉳ᅉࡍࡿၥ㢟ࢆᢤᮏⓗ࡟ゎỴࡋ, 㧗ぶ࿴ຊࡢኚ␗ᢠయࣇࣛࢢ࣓ ࣥࢺࢆᚓࡿࡓࡵ࡟ࡣ, ಶࠎࡢࣇ࢓࣮ࢪࢡ࣮ࣟࣥࢆಶู࡟ᢠཎ࡜཯ᛂࡉࡏ, ࡑࡢ⤖ྜ⬟ࢆ ⥙⨶ⓗ࠿ࡘຠ⋡ⓗ࡟ࢫࢡ࣮ࣜࢽࣥࢢࡍࡿࡇ࡜ࡀ☜ᐇ࡞࢔ࣉ࣮ࣟࢳἲ࡛࠶ࡿ. ࡑࡇ࡛ⴭ ⪅ࡣ, ᅗ 6 ࡟♧ࡍࠕ࢔ࣞ࢖ᆺࣉࣟࣇ࢓࢖ࣜࣥࢢࠖࢩࢫࢸ࣒ࢆ⪃᱌ࡋࡓ. ࡍ࡞ࢃࡕ, ࠶ࡽ ࠿ࡌࡵ࣊ࣝࣃ࣮ࣇ࢓࣮ࢪࢆྵࡴᇵᆅࢆศὀࡋࡓᢠཎᅛᐃ໬ࣉ࣮ࣞࢺ ࡢ࣐࢖ࢡ࢙ࣟ࢘ࣝ ࡟, scFv㑇ఏᏊࢆಖᣢࡍࡿᙧ㉁㌿᥮⳦ࢡ࣮ࣟࣥࢆಶู࡟᥋✀ࡋ࡚ࠕࢥࣟࢽ࣮࢔ࣞ࢖ࠖࢆ సᡂࡋ, ࢙࢘ࣝෆ࡛ᇵ㣴ࡋ࡚ࣇ࢓࣮ࢪࢆ⏘⏕ࡉࡏ, ᢠཎ࡟⤖ྜࡋࡓ scFv ᥦ♧ࣇ࢓࣮ࢪ ࢆᢠࣇ࢓࣮ࢪᢠయࢆ⏝࠸᳨࡚ฟࡍࡿࡶࡢ ࡛࠶ࡿ. ࡑࡢᐇ⌧ࢆ┠ᶆ࡜ࡋ࡚, ࡲࡎ, ᨵⰋᆺ ኚ␗యࡢ༢㞳ຠ⋡ࡢホ౯࡟⏝࠸࠺ࡿኚ␗ scFv ࣛ࢖ࣈ࣮ࣛࣜࢆస〇ࡋ, ᪂つ㑅ᢥἲ࡜ࡢ ẚ㍑ࡢࡓࡵ࡟ᚑ᮶ࡢࣃࣥࢽࣥࢢ࡟ࡼࡿᨵⰋศᏊ✀ࡢ⋓ᚓࢆヨࡳࡓ (➨1❶). ࡘ࠸࡛, ᪂ つ㑅ᢥἲࡢᵓ⠏࡟ᚲ㡲࡛࠶ࡿ⧄⥔≧ࣇ࢓࣮ࢪ࡟ᑐࡍࡿࣔࣀࢡ࣮ࣟࢼࣝᢠయࢆᶞ❧ࡋࡓ (➨2❶). ࡉࡽ࡟ᮏᢠయࢆscFv໬ࡋ, 㧗ឤᗘ࡞Ⓨග᳨ฟࡀྍ⬟࡞ Gaussia ࣝࢩࣇ࢙࣮ࣛ ࢮ࡜ࡢ⼥ྜࢱࣥࣃࢡ㉁ࢆస〇ࡋࡓ (➨2❶). ࡑࡋ࡚, ࡇࡢ⼥ྜࢱࣥࣃࢡ㉁ࢆ࣮࢟ヨ⸆࡜ ࡋ࡚ά⏝ࡋ, ୖグࡢ࢔ࣞ࢖ᆺࣉࣟࣇ࢓࢖ࣜࣥࢢࢩࢫࢸ࣒ࢆᵓ⠏ࡋࡓ (➨3❶). ྛࢫࢸࢵ ࣉࡢㅖ᮲௳ࢆ᭱㐺໬ࡋࡓࡢࡕ, ➨ 1 ❶࡛స〇ࡋࡓࣛ࢖ࣈ࣮ࣛࣜࢆ⏝࠸࡚, ࡑࡢ㑅ᢥຠ ⋡ࢆࣃࣥࢽࣥࢢ࡜ࡢẚ㍑ࡢほⅬ࠿ࡽホ౯ࡋࡓ (➨3❶). ᅗ 6. ࢔ࣞ࢖ᆺࣉࣟࣇ࢓࢖ࣜࣥࢢࢩࢫࢸ࣒ࡢᴫ␎ᅗ  ኚ␗scFv㑇ఏᏊࣛ࢖ࣈ࣮࡛ࣛࣜᙧ㉁㌿᥮ࡋࡓ኱⭠⳦ࢆᐮኳᇵᆅ࡟ሬᕸࡋ࡚ᇵ㣴ࡋ, ᙧᡂࡋࡓࢥࣟࢽ ࣮ࢆ, ࠶ࡽ࠿ࡌࡵ࣊ࣝࣃ࣮ࣇ࢓࣮ࢪࢆྵࡴᇵᆅࢆศὀࡋࡓᢠཎᅛᐃ໬࣐࢖ࢡࣟࣉ࣮ࣞࢺࡢྛ࢙࢘ࣝ࡟ᠱ ⃮ࡍࡿ. ࡑࡢᚋ, ༑ศ࡞᫬㛫᣺࡜࠺ᇵ㣴ࡍࡿࡇ࡜࡛, ࢙࢘ࣝෆ࡛scFvᥦ♧ࣇ࢓࣮ࢪࡀ⏘⏕ࡉࢀࡘࡘ, ᢠ ཎ࡟ᤕᤊࡉࢀࡿ. Ὑίᚋ, ᅛ┦ୖࡢࣇ࢓࣮ࢪࢆ᳨ฟࡍࡿࡇ࡜࡛, ᢠཎ≉␗ⓗ࡞ࣇ࢓࣮ࢪࡀᏑᅾࡍࡿ࢙࢘ ࣝࢆ≉ᐃࡍࡿ.

8 ᮏㄽ ➨❶ 㧗ᛶ⬟ኚ␗య༢㞳ຠ⋡ࡢホ౯⣔࡜ࡋ࡚ࡢᢠయࣇࣛࢢ࣓ࣥࢺࣛ࢖ࣈ࣮ࣛࣜࡢᵓ ⠏ ➨⠇ ᗎ ࣇ࢓࣮ࢪᥦ♧ἲ࡟ࡼࡿヨ㦂⟶ෆぶ࿴ᛶᡂ⇍ࡣ, 㧗ศᏊᢠཎࢆᶆⓗ࡜ࡍࡿᢠయ࡟ࡘ࠸ ࡚ࡣᡂຌ౛ࡀ」ᩘሗ࿌ࡉࢀ࡚࠸ࡿ. 13) ࡜ࡇࢁࡀ, ᢠࣁࣉࢸࣥᢠయ࡟ࡘ࠸࡚ࡣᡂຌ౛࡟ஈ ࡋ࠸. 28–34) ᐇ㝿, ⴭ⪅ࡢᡤᒓ◊✲ᐊ࡛ࡣ4✀ࡢࣁࣉࢸࣥ࡟ࡘ࠸࡚㧗ぶ࿴ຊኚ␗scFvࡢ ๰〇࡟ᡂຌࡋ࡚࠸ࡿࡶࡢࡢ, ࡑࡢ༢㞳࡟ࡣከ኱࡞ປຊ࡜᫬㛫ࢆ㈝ࡸࡋ࡚࠸ࡿ. 32–36) ࡇࢀࡣ⥴ㄽ࡛ゝཬࡋࡓࣃࣥࢽࣥࢢᅛ᭷ࡢㅖၥ㢟ࡀཎᅉ࡜⪃࠼ࡽࢀࡿ. ࢔ࣞ࢖ᆺࣉࣟ ࣇ࢓࢖ࣜࣥࢢࢩࢫࢸ࣒ࡣࡇࢀࡽࡢၥ㢟ࢆᢤᮏⓗ࡟ゎỴࡋ, ᨵⰋศᏊ✀ࡢ⋓ᚓຠ⋡ࢆ㣕 ㌍ⓗ࡟ྥୖࡉࡏࡿࡶࡢ࡜ᮇᚅࡉࢀࡿ. ᮏἲࡣᢠཎࡢ✀㢮࡟࠿࠿ࢃࡽࡎ, ࠶ࡽࡺࡿᢠయࡢ ヨ㦂⟶ෆぶ࿴ᛶᡂ⇍࡟᭷ຠ࡜⪃࠼ࡽࢀࡿࡀ, ᮏ◊✲࡛ࡣ, ࡑࡢᵓ⠏࡟࠶ࡓࡾ, ࠶࠼࡚ࣃ ࣥࢽࣥࢢࡢᡂຌ౛ࡀᑡ࡞࠸ࣁࣉࢸࣥᢠཎ࡟ᑐࡍࡿᢠయࡢᨵኚ࡟ᛂ⏝ࡍࡿࡇ࡜࡟ࡼࡾᛶ ⬟ࢆホ౯ࡍࡿࡇ࡜࡜ࡋࡓ. ࣔࢹࣝࣁࣉࢸࣥ࡜ࡋ࡚, ࢥࣝࢳࢰ࣮ࣝ (cortisol; CS, M_r 362.5) ࢆ㑅ࢇࡔ. CSࡣࣄࢺ࡟ ࠾ࡅࡿ୺せ࡞⢾㉁ࢥࣝࢳࢥ࢖ࢻ࡛࠶ࡾ, どᗋୗ㒊-ୗᆶయ-๪⭈⣔ᶵ⬟ࡢホ౯࡟㔜⏝ࡉ ࢀࡿࡓࡵ, 37) デ᩿ヨ⸆࡜ࡋ࡚ࡢᢠCSᢠయࡢ㟂せࡣᜏᖖⓗ࡟㧗࠸. ࡋ࠿ࡋ࡞ࡀࡽ, ື≀ ࡟ච␿ࡍࡿᚑ᮶ἲ࡟ࡼࡾCS࡟ᑐࡋ࡚‶㊊ࡢ࠸ࡃࣔࣀࢡ࣮ࣟࢼࣝᢠయࢆᚓࡿࡇ࡜ࡣᐜ ࡛᫆ࡣ࡞ࡃ, ᡂຌ౛ࡣᑡ࡞࠸. 38–40) ࡑࡇ࡛, CSࢆᢠཎ࡜ࡍࡿscFvࡢኚ␗యࣛ࢖ࣈࣛࣜ ࣮ࢆస〇ࡍࡿࡇ࡜࡟ࡋࡓ. ࡑࡢࡓࡵ࡟ࡣ, ࣉࣟࢺࢱ࢖ࣉ࡜࡞ࡿኳ↛ᆺᢠయྍኚ㒊ࡢ㑇ఏᏊࡀᚲせ࡟࡞ࡿ. ࡑࡇ ࡛, ࡲࡎ, ࣁ࢖ࣈࣜࢻ࣮࣐ἲ࡟ࡼࡾᢠCSᢠయ⏘⏕⣽⬊ᰴࡢᶞ❧ࢆ௻࡚ࡓ (➨2⠇). ࡑ ࡢ⤖ᯝ, ぶ࿴ຊࡢ␗࡞ࡿ2✀㢮ࡢᢠCSࣔࣀࢡ࣮ࣟࢼࣝᢠయࡀᚓࡽࢀࡓ. ḟ࠸࡛, ࡇࢀ ࡽኳ↛ᆺᢠయࡢྍኚ㒊㑇ఏᏊࢆࢡ࣮ࣟࢽࣥࢢࡋ, 㑇ఏᏊ᧯స࡟㐺ࡍࡿపศᏊ㔞ࣇࣛࢢ ࣓ࣥࢺ, scFv࡟ኚ᥮ࡋࡓ (➨3⠇). ᚓࡽࢀࡓ2✀ࡢscFvࡢ࠺ࡕ, ࠶࠼࡚ぶ࿴ຊࡢప࠸ scFvࢆヨ㦂⟶ෆぶ࿴ᛶᡂ⇍ࡢฟⓎ≀㉁ (㔝⏕ᆺ; wt-scFv) ࡜ࡋ, error-prone polymerase chain reaction (PCR) ࡟ࡼࡾࣛࣥࢲ࣒ኚ␗ࢆᑟධࡋࡓࣛ࢖ࣈ࣮ࣛࣜࢆస〇ࡋࡓ (➨4⠇). ࡑࡋ࡚, ࢔ࣞ࢖ᆺࣉࣟࣇ࢓࢖ࣜࣥࢢࢩࢫࢸ࣒࡟ᑐࡍࡿᑐ↷ᐇ㦂࡜ࡋ࡚, ᮏࣛ࢖ࣈ࣮ࣛࣜ ࠿ࡽᶆ‽ⓗ࡞ࣃࣥࢽࣥࢢ࡟ࡼࡾ㧗ぶ࿴ຊኚ␗యࢆ༢㞳ࡍࡿࡇ࡜ࢆヨࡳࡓ (➨4⠇).

9 ➨⠇ ᢠࢥࣝࢳࢰ࣮ࣝࣔࣀࢡ࣮ࣟࢼࣝᢠయࡢㄪ〇࡜ㅖᛶ㉁ ࣁࣉࢸࣥ࡜ࡣࡑࢀ⮬య࡛ච␿ཎᛶࢆᣢࡓ࡞࠸పศᏊ໬ྜ≀ࡢ⥲⛠࡛࠶ࡾ, ࡑࡢࡲࡲ ື≀࡟ᢞ୚ࡋ࡚ࡶᢠయࡣ⏘⏕ࡉࢀ࡞࠸. ࡋ࠿ࡋ, 㐺ษ࡞㧗ศᏊ࣮࢟ࣕࣜࣖ࡜ඹ᭷⤖ྜࡋ ࡓࡢࡕ࡟ᢞ୚ࡍࡿ࡜, ࣮࢟ࣕࣜࣖୖࡢࣁࣉࢸࣥᇶࡀ࢚ࣆࢺ࣮ࣉ࡜ࡋ࡚ാࡁ, ᢠయࡀᚓࡽ ࢀࡿࡼ࠺࡟࡞ࡿ. ࡑࡢ㝿, ࣁࣉࢸࣥศᏊࡢ࡝ࡢ఩⨨ࢆ࣮࢟ࣕࣜࣖ࡜⤖ྜࡉࡏࡿ࠿ࡀ, ⏘ ⏕ࡉࢀࡿᢠయࡢ≉␗ᛶࢆỴᐃࡍࡿ㔜せ࡞せ⣲࡛࠶ࡿ. 41) ࡍ࡞ࢃࡕ, ୍⯡࡟, ࣮࢟ࣕࣜࣖ ⤖ྜ㒊఩࠿ࡽ㐲ࡃ, ࣮࢟ࣕࣜࣖ࡟ࡼࡿ❧య㞀ᐖࢆཷࡅ࡟ࡃ࠸㒊ศᵓ㐀ࢆࡼࡃㄆ㆑ࡍࡿ ᢠయࡀᚓࡽࢀ࡚ࡃࡿ. ᅗ7a࡟CS ࡢᵓ㐀ࢆ♧ࡍ. 11఩ࡢࣄࢻࣟ࢟ࢩᇶ࡜17 ఩ࡢഃ㙐ࡀ CS ࡢᵓ㐀ࢆ≉ᚩ࡙ࡅࡿᐁ⬟ᇶ࡛࠶ࡾ, ࡇࢀࡽࡢㄆ㆑࡟ඃࢀࡿࡇ࡜ࡀᐇ⏝ⓗ࡞ᢠయࡢ᮲ ௳࡛࠶ࡿ. ࡇࡢⅬࢆ⪃៖ࡋ࡚, CSࡢ3఩࡟࣮࢟ࣕࣜࣖ⤖ྜ⏝ࣈࣜࢵࢪࢆᣢࡘㄏᑟయ, CS 3-(O-carboxymethyl)oxime (CS-3CMO) (ᅗ 7b) ࢆ ⏝ ࠸ ࡚, ࢘ ࢩ ⾑ Ύ ࢔ ࣝ ࣈ ࣑ ࣥ (bovine serum albumin; BSA) ࡜⤖ྜࡉࡏࡓࣁࣉࢸࣥ-࣮࢟ࣕࣜࣖ⤖ྜయ (CS-BSA) (ᅗ7c) ࢆㄪ

〇ࡋ, ච␿ཎ࡟⏝࠸ࡓ.

35,42)

࡞࠾, ྠ᫬࡟⏘⏕ࡉࢀࡿ࡜᝿ᐃࡉࢀࡿ BSA ࡟≉␗ⓗ࡞ᢠయ ࡢ ᙳ 㡪 ࢆ 㑊 ࡅ ࡿ ࡓࡵ, BSA ࢆ ༸ ⓑ ࢔ ࣝ ࣈ࣑ ࣥ (ovalbumin; OVA) ࡟ ⨨ ࡁ᥮ ࠼ ࡓ ⤖ ྜయ (CS-OVA) ࡶేࡏ࡚స〇ࡋ, ⾑Ύ୰ࡢᢠయ౯ࡢẚ㍑ࡸ, ࣁ࢖ࣈࣜࢻ࣮࣐ࡢࢫࢡ࣮ࣜࢽࣥ

ࢢ࡟⏝࠸ࡓ.

CS-BSAࢆච␿ཎ࡜ࡋ࡚, BALB/c࣐࢘ࢫࢆ⧞ࡾ㏉ࡋච␿ࡋࡓࡢࡕ, ⾑୰ࡢᢠCSᢠయ

౯ࡢୖ᪼ࡋࡓ࣐࢘ࢫࢆ enzyme-linked immunosorbent assay (ELISA) ࡟ࡼࡾ≉ᐃࡋ, ࡑࢀ ࡽ࣐࢘ࢫࡢ⭁⣽⬊ࢆ P3/NS1/1-Ag4-1 (NS1) ࣑࢚࣮࣐ࣟ⣽⬊࡜⼥ྜࡉࡏࡓ. ᚓࡽࢀࡿᢠ

ᅗ 7. ࢥࣝࢳࢰ࣮ࣝ࠾ࡼࡧࡑࡢㄏᑟయࡢᵓ㐀

10

CS ᢠయ⏘⏕ࣁ࢖ࣈࣜࢻ࣮࣐ࢆ㝈⏺ᕼ㔘ἲ࡟ࡼࡾࢡ࣮ࣟࢽࣥࢢࡋ, ᢠ CS ᢠయ⏘⏕⣽⬊

ᰴ#3࠾ࡼࡧ#10ࢆᚓࡓ. ࡇࢀࡽ⣽⬊ᰴࡀศἪࡍࡿᢠయAb-CS#3࠾ࡼࡧAb-CS#10ࡢH㙐 ࠾ࡼࡧL㙐ࡢ࢔࢖ࢯࢱ࢖ࣉࢆㄪ࡭ࡓ࡜ࡇࢁ, H㙐ࡣAb-CS#3࡛ࡣg1, Ab-CS#10࡛ࡣg2b ࡛࠶ࡾ, L㙐ࡣ࡜ࡶ࡟ȡ࡛࠶ࡗࡓ.

ḟ࠸࡛, Ab-CS#3࠾ࡼࡧAb-CS#10ࡢK_aࢆ, [3H]-CSࢆࢺ࣮ࣞࢧ࣮࡟⏝࠸ࡿScatchardἲ

43) ࡟ࡼࡾ⟬ฟࡋࡓ࡜ࡇࢁ, ࡑࢀࡒࢀ4.7™10 7_{, 4.0} ™10 10 _M-1 ࡛࠶ࡗࡓ. Ab-CS#10ࡣCS࡟ᑐ ࡋ࡚ࡍ࡛࡟༑ศ࡟኱ࡁ࠸ぶ࿴ຊࢆࡶࡘࡇ࡜࠿ࡽ, ヨ㦂⟶ෆぶ࿴ᛶᡂ⇍ࡢฟⓎ≀㉁࡜ࡋ ࡚ࡣAb-CS#3ࡀࡴࡋࢁዲ㐺࡜ุ᩿ࡋࡓ. ࡑࡇ࡛, Ab-CS#3ࡢ➇ྜELISA࡟࠾ࡅࡿឤᗘࢆ ㄪ࡭ࡓ. ࡍ࡞ࢃࡕ, CS-BSAࢆᅛᐃ໬ࡋࡓ࣐࢖ࢡࣟࣉ࣮ࣞࢺ࡟Ab-CS#3࠾ࡼࡧྛ✀⃰ᗘ ࡢ CS ᶆ ‽ ရ ࢆ ຍ ࠼ ࡚ ➇ ྜ ཯ ᛂ ࢆ ⾜ ࠸, ᅛ ┦ ࡟ ṧ ࡿ Ab-CS#3 ࢆ ࣌ ࣝ ࢜ ࢟ ࢩ ࢲ ࣮ ࢮ (peroxidase; POD) ᶆ㆑ᢠ࣐࢘ࢫᢠయ࡟ࡼࡾᤕᤊࡋࡓ. ᅛ┦ୖࡢPODάᛶࡣ, o-ࣇ࢙ࢽࣞ

ࣥ ࢪ ࢔ ࣑ ࣥ/㐣 㓟 ໬ Ỉ ⣲ ࢆ ⏝ ࠸, ẚ Ⰽ ἲ ࡛   ᐃ ࡋ ࡓ. ⏝ 㔞 స ⏝ ᭤ ⥺ ࢆ స ᡂ ࡋ ࡓ ࡜ ࡇ ࢁ, midpoint (50%㜼ᐖ ࡟せࡍ ࡿ CS 㔞) ࡣ 28 ng (ᅗ 8) ࡛ ࠶ࡗࡓ. CS ࡢ⾑ ୰⃰ᗘᇶ ‽್ (2.7~15.5 mg/dL)44) ࢆ⪃៖ࡍࡿ࡜, ⮫ᗋヨ㦂࡬㐺⏝ࡍࡿࡓࡵ࡟ࡣ, ࠾ࡼࡑ1~10 ng/assayࡢ  ᐃ⠊ᅖࡀồࡵࡽࢀࡿࡀ, ࡇࡢ⤖ᯝࡣ᫂ࡽ࠿࡟୙༑ศ࡛࠶ࡿ. ᅗ 8. Ab-CS#3ࢆ⏝࠸ࡿCSࡢ➇ྜELISA CS-BSAࢆᅛᐃ໬ࡋࡓ࣐࢖ࢡࣟࣉ࣮ࣞࢺ࡟Ab-CS#3࠾ࡼࡧCSᶆ‽ရࢆຍ࠼࡚➇ྜ཯ᛂࢆ⾜࠸, ᚓࡽ ࢀࡓ⏝㔞స⏝᭤⥺ࢆ♧ࡍ. ྛⅬࡢ࢚࣮ࣛࣂ࣮ࡣ4㔜 ᐃࡢᶆ‽೫ᕪࢆ♧ࡍ.

11 ➨⠇ ᢠࢥࣝࢳࢰ࣮ࣝᢠయ୍ᮏ㙐 )Y ࣇࣛࢢ࣓ࣥࢺࡢㄪ〇࡜ㅖᛶ㉁ ⥴ㄽ࡛㏙࡭ࡓࡼ࠺࡟, ᢠయࡢ㑇ఏᏊ᧯స࡛ࡣ, ᨵኚࢆຍ࠼ࡿᑐ㇟ࡢᢠయࢆ scFv ࡜ࡋ ࡚ྲྀࡾᢅ࠺ࡢࡀዲ㒔ྜ࡛࠶ࡿ. ࡑࡇ࡛, ๓⠇࡛స〇ࡋࡓ Ab-CS#3 ࢆ scFv ࡟ኚ᥮ࡋ, ࡑ ࡢㅖᛶ㉁࡟᳨ウࢆຍ࠼ࡓ. ࡲࡓ, ࢔ࣞ࢖ᆺࣉࣟࣇ࢓࢖ࣜࣥࢢࢩࢫࢸ࣒ࢆᵓ⠏ࡍࡿ࡟࠶ࡓ ࡾ, ྛࢫࢸࢵࣉࡢ᮲௳ࢆタᐃࡍࡿ࠺࠼࡛㧗ぶ࿴ຊᢠయࡶᚲせ࡜࡞ࡿࡇ࡜ࡀ᝿ᐃࡉࢀࡿ. ࡑࢀ࡟ഛ࠼࡚Ab-CS#10ࡢscFv໬ࡶేࡏ࡚⾜ࡗࡓ. scFv ໬࡟ࡣ, ᢠయྍኚ㒊ࢻ࣓࢖ࣥ (V ࢻ࣓࢖ࣥ) ࡢ㑇ఏᏊ (V 㑇ఏᏊ) ࡢࢡ࣮ࣟࢽࣥ ࢢࡀᚲ㡲࡛࠶ࡿ. V 㑇ఏᏊࡢࢡ࣮ࣟࢽࣥࢢἲ࡜ࡋ࡚ࡣ, ᢠయ⏘⏕ࣁ࢖ࣈࣜࢻ࣮࣐࠿ࡽ⥲ RNAࢆᢳฟࡋ, ㏫㌿෗㓝⣲ࢆ⏝࠸࡚complementary deoxyribonucleic acid (cDNA) ࢆྜᡂ

ࡋࡓࡢࡕ, ྵࡲࢀࡿV㑇ఏᏊࢆPCR࡟ࡼࡾቑᖜ [ࡍ࡞ࢃࡕ, reverse transcriptase-PCR (RT-PCR)] ࡍࡿࡢࡀ୍⯡ⓗ࡛࠶ࡿ. V 㑇ఏᏊࡢ 3’ᮎ➃࡟ࡣ, ᢠయࡢ࢔࢖ࢯࢱ࢖ࣉࡈ࡜࡟࡯࡜ ࢇ࡝୍ᐃࡢ㓄ิࢆࡶࡘᐃᖖ㒊㑇ఏᏊ (C 㑇ఏᏊ) ࡀ㞄᥋ࡍࡿ. ࡋࡓࡀࡗ࡚, ┠ⓗࡢᢠయ ࡢ࢔࢖ࢯࢱ࢖ࣉࡀྠᐃࡉࢀ࡚࠸ࡿ࡞ࡽࡤ, 3’ࣉࣛ࢖࣐࣮ࡢ㑅ᐃࡣᐜ࡛᫆࠶ࡿ. ࡇࢀ࡟ᑐ ࡋ࡚, 5’ࣉࣛ࢖࣐࣮ࡢ㑅ᢥࡣ㞴ࡋ࠸. ࡑࡢ⤖ྜ㒊఩࡜ࡋ࡚, ྍኚ㒊 N ᮎ➃࡟఩⨨ࡍࡿᯟ ⤌ࡳ㓄ิ (framework region; FR) 1 (ᅗ 2) ࠶ࡿ࠸ࡣࡉࡽ࡟ୖὶࡢ࣮ࣜࢲ࣮㓄ิࡢሷᇶ㓄 ิࢆ฼⏝ࡍࡿࡇ࡜࡟࡞ࡿࡀ, ࠸ࡎࢀࡶ኱ࡁ࡞ከᵝᛶࢆ♧ࡍࡓࡵ࡛࠶ࡿ. ࡇࡢࡼ࠺࡞ሙྜ, 5’RACE (rapid amplification of cDNA ends) ἲ

45)

ࡀ᭷ຠ࡛࠶ࡿ. ᮏἲ࡛ࡣ, cDNA ࡢ3’ᮎ ➃࡟ terminal deoxynucleotidyl transferase (TdT) ࡜deoxycytidine 5’-triphosphate (dCTP) ࢆ ാ࠿ࡏ࡚࣏ࣜࢩࢳࢪࣝ㓟 (࣏ࣜ C) 㓄ิࢆ௜ຍࡋ, ࡇࢀࢆ 5’ࣉࣛ࢖࣐࣮ࡢ⤖ྜ㒊఩࡜ࡋ ࡚PCR ࢆ⾜࠺ (ᅗ 9). ࡋࡓࡀࡗ࡚, 3’ᮎ➃ഃࡢ㓄ิࡉ࠼᪤▱࡛࠶ࢀࡤ, ࡑࡢୖὶࡢ㑇ఏ Ꮚࡢሷᇶ㓄ิࢆቑᖜࡍࡿࡇ࡜ࡀ࡛ࡁࡿ. ᅗ 9. 5’RACE ἲ ࡟ ᇶ ࡙ ࡃ ᢠ య ྍ ኚ 㒊 㑇 ఏ Ꮚ ࢡ ࣟ ࣮ ࢽࣥࢢࡢᴫせ ᢠయ⏘⏕ࣁ࢖ࣈࣜࢻ࣮࣐⣽⬊ ࠿ࡽᢳฟࡋࡓmRNAࢆ㗪ᆺ࡜ ࡋ࡚, C㡿ᇦ࡟┦⿵ⓗ࡞GSP-1 ࣉࣛ࢖࣐࣮࠾ࡼࡧ㏫㌿෗㓝⣲ࢆ ⏝࠸࡚V㑇ఏᏊࢆྵࡴcDNAࢆ ྜᡂࡋࡓࡢࡕ, TdT࠾ࡼࡧdCTP ࢆస⏝ࡉࡏ࡚ࡑࡢ3’ᮎ➃࡟࣏ࣜ C㓄ิࢆ௜ຍࡍࡿ. ⥆࠸࡚, GSP-1ࡼࡾࡶୖὶࡢC㡿ᇦ࡟┦⿵ⓗ ࡞GSP-2ࣉࣛ࢖࣐࣮࡜AAPࢆ ⏝࠸࡚PCRࢆ⾜࠺. AAP୰ࡢI ࡣ࢖ࣀࢩࣥࢆ♧ࡍ. ࡑࡢቑᖜ⏘ ≀ࢆ㗪ᆺ࡟, ไ㝈㓝⣲㓄ิࢆྵ ࡴࣉࣛ࢖࣐࣮ (AUAP࠾ࡼࡧ GSP-3) ࢆ⏝࠸࡚nested PCRࢆ ⾜࠺. ᚓࡽࢀࡓ⏘≀ࢆไ㝈㓝⣲ ࡛ᾘ໬ᚋ, ࣋ࢡࢱ࣮࡬ᤄධࡍࡿ.

12

๓⠇࡟グࡋࡓࡼ࠺࡟, Ab-CS#3࠾ࡼࡧAb-CS #10ࡢH㙐࠾ࡼࡧL㙐ࡢ࢔࢖ࢯࢱ࢖ࣉࡣ ྠᐃࡉࢀ࡚࠸ࡿ. ࡑࡇ࡛, ࡲࡎ, 5’RACEἲ࡟ࡼࡿV㑇ఏᏊࡢࢡ࣮ࣟࢽࣥࢢࢆヨࡳࡓ (ᅗ 9). ๓⠇࡛ᶞ❧ࡋࡓࣁ࢖ࣈࣜࢻ࣮࣐ᰴࡼࡾ⥲ RNA ࢆᢳฟࡋ, ࡑࢀࡒࢀࡢ C 㑇ఏᏊ࡟≉

␗ⓗ࡞ࣉࣛ࢖࣐࣮ [group specific primer (GSP)-1] ࡜㏫㌿෗㓝⣲ࢆస⏝ࡉࡏࡿࡇ࡜࡟ࡼ ࡾ, V 㑇ఏᏊࢆྵࡴcDNAࢆྜᡂࡋࡓ. ḟ࠸࡛, ࡇࢀࡽࡢcDNAࡢ3’ᮎ➃࡟ୖ㏙ࡢࡼ࠺ ࡟ ࣏ ࣜ C 㓄 ิ ࢆ ௜ ຍ ࡋ ࡓ ࡢ ࡕ, ࣏ ࣜ C 㓄 ิ ࡟ ┦ ⿵ ⓗ ࡞ ࣉ ࣛ ࢖ ࣐ ࣮ (abridged anchor primer; AAP) ࡜, GSP-2 (C㑇ఏᏊୖ, GSP-1ࡼࡾࡶ5’ഃࡢ㓄ิ࡟┦⿵ᛶࢆ♧ࡍ) ࢆ⏝࠸

࡚ PCR ࢆ ⾜ࡗ ࡓ. ࡉ ࡽ࡟ ቑᖜ ࡢ≉␗ ᛶࢆ 㧗ࡵࡿ ࡓࡵ ࡟, ᚓ ࡽࢀࡓ ⏘≀ࢆ 㗪ᆺ ࡜ࡋ࡚, AAP ࡢ 5’ഃ ࡜ ྠ ࡌ 㓄 ิ ࢆ ᭷ ࡍ ࡿ ࣉ ࣛ ࢖ ࣐ ࣮ (abridged universal amplification primer; AUAP) ࡜GSP-3 (C㑇ఏᏊୖ, GSP-2ࡼࡾࡶ5’ഃࡢ㓄ิ࡟┦⿵ᛶࢆ♧ࡍ) ࣉࣛ࢖࣐࣮ࢆ

⏝࠸ࡿnested PCRࢆ⾜ࡗࡓ࡜ࡇࢁ, Ab-CS#3ࡢV_L㑇ఏᏊ, Ab-CS#10ࡢV_H࠾ࡼࡧV_L㑇ఏ ᏊࢆྵࡴDNA᩿∦ࡀᚓࡽࢀࡓ.

࡜ࡇࢁࡀ, Ab-CS#3 ࡢ V_H㑇ఏᏊࢆྵࡴ DNA᩿∦ࡣᚓࡽࢀ࡞࠿ࡗࡓࡓࡵ, Jones ࡽ

46) ࡀ㛤Ⓨࡋࡓࣘࢽࣂ࣮ࢧࣝࣉࣛ࢖࣐࣮ࢭࢵࢺࢆ⏝࠸ࡿࢡ࣮ࣟࢽࣥࢢࢆヨࡳࡓ (ᅗ 10). ࡇ ࡢࣉࣛ࢖࣐࣮ࢭࢵࢺࡣ, 12✀㢮ࡢ⦰㔜ࣉࣛ࢖࣐࣮ (MHV-1~12) ࠿ࡽᵓᡂࡉࢀ, ከࡃࡢሙ ྜ, ࡇࢀࡽࡢ࠸ࡎࢀ࠿ࡀᶆⓗࡢVH㑇ఏᏊࡢ࣮ࣜࢲ࣮㓄ิ࡟┦⿵ᛶࢆ♧ࡍ. ୖグࡢcDNA ࢆ㗪ᆺ࡜ࡋ, ࡇࢀࡽࣉࣛ࢖࣐࣮ࡢ࠸ࡎࢀ࠿࡜ H㙐ᐃᖖ㒊࡟┦⿵ⓗ࡞ࣉࣛ࢖࣐࣮ (MHC) ࢆ⏝࠸ࡿ12✀ࡢRT-PCRࢆヨࡳࡓ࡜ࡇࢁ, MHV-2ࣉࣛ࢖࣐࣮ࢆ⏝࠸ࡓ࡜ࡁ࡟┠ⓗࡢ㑇 ఏᏊ᩿∦ࢆᚓࡿࡇ࡜ࡀ࡛ࡁࡓ. ᅗ 10. ࣘࢽࣂ࣮ࢧࣝࣉࣛ࢖࣐࣮ࢭࢵࢺࢆ⏝࠸ࡿV_H㑇ఏᏊࡢࢡ࣮ࣟࢽࣥࢢ Jonesࡽ࡟ࡼࡗ࡚ሗ࿌ࡉࢀࡓࣘࢽࣂ࣮ࢧࣝࣉࣛ࢖࣐࣮ࢭࢵࢺࢆ♧ࡍ.46) ࡇࢀࡽࡢࣉࣛ࢖࣐࣮ࢆ⏝࠸࡚PCR ࢆ⾜࠺ࡇ࡜࡟ࡼࡾ, ┠ⓗࡢVH㑇ఏᏊࢆ≉␗ⓗ࡟ቑᖜࡋ, ࢡ࣮ࣟࢽࣥࢢࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡿ. ࣉࣛ࢖࣐࣮ ୰ࡢY, R, W, K, M, SࡣࡑࢀࡒࢀCࡲࡓࡣT, AࡲࡓࡣG, AࡲࡓࡣT, GࡲࡓࡣT, AࡲࡓࡣC, CࡲࡓࡣGࢆ ♧ࡍ. CHࡣH㙐ᐃᖖ㒊ࢆ♧ࡍ.

13 ᘬࡁ⥆ࡁ, ᚓࡽࢀࡓV_H, V_Lྛ㑇ఏᏊ᩿∦ࢆpBluescript II࣋ࢡࢱ࣮࡬ࢧࣈࢡ࣮ࣟࢽࣥࢢ ࡋ, ኱⭠⳦ XL1-Blue ⣽⬊࡬ᑟධࡋࡓ. ᙧ㉁㌿᥮⳦ࡢࢡ࣮ࣟࣥࢆࢥࣟࢽ࣮PCR ࡟௜ࡋ࡚ V㑇ఏᏊࢆಖᣢࡍࡿࢡ࣮ࣟࣥࢆ≉ᐃࡋ, ⤌᥮࠼ࣉࣛࢫ࣑ࢻࢆᢳฟࡋ࡚, DNAሷᇶ㓄ิࢆ ゎᯒࡋࡓ. ᑟ࠿ࢀࡿ࢔࣑ࣀ㓟㓄ิࢆ Kabat ࡢᢠయࢩ࣮ࢡ࢚ࣥࢫࢹ࣮ࢱ࣮࣋ࢫ 47) ࡜↷ྜ ࡋࡓ࡜ࡇࢁ, Ab-CS#3࠾ࡼࡧAb-CS#10ࡢV_Hࢻ࣓࢖ࣥ (ࡑࢀࡒࢀ115࢔࣑ࣀ㓟, 118࢔࣑ ࣀ㓟࠿ࡽ࡞ࡿ), V_Lࢻ࣓࢖ࣥ (ࡑࢀࡒࢀ 113 ࢔࣑ࣀ㓟, 114 ࢔࣑ࣀ㓟࠿ࡽ࡞ࡿ) ࢆྠᐃࡋ ࡓ. V_H, V_L ࡟ 3 ࢝ ᡤ ࡎ ࡘ Ꮡ ᅾ ࡋ, ᢠ ཎ ࡜ ┤ ᥋ ┦ ஫ స ⏝ ࡍ ࡿ ࡜ ࡉ ࢀ ࡿ ┦ ⿵ ᛶ Ỵ ᐃ 㒊 (complementarity-determining region; CDR) ࡢ 㓄 ิ ࡶ Ỵ ᐃ ࡍ ࡿ ࡇ ࡜ ࡀ ࡛ ࡁ ࡓ. ࡜ ࡇ ࢁ ࡛, ᢠయࡢV ࢻ࣓࢖ࣥ඲యࡢ࢔࣑ࣀ㓟㓄ิࡣ኱ࡁ࡞ከᵝᛶࢆ♧ࡍࡀ, FR ࡢከᵝᛶࡣẚ㍑ⓗ ᑠࡉࡃ, Kabatࡽࡣࡇࢀࡽࢆᇶ࡟V_Hࢆ11✀, V_Lࢆ7✀ࡢࢧࣈࢢ࣮ࣝࣉ࡟ศ㢮ࡋ࡚࠸ࡿ. 47)

Ab-CS#3࡟ࡘ࠸࡚ࡣ V_Hࢆࢧࣈࢢ࣮ࣝࣉIIB, V_Lࢆࢧࣈࢢ࣮ࣝࣉIII࡟, Ab-CS#10࡟ࡘ࠸ ࡚ࡣ V_HࢆIB, V_LࢆII࡟ᖐᒓࡍࡿࡇ࡜ࡀ࡛ࡁࡓ. ḟ࡟, ᚓࡽࢀࡓሷᇶ㓄ิࢆࡶ࡜࡟, V_H, V_Lྛ㑇ఏᏊࡢ 5’ᮎ➃࡜ 3’ᮎ➃࡟┦⿵ⓗ࡞ࣉࣛ ࢖࣐࣮ࢆࡑࢀࡒࢀタィࡋ࡚PCRࢆ⾜࠸, scFv㑇ఏᏊࢆᵓ⠏ࡍࡿࡓࡵࡢDNAࣇࣛࢢ࣓ࣥ ࢺࢆㄪ〇ࡋࡓ. Ab-CS#3࡟ࡘ࠸࡚ࡣ, ቑᖜࡋࡓV_H, V_Lྛ㑇ఏᏊࢆscFvࣇ࢓࣮ࢪᥦ♧⏝࣋ ࢡࢱ࣮ (pEXmide 7) ࡢV_Hࢡ࣮ࣟࢽࣥࢢࢧ࢖ࢺ, V_Lࢡ࣮ࣟࢽࣥࢢࢧ࢖ࢺ࡟㡰ḟ⤌ࡳ㎸ࡳ, ᑐᛂࡍࡿscFv (CS#3-scFv) ࡢ㑇ఏᏊࢆࣉࣛࢫ࣑ࢻෆ࡟ᵓ⠏ࡋࡓ (ᅗ11). pEXmide 7࡟ࡣ, VH࠾ࡼࡧ V_Lࡢྛࢡ࣮ࣟࢽࣥࢢࢧ࢖ࢺࡢ㛫࡟, ࣮ࣜࣥ࢝࣌ࣉࢳࢻ [VSS(GGGGS)₃T] (࢔ ࣑ࣀ㓟㓄ิࢆ୍ᩥᏐ⾲グ࡛♧ࡍ) ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡀ࠶ࡽ࠿ࡌࡵᑟධࡉࢀ࡚࠸ࡿ. ࡞ ࠾, ࡇࡢ㓄ิࡢ࠺ࡕ, (GGGGS)₃ࡣscFvㄪ〇࡟࠾ࡅࡿᶆ‽ⓗ࡞࣮ࣜࣥ࢝㓄ิ࡛, 21,48) ୧➃ ࡢVSS࡜TࡣV_H, V_Lࢆᑟධࡍࡿࡓࡵࡢไ㝈㓄ิ࡟⏤᮶ࡍࡿࡶࡢ࡛࠶ࡿ. ୍᪉, Ab-CS#10 ࢆscFv໬ࡍࡿࡓࡵ࡟, V_Hቑᖜࡢ3’ࣉࣛ࢖࣐࣮ (CS#10V_H-For) ࡜V_Lቑᖜࡢ5’ࣉࣛ࢖࣐࣮ ᅗ 11. CS#3-scFv㑇ఏᏊࡢᵓ⠏࠾ࡼࡧpEXmide 7࡜pEXmide 7’ࡢࢡ࣮ࣟࢽࣥࢢࢧ࢖ࢺ

PCR࡟ࡼࡾቑᖜࡋࡓྍኚ㒊㑇ఏᏊࡢ࠺ࡕ, V_HࡣNco I࠾ࡼࡧXho I࡛, V_LࡣSal I࠾ࡼࡧNot I࡛ࡑࢀࡒࢀ ᾘ໬ࡋ, 㡰ḟpEXmide 7࣋ࢡࢱ࣮࡟⤌ࡳ㎸ࡴࡇ࡜࡛CS#3-scFv㑇ఏᏊࢆᵓ⠏ࡋࡓ. ࣋ࢡࢱ࣮ෆ࡛᏶ᡂࡋࡓ scFv㑇ఏᏊࡣࣇ࢓࣮ࢪgIII࡜┤⤖ࡍࡿࡓࡵ, scFv-pIII⼥ྜࢱࣥࣃࢡ࡜ࡋ࡚Ⓨ⌧ࡉࢀ࡚ࣇ࢓࣮ࢪ⢏Ꮚ࡟⤌ࡳ ㎸ࡲࢀࡿ. ࡇࢀ࡟ᑐࡋ࡚ pEXmide 7’ࡣ, pEXmide 7ࡢV_Lࢡ࣮ࣟࢽࣥࢢࢧ࢖ࢺࡢୗὶ࡟FLAG࠾ࡼࡧ2㔜ࡢ ⤊Ṇࢥࢻࣥ (TAATGA) ࢆᤄධࡋࡓࡶࡢ࡛࠶ࡾ, scFv㑇ఏᏊࡣྍ⁐ᆺscFvࢱࣥࣃࢡ㉁࡜ࡋ࡚Ⓨ⌧ࡉࢀࡿ.

14 (CS#10VL-Rev) ࡟, ࡇ ࢀࡽ ࡢ 㐃 ⤖ 㒊 ࡜ ࡞ ࡿࣜ ࣥ࢝ ࣮ ࣌ ࣉ ࢳ ࢻ [(GGGGS)₃] ࢆࢥ ࣮ ࢻ ࡍ ࡿ㓄ิࡢ୍㒊ࢆᑟධࡋࡓ. ࡇࡢ㓄ิࡢ┦⿵ᛶࢆ฼⏝ࡍࡿ overlap extension PCR ࡟ࡼࡾ, VH࡜V_Lࢆ㐃⤖ࡋ࡚CS#10-scFv㑇ఏᏊࢆᵓ⠏ࡋ (ᅗ12), scFvࣇ࢓࣮ࢪᥦ♧⏝pEXmide 5 ࣋ࢡࢱ࣮ 49) ࡟ࢧࣈࢡ࣮ࣟࢽࣥࢢࡋࡓ. ࡇࢀࡽࡢ⤌᥮࠼ࣉࣛࢫ࣑ࢻࢆ኱⭠⳦XL1-Blue ⣽⬊࡟ᑟධࡋ, ࢥࣟࢽ࣮PCR࡟ࡼࡾ┠ ⓗࡢscFv㑇ఏᏊ (CS#3-scFv࠾ࡼࡧCS#10-scFv) ࢆಖᣢࡍࡿࢡ࣮ࣟࣥࢆྠᐃࡋࡓ. ࡑࡢ ሷᇶ㓄ิࢆゎᯒࡋࡓ࡜ࡇࢁ, ᭱ึ࡟ྠᐃࡋࡓVH࡜VLࡢሷᇶ㓄ิࡀኚ␗ࢆక࠺ࡇ࡜࡞ࡃ ಖᣢࡉࢀ࡚࠸ࡿࡇ࡜ࡀศ࠿ࡗࡓ. ࡲࡓ, CS#3-scFv 㑇ఏᏊࢆ pEXmide 7’࣋ࢡࢱ࣮࡟⤌ࡳ ㎸ࡳ, ኱⭠⳦XL1-Blue⣽⬊࡟ᑟධࡋࡓ. pEXmide 7’࣋ࢡࢱ࣮࡜ࡣ, pEXmide 7࣋ࢡࢱ࣮ ࢆྍ⁐ᆺ scFv (ࣇ࢓࣮ࢪ࡟㐃⤖ࡉࢀ࡚࠸࡞࠸ scFv ࢆࡇࡢࡼ࠺࡟࿧ࡪ) ࡢⓎ⌧⏝࡟ᨵኚ ࡋ ࡓ ࡶ ࡢ ࡛ ࠶ ࡿ (ᅗ 11). ࡇ ࡢ ࣋ ࢡ ࢱ ࣮ ࡢ VL㑇 ఏ Ꮚ ࡢ 3’ᮎ ➃ 㒊 ศ ࡟ ࡣ FLAG ࢱ ࢢ (DYKDDDDK ࡢ ࢔ ࣑ ࣀ 㓟 㓄 ิ) 50) ࢆ ࢥ ࣮ ࢻ ࡍ ࡿ ሷ ᇶ 㓄 ิ ࠾ ࡼ ࡧ 2 㔜 ࡢ ⤊ Ṇ ࢥ ࢻ ࣥ (TAATGA) ࡀ㓄⨨ࡉࢀ࡚࠾ࡾ, ᤄධࡉࢀࡓscFv 㑇ఏᏊࡣ, lac ࣉ࣮ࣟࣔࢱ࣮ࡢᨭ㓄ୗ࡛ ྍ⁐ᆺࢱࣥࣃࢡ㉁࡜ࡋ࡚Ⓨ⌧ࡉࢀ, pelB࣮ࣜࢲ࣮࣌ࣉࢳࢻࡢാࡁ࡟ࡼࡾ࣌ࣜࣉࣛࢬ࣒㛫 㝽࡟ᨺฟࡉࢀࡿ. 51) ௚᪉, CS#10-scFv࡟ࡘ࠸࡚ࡣ, CS#10VH-Rev࠾ࡼࡧFLAG-TAA2ࣉࣛ ࢖࣐࣮ࢆ⏝࠸ࡓPCR ࡟ࡼࡾྍ⁐ᆺscFvⓎ⌧⏝ࡢ㑇ఏᏊࢆቑᖜࡋ, pEXmide 5࣋ࢡࢱ࣮ ࡟⤌ࡳ㎸ࢇࡔࡢࡕ, ኱⭠⳦ XL1-Blue ⣽⬊࡟ᑟධࡋࡓ. ᚓࡽࢀࡓࡇࢀࡽࡢᙧ㉁㌿᥮⳦ࢆ, ࢖ࢯࣉࣟࣆࣝ b-D-ࢳ࢜࢞ࣛࢡࢺࣆࣛࣀࢩࢻ (isopropyl b-D-1-thiogalactopyranoside; IPTG) ࡜ࢫࢡ࣮ࣟࢫࡢᏑᅾୗ࡛ᇵ㣴ࡋ, ᾐ㏱ᅽࢩࣙࢵࢡἲ࡟ࡼࡾ, ྍ⁐ᆺ CS#3-scFv ࠾ࡼࡧ CS#10-scFvࡀྵࡲࢀࡿ࣌ࣜࣉࣛࢬ࣒ᢳฟ≀ࢆᚓࡓ. ࡇࢀࡽ scFv ࡢ K_aࢆ Scatchard ἲ࡟ࡼࡾ⟬ฟࡋࡓ࡜ࡇࢁ, CS#3-scFv ࡣ 3.4™108 M-1, CS#10-scFvࡣ1.7™1010 M-1࡛࠶ࡗࡓ. ࡲࡓ, ぶ࿴ᛶᡂ⇍ࡢฟⓎ≀㉁࡜࡞ࡿCS#3-scFvࢆ ᅗ 12. Overlap extension PCR࡟ࡼࡿCS#10-scFv㑇ఏᏊࡢᵓ⠏ Ab-CS#10ࡢV_H, V_L㑇ఏᏊࢆPCR࡟ࡼࡾቑᖜࡍࡿ. ࡇࡢ࡜ࡁ, V_Hቑᖜࡢ3’ࣉࣛ࢖࣐࣮࡜V_Lቑᖜࡢ5’ࣉ ࣛ࢖࣐࣮࡟࣮ࣜࣥ࢝࣌ࣉࢳࢻ [(GGGGS)₃] ࡢ୍㒊ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࢆᑟධࡋ࡚࠾ࡃ. ቑᖜࡉࢀࡓྛ㑇 ఏᏊ᩿∦ࡢ࠺ࡕ࣮ࣜࣥ࢝࣌ࣉࢳࢻ㓄ิࡢ୍㒊ࡀ┦⿵ⓗ࡛࠶ࡿࡓࡵ, ୧⪅ࢆΰྜࡋ࡚PCRࢆ⾜࠺ࡇ࡜࡟ࡼ ࡾscFv㑇ఏᏊࡀᵓ⠏ࡉࢀࡿ. ࡇࡢ㑇ఏᏊࢆไ㝈㓝⣲࡛ฎ⌮ࡋ࡚pEXmide 5࣋ࢡࢱ࣮࡟⤌ࡳ㎸ࢇࡔ.

15 ๓⠇࡜ྠᵝࡢ➇ྜELISA࡟௜ࡋࡓ࡜ࡇࢁ, CSࡢቑ㔞࡟ᛂࡌࡓࢩࢢࢼࣝࡢῶᑡࡀㄆࡵࡽ ࢀࡓ. ⏝㔞స⏝᭤⥺ࡢmidpointࡣ720 pg࡛࠶ࡾ, Ab-CS#3 (28 ng; ๓⠇ཧ↷) ࡟ẚ࡭࡚኱ ᖜ࡟㧗ឤᗘ໬ࡋ࡚࠸ࡓ. CS#3-scFvࡀ, ࠕぶᢠయࠖ࡜࡞ࡿAb-CS#3 (K_a; 4.7™10 7 _M-1₎ ࡼࡾ ࡶぶ࿴ຊࡀቑ኱ࡋࡓ⌮⏤ࡣᐃ࠿࡛ࡣ࡞࠸ࡶࡢࡢ, ➇ྜELISA࡟࠾ࡅࡿឤᗘࡢᨵၿࡣ, ぶ ࿴ຊࡢኚ໬ࡔࡅ࡛࡞ࡃ, ᢠయ1ศᏊෆࡢࣃࣛࢺ࣮ࣉᩘ (IgG࡛ࡣ2, scFv࡛ࡣ1) ࡟ࡼࡿ ᙳ㡪ࡶ཯ᫎࡋࡓࡶࡢ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿ.

16 ➨⠇ ኚ␗ᢠయࣇࣛࢢ࣓ࣥࢺࣛ࢖ࣈ࣮ࣛࣜࡢᵓ⠏࡜ࣃࣥࢽࣥࢢ࡟ࡼࡿ㧗ぶ࿴ຊኚ␗ యࡢ᥈⣴ ヨ㦂⟶ෆぶ࿴ᛶᡂ⇍࡛ࡣ, ࣉࣟࢺࢱ࢖ࣉ࡜࡞ࡿᢠయࣇࣛࢢ࣓ࣥࢺࡢ㑇ఏᏊ࡟ኚ␗ࢆ ᑟධࡋ࡚ᚓࡽࢀࡿኚ␗㑇ఏᏊ⩌ࢆ, 㐺ษ࡞ᐟ୺࡟Ⓨ⌧ࡉࡏ࡚ࣛ࢖ࣈ࣮ࣛࣜࢆᵓ⠏ࡋ, അ ↛࡟⏕ࡌࡿࡈࡃࢃࡎ࠿࡞ᨵⰋᆺศᏊ✀ࢆ㑅ᢥࡋ࡚༢㞳ࡍ ࡿ. ኚ␗ࡢᑟධ᪉ἲ࡜ࡋ࡚ࡣ, PCR ࡢᛅᐇᗘ (ࣇ࢕ࢹࣜࢸ࢕) ࢆᨾព࡟పୗࡉࡏ࡚⾜࠺ࡇ࡜࡛Ⅼኚ␗ࢆᑟධࡍࡿ᪉ἲ (error-prone PCR)52) ࡸ, CDR࡞࡝ᶵ⬟ᨵኚ࡟᭷ຠ࡜⪃࠼ࡽࢀࡿ㒊఩࡟⦰㔜㓄ิࢆࡶࡘ໬ Ꮫྜᡂ࢜ࣜࢦDNAࢆᑟධࡍࡿ᪉ἲ 53) ࡀ⏝࠸ࡽࢀࡿ. ๓⪅ࡣ᧯సࡶ⡆౽࡛࠶ࡾ, ᶆⓗ㑇 ఏᏊࡢᗈ࠸⠊ᅖ࡟ࢃࡓࡗ࡚㐺ᗘ࡞㢖ᗘࡢኚ␗ࢆຍ࠼ࡿࡢ࡟㐺ࡋ࡚࠸ࡿ. ࡑࡇ࡛ᮏ◊✲࡛ࡣ, error-prone PCR࡟ࡼࡾ, CS#3-scFv (ࡇࢀࢆ㔝⏕ᆺscFv࡜ࡋ, ௨ᚋ wt-scFv ࡜␎ࡍ) ࡢ඲㛗࡟ࣛࣥࢲ࣒ኚ␗ࢆᑟධࡋࡓࣛ࢖ࣈ࣮ࣛࣜࢆస〇ࡋࡓ. ᮏἲ࡛ࡣ PCR཯ᛂᾮ࡟Mn 2+ ࢆῧຍࡋ, ࡉࡽ࡟4 ✀ࡢdeoxynucleoside triphosphate (dNTP) ࢆ୙ᆒ ➼࡞๭ྜ࡛ῧຍࡍࡿࡇ࡜࡟ࡼࡾ, ࣇ࢕ࢹࣜࢸ࢕ࢆపୗࡉࡏࡿ. wt-scFv㑇ఏᏊࡀ⤌ࡳ㎸ࡲ ࢀ ࡓ pEXmide 7 ࣋ ࢡ ࢱ ࣮ ࢆ 㗪 ᆺ ࡜ ࡋ ࡚, 0.10 mM Mn 2+ Ꮡ ᅾ ୗ, deoxyadenosine 5’-triphosphate (dATP) ࡢࡳࢆ௚ 3 ✀ࡢ 1/5 ࡟ῶࡌࡓ᮲௳࡛ PCR ࢆ⾜ࡗࡓ. ቑᖜ㓝⣲࡟ࡣ, ධᡭࡀᐜ᫆࡞ࡶࡢࡢ࡞࠿࡛ࡣࣇ࢕ࢹࣜࢸ࢕ࡀඖ᮶ప࠸AmpliTaq DNA࣏࣓࣮ࣜࣛࢮࢆ⏝ ࠸ࡓ. ࣉࣛ࢖࣐࣮ࡣ, 5’ࣉࣛ࢖࣐࣮࡟pEX7-V_H-CP5 (V_Hࡢୖὶ࡟఩⨨ࡍࡿpelB㓄ิ࡟┦ ⿵ᛶࢆ♧ࡍ), 3’ࣉࣛ࢖࣐࣮࡟ pEX7-V_L-CP3 (V_Lࡢୗὶ࡟఩⨨ࡍࡿ gIII ࡟┦⿵ᛶࢆ♧ࡍ) ࢆ⏝࠸, ࣮ࣜࣥ࢝ࢆྵࡵࡓscFv඲㛗࡟ኚ␗ࢆᑟධࡋࡓ. ᚓࡽࢀࡓቑᖜ⏘≀ࢆpEXmide 7࣋ࢡࢱ࣮࡟⤌ࡳ㎸ࡳ, 㟁Ẽ✸Ꮝἲ (electroporation) ࡟ ࡼࡾ኱⭠⳦ TG1 ࡟ᑟධࡋ, ᐮኳᇵᆅୖ୍࡛ᬌᇵ㣴ࡋ࡚ᙧ㉁㌿᥮⳦ࡢࣛ࢖ࣈ࣮ࣛࣜ (5.9 ™10 5 ࢡ ࣟ ࣮ ࣥ ࢆ ྵ ࡴ) ࢆ స 〇 ࡋ ࡓ. ᐮ ኳ ᇵ ᆅୖࡢࡍ࡭࡚ࡢࢥࣟࢽ࣮ࢆΰྜࡋ࡚ᆒ୍࡞ࢢ ࣜࢭ࣮ࣟࣝಖᏑᾮ࡜ࡋࡓࡢࡕ, ࡑࡢ୍㒊ࢆ 6 ศ๭ࡋ࡚, ࡑࢀࡒࢀࢆᑐᩘቑṪᮇࡲ࡛ᇵ㣴ࡋ, ࣊ ࣝ ࣃ ࣮ ࣇ ࢓ ࣮ ࢪ KM13 54) ࢆ ឤ ᰁ ࡉ ࡏ ࡚ 6 ࡘ ࡢ ࣇ ࢓ ࣮ ࢪ ࣛ ࢖ ࣈ ࣛ ࣜ ࣮ ࢆ స 〇 ࡋ ࡓ (ᅗ 13). ࡞࠾, ᐟ୺࡜ࡋ࡚ TG1, ࣊ࣝࣃ࣮ࣇ࢓࣮ ᅗ 13. ࣃࣥࢽࣥࢢ࡟ࡼࡿᨵⰋᆺኚ␗scFvࡢ㑅ᢥ ࣛࣥࢲ࣒ኚ␗ࢆᑟධࡋࡓኚ␗scFvࣛ࢖ࣈ࣮࡛ࣛࣜᙧ ㉁㌿᥮ࡋࡓ኱⭠⳦ࡢಖᏑᾮࢆ6ࡘ࡟ศࡅ, ࡑࢀࡒࢀ࣊ࣝ ࣃ࣮ࣇ࢓࣮ࢪࢆឤᰁࡉࡏ࡚ࣇ࢓࣮ࢪࣛ࢖ࣈ࣮ࣛࣜࢆᵓ⠏ ࡋࡓ. ࡇࢀࡽࢆࡑࢀࡒࢀCS-BSAᅛᐃ໬ࢳ࣮ࣗࣈ (#1~6) ࡟ຍ࠼, ྛࢳ࣮ࣗࣈ࡟ࡘ࠸࡚ࣃࣥࢽࣥࢢࡢ᧯సࢆ3ᅇࡎ ࡘ⧞ࡾ㏉ࡋࡓ. ᅛ┦࡟྾╔ࡋࡓࣇ࢓࣮ࢪࡢᅇ཰ࡢ㝿, #1~3࡟ࡣࢢࣜࢩࣥሷ㓟⦆⾪ᾮ (pH 2.2), #4~6࡟ࡣࢺ࢚ࣜ ࢳࣝ࢔࣑ࣥỈ⁐ᾮ (pH 12) ࢆ⏝࠸ࡓ. 300ࢡ࣮ࣟࣥࢆゎ ᯒࡋࡓ⤖ᯝ, 32ࢡ࣮࡛ࣟࣥ㝧ᛶࢩࢢࢼࣝࡀㄆࡵࡽࢀࡓࡀ, ࡑࡢ࠺ࡕᅗ࡟♧ࡋࡓ6✀ࡢኚ␗scFvࡀwt-scFvࡼࡾࡶ኱ ࡁ࡞Ka್ࢆ♧ࡋࡓ.

17 ࢪ࡜ࡋ࡚KM13ࢆ⏝࠸ࡓࡢࡣ, ➨3❶࡛㏙࡭ࡿࡼ࠺࡟, ࢔ࣞ࢖ᆺࣉࣟࣇ࢓࢖ࣜࣥࢢࢩࢫ ࢸ࣒ࡢᵓ⠏࡟࠾࠸࡚ࡇࡢ⤌ࡳྜࢃࡏࢆ᥇⏝ࡋࡓࡇ࡜࡟ࡼࡿ. ᚓࡽࢀࡓࣇ࢓࣮ࢪࣛ࢖ࣈ ࣮ࣛࣜࢆ, ࡑࢀࡒࢀCS-BSAᅛᐃ໬ࢳ࣮ࣗࣈ (#1~6) ࡟ຍ࠼, 37Υ࡛1᫬㛫཯ᛂࡉࡏࡓ. ᮍ ཯ ᛂ ࡢ ࣇ ࢓ ࣮ ࢪ ࠾ ࡼ ࡧ 㠀 ≉ ␗ ⓗ ࡟ ᙅ ࡃ ⤖ ྜ ࡋ ࡓ ࣇ ࢓ ࣮ ࢪ ࢆ Ὑ ί ࡋ ࡚ 㝖 ཤ ࡋ ࡓ ࡢ ࡕ, ࢳ࣮ࣗࣈ#1~3࡟ࡣࢢࣜࢩࣥሷ㓟⦆⾪ᾮ (pH 2.2) ࢆ, #4~6࡟ࡣࢺ࢚ࣜࢳࣝ࢔࣑ࣥỈ⁐ᾮ (pH 12) ࢆຍ࠼࡚ᅛ┦࡟ṧࡗࡓࣇ࢓࣮ࢪࢆ⁐ฟ࣭ᅇ཰ࡋ, TG1࡟ឤᰁࡉࡏ࡚」〇ࡉࡏࡓ. ࡇࡢࢧ࢖ࢡࣝࢆ 3 ᅇ⧞ࡾ㏉ࡋ, ྛࢳ࣮ࣗࣈ࠿ࡽ᭱⤊ⓗ࡟ᅇ཰ࡉࢀࡓࣇ࢓࣮ࢪ࡟ࡘ࠸࡚ 50ࢡ࣮ࣟࣥࡎࡘ (ࡍ࡞ࢃࡕ, ィ300ࢡ࣮ࣟࣥ) ࢆ㑅ᢥࡋࡓ. ࡇࢀࡽ࡟ࡘ࠸࡚,ࠕࣇ࢓࣮ࢪ ELISAࠖ(CS-BSA ᅛᐃ໬ࣉ࣮ࣞࢺෆ࡛ CS ࡜ࡢ➇ྜ཯ᛂࢆ⾜࠸, ᅛ┦ୖࡢࣇ࢓࣮ࢪࢆ PODᶆ㆑ᢠࣇ࢓࣮ࢪᢠయ᳨࡛ฟࡍࡿ) ࡟ࡼࡾCS⤖ྜ⬟ࢆ⢭ᰝࡋࡓ࡜ࡇࢁ, wt-scFvࡼࡾ ࡶ㧗ឤᗘ࡞ᛂ⟅ࢆ♧ࡋࡓࣇ࢓࣮ࢪࢡ࣮ࣟࣥࡣ6 ✀ࡢࡳ࡛࠶ࡗࡓ. ྛࢳ࣮ࣗࣈ (#1~6) ࠿ ࡽᚓࡽࢀࡓࢡ࣮ࣟࣥࢆᅗ 14 ࡟♧ࡍ. ྠ୍ࡢᙧ㉁㌿᥮⳦ࣛ࢖ࣈ࣮ࣛࣜࢆฎ⌮ࡋࡓ࡟ࡶ࠿ ࠿ࢃࡽࡎ, ࢳ࣮ࣗࣈ#3 ࡜#6࠿ࡽࡣᨵⰋᆺࢡ࣮ࣟࣥࡣᚓࡽࢀ࡚࠸࡞࠸. ࡋ࠿ࡶ, ࡇࢀࡽ6 ✀࡟ࡘ࠸࡚ᥦ♧ࡍࡿscFv ࢆྍ⁐ᆺࢱࣥࣃࢡ㉁࡟ኚ᥮ࡋ࡚, ࡑࡢ K_aࢆ Scatchard ἲ࡟ࡼ ࡾ⟬ฟࡋࡓ࡜ࡇࢁ 4.1~19™10 8 _M-1 ࡢ⠊ᅖ࡛࠶ࡾ, wt-scFv ࠿ࡽࡢୖ᪼⋡ࡣ 1.2~5.6 ಸ࡟ 㐣ࡂ࡞࠿ࡗࡓ (ᅗ14). ᅗ 14. ࣃࣥࢽࣥࢢ࡛ᚓࡽࢀࡓscFv ࢡ࣮ࣟࣥࡢ1ḟᵓ㐀 (ᶍᘧᅗ) ࡜ Scatchardἲ࡟ࡼࡿ⤖ྜ ᐃᩘࡢ⟬ฟ ᮏἲ࡟ࡼࡾᚓࡽࢀࡓࢡ࣮ࣟࣥࡢ1ḟᵓ㐀࠾ࡼࡧ⟬ฟࡉࢀࡓKaࢆ♧ࡍ. ᫍ༳ࡣࡑࡢ఩⨨ࡢ࢔࣑ࣀ㓟ࡀ⨨ ᥮ࡉࢀࡓࡇ࡜ࢆព࿡ࡍࡿ. scFv#mc1-7ࡢVH 62␒┠࠾ࡼࡧscFv#mc2-21ࡢVL 103␒┠࡟グࡉࢀ࡚࠸ࡿ ࠕ*ࠖࡣ, ⤊Ṇࢥࢻࣥ (࢔ࣥࣂ࣮ࢥࢻࣥ; TAG) ࢆ♧ࡍ. ࡞࠾, ࡇࢀࡽࡢ఩⨨ࡣ㉁㔞ศᯒ࡟ࡼࡾQ࡟⩻ヂࡉ ࢀ࡚࠸ࡿࡇ࡜ࡀุ᫂ࡋ࡚࠸ࡿ. ࣃࣥࢽࣥࢢࢢ࡛࡛ᚓࡽᚓࡽࢀࡓscFv ࢡࣟࢡ࣮ࣟࣥࡢ1ḟᵓ㐀㐀 (ᶍᘧᶍᘧᅗᅗ) ࡜ Scatchardἲ࡟ࡼࡿࡼࡿ

18 ➨⠇ ⪃ᐹ CSࢆᶆⓗᢠཎ࡜ࡍࡿscFv࡟error-prone PCR࡟ࡼࡾࣛࣥࢲ࣒ኚ␗ࢆᑟධࡋ࡚ኚ␗scFv ࣛ࢖ࣈ࣮ࣛࣜࢆస〇ࡋ, ᶆ‽ⓗ࡞ࣃࣥࢽࣥࢢ࡟ࡼࡾぶ࿴ຊࡢྥୖࡋࡓࢡ࣮ࣟࣥࢆ᥈⣴ ࡋࡓ. ᚓࡽࢀࡓ scFv 㑇ఏᏊࡢሷᇶ㓄ิࢆゎᯒࡋ࡚࢔࣑ࣀ㓟㓄ิࢆ᥎ᐃࡋ, ྛኚ␗ scFv ࡢ࢔࣑ࣀ㓟⨨᥮ࢆ≉ᐃࡋࡓ࡜ࡇࢁ, ⨨᥮ᩘࡣࢡ࣮ࣟࣥ㛫࡛኱ࡁࡃ␗࡞ࡗ࡚࠸ࡓࡀ (1~7 ࢝ᡤ), ࠸ࡎࢀࡢኚ␗య࡛ࡶwt-scFvࡢV_L 49␒┠࡟Ꮡᅾࡋࡓࢩࢫࢸ࢖ࣥࡀ⨨᥮ࡉࢀ࡚࠸ ࡓ (ᅗ14). ᢠయࡢ V_H࠾ࡼࡧV_L࡟ࡣ, 㙐ෆࢪࢫࣝࣇ࢕ࢻ⤖ྜ࡟୚ࡿࢩࢫࢸ࢖ࣥࡀ 2 ࢝ ᡤ (V_H࡛ࡣ22࠾ࡼࡧ92␒┠, V_L࡛ࡣ23࠾ࡼࡧ88␒┠) ࡟Ꮡᅾࡍࡿࡀ, ࡑࢀ௨እࡢ఩ ⨨࡟ࢩࢫࢸ࢖ࣥࡀᏑᅾࡍࡿࡇ࡜ࡣ⛥࡛࠶ࡿ. V_L 49␒┠ࡢࠕ୙⮬↛࡞ࠖࢩࢫࢸ࢖ࣥࢆ㝖ࡃ ࡇ࡜ࡀぶ࿴ຊࡢྥୖ࡟ᐤ୚ࡋ࡚࠸ࡿࡇ࡜ࡣ, ⯆࿡῝࠸. ୍᪉࡛, ィ300ࡶࡢࢡ࣮ࣟࣥࡢᢠཎ⤖ྜ⬟ࢆ⢭ᰝࡋࡓ࡟ࡶ࠿࠿ࢃࡽࡎ, K_aࡢቑ኱ࡋࡓ ኚ␗scFvࢡ࣮ࣟࣥࡣࢃࡎ࠿6✀ࡋ࠿ᚓࡽࢀࡎ, ࡋ࠿ࡶ, K_aࡢୖ᪼⋡ࡣ᭱኱࡛ࡶ5.6ಸ࡟ 㐣ࡂ࡞࠿ࡗࡓ. ࡇࡢヨ㦂࡛ࡣ, ࣃࣥࢽࣥࢢࢆ6㔜࡟⾜ࡗ࡚ࡑࡢ෌⌧ᛶ࡟ࡘ࠸࡚ࡶ㓄៖ࡋ, ᅛ┦࠿ࡽࡢ⁐ฟ᮲௳࡟ࡘ࠸࡚ࡶ2✀ (㓟ฎ⌮࡜ሷᇶฎ⌮) ࢆヨࡳࡓࡀ, ࢳ࣮ࣗࣈ#3࠾ࡼ ࡧ#6 ࡛ࡣ᭷ᮃ࡞ೃ⿵ࢡ࣮ࣟࣥࢆぢฟࡏ࡞࠿ࡗࡓ. ࡇࡢ⤖ᯝࡣ, ࣃࣥࢽࣥࢢࡢᡂྰࡀ, ไ ᚚࡢᅔ㞴࡞ᅉᏊ࡟ࡼࡾ኱ࡁࡃኚືࡍࡿࡇ࡜ࢆ♧၀ࡍࡿ. ࡲࡓ, scFv#mc1-7, 2-17, ࠾ࡼࡧ 2-21 ࡣ␗࡞ࡿࢳ࣮ࣗࣈ࠿ࡽඹ㏻ࡋ࡚ᚓࡽࢀ࡚࠾ࡾ, ≉ᐃࡢࢡ࣮ࣟࣥࡀ㧗㢖ᗘ࡛༢㞳ࡉ ࢀ࠺ࡿࡇ࡜ࡀ♧ࡉࢀࡓ. ࡇࢀࡽࡢࢡ࣮ࣟࣥࡣ, ⥴ㄽ࡛ゝཬࡋࡓࡼ࠺࡟, ᥦ♧ࡍࡿscFvࡢ ぶ࿴ຊࡼࡾࡶቑṪ⋡ࡢ㧗ࡉ (ࡍ࡞ࢃࡕ, ࣇ࢓࣮ࢪᥦ♧ࡢࡓࡵࡢ2ḟᇵ㣴࡟࠾ࡅࡿᙧ㉁㌿ ᥮⳦ࡢቑṪ࠶ࡿ࠸ࡣࣇ࢓࣮ࢪࡢࣃࢵࢣ࣮ࢪࣥࢢ࡟࠾ࡅࡿඃ఩ᛶ) ࡟ࡼࡾ㑅ᢥࡉࢀࡓྍ ⬟ᛶࡀ࠶ࢁ࠺. ௨ୖࡢᐇ㦂⤖ᯝࡣࠕᮏࣛ࢖ࣈ࣮ࣛࣜ࡟ࡘ࠸࡚ࣃࣥࢽࣥࢢࢆఱ㔜࡟⾜ࡗࡓ࡜ࡋ࡚ࡶ, , ࡑ ࡢ Kaࡀ 10 10 _M-1 ࢆ㉸࠼ࡿࡼ࠺࡞㧗ぶ࿴ຊࢡ࣮ࣟࣥࢆᚓࡿࡇ࡜ࡣᅔ㞴ࠖ࡜ᛮࢃࡏࡿࡶࡢ ࡛࠶ࡗࡓ. ࡇࢀࡣ, ࡇࡢࣛ࢖ࣈ࣮ࣛࣜ࡟ᮏᙜ࡟㧗ぶ࿴ຊࢡ࣮ࣟࣥࡀᏑᅾࡋ࡞࠸ࡓࡵ࡞ࡢ ࠿, ࠶ࡿ࠸ࡣࡸࡣࡾࣃࣥࢽࣥࢢࡢ㑅ᢥຠ⋡࡟ᇶ࡙ࡃࡶࡢ࡞ࡢ࠿? ᚋ⪅࡞ࡽࡤ, ࢔ࣞ࢖ᆺ ࣉࣟࣇ࢓࢖ࣜࣥࢢࢩࢫࢸ࣒ࡢᑟධ࡟ࡼࡾゎỴࡉࢀࡿྍ⬟ᛶࡀ࠶ࡿ, ࡜ᮇᚅࡉࢀࡓ.

19 ➨❶ ⧄⥔≧ࣇ࢓࣮ࢪࡢ㧗ឤᗘ᳨ฟࢆ┠ⓗ࡜ࡍࡿᢠࣇ࢓࣮ࢪᢠయࣇࣛࢢ࣓ࣥࢺ-ࣝࢩ ࣇ࢙࣮ࣛࢮ⼥ྜࢱࣥࣃࢡ㉁ࡢ๰〇 ➨⠇ ᗎ ᮏ◊✲࡛ࡑࡢᵓ⠏ࢆ┠ᣦࡍ࢔ࣞ࢖ᆺࣉࣟࣇ࢓࢖ࣜࣥࢢࢩࢫࢸ࣒࡛ࡣ, ࣐࢖ࢡ࢙ࣟ࢘ ࣝෆ࡛⏕ᡂࡋ, ᢠཎ࡟ᤕᤊࡉࢀࡿscFvᥦ♧ࣇ࢓࣮ࢪࢆ㧗ឤᗘ࡟᳨ฟࡍࡿᚲせࡀ࠶ࡿ. ࡑ ࡢᡭẁ࡜ࡋ࡚, ࡈࡃᚤ㔞᳨࡛ฟࡀྍ⬟࡞࣐࣮࣮࢝ศᏊࢆᶆ㆑ࡋࡓࣇ࢓࣮ࢪ≉␗ⓗᢠయ ࡀ᭱㐺࡛࠶ࡿ. ࣐࣮࣮࢝ศᏊ࡜ࡋ࡚ࡣ, ቑᖜຠᯝࡀᚓࡽࢀ, 㧗ឤᗘ࡞᳨ฟࡀྍ⬟࡞ࡇ࡜ ࠿ࡽ㓝⣲ࡀᮃࡲࡋ࠸. 㓝⣲άᛶࡢ᳨ฟἲ࡜ࡋ࡚ࡣ, ẚⰍἲ࠶ࡿ࠸ࡣ⺯ගἲࡀᶆ‽ⓗ࡛࠶ ࡿࡀ, Ⓨග᳨ฟࡢ฼⏝ࡀྍ⬟࡞ࡽࡤࡉࡽ࡟㧗ឤᗘ࡞᳨ฟࡀᮇᚅ࡛ࡁࡿ. Gaussia ࣝࢩࣇ ࢙࣮ࣛࢮ (GLuc) ࡣ, ᾏὒᛶ࢝࢖࢔ࢩ㢮࡟⏤᮶ࡍࡿ㓝⣲࡛࠶ࡾ, ᇶ㉁࡛࠶ࡿࢭࣞࣥࢸࣛ ࢪࣥࢆ㓟໬ࡍࡿ㝿࡟480 nm ࢆᴟ኱Ἴ㛗࡜ࡍࡿගࢆ⏕ࡌࡿ. ᅾ᮶ࡢ࣍ࢱࣝࣝࢩࣇ࢙࣮ࣛ ࢮ (firefly luciferase; FLuc) ࡸ࣑࢘ࢩ࢖ࢱࢣࣝࢩࣇ࢙࣮ࣛࢮ (Renilla reniformis luciferase; RLuc) ࡼࡾ㧗ឤᗘ࡞Ⓨග᳨ฟࡀྍ⬟࡛࠶ࡾ, 55–57) ච␿ ᐃἲ࡟࠾ࡅࡿᶆ㆑㓝⣲࡜ࡋ࡚ࡶ ᭷⏝࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀ࡚࠸ࡿ. 58) 㓝⣲ࢆᢠయ࡟ᶆ㆑ࡍࡿሙྜ, ᚑ᮶ࡣᯫᶫヨ⸆ࢆ⏝࠸࡚୧⪅ࢆ໬Ꮫⓗ࡟㐃⤖ࡋ࡚࠸ࡓ ࡀ, ௒᪥࡛ࡣ㑇ఏᏊ᧯స࡟ࡼࡾ, ᢠయࢆscFv ໬ࡋࡓࡢࡕࡑࡢNᮎ➃࠶ࡿ࠸ࡣ Cᮎ➃࡟ 㓝⣲ࢆ⼥ྜࡉࡏࡿࡇ࡜ࡀྍ⬟࡛࠶ࡿ. scFv໬࡟ࡼࡾᢠయ࡜ࡋ࡚ࡢศᏊ㔞ࡀపୗ (IgG ࡢ ⣙1/6) ࡍࡿࡓࡵ, ࣇ࢓࣮ࢪ1ࣅࣜ࢜ࣥ࡟ከᩘࡢGLucࢆ⤖ྜࡉࡏࡿࡇ࡜ࡀྍ⬟࡜ᮇᚅࡉ ࢀ, ᳨ฟឤᗘྥୖࡢほⅬ࠿ࡽࡶ᭷฼࡛࠶ࡿ. ௨ ୖ ࡢ ほ Ⅼ ࠿ ࡽ, ࢔ ࣞ ࢖ ᆺ ࣉ ࣟ ࣇ ࢓ ࢖ ࣜ ࣥ ࢢ ࢩ ࢫ ࢸ ࣒ ࡟ 㐺 ࡍ ࡿ ᢠ ࣇ ࢓ ࣮ ࢪ ᢠ య scFv-GLuc⼥ྜࢱࣥࣃࢡ㉁ࡢ๰〇ࢆヨࡳࡓ. ⧄⥔≧ࣇ࢓࣮ࢪ࡟ᑐࡍࡿᢠࣇ࢓࣮ࢪᢠయࡣ ᕷ㈍ࡉࢀ࡚࠸ࡿࡶࡢࡢ, ࡑࡢ㓄ิ᝟ሗࡣᚓࡽࢀ࡞࠸ࡓࡵ, ࡲࡎࡣᢠࣇ࢓࣮ࢪᢠయࢆ᪂つ ࡟ㄪ〇ࡋ (➨ 2 ⠇), ᚓࡽࢀࡓᢠయࡢྍኚ㒊㑇ఏᏊࢆࢡ࣮ࣟࢽࣥࢢࡋ࡚ᢠࣇ࢓࣮ࢪ scFv ࢆᵓ⠏ࡋࡓ (➨3⠇). ḟ࠸࡛, ┠ⓗࡢ⼥ྜࢱࣥࣃࢡ㉁࡟ඛࡔࡗ࡚, ➨1 ❶࡛స〇ࡋࡓ㧗 ぶ࿴ຊࡢᢠCS ᢠయࣇࣛࢢ࣓ࣥࢺ࡛࠶ࡿ CS#10-scFv ࢆGLuc ࡜㐃⤖ࡋࡓ⼥ྜࢱࣥࣃࢡ ㉁ࢆㄪ〇ࡋ, ࡑࡢⓎග≉ᛶࡸ᭷⏝ᛶࢆ᳨ウࡋࡓ. ᘬࡁ⥆ࡁ, ୖグࡢᢠࣇ࢓࣮ࢪ scFv ࡟ GLuc ࢆ㐃⤖ࡉࡏࡓ⼥ྜࢱࣥࣃࢡ㉁ (M13-scFv-GLuc) ࢆస〇ࡋ, ࣉࣟࣇ࢓࢖ࣜࣥࢢࢩ ࢫࢸ࣒࡟࠾ࡅࡿ᭷⏝ᛶࢆホ౯ࡋࡓ (➨4⠇).

20 ➨⠇ ᢠࣇ࢓࣮ࢪࣔࣀࢡ࣮ࣟࢼࣝᢠయࡢㄪ〇࡜ㅖᛶ㉁ ⧄⥔≧ࣇ࢓࣮ࢪࡣ㛗࠸⟄≧ࡢ࢝ࣉࢩࢻࡢ࡞࠿࡟⎔≧୍ᮏ㙐DNAࢆࢤࣀ࣒࡜ࡋ࡚ಖᣢ ࡋ, ኱⭠⳦㞝ᰴ࡟ࡑࡢF⥺ẟࢆ௓ࡋ࡚ឤᰁࡍࡿࣂࢡࢸࣜ࢜ࣇ࢓࣮ࢪ࡛࠶ࡾ, Ffࣇ࢓࣮ࢪ ࡜ࡶ⥲⛠ࡉࢀࡿ. M13, f1, ࠾ࡼࡧ fdࡀ௦⾲ⓗ࡛࠶ࡿࡀ, ࡞࠿࡛ࡶ M13 ࣇ࢓࣮ࢪࡣࣇ࢓ ࣮ࢪᥦ♧ἲ࡟ከ⏝ࡉࢀ࡚࠸ࡿ. 59,60) ᅗ 15 ࡟ Ff ࣇ࢓࣮ࢪࡢᵓ㐀ࢆ♧ࡍ. ᢠࣇ࢓࣮ࢪᢠయ ࢆ⏝࠸࡚ࣇ࢓࣮ࢪࢆ᳨ฟࡍࡿሙྜ, ࣇ࢓࣮ࢪ1ࣅࣜ࢜ࣥ࡟ᑐࡋ࡚ࡼࡾከࡃࡢᢠయࢆ⤖ྜ ࡉࡏࡿࡇ࡜ࡀ࡛ࡁࢀࡤ, ࡼࡾ㧗ឤᗘ࡞᳨ฟ⣔ࡀᵓ⠏࡛ࡁࡿ. Ff ࣇ࢓࣮ࢪࡢ࢝ࣉࢩࢻࡣ୺ ࡟ࢥ࣮ࢺࢱࣥࣃࢡ㉁pVIII࠿ࡽᵓᡂࡉࢀ, 1ࣅࣜ࢜ࣥ୰࡟⣙2700ศᏊࡢpVIIIࡀྵࡲࢀ ࡿ (ᅗ15). 61) ࡋࡓࡀࡗ࡚, ࣇ࢓࣮ࢪࢆ࣐ࣝࢳࣉࣝ࡟ᶆ㆑ࡍࡿࡓࡵ࡟ࡣ, pVIIIࢆㄆ㆑ࡍࡿ ᢠయࢆㄪ〇ࡍ࡭ࡁ, ࡜⪃࠼ࡓ. ࡣࡌࡵ࡟, ⤌᥮࠼ pVIII ࢱࣥࣃࢡ㉁ࢆච␿ཎ࡜ࡍࡿࡇ࡜ࢆ௻࡚ࡓ. ࢹ࣮ࢱ࣮࣋ࢫ࠿ࡽ ᚓࡽࢀࡓpVIII㑇ఏᏊ (gVIII) ࡢሷᇶ㓄ิ 62) ࢆⓎ⌧࣋ࢡࢱ࣮࡟⤌ࡳ㎸ࡳ, ኱⭠⳦࡟ᑟධ ࡋ࡚⤌᥮࠼pVIIIࢱࣥࣃࢡ㉁ࢆస〇ࡋࡓࡀ, ᕷ㈍ࡢᢠM13ࣇ࢓࣮ࢪᢠయ࡟ࡣ཯ᛂᛶࢆ♧ ࡉࡎ, ࣇ࢓࣮ࢪ⢏Ꮚࢆᵓ⠏ࡋ࡚࠸ࡿ pVIII ࡜ࡣ❧యᵓ㐀ࡀ኱ࡁࡃ␗࡞ࡿࡶࡢ࡜⪃࠼ࡽࢀ ࡓ. ࡑࡇ࡛, VCSM13ࣇ࢓࣮ࢪࡑࡢࡶࡢ࡛࣐࢘ࢫࢆච␿ࡋࡓ. VCSM13ࡣ, M13ࣇ࢓࣮ࢪ ࡢࢤࣀ࣒ (」〇㉳Ⅼ) ࡟ேᕤⓗ࡞ᨵኚࢆຍ࠼࡚ ឤᰁ⳦ෆ࡛ࡢ」〇ࢆ㜼 ᐖࡋࡓኚ␗య࡛ ࠶ࡿ. ࣇ࢓࣮ࢪ࣑ࢻ࣋ࢡࢱ࣮ࢆ⏝࠸ࡓࣇ࢓࣮ࢪᥦ♧ἲ (➨3❶➨2⠇࡛㏙࡭ࡿ) 63) ࡟࠾ ࠸࡚࣊ࣝࣃ࣮ࣇ࢓࣮ࢪ࡜ࡋ࡚ከ⏝ࡉࢀࡿࡀ, 60,64) ࡑࡢpVIIIࡢ୍ḟᵓ㐀ࡣ㔝⏕ᆺM13ࡢ ࡑࢀ࡜ྠ୍࡛࠶ࡿ. 62,65)

࣏࢚ࣜࢳࣞࣥࢢࣜࢥ࣮ࣝ (polyethylene glycol; PEG) ỿẊἲ࡟ࡼ ࡾ⢭〇ࡋࡓVCSM13ࢆỈ⁐ᛶࡢABISCO-100࢔ࢪࣗࣂࣥࢺ 66) ࡟ᠱ⃮ࡋ, BALB/c ࣐࢘ ࢫࡢ⓶ୗ࡟⧞ࡾ㏉ࡋᢞ୚ࡋࡓࡢࡕ, ⾑୰ᢠయ౯ࡢୖ᪼ࢆẚ㍑ࡋࡓ. ᭱ࡶⰋዲ࡞ච␿ᛂ⟅ ࢆ♧ࡋࡓ࣐࢘ࢫ࡟VCSM13ࢆ᭱⤊ච␿ࡋ, ࡑࡢ3᪥ᚋ࡟⭁⮚ࢆ᦬ฟࡋࡓ. ᚓࡽࢀࡓ⭁⣽ ⬊ࢆNS1 ࣑࢚࣮࣐ࣟ⣽⬊࡜⼥ྜࡉࡏ, ⏕ᡂࡋࡓᢠ VCSM13ᢠయ⏘⏕ࣁ࢖ࣈࣜࢻ࣮࣐ࢆ 㝈⏺ᕼ㔘ἲ࡟ࡼࡾࢡ࣮ࣟࣥ໬ࡋ, ⣽⬊ᰴ (#71) ࢆᶞ❧ࡋࡓ. 67) ᮏ⣽⬊ᰴࡀศἪࡍࡿᢠVCSM13 ᢠయ (Ab-M13#71) ࡢH 㙐࡜L㙐ࡢ࢔࢖ࢯࢱ࢖ࣉࢆ ㄪ࡭ࡓ࡜ࡇࢁ, ࡑࢀࡒࢀg2b, k࡛࠶ࡗࡓ. ᮏᢠయࡢ࢚ࣆࢺ࣮ࣉࢆ, ࢖࣒ࣀࣈࣟࢵࢺ࡟ࡼࡾ ᅗ 15. ⧄⥔≧ࣇ࢓࣮ࢪ (Ffࣇ࢓࣮ࢪ) ࡢᵓ㐀 ⣙2700ศᏊࡢpVIIIࢱࣥࣃࢡ㉁ (50࢔࣑ࣀ㓟, Mr; 5235) ࠿ࡽ࡞ࡿ⟄≧ࡢ࢝ࣉࢩࢻෆ࡟, 11ࡢ㑇ఏᏊࢆྵ ࡴࢤࣀ࣒ࢆಖᣢࡋ࡚࠸ࡿ. ᅗࡢᕥ➃࡟ࡣpIII (406࢔࣑ࣀ㓟, Mr; 42522) ࠾ࡼࡧ pVI (112࢔࣑ࣀ㓟, Mr; 12342), ྑ➃࡟ࡣpVII (33࢔࣑ࣀ㓟, Mr; 3599) ࠾ࡼࡧpIX (32࢔࣑ࣀ㓟, Mr; 3650) ࡀ఩⨨ࡋ࡚࠸ࡿ.

21

ㄪ࡭ࡓ. ࡍ࡞ࢃࡕ, ຍ⇕ฎ⌮ࡋࡓ VCSM13 ࢆࢻࢹࢩࣝ◲㓟ࢼࢺ࣒ࣜ࢘ (sodium dodecyl sulfate; SDS) ࡢ Ꮡ ᅾ ୗ ࡛ ࣏ ࣜ ࢔ ࢡ ࣜ ࣝ ࢔ ࣑ ࢻ ࢤ ࣝ 㟁 Ẽ Ὃ ື (polyacrylamide gel electrophoresis; PAGE) ࡟ ௜ ࡋ, ศ 㞳 ࡉ ࢀ ࡓ ࢱ ࣥ ࣃ ࢡ ㉁ ࢆ ࣏ ࣜ ࣇ ࢵ ໬ ࣅ ࢽ ࣜ ࢹ ࣥ [poly(vinylidene fluoride); PVDF] ⭷࡟㌿෗ࡋࡓࡢࡕ, Ab-M13#71 ࢆ཯ᛂࡉࡏࡓ. ḟ࠸࡛, PODᶆ㆑➨2 ᢠయ࡟ࡼࡾ⭷ୖࡢᢠయࢆᤕᤊࡋྍど໬ࡋࡓ࡜ࡇࢁ, pVIIIࡢศᏊ㔞࡟┦ᙜ

ࡍࡿ5.2 kDa௜㏆࡟ࣂࣥࢻࡀㄆࡵࡽࢀ, Ab-M13#71ࡣࣇ࢓࣮ࢪࡢpVIIIࢆㄆ㆑ࡍࡿࡇ࡜ ࡀ♧ࡉࢀࡓ (ᅗ 16). ࡉࡽ࡟, ࣇ࢓࣮ࢪ࡬ࡢ཯ᛂᛶࢆ➇ྜ ELISA ࡟ࡼࡾㄪ࡭ࡓ. ࡍ࡞ࢃ ࡕ, VCSM13ࢆᅛᐃ໬ࡋࡓ࣐࢖ࢡࣟࣉ࣮ࣞࢺ࡟, Ab-M13#71࠾ࡼࡧVCSM13ࢆຍ࠼, ➇ ྜ཯ᛂࢆ⾜ࡗࡓ. ᅛ┦࡟ṧࡿ Ab-M13#71 ࢆPOD ᶆ㆑ᢠ࣐࢘ࢫᢠయ࡛ᤕᤊࡋ, o-ࣇ࢙ࢽ ࣞࣥࢪ࢔࣑ࣥࢆ⏝࠸࡚ẚⰍ ᐃࡋࡓ. ࡑࡢ⤖ᯝ, VCSM13 ࡢῧຍ㔞ࡢቑຍ࡟క࠸, ࢩࢢ ࢼࣝࡀῶᑡࡋ, ࣇ࢓࣮ࢪ࡬ࡢ཯ᛂࡣ≉␗ⓗ࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀࡓ (ᅗ17). ᅗ 16. VCSM13ࢆ⏝࠸ࡿAb-M13#71ࡢ࢖࣒ࣀࣈࣟࢵࢺゎᯒ Ab-M13#71ࡢ≉␗ᛶࢆ᥎ᐃࡍࡿࡓࡵࡢ࢖࣒ࣀࣈࣟࢵࢺࡢ⤖ᯝࢆ♧ ࡍ. lane1࠾ࡼࡧ2ࡣࡑࢀࡒࢀ1™1010ࡲࡓࡣ1™109 pfuࡢࣇ࢓࣮ࢪࢆ ⏝࠸ࡓ཯ᛂ, MࡣMr࣐࣮࣮࡛࢝࠶ࡿ. ᅗ 17. Ab-M13#71ࢆ⏝࠸ࡿVCSM13 ࣇ࢓࣮ࢪ ࡢ➇ྜELISA VCSM13ࣇ࢓࣮ࢪࢆᅛᐃ໬ࡋࡓ࣐࢖ࢡࣟࣉ࣮ࣞࢺ࡟, 㐟㞳ࡢVCSM13ࣇ࢓࣮ࢪ࠾ࡼࡧAb-M13#71ࢆຍ࠼࡚ ➇ྜ཯ᛂࢆ⾜ࡗࡓ⤖ᯝࢆ♧ࡍ. ⏝㔞స⏝᭤⥺ࡢ midpoint (50%㜼ᐖ࡟せࡍࡿVCSM13㔞) ࡣ2.0™106 pfu/assay࡛࠶ࡗࡓ. ᅗ ࡢ➇ྜ➇ྜ VC 㐟㞳 㐟㞳 ➇ྜ ➇ྜ midp pfu/

22 ➨⠇ ᢠࣇ࢓࣮ࢪᢠయ୍ᮏ㙐 )Y ࣇࣛࢢ࣓ࣥࢺࡢㄪ〇࡜ㅖᛶ㉁ ┠ⓗࡢࣇ࢓࣮ࢪ᳨ฟヨ⸆ࢆస〇ࡍࡿࡓࡵ࡟, ๓⠇࡛ᚓࡽࢀࡓ࣐࢘ࢫᢠయ Ab-M13#71 ࡢྍኚ㒊㑇ఏᏊ (V 㑇ఏᏊ) ࢆࢡ࣮ࣟࢽࣥࢢࡋ࡚ scFv ࢆᵓ⠏ࡋ, ࡑࡢ⤖ྜ⬟ࢆ⢭ᰝࡋ ࡓ. ࡲࡎ, ➨1 ❶➨ 3 ⠇࡜ྠᵝࡢ5’RACE ἲ࡟ࡼࡿV 㑇ఏᏊࡢࢡ࣮ࣟࢽࣥࢢࢆヨࡳࡓ. ๓⠇࡟グࡋࡓࡼ࠺࡟, Ab-M13#71ࡢH㙐ࡢ࢔࢖ࢯࢱ࢖ࣉࡣg2b࡜ྠᐃࡉࢀ࡚࠸ࡿ. ࡑࡇ ࡛, ๓⠇࡛ᶞ❧ࡋࡓࣁ࢖ࣈࣜࢻ࣮࣐ᰴ#71ࡼࡾ⥲RNAࢆᢳฟࡋ, g2b࡟≉␗ⓗ࡞ࣉࣛ࢖ ࣐࣮ (mg2b-GSP-1) ࡜㏫㌿෗㓝⣲ࢆస⏝ࡉࡏࡿࡇ࡜࡟ࡼࡾ, V_H㑇ఏᏊࢆྵࡴcDNAࢆྜ ᡂࡋࡓ. ḟ࠸࡛, ᮏcDNAࢆ㗪ᆺ࡜ࡋ࡚, ᅗ9࡟♧ࡍPCRࢆ㡰ḟ⾜࠸, V_H㑇ఏᏊࢆྵࡴ DNA᩿∦ࢆᚓࡓ. ྠᵝࡢ᧯సࢆL㙐࡟ࡘ࠸࡚ࡶ⾜ࡗࡓࡀ, V_L㑇ఏᏊࡣᚓࡽࢀ࡞࠿ࡗࡓ. ࡑࡢཎᅉ࡜ࡋ࡚, 5’RACEἲ࡟ࡼࡿk㙐ྍኚ㒊㑇ఏᏊ (Vk㑇ఏᏊ) ࡢࢡ࣮ࣟࢽࣥࢢࡣ, ࣁ࢖ࣈࣜࢻ࣮࣐࠿ࡽ ᚓࡽࢀࡿRNA୰࡟NS1࣑࢚࣮࣐ࣟ⣽⬊ࡀඖ᮶ಖ᭷ࡍࡿk㙐ࡢmRNAࡀΰධࡋࡓሙྜ࡟ ጉᐖࢆ࠺ࡅࡸࡍ࠸ࡇ࡜ࡀᣲࡆࡽࢀࡿ. 68) ࡑࡇ࡛, ᮏᢠయࡢ V_L㑇ఏᏊ࡟ࡘ࠸࡚ࡣ Nichols ࡽ 69) ࡟ࡼࡾ㛤Ⓨࡉࢀࡓࣘࢽࣂ࣮ࢧࣝࣉࣛ࢖࣐࣮ࢭࢵࢺࢆ⏝࠸ࡿࢡ࣮ࣟࢽࣥࢢࢆヨࡳࡓ (ᅗ18). ከᵝᛶࢆ♧ࡍVk㑇ఏᏊࡢ5’ᮎ➃ (FR1ࡢ୍㒊ࢆࢥ࣮ࢻࡍࡿ) ࡟ᑐᛂࡍࡿ6✀㢮 ࡢ⦰㔜ࣉࣛ࢖࣐࣮ࢆ5’ࣉࣛ࢖࣐࣮࡜ࡋ࡚PCRࢆ⾜࠺ࡶࡢ࡛࠶ࡿ. mk-GSP-1ࢆࣉࣛ࢖࣐ ࣮࡜ࡋ࡚ྜᡂࡋࡓ cDNA ࢆ㗪ᆺ࡜ࡋ, 6 ✀ࡢ 5’ࣉࣛ࢖࣐࣮ࡢ࠸ࡎࢀ࠿࡜k㙐ᐃᖖ㒊࡟┦ ⿵ⓗ࡞3’ࣉࣛ࢖࣐࣮ (mk-GSP-3) ࢆ⤌ࡳྜࢃࡏࡿ6✀ࡢPCRࢆヨࡳࡓ࡜ࡇࢁ, 5’ࣉࣛ࢖ ࣐࣮࡜ࡋ࡚VL-I/IIIࣉࣛ࢖࣐࣮ࢆ⏝࠸ࡓ࡜ࡁ࡟, V_Lࢆྵࡴ┠ⓗࡢ㑇ఏᏊ᩿∦ࢆᚓࡿࡇ࡜ ࡀ࡛ࡁࡓ. ᅗ 18. ࣘࢽࣂ࣮ࢧࣝࣉࣛ࢖࣐࣮ࢭࢵࢺࢆ⏝࠸ࡿkࢱ࢖ࣉV_L㑇ఏᏊ (Vk) ࡢࢡ࣮ࣟࢽࣥࢢ ᮏ◊✲࡛⣽⬊⼥ྜ࡟⏝࠸ࡓ࣑࢚࣮࣐ࣟ⣽⬊ (P3/NS1/1-Ag-4) ࡣ࣐࢘ࢫᢠయk㙐ࡢഇ㑇ఏᏊࢆ᭷ࡍࡿࡓ ࡵ, 5’RACEἲ࡟ࡼࡿࢡ࣮ࣟࢽࣥࢢ࡟ࡣᅔ㞴ࢆక࠺ࡇ࡜ࡀ࠶ࡿ. Nichollsࡽ࡟ࡼࡗ࡚ሗ࿌ࡉࢀࡓࣘࢽࣂ࣮ ࢧࣝࣉࣛ࢖࣐࣮ࢭࢵࢺ69) ࢆ5’ࣉࣛ࢖࣐࣮࡟⏝࠸ࡓPCRࢆ⾜࠺ࡇ࡜࡟ࡼࡾ, ┠ⓗࡢVk㑇ఏᏊࢆ≉␗ⓗ ࡟ቑᖜࡋ, ࢡ࣮ࣟࢽࣥࢢࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡿ. ࣉࣛ࢖࣐࣮୰ࡢY, R, W, K, M, SࡣࡑࢀࡒࢀCࡲࡓࡣ T, AࡲࡓࡣG, AࡲࡓࡣT, GࡲࡓࡣT, AࡲࡓࡣC, CࡲࡓࡣGࢆ♧ࡍ. CLࡣL㙐ᐃᖖ㒊ࢆ♧ࡍ.

23 ᘬࡁ⥆ࡁ, ᚓࡽࢀࡓ㑇ఏᏊ᩿∦ࢆpBluescript II࣋ࢡࢱ࣮࡬ࢧࣈࢡ࣮ࣟࢽࣥࢢࡋ࡚኱⭠ ⳦XL1-Blue⣽⬊࡬ᑟධࡋ, V㑇ఏᏊࢆಖᣢࡍࡿࢡ࣮ࣟࣥࢆ≉ᐃࡋࡓ. ⤌᥮࠼ࣉࣛࢫ࣑ࢻ ࢆᢳฟࡋ࡚DNAሷᇶ㓄ิࢆゎᯒࡋ, ᑟ࠿ࢀࡿ࢔࣑ࣀ㓟㓄ิࢆ Kabat ࡢᢠయࢩ࣮ࢡ࢚ࣥ ࢫࢹ࣮ࢱ࣮࣋ࢫ 47) ࡜↷ྜࡋࡓ. ࡑࡢ⤖ᯝ, V_Hࢻ࣓࢖ࣥ (125࢔࣑ࣀ㓟࠿ࡽ࡞ࡿ), V_Lࢻ࣓ ࢖ ࣥ (109 ࢔ ࣑ ࣀ 㓟 ࠿ࡽ ࡞ ࡿ) ࢆ ྠ ᐃ ࡋ, CDR ࡢ 㓄 ิ (V_H-CDR1, NTYVH; V_H-CDR2, RIDPANGKTKYDPNFQD; VH-CDR3, DFPYYIGSTYVSFFDV; VL-CDR1,

KASQNVRTDVA; VL-CDR2, LASKRHT; VL-CDR3, LQHRSYPLT) ࡶỴᐃࡍࡿࡇ࡜ࡀ࡛ࡁ ࡓ. ࡲࡓ, ᮏᢠయ࡟ࡘ࠸࡚ࡣ, V_Hࢆࢧࣈࢢ࣮ࣝࣉIIC, V_Lࢆࢧࣈࢢ࣮ࣝࣉI࡟ᖐᒓࡍࡿࡇ ࡜ࡀ࡛ࡁࡓ. 67) ḟ࡟, ᚓࡽࢀࡓሷᇶ㓄ิࢆࡶ࡜࡟, V_H, V_Lྛ㑇ఏᏊࡢ 5’ᮎ➃࡜ 3’ᮎ➃࡟┦⿵ⓗ࡞ࣉࣛ ࢖࣐࣮ࢆࡑࢀࡒࢀタィࡋ࡚PCRࢆ⾜࠸, scFv㑇ఏᏊࢆᵓ⠏ࡍࡿࡓࡵࡢDNAࣇࣛࢢ࣓ࣥ ࢺࢆㄪ〇ࡋࡓ. ࡇࡢ࡜ࡁ, V_Hቑᖜࡢ 3’ࣉࣛ࢖࣐࣮ (M13#71V_H-For) ࡜ V_Lቑᖜࡢ 5’ࣉࣛ ࢖࣐࣮ (M13#71V_L-Rev) ࡟ࡘ࠸࡚ࡣ, V_H࡜V_Lࡢ㐃⤖࡟⏝࠸ࡿᶆ‽ⓗ࡞࣮ࣜࣥ࢝࣌ࣉࢳ ࢻ [(GGGGS)₃; linker1] 21) ࢆࢥ࣮ࢻࡍࡿሷᇶ㓄ิࡢ୍㒊ࢆᑟධࡋࡓ. ࡇࡢ㓄ิࡢ┦⿵ᛶ ࢆ฼⏝ࡍࡿoverlap extension PCR࡟ࡼࡾ, V_H࡜V_Lࢆ㐃⤖ࡋ࡚scFv㑇ఏᏊࢆᵓ⠏ࡋࡓ. ࡞ ࠾, V_Lࡢ3’ࣉࣛ࢖࣐࣮ (FLAG-TAA2) ࡟FLAG ࢱࢢ (DYKDDDDK)

50)

ࢆࢥ࣮ࢻࡍࡿሷ ᇶ㓄ิ࠾ࡼࡧ 2 㔜ࡢ⤊Ṇࢥࢻࣥ (TAATAA) ࢆ௜ຍࡋࡓ. ᚓࡽࢀࡓ scFv 㑇ఏᏊ

(M13-scFv; 5’-VH-linker1-VL-FLAG-TAATAA-3’) ࢆ, pEXmide 5࣋ࢡࢱ࣮

49) ࡟ࢧࣈࢡ࣮ࣟࢽࣥࢢ ࡋ, ኱⭠⳦XL1-Blue⣽⬊࡟ᑟධࡋࡓ. ࢥࣟࢽ࣮PCR࡟ࡼࡾM13-scFv㑇ఏᏊࢆಖᣢࡍࡿ ࢡ࣮ࣟࣥࢆྠᐃࡋ, ࡑࡢሷᇶ㓄ิࢆㄪ࡭࡚, ᢠయ Ab-M13#71 ࡢV_H࡜V_Lࡢ㓄ิࡀಖᣢ ࡉࢀ࡚࠸ࡿࡇ࡜ࢆ☜ㄆࡋࡓ. ᚓ ࡽ ࢀ ࡓ ᙧ ㉁ ㌿ ᥮ ⳦ ࢆ ᇵ 㣴 ࡋ, ྍ ⁐ ᆺ scFv (M13-scFv) ࡀྵࡲࢀࡿ࣌ࣜࣉࣛࢬ࣒ᢳ ฟ ≀ ࢆ ᚓ ࡓ. ࡇ ࢀ ࢆ ➨ 2 ⠇ ࡜ ྠ ᵝ ࡢ ➇ ྜ ELISA࡟௜ࡋࡓ࡜ࡇࢁ, VCSM13ࡢῧຍ㔞ࡢ ቑ ຍ ࡟ ᛂ ࡌ ࡓ ࢩ ࢢ ࢼ ࣝ ࡢ ῶ ᑡ ࡀ ㄆ ࡵ ࡽ ࢀ, ࣇ ࢓ ࣮ ࢪ ࡬ ࡢ⤖ ྜ ⬟ ࡣಖ ᣢ ࡉ ࢀ ࡚ ࠸ࡿ ࡇ ࡜ ࡀ♧ ࡉࢀࡓ (ᅗ 19). ࡲࡓ, ᚓ ࡽࢀࡓ࣌ ࣜࣉ ࣛࢬ࣒ᢳฟ≀ࢆ, ᢠ FLAG ᢠయᅛᐃ໬࢔࢞ ࣮ࣟࢫࢆ⏝࠸࡚࢔ࣇ࢕ࢽࢸ࢕⢭〇ࡋࡓࡢࡕ, SDS-PAGE࡟௜ࡋࡓ࡜ࡇࢁ, ண᝿ࡉࢀࡿศᏊ 㔞 (Mr; 27878.8) ௜㏆࡟༢୍ࡢࣂࣥࢻࡀㄆࡵ ࡽࢀࡓ (ᅗ20). ࡉࡽ࡟, M13-scFvࡢ Kaࢆࣂ࢖࢜ࣞ࢖࣮ࣖ ᖸ ΅ ἲ ࡟ ࡼ ࡾ ⟬ ฟ ࡋ ࡓ. ᮏ ἲ ࡣ ࢭ ࣥ ࢧ ࣮ ඛ ➃ ⾲ 㠃 ୖ ࡢ ࣜ࢞ ࣥ ࢻ ࡟ᑐ ࡍ ࡿ ࢔ ࢼ ࣛ࢖ ࢺ ࡢ ᅗ 19. M13-scFvࢆ⏝࠸ࡿVCSM13ࣇ࢓࣮ ࢪࡢ➇ྜELISA VCSM13ࣇ࢓࣮ࢪࢆᅛᐃ໬ࡋࡓ࣐࢖ࢡࣟࣉ࣮ࣞ ࢺ࡟, 㐟㞳ࡢVCSM13ࣇ࢓࣮ࢪ࠾ࡼࡧM13-scFv ࢆຍ࠼࡚37Υ࡛1᫬㛫➇ྜ཯ᛂࡋࡓࡢࡕ࡟, ᅛ┦ ୖࡢM13-scFv㔞ࢆPODᶆ㆑ᢠFLAGᢠయ᳨࡛ฟ ࡋࡓ.