᪥, ᢠయࡢศᏊᵓ㐀ࢆ㑇ఏᏊ࡛ࣞ࣋ࣝᨵኚࡍࡿࠕᢠయᕤᏛࠖࡼࡾ, ື≀ࡀ⏘⏕ࡋ ᚓ࡞࠸ඃࢀࡓኚ␗ᢠయࢆ㎿㏿ฟࡍࡿࡇࡀྍ⬟どࡉࢀ࡚࠸ࡿ. ᚑ᮶ἲ࡛ࡣᚓ㞴࠸
㧗ぶຊᢠయࢆฟ࡛ࡁࡿ࡞ࡽࡤ, ච ᐃἲࡢ㧗ឤᗘ㈨ࡍࡿࡇࢁᮇᚅࡉ
ࢀࡿ. ከᵝᛶᐩࡴኚ␗ scFv ࣛࣈ࣮ࣛࣜࡽ, ᢠཎ⤖ྜ⬟ࡢྥୖࡋࡓኚ␗యࢆຠ⋡
ࡼࡃ㑅ูࡍࡿࡓࡵ, ࣇ࣮ࢪᥦ♧ἲࡀỗ⏝ࡉࢀࡿ. ᮏἲ࡛ࡣ, അ↛⏕ᡂࡋࡓᕼᑡ࡞
ᨵⰋᆺ scFv ࢆᥦ♧ࡍࡿࣇ࣮ࢪࢆ, ࣃࣥࢽࣥࢢ (ᢠཎ⤖ྜ⬟ᇶ࡙ࡃࣇࢽࢸᢳ ฟ) ࠸࠺ᡭἲ࡛, 㔞⏕ࡍࡿᨵᝏᆺscFvᥦ♧ࣇ࣮ࢪࡽศ㞳ࡋ࡚༢㞳ࡍࡿ. ࡋ
ࡋ, ࣃࣥࢽࣥࢢࡣ⥴ㄽ࡛㏙ࡓၥ㢟ࢆෆໟࡍࡿࡓࡵ, ‶㊊ࡢ࠸ࡃኚ␗యࢆᚓࡿࡲ࡛
ከࡢປຊࢆせࡍࡿࡢࡀᐇ࡛࠶ࡗࡓ.
ᮏ◊✲࡛ࡣ, ࡇࡢၥ㢟ࡢᢤᮏⓗ࡞ゎỴࢆ┠ᣦࡋ࡚, ࣞᆺࣉࣟࣇࣜࣥࢢࢩࢫࢸ
࣒ࢆᵓ⠏ࡋࡓ. ➨1 ❶࡛ࡣ, CS ࢆࣔࢹࣝᶆⓗᢠཎࡋ࡚ኚ␗ scFv ࣛࣈ࣮ࣛࣜࢆస〇 ࡋ, ᪂つ㑅ᢥἲࡢ༢㞳ຠ⋡ࢆホ౯ࡍࡿࡓࡵࡢᑐ↷ᐇ㦂ࡋ࡚, ᚑ᮶࠾ࡾࡢࣃࣥࢽࣥࢢ
ࡼࡿ≉␗ᢠయࡢ㑅ูࢆ⾜ࡗࡓ. ᛕࡢࡓࡵ, CSࢆᅛᐃࡋࡓᅛ┦ࡽࡢ⁐ฟ᮲௳ࡣ㓟ᛶ
ሷᇶᛶࡢ2 ᮲௳ࡋ, ࡑࢀࡒࢀࡘ࠸࡚ 3 㔜ࡢᐇ㦂ࢆే⾜ࡋ࡚⾜ࡗࡓ. ࡋࡋ, 㔝⏕
ᆺࡢwt-scFvࡼࡾࡶぶຊࡢྥୖࡋࡓࢡ࣮ࣟࣥࡣ, ᚓࡽࢀࡓࡶࡢࡢ Kaࡋ࡚109 M-1ࡢ
ࣞ࣋ࣝࡲࡗࡓ. ࡋࡶ, ༢㞳ࡉࢀࡓࢡ࣮ࣟࣥࡣከᵝᛶஈࡋࡃ, ྠ୍ࡢࢡ࣮ࣟࣥ
ࡀ 」 ᩘ ᚓ ࡽ ࢀ ࡓ. ➨ 2 ❶ ࡛ ࡣ ᮏ ἲ ࡢ ᵓ ⠏ ᚲ 㡲 ࡛ ࠶ ࡿ ࠕ ࣇ ࣮ ࢪ ᳨ ฟ ヨ ⸆ ࠖ
(M13-scFv-GLuc) ࢆస〇ࡋࡓ. ࣇ࣮ࢪᥦ♧ᖖ⏝ࡉࢀࡿM13ࣇ࣮ࢪᑐࡍࡿࣔࣀࢡ࣮ࣟ
ࢼࣝᢠయࢆ᪂つస〇ࡋ, ࡇࢀࢆ scFv ࡋ࡚Ⓨග㓝⣲ GLucࡢ⼥ྜࢱࣥࣃࢡ㉁ࡋࡓ.
࣏࣮ࣞࢱ࣮ศᏊࡋ࡚㑅ᢥࡋࡓGLucࡣ㧗ឤᗘ᳨ฟࡀྍ⬟࡛࠶ࡿࡇຍ࠼, ᇶ㉁ࡢῧ ຍᚋ, ┤ࡕⓎගࡍࡿᛶ㉁ࢆഛ࠼, ┠ⓗࡢࣉࣟࣇࣜࣥࢢࢩࢫࢸ࣒ࢆᵓ⠏ࡍࡿ࠺࠼࡛
ከࡃࡢⅬࢆ᭷ࡋ࡚࠸ࡓ. ≉, 㓟ࡸࣝ࢝ࣜ࡞ࡢ㓝⣲ᛂṆᾮࢆຍ࠼ࡿᚲせࡀ࡞
࠸ࡓࡵ, ᇶ㉁ࢆᗘῧຍࡍࡿࡇࡼࡾ⧞ࡾ㏉ࡋⓎගࡉࡏࡿࡇࡀྍ⬟࡛࠶ࡾ, off-rate
ࡼࡿ㑅ᢥࢆᐇࡍࡿࡓࡵᴟࡵ࡚᭷࡛࠶ࡗࡓ.
➨3❶࡛ࡣ✀ࠎࡢᐇ㦂᮲௳ࢆᩚ࠼ࡓ⤖ᯝ, ࣞᆺࣉࣟࣇࣜࣥࢢࢩࢫࢸ࣒ࡢᵓ⠏
ᡂຌࡋࡓ. ᮏἲࡣᢠཎ≉␗ⓗ࡞ᢠయᥦ♧ࣇ࣮ࢪࡢ⏘⏕ ⬟ࢆᙧ㉁㌿⳦ࢡ࣮ࣟࣥࡈ
ࢫࢡ࣮ࣜࢽࣥࢢࡍࡿࡶࡢ࡛࠶ࡾ, 1990 ᖺ௦ሗ࿌ࡉࢀࡓ colony-lift assay (CLA; ᐮ ኳᇵᆅୖᒎ㛤ࡉࢀࡓᙧ㉁㌿⳦ࢥࣟࢽ࣮ࡢࣞࣉࣜ࢝ࢆ࣓ࣥࣈࣛࣥୖస〇ࡋ, ࡶ࠺ 1 ᯛࡢ࣓ࣥࣈࣛࣥࢆ㔜ࡡ࡚ྛࢥࣟࢽ࣮ࡀ⏘⏕ࡍࡿᢠయࢆࢫ࣏ࢵࢺࡋ࡚㌿ࡋ, ྛࢫ࣏
ࢵࢺࡢᢠཎ⤖ྜ⬟ࢆྍどࡍࡿࡶࡢ)86) 㢮ఝࡢࢥࣥࢭࣉࢺᇶ࡙࠸࡚࠸ࡿゝ࠼ࡿ. ࡋࡋ, CLA ࡛ࡣឤᗘࡸ⌧ᛶ㞴ࡀ࠶ࡿ࠺࠼, 㑇ఏᆺ (ࢥࣟࢽ࣮) –⾲⌧ᆺ (ᢠయ) ࡢ ᑐᛂࡢ⥔ᣢࡶࢫࢺࣞࢫࢆక࠸, ᬑཬࡣ⮳ࡽ࡞ࡗࡓ. ࣞᆺࣉࣟࣇࣜࣥࢢࢩ ࢫࢸ࣒࡛ࡣ, ࣇ࣮ࢪࡢ」〇⬟ຊ㧗ឤᗘ࡞ࣇ࣮ࢪ᳨ฟヨ⸆ (M13-scFv-GLuc) ࢆά
⏝ࡋ, ᙧ㉁㌿⳦ࡢࢡ࣮ࣟࢽࣥࢢቑṪ, ࣇ࣮ࢪࡢ⏘⏕, ࡑࡋ࡚ࡑࡢᢠཎ⤖ྜ⬟ࡢホ
49
౯ࡲ࡛ࢆྠ୍ࡢ࣐ࢡ࢙࡛ࣟ࢘ࣝ⾜࠺ࡇࡼࡾ, ୖグࡢḞⅬࢆඞ᭹ࡍࡿࡇᡂຌ
ࡋࡓ. ᮏ❶࡛ᚓࡽࢀࡓ scFv ࢡ࣮ࣟࣥࡣ, ྠ୍ࡢࣛࣈ࣮ࣛࣜࡽ㑅ᢥ᧯సࢆ⾜ࡗ࡚࠸
ࡿࡶࢃࡽࡎ, ➨1❶࡛ᚓࡽࢀࡓኚ␗scFvࢡ࣮ࣟࣥࡢඹ㏻ᛶࡣࡃぢࡽࢀࡎ, ᫂
ࡽぶຊඃࢀ࡚࠸ࡓ. ື≀ࢆචࡍࡿᚑ᮶ࡢ᪉ἲ࡛, CSᑐࡋ࡚1010࣮࢜ࢲ࣮ࡢ Kaࢆ♧ࡍ㧗ぶຊᢠయࢆస〇ࡋࡓࡣᑡ࡞࠸.42) ᮏࣉࣟࣇࣜࣥࢢࢩࢫࢸ࣒࡛ࡣ, ➨ 3❶➨3⠇➨4⠇ࡢࢃࡎ2ᅇࡢヨ⾜࡛, ࡋࡶ~105ࢡ࣮ࣟࣥࣞ࣋ࣝࡢᑠࡉ࠸ࣛࣈ
࣮ࣛࣜࡢ3%ࢆฎ⌮ࡋࡓࡔࡅ࡛, 1010࣮࢜ࢲ࣮ࡢኚ␗ᢠCSᢠయࣇࣛࢢ࣓ࣥࢺࡀィ5✀ࡶ
ᚓࡽࢀ࡚࠸ࡿ. ࢩࢫࢸ࣒ࢆ⮬ືࡍࡿࡇ࡛, ࣛࣈ࣮ࣛࣜࡢࢡ࣮ࣟࣥࡢฎ⌮ࡀྍ⬟
࡞ࡗࡓࡁࡣ, ᩘ༑ࢆ㉸࠼ࡿ㧗ぶຊኚ␗ scFv ࡀ୍ᣲᚓࡽࢀࡿࡶࡢᮇᚅࡉࢀ
ࡿ.
ᮏ◊✲࡛ࡣ, ࣞᆺࣉࣟࣇࣜࣥࢢࢩࢫࢸ࣒ࡀ, ᚑ๓ࡢࣃࣥࢽࣥࢢ࡛ࡣᅇࡀᅔ 㞴࡞ᨵⰋศᏊ✀ࡢ⋓ᚓࢆྍ⬟ࡍࡿࡇࢆᐇドࡋࡓ. ᚋ, ᮏࢩࢫࢸ࣒ࡀ, 㧗ศᏊᢠཎ
ࢆྵࡵࡓᵝࠎ࡞ᶆⓗྜ≀ᑐࡍࡿᐇ⏝ᢠయࡢ⏘⏕㈉⊩ࡍࡿࡇࢆᮇᚅࡍࡿ.
50
ㅰ㎡
ᮏ◊✲ࡢᶵࢆ࠼࡚࠸ࡓࡔࡁ, ⤊ጞᚚ᠓⠜࡞ᚚᣦᑟ, ᚚ㠴᧡ࢆ㈷ࡾࡲࡋࡓᮏᏛ⏕
ศᯒᏛ◊✲ᐊᩍᤵ ᑠᯘ⿱ඛ⏕ㅽࢇ࡛ឤㅰ⏦ࡋୖࡆࡲࡍ.
ᮏ ◊ ✲ 㝿 ࡋ, ᭷ ┈ ࡞ ᚚ ຓ ゝ ᚚ ༠ ຊ ࢆ ㈷ ࡾ ࡲ ࡋ ࡓ ᮏ Ꮫ ⏕ ศ ᯒ Ꮫ ◊ ✲ ᐊ ㅮ ᖌ
ᒣᾈஅඛ⏕, ྠຓᡭ ᳃⏣࠸ࡎࡳඛ⏕῝ㅰ࠸ࡓࡋࡲࡍ.
ᮏ◊✲ࡢㄽᩥసᡂ㝿ࡋ, ㈗㔜࡞ᚚຓゝ, ᚚᣦᑟࢆ㈷ࡾࡲࡋࡓᮏᏛ་⸆⣽⬊⏕≀Ꮫ◊
✲ᐊᩍᤵ ኈఙඛ⏕, ྠ⸆Ꮫ◊✲ᐊᩍᤵ ዟ⏣ඛ⏕, ྠ⏕Ꮫ◊✲ᐊᩍᤵ
ℿ୰㔛⨾ඛ⏕ཌࡃ࠾♩⏦ࡋୖࡆࡲࡍ.
ᮏ ◊ ✲ ࢆ ⾜ ࠺ ࠶ ࡓ ࡾ, ከ ࡞ ᚚ ༠ ຊ ࢆ ࠸ ࡓ ࡔ ࡁ ࡲ ࡋ ࡓ ᮏ Ꮫ ⏕ ศ ᯒ Ꮫ ◊ ✲ ᐊ
࡛◊✲⏕άࢆඹࡋࡓ༞ᴗ⏕࡞ࡽࡧᏛ⏕ㅖẶᚰࡽឤㅰ࠸ࡓࡋࡲࡍ.
ᮏ◊✲࡛ࡢ scFv ࢱࣥࣃࢡ㉁ࡢ࣑ࣀ㓟ศᯒࡘࡁࡲࡋ࡚, ᮏᏛ୰ኸศᯒᩍᤵ ➉ ෆᩔᏊඛ⏕ከࡢ࠾ຊῧ࠼ࢆ࠸ࡓࡔࡁࡲࡋࡓ. ࡇࡇឤㅰࡢពࢆ⾲ࡋࡲࡍ.
➨1❶࠾ࡼࡧ➨2❶ࡢ◊✲࠾࠸࡚, ᬮ࠸ᚚᨭࢆ㈷ࡾࡲࡋࡓᮾᏛᏛ㝔་Ꮫ
⣔◊✲⛉ᩍᤵ ⩚ಇᩥඛ⏕ឤㅰ࠸ࡓࡋࡲࡍ. ࡲࡓ, pEXmide 5 ࣋ࢡࢱ࣮ࢆᚚᜨ
ୗࡉ࠸ࡲࡋࡓࢫ࢙࣮࢘ࢹࣥ Lund Ꮫᩍᤵ Carl A. K. Borrebaeck ඛ⏕, ࢫ࢙࣮࢘ࢹࣥ
Avena Partners AB Eskil Söderlind༤ኈ῝ㅰ࠸ࡓࡋࡲࡍ.
᭱ᚋ, ༤ኈㄢ⛬ᅾ⡠୰, ࠶ࡽࡺࡿ㠃࡛⚾ࢆᨭ࠼࡚ࡃࢀࡓ୧ぶឤㅰ࠸ࡓࡋࡲࡍ.
51
ᐇ㦂ࡢ㒊
◊✲⯡㛵ࡍࡿ㡯┠
ᮏ◊✲⏝࠸ࡓせ࡞⨨, ࢯࣇࢺ࢙࢘, ჾᮦ࣭ヨ⸆࡞ྛ✀ᐇ㦂ࡢᆺᘧ, ᛶ≧, ㉎ ධඛ࡞ࡘ࠸࡚௨ୗ୍ᣓࡍࡿ. ື≀ᐇ㦂ࡣ, ⚄ᡞ⸆⛉Ꮫື≀ᐇ㦂ጤဨࡢᢎㄆࢆ
ᚓࡓࡢࡕ, ⚄ᡞ⸆⛉Ꮫື≀ᐇ㦂ᐇつ⛬ࢆ㑂Ᏺࡋ, ື≀ᚲせ࡞ⱞ③ࢆ࠼ࡠࡼ࠺,
༑ศ㓄៖ࡋ࡚⾜ࡗࡓ. ࡲࡓ, ⤌࠼DNAᐇ㦂ࡣ⚄ᡞ⸆⛉Ꮫ⤌࠼DNAᐇ㦂Ᏻጤ ဨࡢᢎㄆࢆᚓࡓࡢࡕ, ྠጤဨࡢᣦ㔪࠾ࡼࡧࠕ㑇ఏᏊ⤌࠼⏕≀➼ࡢ⏝➼ࡢつไ
ࡼࡿ⏕≀ࡢከᵝᛶࡢ☜ಖ㛵ࡍࡿἲᚊࠖࢆ㑂Ᏺࡋ࡚⾜ࡗࡓ.
i. ⨨
⣸እྍど྾ගᗘࡢ ᐃ (DNA ࠾ࡼࡧࢱࣥࣃࢡ㉁ࡢᐃ㔞, ⭠⳦ᐦᗘࡢ ᐃ࡞) ࡣ, Ultrospec 2100ศගගᗘィ (Amersham Bioscience) ࢆ⏝࠸ࡓ.
PCRࡼࡿDNAࡢቑᖜࡣ, T100 Thermal Cycler (Bio-Rad) ࢆ⏝࠸ࡓ.
ᢠయࣇࣛࢢ࣓ࣥࢺࡢSDS-PAGEࡣ, X Cell SureLock (Invitrogen) ࢆ⏝࠸ࡓ. ࡲࡓ, ࣈ
ࣟ ࢵ ࢸ ࣥ ࢢ ศ ᯒ ࠾ ࡅ ࡿ ࢱ ࣥ ࣃ ࢡ ㉁ ࡢ ㌿ ࡣ, iBlot Gel Transfer System (Invitrogen) ࢆ⏝࠸ࡓ.
⭠⳦ࡢ㟁Ẽ✸Ꮝἲࡼࡿࣉࣛࢫ࣑ࢻࡢᑟධࡣ, ECM 630 (BTX) ࢆ⏝࠸ࡓ.
ELISA࠾ࡅࡿPODάᛶࡢྍど㒊྾ගᗘ ᐃ (ẚⰍ ᐃ) ࡣ, iMark࣐ࢡࣟࣉࣞ
࣮ࢺ࣮ࣜࢲ࣮ (Bio-Rad) ࢆ⏝࠸ࡓ.
GLucάᛶࡢ ᐃ (Ⓨග ᐃ) ࡣ, Synergy HTXࣉ࣮ࣞࢺ࣮ࣜࢲ࣮ (BioTek) ࡲࡓࡣ FLUOstar OPTIMAࣉ࣮ࣞࢺ࣮ࣜࢲ࣮ (BMG Labtech) ࢆ⏝࠸ࡓ.
ࢺ ࣜ ࢳ ࢘ ࣒ ᶆ ㆑ య ࢆ ⏝ ࠸ ࡿ Scatchard ゎ ᯒ ࠾ ࡅ ࡿ ᨺ ᑕ ⬟ ࡢ ᐃ ࡣ, Tri-Carb 2900TRᾮయࢩࣥࢳ࣮ࣞࢩࣙࣥ࢝࢘ࣥࢱ࣮ (Perkin Elmer) ࢆ⏝࠸ࡓ.
ࣂ ࢜ ࣞ ࣖ ࣮ ᖸ ΅ ἲ ࡼ ࡿ ᢠ య ࣇ ࣛ ࢢ ࣓ ࣥ ࢺ ࡢ ⤖ ྜ ࣃ ࣛ ࣓ ࣮ ࢱ ࣮ ࡢ ᐃ ࡣ, BLItz (Pall ForteBio) ࢆ⏝࠸ࡓ.
ii. ჾᮦ
ẚⰍ ᐃࡼࡿELISA⏝࠸ࡓCostar 96࢙࣐࢘ࣝࢡࣟࣉ࣮ࣞࢺ (No. 3590) ࠾ࡼ
ࡧࣞᆺࣉࣟࣇࣜࣥࢢ⏝࠸ࡓ Costar ⓑⰍ96࢙࣐࢘ࣝࢡࣟࣉ࣮ࣞࢺ (No.
3922) ࡣ, Corningࡽ㉎ධࡋࡓ.
ᢠయࣛࣈ࣮ࣛࣜࡢࣃࣥࢽࣥࢢ⏝࠸ࡓࢳ࣮ࣗࣈ (75 mm12 mm͆Maxisorp͇࣏ࣜ
ࢫࢳࣞࣥヨ㦂⟶) ࡣ, Nuncࡽ㉎ධࡋࡓ.
⣽⬊ᇵ㣴⏝࠸ࡓ⁛⳦῭ࡳࢹࢫ࣏࣮ࢨࣈࣝࣇࣛࢫࢥ, ࢩ࣮ࣕࣞ, ࢡࣛࢫࢱ࣮ࢹ
ࢵࢩࣗ, ࣆ࣌ࢵࢺ㢮ࡣ, Corningࡲࡓࡣᮾὒჾᮦࡽ㉎ධࡋࡓ.
ᨺᑕ⬟ ᐃ⏝࠸ࡓࢩࣥࢳ࣮ࣞࢩࣙࣥࣂࣝࡣ, Iwaki glassࡽ㉎ධࡋࡓ.
52 iii. ࢯࣇࢺ࢙࢘
PCR⏝ࣉ࣐࣮ࣛ࠾ࡼࡧᏛྜᡂscFv㑇ఏᏊࡢᵓ⠏⏝࠸ࡿ୍ᮏ㙐࢜ࣜࢦDNAࡢ タィࡣ, OligoTM program version 4.0 (National Bioscience) ࢆ⏝࠸ࡓ.
DNAࡢሷᇶ㓄ิࢹ࣮ࢱࡢゎㄞࡣ, Sequence Scanner Software 2 (Applied Biosystems) ࢆ
⏝࠸ࡓ.
࣑ࣀ㓟㓄ิࡢ᥎ᐃࡣ, ExPASy Translate tool (https://web.expasy.org/translate/) ࢆ
⏝ࡋࡓ.
ᢠ య ࣇ ࣛ ࢢ ࣓ ࣥ ࢺ ࡢ ❧ య ᵓ 㐀 ࣔ ࢹ ࣜ ࣥ ࢢ ࡣ, SWISS-MODEL ࢧ ࣮ ࣂ ࣮ (http://swissmodel.expasy.org)83) ࢆ⏝ࡋࡓ.
ᢠ య ࣇ ࣛ ࢢ ࣓ ࣥ ࢺ ᢠ ཎ ࡢ ࢻ ࢵ ࢟ ࣥ ࢢ ࣔ ࢹ ࣝ ࡢ స 〇 ࡣ, SwissDock ࢧ ࣮ ࣂ ࣮ (http://www.swissdock.ch)84) ࢆ⏝ࡋࡓ.
iv. ⦆⾪ᾮ
௨ୗࡢ␎ྕ࡛⾲グࡍࡿྛ⦆⾪ᾮࡢ⤌ᡂࢆ♧ࡍ. PB: 50 mM NaH2PO4-Na2HPO4 (pH 7.3).
PBS: 9.0 g/L NaClࢆྵࡴPB.
PBS-2: NaCl (137 mM), Na2HPO4 (10.0 mM), KCl (2.68 mM), ࠾ࡼࡧKH2PO4 (1.76 mM) ࡢỈ⁐ᾮࢆpH 7.4ㄪᩚࡋ, ࣮࢜ࢺࢡ࣮ࣞࣈ⁛⳦ࡋࡓࡶࡢ.
G-PBS: 1.0 g/LࡢࢮࣛࢳࣥࢆྵࡴPBS-2.
PVG-PBS: 1.0 g/Lࡢ࣏ࣜࣅࢽࣝࣝࢥ࣮ࣝ (ᖹᆒ㔜ྜᗘ500) ࢆྵࡴG-PBS.
T-PBS: 0.050 (v/v) %ࡢTween 20ࢆྵࡴPBS.
T-PBS-2: 0.10 (v/v) %ࡢTween 20ࢆྵࡴPBS-2.
M-PBS: 20 g/Lࡢࢫ࣒࣑࢟ࣝࢡࢆྵࡴPBS.
M-PBS-2: 20 g/Lࡢࢫ࣒࣑࢟ࣝࢡࢆྵࡴPBS-2.
TBS: NaCl (137 mM), KCl (2.68 mM), ࠾ ࡼ ࡧ tris(hydroxymethyl)aminomethane (Tris) (24.8 mM) ࡢỈ⁐ᾮࢆ3 M HCl࡛pH 7.4ㄪᩚࡋࡓࡶࡢ.
CB: 0.1 M NaHCO3-Na2CO3 (pH 8.6).
TAE⦆⾪ᾮ: ࢼ࢝ࣛࢸࢫࢡࡽ㉎ධࡋࡓ.
ᾐ ㏱ ᅽ ࢩ ࣙ ࢵ ࢡ ⏝ ⦆ ⾪ ᾮ: ࢫ ࢡ ࣟ ࣮ ࢫ (584 mM), ethylenediaminetetraacetic acid (EDTA, 1.0 mM), ࠾ࡼࡧTris-HCl (50 mM; pH 8.0) ࡢỈ⁐ᾮࢆࢁ㐣⁛⳦ࡋࡓࡶࡢ.
v. ࢫࢸࣟࢻ㢮
CS࠾ࡼࡧCSࣛ࣋ࣝ⏝ㄏᑟయ [CS 3-(O-carboxymethyl)oxime (CS-3CMO)] ࡣ, Sigma-Aldrichࡽ㉎ධࡋࡓ.
53 vi. ࢺࣜࢳ࣒࢘ᶆ㆑ྜ≀
[1,2,6,7-3H (N)]-CS (3.44 TBq/mmol) ࡣ, PerkinElmerࡽ㉎ධࡋࡓ.
vii. ᢠయ㢮࠾ࡼࡧᢠయ㛵㐃ヨ⸆
ᢠFLAGᢠయᅛᐃ࣮࢞ࣟࢫࡣ, Sigma-Aldrichࡽ㉎ධࡋࡓ. PODᶆ㆑ᢠ࣐࢘ࢫIgGᢠయࡣ, Jackson ImmunoResearchࡽ㉎ධࡋࡓ. PODᶆ㆑ᢠM13ࣔࣀࢡ࣮ࣟࢼࣝᢠయࡣ, GE Healthcareࡽ㉎ධࡋࡓ. PODᶆ㆑ᢠFLAGᢠయࡣ, Sigma-Aldrichࡽ㉎ධࡋࡓ.
viii. ᢠయ௨እࡢචᏛ㛵㐃ヨ⸆
AbISCO-100ࢪࣗࣂࣥࢺࡣ, Isconova ABࡽ㉎ධࡋࡓ.
EZ-Link NHS-LC-Biotin (ࣅ࢜ࢳࣥᶆ㆑ヨ⸆) ࡣ, Thermo Fisher Scientificࡽ㉎ධࡋࡓ. Biotin N-succinimidyl ester (ࣅ࢜ࢳࣥᶆ㆑ヨ⸆) ࡣ, Sigma-Aldrichࡽ㉎ධࡋࡓ. ࢭࣞࣥࢸࣛࢪࣥࡣ, NanoLightࡲࡓࡣPromegaࡽ㉎ධࡋࡓ.
ix. 㓝⣲㢮
ไ㝈㓝⣲㢮: Xma I, Sal I, Nco I, Xho I, Not I, Spe I, Sfi I (ྛ10 U/mL) ࡣ, New England Biolabsࡽ㉎ධࡋࡓ.
DNA ࣏࣓࣮ࣜࣛࢮ㢮: AmpliTaq ࣏࣓࣮ࣜࣛࢮ (5 U/ mL) ࡣ Thermo Fisher Scientific
ࡽ, Ex Taq DNA࣏࣓࣮ࣜࣛࢮ (5 U/ mL) ࡣ TaKaRaࡽ, KOD࣏࣓࣮ࣜࣛࢮ (2.5 U/ mL) ࠾ࡼࡧKOD Fx࣏࣓࣮ࣜࣛࢮ (1 U/ mL) ࡣ TOYOBOࡽ, Pfu DNA࣏࣓ࣜࣛ
࣮ࢮࡣPromegaࡽ, ࡑࢀࡒࢀ㉎ධࡋࡓ.
⤌࠼DNAᐇ㦂⏝࠸ࡓࡑࡢࡢ㓝⣲: Superscript II reverse transcriptase (200 U/mL)
ࡣInvitrogenࡽ, T4 DNA࣮ࣜ࢞ࢮ (400 U/mL) ࡣNew England Biolabsࡽ, ࡑࢀࡒ
ࢀ㉎ධࡋࡓ.
x. ᇶ㉁⁐ᾮ
ẚⰍ ᐃ⏝PODᇶ㉁⁐ᾮ: ࢡ࢚ࣥ㓟 (25 mM) ࠾ࡼࡧNa2HPO4 (50 mM) ࡢྛỈ⁐ᾮ
ࢆΰྜࡋ࡚pH 5.0ㄪᩚࡋࡓ⁐ᾮ, 30% (w/v) H2O2ࢆయ✚ẚ5000 : 3࡛ΰྜࡋࡓ⁐
ᾮ (25 mL) , o-ࣇ࢙ࢽࣞࣥࢪ࣑࣭ࣥ2ሷ㓟ሷ (10 mg) ࢆ⁐ゎࡋࡓࡶࡢ.
Ⓨග ᐃ⏝GLucᇶ㉁⁐ᾮ: ࢭࣞࣥࢸࣛࢪࣥ (5.0 mg) ࢆ0.10 M HClࢆྵࡴ࣓ࢱࣀ࣮
ࣝ (1.18 mL) ⁐ゎࡋࡓࡢࡕ, ࢭࣞࣥࢸࣛࢪࣥࡋ࡚5.0ࡲࡓࡣ 20 mM ࡞ࡿࡼ࠺
PBS࡛ᕼ㔘ࡋࡓࡶࡢ.
xi. ࡑࡢࡢヨ⸆࣭ჾᮦ
ࢮࣛࢳࣥ, SDS, ࣥࣆࢩࣜࣥࢼࢺ࣒ࣜ࢘, ◲㓟࢝ࢼ࣐ࢩࣥ, TMB, Tween 20, ࣏ࣜ
54
ࣅࢽࣝࣝࢥ࣮ࣝ (㔜ྜᗘ⣙500), ࢡࣜࢰࣝII, ࠾ࡼࡧIPTG ࡣ, ࢼ࢝ࣛࢸࢫࢡ
ࡽ㉎ධࡋࡓ.
o-ࣇ࢙ࢽࣞࣥࢪ࣑࣭ࣥ2ሷ㓟ሷ, BSA, OVA, ࠾ࡼࡧPEG8000ࡣ, Sigma-Aldrichࡽ
㉎ධࡋࡓ.
PEG4000ࡣ, Merckࡽ㉎ධࡋࡓ.
ࢫ࣒࣑࢟ࣝࢡࡣ, BD Biosciencesࡽ㉎ධࡋࡓ.
ࣈࣟࢵࢡ࢚࣮ࢫ⢊ᮎࡣ, DSࣇ࣮࣐ࣂ࣓࢜ࢹ࢝ࣝࡽ㉎ධࡋࡓ.
ࡑࡢࡢ⏕Ꮫ⏝ヨ⸆, ศᏊ⏕≀Ꮫ⏝ヨ⸆࡞ࡽࡧ᭷ᶵྜᡂ⏝ヨ⸆ࡣヨ ⸆≉⣭ࢆ
⏝࠸ࡓ.
xii. ᇵᆅ㢮
A) ࣐࢘ࢫ⣽⬊⏝ᇵᆅ
ᇶᮏᇵᆅ: 1.0 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) -NaOH⦆⾪
ᾮ (pH 7.3) [1% (v/v)] ࠾ࡼࡧ◲㓟࢝ࢼ࣐ࢩࣥ (100 mM) ࢆྵࡴRPMI-1640.
ࣁࣈࣜࢻ࣮࣐⏝ᇵᆅ:࢘ࢩ⫾ඣ⾑Ύ [10% (v/v)], 2-࣓ࣝ࢝ࣉࢺ࢚ࢱࣀ࣮ࣝ (50 mM),
L-ࢢࣝࢱ࣑ࣥ (2.0 mM), ࠾ࡼࡧࣆࣝࣅࣥ㓟ࢼࢺ࣒ࣜ࢘ (1.0 mM) ࢆྵࡴᇶᮏᇵᆅ. Hypoxanthine-thymidine (HT) ᇵᆅ: ࣄ࣏࢟ࢧࣥࢳࣥ (0.10 mM) ࠾ࡼࡧࢳ࣑ࢪࣥ (16
mM) ࢆྵࡴࣁࣈࣜࢻ࣮࣐⏝ᇵᆅ.
HATᇵᆅ: ࣑ࣀࣉࢸࣜࣥ (0.40 mM) ࢆྵࡴHTᇵᆅ.
࡞࠾, RPMI-1640ᾮయᇵᆅ࠾ࡼࡧ࢘ࢩ⫾ඣ⾑ΎࡣGIBCOࡽ, HATᇵᆅࢧࣉ࣓ࣜࣥࢺ
࠾ࡼࡧHTᇵᆅࢧࣉ࣓ࣜࣥࢺࡣSigma-Aldrichࡽ, ࡑࢀࡒࢀ㉎ධࡋࡓ. B) ⭠⳦࠾ࡼࡧࣇ࣮ࢪ⏝ᇵᆅ
2YTᇵᆅ: Bacto tryptone (16 g/L), Bacto yeast extract (10 g/L), ࠾ࡼࡧNaCl (5.0 g/L) ࡢỈ⁐ᾮࢆpH 7.0ㄪᩚࡋ, ࣮࢜ࢺࢡ࣮ࣞࣈ⁛⳦ࡋࡓࡶࡢ.
2YT-AG (1%) ᇵ ᆅ: ࣥ ࣆ ࢩ ࣜ ࣥ ࢼ ࢺ ࣜ ࢘ ࣒ (100 mg/L) ࠾ ࡼ ࡧ D-ࢢ ࣝ ࢥ ࣮ ࢫ (10 g/L) ࢆྵࡴ2YTᇵᆅ.
2YT-AG (2%) ᇵ ᆅ: ࣥ ࣆ ࢩ ࣜ ࣥ ࢼ ࢺ ࣜ ࢘ ࣒ (100 mg/L) ࠾ ࡼ ࡧ D-ࢢ ࣝ ࢥ ࣮ ࢫ (20 g/L) ࢆྵࡴ2YTᇵᆅ.
2YT-AK (1%) ᇵᆅ: ࣥࣆࢩࣜࣥࢼ ࢺ࣒ࣜ࢘ (100 mg/L) ࠾ࡼࡧ◲㓟࢝ࢼ࣐ࢩ
ࣥ (50 mg/L) ࢆྵࡴ2YTᇵᆅ.
2YT-ATG (1%) ᇵᆅ: ࣥࣆࢩࣜࣥࢼࢺ࣒ࣜ࢘ (100 mg/L), ࢸࢺࣛࢧࢡࣜࣥሷ㓟 ሷ (10 mg/L), ࠾ࡼࡧD-ࢢࣝࢥ࣮ࢫ (10 g/L) ࢆྵࡴ2YTᇵᆅ.
2YT-ATG (2%) ᇵᆅ: ࣥࣆࢩࣜࣥࢼࢺ࣒ࣜ࢘ (100 mg/L), ࢸࢺࣛࢧࢡࣜࣥሷ㓟 ሷ (10 mg/L), ࠾ࡼࡧD-ࢢࣝࢥ࣮ࢫ (20 g/L) ࢆྵࡴ2YTᇵᆅ.
SOB (-) ᇵᆅ: Bacto tryptone (20 g/L), Bacto yeast extract (5.0 g/L), NaCl (0.50 g/L), ࠾ࡼ
ࡧKCl (0.186 g/L) ࡢỈ⁐ᾮࢆpH 7.0ㄪᩚࡋ, ࣮࢜ࢺࢡ࣮ࣞࣈ⁛⳦ࡋࡓࡶࡢ.
55
SOCᇵᆅ: MgCl2 (5.0 mM), MgSO4 (5.0 mM), ࠾ࡼࡧD-ࢢࣝࢥ࣮ࢫ (20 mM) ࢆSOB (-) ᇵᆅΰྜࡋࡓࡶࡢ.
ࢱࣥࣃࢡ㉁Ⓨ⌧⏝ㄏᑟᇵᆅ: ࢫࢡ࣮ࣟࢫ (0.40 M), IPTG (0.10 mM), ࠾ࡼࡧࣥࣆࢩ
ࣜࣥࢼࢺ࣒ࣜ࢘ (100 mg/L) ࢆྵࡴ2YTᇵᆅ.
ࢺࢵࣉ࣮࢞: Bacto agar (7.0 g/L) ࢆྵࡴ2YTᇵᆅࢆ࣮࢜ࢺࢡ࣮ࣞࣈ⁛⳦ࡋࡓࡶࡢ. TYE࣮࢞: Bacto tryptone (10 g/L), Bacto yeast extract (5.0 g/L), Bacto agar (15 g/L), ࠾
ࡼࡧNaCl (8.0 g/L) ࡢỈ⁐ᾮࢆ࣮࢜ࢺࢡ࣮ࣞࣈ⁛⳦ࡋࡓࡶࡢ.
࡞࠾, Bacto tryptone, Bacto yeast extract, ࠾ࡼࡧBacto agarࡣBD Biosciencesࡽ㉎ධ ࡋࡓ.
xiii. ⣽⬊ࣇ࣮ࢪ
P3/NS1/1-Ag4-1࣑࢚࣮࣐ࣟ⣽⬊ࡣ, ࣄ࣮࣐ࣗࣥࢧ࢚ࣥࢫ㈨※ࣂࣥࢡࡼࡾධᡭࡋࡓ.
⭠⳦XL1-Blue⣽⬊࠾ࡼࡧTG1⣽⬊ࡣ, Agilent Technologiesࡽ㉎ධࡋࡓ.
⭠⳦XLOLR⣽⬊࠾ࡼࡧVCSM13࣊ࣝࣃ࣮ࣇ࣮ࢪࡣ, Stratageneࡽ㉎ධࡋࡓ.
⭠⳦ XL1-Blue࠾ࡼࡧ XLOLR ࢚ࣞࢡࢺࣟࢥࣥࣆࢸࣥࢺ⣽⬊ࡣ, ᖖἲ
87)
ᚑࡗ࡚
ㄪ〇ࡋࡓ.
KM13࣊ࣝࣃ࣮ࣇ࣮ࢪࡣ, ⱥᅜMedical Research CouncilࡢGregory Paul Winter, Ian
M. Tomlinson୧༤ኈࡼࡾ౪ࡉࢀࡓࡶࡢࢆ⏝࠸ࡓ.
xiv. ࣋ࢡࢱ࣮DNA
pBluescript IIࡣ, Invitrogenࡽ㉎ධࡋࡓ.
pEXmide 5ࡣࢫ࢙࣮࢘ࢹࣥAvena Partners AB࣭Eskil Söderlind༤ኈ, ࢫ࢙࣮࢘ࢹࣥLund
Ꮫ࣭Carl A. K. Borrebaeck༤ኈࡽ౪ࡉࢀࡓ.
pEXmide 7ࡣ, Avena Partners AB࣭Eskil Söderlind༤ኈ, ࢫ࢙࣮࢘ࢹࣥLundᏛ࣭Carl
A. K. Borrebaeck༤ኈࡢチㅙࡢࡶ, VH㑇ఏᏊVL㑇ఏᏊࢆ⊂❧ࢡ࣮ࣟࢽࣥࢢࡋࡓ
ࡢ ࡕ ࡇ ࢀ ࡽ ࡽ ᵓ ᡂ ࡉ ࢀ ࡿ scFv 㑇 ఏ Ꮚ ࡀ ࣇ ࣮ ࢪ ᥦ ♧ ࡉ ࢀ ࡿ ࡼ ࠺, ⴭ ⪅ ࡽ ࡀ
pEXmide 5ࢆᨵኚࡋ࡚స〇ࡋࡓ.
pEXmide 7’ࡣ, Avena Partners AB࣭Eskil Söderlind༤ኈ, ࢫ࢙࣮࢘ࢹࣥLundᏛ࣭Carl
A. K. Borrebaeck༤ኈࡢチㅙࡢࡶ, VH㑇ఏᏊVL㑇ఏᏊࢆ⊂❧ࢡ࣮ࣟࢽࣥࢢࡋࡓ
ࡢࡕࡇࢀࡽࡽᵓᡂࡉࢀࡿ scFv 㑇ఏᏊࡀྍ⁐ᆺࢱࣥࣃࢡ㉁ࡋ࡚Ⓨ⌧ࡉࢀࡿࡼ࠺,
ⴭ⪅ࡽࡀpEXmide 5ࢆᨵኚࡋ࡚స〇ࡋࡓ.
pMCS-Gaussia Luc vectorࡣ, Thermo Fisher Scientificࡽ㉎ධࡋࡓ.
xv. ࣉ࣐࣮ࣛ
PCR࠾ࡼࡧDNAࡢሷᇶ㓄ิࡢỴᐃ⏝࠸ࡓࣉ࣐࣮ࣛࡣ, ࡘࡃࡤ࢜ࣜࢦࢧ࣮ࣅࢫ࡛
Ꮫྜᡂࡉࢀ, ㏫┦࣮࢝ࢺࣜࢵࢪ⢭〇 ရࡲࡓࡣ㧗㏿ᾮయࢡ࣐ࣟࢺࢢࣛࣇ࣮⢭〇ရ
56
ࡋ࡚౪ࡉࢀࡓࡶࡢࢆ⏝࠸ࡓ. ௨ୗ, ᮏᩥ୰グ㍕ࡉࢀࡓࣉ࣐࣮ࣛࡢ␎ྕࡑࡢሷ ᇶ㓄ิ (ࣥࢲ࣮ࣛࣥࡣቑᖜ⏘≀ࡢࢡ࣮ࣟࢽࣥࢢ⏝࠸ࡓไ㝈㓄ิ) ࢆ♧ࡍ.
mg1-GSP-1: 5’-GCTGGCCGGGTGGGCAAC-3’
mg1-GSP-2: 5’-ACACCGCTGGACAGGGAT-3’
mg1-GSP-3: 5’-GGATCCCGGGAATAGCCCTTGACCAGGC-3’ (Xma I) mg2b-GSP-1: 5’-GCTGGCCGGGTGGGCAAC-3’
mg2b-GSP-2: 5’-ACACTGCTGGACAGGGAT-3’
mg2b-GSP-3: 5’-GGATCCCGGGAGTACCCCTTGACCAGGC-3’ (Xma I) mk-GSP-1: 5’-GTTGAAGCTCTTGACAAT-3’
mk-GSP-2: 5’-ATTTTGTCGTTCACTGCC-3’
mk-GSP-3: 5’-GGATCCCGGGTGGATGGTGGGAAGATG-3’ (Xma I) AAP: 5’-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3’ (Sal I) AUAP: 5’-GGCCACGCGTCGACTAGTAC-3’ (Sal I)
MHC: 5’-GGATCCCGGGCCAGTGGATAGACHGATG-3’ (Xma I)
MHV-2: 5’-ACTAGTCGACATGGGATGGAGCTRTATCATSYTCTT-3’ (Sal I) VL-I/III: 5’-GACATTGTGATGACYCARTCT-3’
KS-reverse: 5’-GGAAACAGCTATGACCATG-3’
KS-forward: 5’-GTAAAACGACGGCCAGT-3’
VHH-cPCR-back: 5’-ATTGTTATTACTCGCGGCCCA-3’
VHH-cPCR-for: 5’-GATTTGGGCTCAACTTTCTTGT-3’
pEX7-VH-CP5: 5’-GGATTGTTATTACTCGCGGC-3’
pEX7-VL-CP3: 5’-CCTGATGTGTGCGTCTTAGT-3’
pEX-cla-3: 5’-GCCATTTGGGAATTAGAGCCA-3’
CS#3VH-Rev: 5’-CTCGCGGCCCAGCCGGCCATGGCCCAGGTCCAACTGCAGCAGCC TG-3’ (Nco I)
CS#3VH-For: 5’-TGAACCGCCTCCACCGCTCGAGACTGCAGAGACAGTGACCAGAG TC-3’ (Xho I)
CS#3VL-Rev: 5’-GGATCCGGCGGTGGCGGGTCGACGGACATTGTGCTGACACAGTC TC-3’ (Sal I)
CS#3VL-For: 5’-GGGCTCAACTTTCTTTGCGGCCGCAGCCCGTTTTATTTCCAGCTTG -3’ (Not I)
CS#10VH-Rev: 5’-ATTGTTATTACTCGCGGCCCAACCGGCCATGGCCCAAGTTACTCT AAAAGAGTCTGGC-3’ (Nco I)
CS#10VH-For: 5’-CCGCCGGATCCACCTCCGCCTGAACCGCCTCCACCGGAGGAGA CGGTGACTGAGGTTCC-3’
CS#10VL-Rev: 5’-CAGGCGGAGGTGGATCCGGCGGTGGCGGATCGGATGTTTTGGT
57 GACCCAAACTC-3’
CS#10VL-For: 5’-GATTTGGGCTCAACTTTCTTGTCGACTTTATCATCATCATCTTTAT AATCAGCCCGTTTAATTTCCAGCTTGG-3’ (Sal I)
VL-For-2: 5’-TTTACCTTCGCTACTTTTGCCGCTACCACTAGTAGAGCCAGCCCGTTT AATTTCCAGCTTG-3’ (Spe I)
GLuc-Rev: 5’-TCTACTAGTGGTAGCGGCAAAAGTAGCGAAGGTAAAGGTAAGCCCA CCGAGAACAACGAAGAC-3’ (Spe I)
GLuc-For: 5’-GGGCTCAACTTTCTTTGCGGCCGCTCATTAATGGTGGTGATGGTGAT GCCCTTTATCATCATCATCTTTATAATCGTCACCACCGGCCCCCTTGATC-3’ (Not I) M13#71VH-Rev: 5’-ATTGTTATTACTCGCGGCCCAACCGGCCATGGCCGAGGTTCAG
CTGCAGCAGTCTG-3’ (Nco I)
M13#71VH-For: 5’-CCGCCGGATCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGA CGGTGACCGTGGTC-3’
M13#71VL-Rev: 5’-CAGGCGGAGGTGGATCCGGCGGTGGCGGATCGGACATTGTGA TGACTCAGTCTCA-3’
M13#71VL-For: 5’-GATTTGGGCTCAACTTTCTTGTCGACTTTATCATCATCATCTTTA TAATCAGCCCGTCTCAGCTCCAGCTTG-3’ (Sal I)
FLAG-TAA2: 5’-GATTTGGGCTCAACTTTCTTGTCGACTTATTATTTATCATCATC ATCTTTATAATC-3’ (Sal I)
xvi. ࢟ࢵࢺ㢮
RNAᢳฟ࢟ࢵࢺ (RNeasy mini kit) ࡣ, Qiagenࡽ㉎ධࡋࡓ.
5’RACE࢟ࢵࢺ (5’RACE system for rapid amplification of cDNA ends, version 2.0) ࡣ, Invitrogenࡽ㉎ධࡋࡓ.
ࣉࣛࢫ࣑ࢻDNAᢳฟ࢟ࢵࢺ (QIAGEN plasmid mini kit) ࡣ, Qiagenࡽ㉎ධࡋࡓ. DNA⢭〇࢟ࢵࢺ (A) (Wizard PCR preps DNA purification system) ࠾ࡼࡧDNA⢭〇࢟
ࢵࢺ (B) (Wizard SV gel and PCR clean-up system) ࡣ, Promegaࡽ㉎ධࡋࡓ. ㉁㔞ศᯒ⏝ࢤࣝෆᾘ࢟ࢵࢺ (XL-Tryp Kit) ࡣࣉࣟࢧ࢚ࣥࢫࡽ㉎ධࡋࡓ. ࢱࣥࣃࢡ㉁ᐃ㔞⏝࠸ࡓࣉࣟࢸࣥࢵࢭ Lowry ࢟ࢵࢺࡣ, ࢼ࢝ࣛࢸࢫࢡࡽ
㉎ධࡋࡓ.
xvii. 㟁ẼὋື⏝ࢤࣝὋື᮲௳
Invitrogen〇ࡢ࣮࢞ࣟࢫࢤࣝࢆ⏝࠸, TAE⦆⾪ᾮ୰, 100 V࡛Ὃືࢆ⾜ࡗࡓ. xviii. ᐇ㦂ື≀
ᢠCS ᢠయ, ᢠ VCSM13 ᢠయࡢస〇㝿ࡋ࡚ච⏝࠸ࡓ BALB/c ࣐࢘ࢫ (8 㐌㱋,
58 㞤) ࡣ, ᪥ᮏSLCࡽධᡭࡋࡓ.
xix. ౫㢗ศᯒ
DNA ࡢሷᇶ㓄ิࡢゎᯒࡣ, TaKaRa ࣂ࢜ࡲࡓࡣ࣮ࣘࣟࣇࣥࢪ࢙ࣀ࣑ࢡࢫࡢཷク DNAࢩ࣮ࢣࣥࢫࢧ࣮ࣅࢫ౫㢗ࡋࡓ.
➨1❶➨4⠇࠾ࡼࡧ➨3❶➨4⠇ࡢ㉁㔞ศᯒࡼࡿscFv㑇ఏᏊෆࡢ⤊Ṇࢥࢻࣥ㒊
ࡢ࣑ࣀ㓟ࡢ≉ᐃࡣ, ࣉࣟࢧ࢚ࣥࢫࡲࡓࡣ⚄ᡞ⸆⛉Ꮫ୰ኸศᯒᐊ౫㢗ࡋࡓ.
59
➨❶ᒓᐇ㦂
➨⠇ᒓᐇ㦂
1.2.1㡯 CS-ࣝࣈ࣑ࣥ⤖ྜయ (CS-BSAࡲࡓࡣCS-OVA) ࡢㄪ〇
CS 3-(O-carboxymethyl)oxime (CS-3CMO) (30 mg; 70 mmol) ࢆ95% 1,4-ࢪ࢜࢟ࢧࣥ (550 mL) ⁐ゎࡋ, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (210 mmol) N-hydroxysuccinimide (210 mmol) ࢆຍ࠼, ᐊ ࡛ 15 ศᛂࡉࡏ࡚, CS 3-(O-carboxymethyl)oxime N-hydroxysuccinimide ester (CS-3CMO-NHS) ࢆస〇ࡋࡓ. CS-3CMO-NHS (18 mmol) ࢆࣆࣜࢪࣥ (1.5 mL) ⁐ゎࡋ, BSAࡲࡓࡣOVA (ྛ0.45 mmol) ࡢPB⁐
ᾮ (3 mL) ΰࡋ࡚, ᐊ ࡛1㛫ᛂࡉࡏࡓࡢࡕ, 4Υ୍࡛ᬌ᧠ᢾࡋࡓ. ࡇࢀࢆ⢭〇
Ỉᑐࡋ࡚4Υ୍࡛ᬌ㏱ᯒࡋࡓࡢࡕ, ᛂᾮࢆ⢭〇Ỉ࡛㔞⣙10 mLㄪᩚࡋ, ị෭ࡋ ࡓࢭࢺࣥ (10~20 mL) ࠾ࡼࡧᑡ㔞ࡢNaClࢆຍ࠼, ịୖ࡛30ศ㛫ᨺ⨨ࡋࡓ. 㐲ᚰศ㞳 (1500g, 15 ศ, 4Υ) ᚋ, ỿ Ẋ ࢆ ⢭ 〇 Ỉ (⣙ 10 mL) ࡛ ࡧ ᠱ ⃮ ࡋ, ị ෭ ࡋ ࡓ ࢭ ࢺ ࣥ
(10~20 mL) ࢆῧຍࡋ࡚ྠᵝࡢ᧯సࢆ⧞ࡾ㏉ࡋࡓ. ᚓࡽࢀࡓỿẊࢆ20% ࣆࣜࢪࣥ⁐ゎ
ࡋࡓࡢࡕ, ⢭〇Ỉᑐࡋ࡚4Υ୍࡛ᬌ㏱ᯒࡋࡓ. ࡉࡽ0.90% NaClᑐࡋ࡚4Υ୍࡛ᬌ
㏱ᯒࡋࡓࡢࡕ㏱ᯒ⭷ෆᐜ≀ࢆᅇࡋ, ┠ⓗࡢ CS-ࣝࣈ࣑ࣥ⤖ྜయࢆ 0.90% NaCl⁐ᾮ
ࡋ࡚ᚓࡓ. CS-BSA࠾ࡅࡿ BSA⃰ᗘࢆHartree ἲ,88) ᪉, CS-OVA࠾ࡅࡿ OVA
⃰ᗘࢆࣉࣟࢸࣥࢵࢭLowry࢟ࢵࢺࢆ⏝࠸࡚ࡑࢀࡒࢀ⟬ฟࡋࡓ. ࡲࡓ, 250 nm࠾
ࡅࡿ྾ගᗘ (CS ṧᇶࡢ⤖ྜࡼࡿ) ࢆ ᐃࡋ࡚, ྛ CS-ࣝࣈ࣑ࣥ⤖ྜయࡢ⤖ྜࣔࣝ
ẚࢆồࡵࡓࡇࢁ, CS/BSA࡛ࡣ12, CS/OVA࡛ࡣ5࡛࠶ࡗࡓ.
1.2.2㡯 ᢠCSᢠయ⏘⏕ࣁࣈࣜࢻ࣮࣐ᰴࡢᶞ❧࠾ࡼࡧᢠCSࣔࣀࢡ࣮ࣟࢼࣝᢠయࡢ⏘
⏕
CS-BSA ࡢ⏕⌮㣗ሷỈ⁐ᾮ (0.5 mg/mL) ࢆ➼య✚ࡢࣇࣟࣥࢺࢪࣗࣂࣥࢺ
ΰྜࡋ࡚࢚࣐ࣝࢪࣙࣥࡋࡓࡢࡕ, 8 㐌㱋ࡢ BALB/c ࣐࢘ࢫ (㞤) ࡢ⫼㒊⓶ୗᢞ (1 ಶయ࠶ࡓࡾ0.2 mL) ࡋࡓ. 2㐌㛫㝸࡛ 4 ᅇ㏣ຍච (ࣇࣟࣥࢺࡢࢪࣗࣂࣥࢺ
ࢆ⏝) ࢆ⾜࠸, ᭱⤊චࡽ7᪥ᚋ, CS-BSA (0.25 mg/mL) ࢆྵࡴ⏕⌮㣗ሷỈ (ィ0.5 mL) ࢆ࣐࢘ࢫࡢ⭡⭍ෆ⭁⮚ᢞࡋ, 3᪥ᚋ⭁⮚ࢆฟࡋࡓ. ᚓࡽࢀࡿ⭁⣽⬊ (⣙1
108 ಶ) ࢆ, ࣏ࣜ-L-ࣝࢠࢽࣥሷ㓟ሷ (10 mg/L) ࠾ࡼࡧ 10% (v/v) dimethyl sulfoxide (DMSO) ࢆྵࡴ PEG4000 (400 g/L) (1.0 mL) ࢆ⼥ྜ⏝࠸࡚, ࣑࢚࣮࣐ࣟ P3/NS1/1-Ag4-1⣽⬊ (⣙2107 ಶ) ⼥ྜࡉࡏࡓ.89) ⼥ྜᚋࡢ⣽⬊ࢆ, 96࢙࢘ࣝࢡࣛࢫࢱ࣮ࢹ
ࢵࢩࣗ୰, HATᇵᆅ࡛⣙2㐌㛫ᇵ㣴ࡍࡿࡇࡼࡾࣁࣈࣜࢻ࣮࣐ࡢ㑅ᢥࢆ⾜ࡗࡓ. ࣁ
ࣈࣜࢻ࣮࣐ࡢቑṪࡀ☜ㄆࡉࢀࡓ࣐ࢡ࢙ࣟ࢘ࣝࡢᇵ㣴ୖΎࢆ, ୗグࡢ ELISA ࡋ
࡚, ᢠ CS ᢠయࡢศἪࡢ᭷↓ࢆㄪࡓ. 㝧ᛶࢆ♧ࡋࡓࣁࣈࣜࢻ࣮࣐⣽⬊ (#3 ࠾ࡼࡧ
#10) ࢆHTᇵᆅ࡛ᣑᇵ㣴ࡋࡓࡢࡕ, 㝈⏺ᕼ㔘ἲࡼࡾࢡ࣮ࣟࣥࡋࡓ.
60
1.2.3㡯 ELISAࡼࡿᇵ㣴ୖΎ୰ᢠCSᢠయࡢ᳨ฟ
CS-OVA (1.0 mg/mL) ࢆྵࡴ0.10 MⅣ㓟⦆⾪ᾮ (pH 8.6) (CB) ࢆELISA⏝96࢙࢘ࣝ
࣐ࢡࣟࣉ࣮ࣞࢺศὀ (100 mL/࢙࢘ࣝ) ࡋ, ᐊ ୍࡛ᬌᨺ⨨ࡋࡓ. ⁐ᾮࢆ྾ᘬ㝖ཤࡋ, PBS࡛3ᅇὙίࡋࡓࡢࡕ, M-PBS (300 mL/࢙࢘ࣝ) ࢆῧຍࡋ, 37Υ࡛1㛫ᨺ⨨ࡋ࡚ࣈࣟ
ࢵ࢟ࣥࢢࢆ⾜ࡗࡓ. ࢙࢘ࣝࢆT-PBS࡛3ᅇὙίࡋࡓࡢࡕ, ᳨య࡛࠶ࡿࣁࣈࣜࢻ࣮࣐ᇵ 㣴ୖΎ (G-PBS࡛5ಸ௨ୖᕼ㔘ࡋࡓࡶࡢ; 100 mL) ࢆຍ࠼࡚37Υ࡛1㛫ᛂࡉࡏࡓ.
ྠᵝ⁐ᾮࢆ㝖ཤ, ࢙࢘ࣝࢆὙίࡋࡓࡢࡕ, POD ᶆ㆑ࣖࢠᢠ࣐࢘ࢫ IgG ࡢ G-PBS ⁐ᾮ (10 mg/mL) ࢆศὀ (100 mL/࢙࢘ࣝ) ࡋ, 37Υ࡛ 1 㛫ᨺ⨨ࡋࡓ. ⁐ᾮࢆ㝖ཤ, ࢙࢘ࣝࢆ
Ὑίࡋࡓࡢࡕ, ྛ࢙࢘ࣝPODᇶ㉁⁐ᾮࢆศὀ (100 mL/࢙࢘ࣝ) ࡋ, ༑ศ࡞ⓎⰍࡀㄆࡵ
ࡽࢀࡿࡲ࡛ (10~30ศ), ᐊ ࡛ᨺ⨨ࡋࡓ. 1.0 M H2SO4ࢆῧຍ (100 mL/࢙࢘ࣝ) ࡋ࡚㓝⣲
ᛂࢆṆࡉࡏࡓࡢࡕ, ࣐ࢡࣟࣉ࣮ࣞࢺ࣮ࣜࢲ࣮ࢆ⏝࠸࡚ 490 nm ࡢ྾ගᗘࢆ ᐃࡋ ࡓ.
1.2.4㡯 Scatchardἲࡼࡿ⤖ྜᐃᩘࡢ⟬ฟ
࢞ࣛࢫヨ㦂⟶ (1275 mm) , G-PBS࡛ྛ✀⃰ᗘᕼ㔘ࡋࡓ CSࡢᶆ‽ᾮ (100 mL), [3H]-CS (1.5104 dpm; 500 mL), ࠾ࡼࡧAb-CS#3ࡲࡓࡣAb-CS#10 (100 mL) ࢆῧຍࡋ࡚
᧠ᢾᚋ, 4Υ୍࡛ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ᛂᾮࢆịୖ࡛ 10 ศ㛫࣮ࣥ࢟ࣗ࣋ࢺࡋ, ࡇ ࡇdextran-coated charcoal ᠱ⃮ᾮ (0.4%ࡢάᛶⅣᮎࢆ 0.01%ࡢࢹ࢟ࢫࢺࣛࣥࢆྵࡴ G-PBS ࡛ᠱ⃮ࡋࡓᾮ) (500 mL) ࢆῧຍࡋ࡚᧠ᢾᚋ, ịୖ࡛ࡉࡽ 20 ศ㛫࣮ࣥ࢟ࣗ࣋ࢺ
ࡋࡓ. ࡇࡢᠱ⃮ᾮࢆ㐲ᚰศ㞳 (800g, 10ศ, 4Υ) ࡋ, ୖΎࢆࢡࣜࢰࣝII (10 mL) ΰ
ࡋ, ᨺᑕ⬟ࢆᾮయࢩࣥࢳ࣮ࣞࢩࣙࣥ࢝࢘ࣥࢱ࣮࡛ ᐃࡋࡓ. ᚓࡽࢀࡓ ᨺᑕ⬟ (dpm) ῧຍࡋࡓCS㔞 (pmol) ࡽScatchard ࣉࣟࢵࢺࢆ⾜࠸,43) Ab-CS#3 ࠾ࡼࡧAb-CS#10ࡢ CSᑐࡍࡿ⤖ྜᐃᩘKaࢆ⟬ฟࡋࡓ.
1.2.5㡯 Ab-CS#3ࢆ⏝࠸ࡿELISA
CS-BSA (0.10 mg/mL) ࢆྵࡴCBࢆELISA⏝96࢙࣐࢘ࣝࢡࣟࣉ࣮ࣞࢺศὀ (100 mL/࢙࢘ࣝ) ࡋ, ᐊ ୍࡛ᬌᨺ⨨ࡋࡓ. ⁐ᾮࢆ྾ᘬ㝖ཤࡋ, PBS ࡛3 ᅇὙίࡋࡓࡢࡕ, M-PBS (300 mL/࢙࢘ࣝ) ࢆῧຍࡋ࡚, 37Υ࡛2㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ⁐ᾮࢆ྾ᘬ㝖ཤࡋ, T-PBS࡛3ᅇὙίࡋࡓ. ࡇࡢࣉ࣮ࣞࢺ, G-PBS࡛ᕼ㔘ࡋࡓAb-CS#3 (100 mL) , ྛ✀
⃰ᗘࡢCSᶆ‽ရࢆྵࡴPVG-PBS⁐ᾮ (50 mL) ࢆຍ࠼࡚ΰࡋ, 4Υ࡛2㛫ࣥ࢟ࣗ
࣮࣋ࢺࡋࡓ. ⁐ᾮࢆ྾ᘬ㝖ཤࡋ, T-PBS࡛3ᅇὙίࡋࡓࡢࡕ, PVG-PBS࡛5000ಸᕼ㔘ࡋ ࡓPODᶆ㆑ᢠ࣐࢘ࢫᢠయ (100 mL) ࢆຍ࠼, 37Υ࡛30ศ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ࡧ⁐ᾮ
ࢆ྾ᘬ㝖ཤࡋ, T-PBS࡛3ᅇὙίࡋࡓࡢࡕ, ྛ࢙࢘ࣝPODᇶ㉁⁐ᾮࢆศὀ (100 mL/࢘
࢙ࣝ) ࡋ࡚ᐊ ࡛30ศᨺ⨨ࡋࡓ. 1.0 M H2SO4ࢆῧຍ (100 mL/࢙࢘ࣝ) ࡋ࡚㓝⣲ᛂࢆ
Ṇࡉࡏࡓࡢࡕ, 490 nmࡢ྾ගᗘࢆ, ࣐ࢡࣟࣉ࣮ࣞࢺ࣮ࣜࢲ࣮ࢆ⏝࠸࡚ ᐃࡋࡓ.
61
➨⠇ᒓᐇ㦂
1.3.1㡯 Ab-CS#3࠾ࡼࡧAb-CS#10ᢠయྍኚ㒊㑇ఏᏊࢆྵࡴcDNAࡢㄪ〇
1.2.2㡯࡛ᶞ❧ࡋࡓࣁࣈࣜࢻ࣮࣐⣽⬊#3࠾ࡼࡧ#10 (ࡑࢀࡒࢀAb-CS#3࠾ࡼࡧ#10ࢆ
ศἪࡍࡿ) (~1107 ಶ) ࡽ, RNAᢳฟ࢟ࢵࢺࢆ⏝࠸࡚, ⥲RNAࢆᢳฟࡋࡓ. ᚓࡽࢀࡓ RNA (2 mg) , dNTPΰྜ≀ (ྛ10 nmol), Superscript II reverse transcriptase (200 U), ࠾ࡼ
ࡧH㙐࡛ࡣ#3ࡢሙྜࡣmg1-GSP-1, #10ࡢሙྜࡣmg2b-GSP-1ࣉ࣐࣮ࣛ, L㙐࡛ࡣࡶ
mk-GSP-1ࣉ࣐࣮ࣛ (ྛ2.5 pmol) ࢆຍ࠼, ᑓ⏝⦆⾪ᾮ୰ (25 mL) ࡛42Υ, 50ศ
࣮ࣥ࢟ࣗ࣋ࢺࡋ, VH㑇ఏᏊࢆྵࡴcDNA (#3VH-cDNA࠾ࡼࡧ#10VH-cDNA), VL㑇ఏᏊࢆྵ
ࡴ cDNA (#3VL-cDNA ࠾ ࡼ ࡧ#10VL-cDNA) ࢆ ࡑ ࢀ ࡒ ࢀ ྜ ᡂ ࡋ ࡓ. ࡇ ࢀ ࡽ ᛂ ᾮ ࢆ,
5’RACE࢟ࢵࢺྵࡲࢀࡿDNA⢭〇࣮࢝ࢺࣜࢵࢪࡋ࡚, VH-cDNA, VL-cDNAࢆྵࡴ
Ỉ⁐ᾮ (~50 mL) ࢆᚓࡓ.
1.3.2㡯 Ab-CS#3ᢠయH㙐ྍኚ㒊㑇ఏᏊ (#3VH) ࢆྵࡴDNA᩿∦ࡢㄪ〇
1.3.1㡯࡛ㄪ〇ࡋࡓ#3VH-cDNAࡢ⁐ᾮࢆ10ಸᕼ㔘ࡋ, ࡑࡢ୍㒊 (1 mL) , Jonesࡽ
ࡼࡾሗ࿌ࡉࢀࡓࣘࢽࣂ࣮ࢧࣝࣉ࣐࣮ࣛ (MHV-1~12ࡢ12✀ࡢ࠺ࡕ࠸ࡎࢀ1✀)46)
࠾ࡼࡧMHCࣉ࣐࣮ࣛ (ྛ1 mM), dNTPΰྜ≀ (ྛ0.2 mM), ࠾ࡼࡧEx Taq DNA࣏ࣜ
࣓࣮ࣛࢮ (25 U/mL) ࢆῧຍࡋ, ᑓ⏝⦆⾪ᾮ୰ (20 mL) ࡛PCRࢆ⾜ࡗࡓ. PCR᮲௳ࡣ, ⇕ ኚᛶ95Υ, 1ศ, ࢽ࣮ࣜࣥࢢ64Υ, 1ศ, ఙ㛗72Υ, 2ศࡋ, ࡇࢀࢆ35ᅇ⧞ࡾ㏉ࡋࡓ ࡢࡕ, 72Υ࡛10ศࡢఙ㛗ᛂࢆຍ࠼ࡓ. ᛂᾮࢆ࣮࢞ࣟࢫࢤࣝ (2%) ࢆ⏝࠸ࡿ㟁ẼὋ
ື ࡛ ศ ᯒ ࡋ ࡓ ࡇ ࢁ, MHV-2 ࣉ ࣛ ࣐ ࣮ ࢆ ⏝ ࠸ ࡿ ࡁ ቑ ᖜ ࡀ ㄆ ࡵ ࡽ ࢀ ࡓ. ࡑ ࡇ ࡛,
MHV-2ࣉ࣐࣮ࣛࢆ⏝࠸ࡿPCRࢆ, ୖグྠࡌ᮲௳࡛100 mLࢫࢣ࣮ࣝࢵࣉࡋ࡚⾜
࠸, ᛂᾮ୰ࡢቑᖜ⏘≀ࢆDNA⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ, #3VHࢆྵࡴDNA᩿
∦ࢆᚓࡓ.
1.3.3㡯 Ab-CS#3ᢠయL㙐ྍኚ㒊㑇ఏᏊ (#3VL) ࢆྵࡴDNA᩿∦ࡢㄪ〇
1.3.1㡯࡛ㄪ〇ࡋࡓ#3VL-cDNA, ᑓ⏝⦆⾪ᾮ୰ (25 mL), TdT (15 U) dCTP (5 nmol)
ࢆຍ࠼࡚37Υ࡛10ศ࣮ࣥ࢟ࣗ࣋ࢺࡋ, ࡑࡢ3’ᮎ➃࣏ࣜC㓄ิࢆຍࡋࡓ. ࡑࡢ
ᛂᾮ (8 mL) ࢆAAP࠾ࡼࡧmk-GSP-2ࢆࣉ࣐࣮ࣛࡍࡿPCRࡋࡓ. ᮏPCRࡣୖ
グࣉ࣐࣮ࣛ (ྛ10 pmol), dNTPΰྜ≀ (ྛ8 nmol), ࠾ࡼࡧEx Taq DNA࣏࣓࣮ࣜࣛࢮ (0.5 U) ࢆྵࡴᑓ⏝⦆⾪ᾮ୰ (20 mL) ࡛⾜࠸, ⇕ኚᛶ 95Υ, 1 ศ, ࢽ࣮ࣜࣥࢢ 64Υ, 1
ศ, ఙ㛗72Υ, 2ศࡋ, ࡇࢀࢆ35ᅇ⧞ࡾ㏉ࡋࡓࡢࡕ, 72Υ࡛10ศࡢఙ㛗ᛂࢆຍ࠼ࡓ. ࡉࡽ, ࡇࡢPCRᛂᾮࡢ୍㒊 (8 mL) ࢆAUAP࠾ࡼࡧmk-GSP-3ࢆࣉ࣐࣮ࣛࡍࡿ
PCRࡋࡓ. ᮏPCRࡣୖグࣉ࣐࣮ࣛ (ྛ100 pmol), dNTPΰྜ≀ (ྛ 40 nmol), ࠾
ࡼࡧEx Taq DNA࣏࣓࣮ࣜࣛࢮ (2.5 U) ࢆྵࡴᑓ⏝⦆⾪ᾮ୰ (100 mL), ྠୖࡢᛂ᮲௳
࡛⾜ࡗࡓ. ࡑࡢᛂᾮ୰ࡢቑᖜ⏘≀ࢆ, DNA⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ, #3VLࢆྵ
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ࡴDNA᩿∦ࢆᚓࡓ.
1.3.4㡯 Ab-CS#10ᢠయྍኚ㒊㑇ఏᏊ (#10VH࠾ࡼࡧ#10VL) ࢆྵࡴDNA᩿∦ࡢㄪ〇
1.3.1㡯࡛ㄪ〇ࡋࡓ#10VH-cDNA࠾ࡼࡧ#10VL-cDNAࢆ, ๓㡯ྠᵝ5’RACE ἲ
ࡋ, #10VH࠾ࡼࡧ#10VL㑇ఏᏊࢆྵࡴDNA᩿∦ࢆᚓࡓ. ࡞࠾, VH㑇ఏᏊቑᖜࡢ㝿ࡢ3’ࣉ
࣐࣮ࣛࡣmg2b-GSP-2࠾ࡼࡧmg2b-GSP-3ࢆ⏝࠸ࡓ.
1.3.5㡯 Ab-CS#3࠾ࡼࡧAb-CS#10ᢠయࡢVHࡲࡓࡣVL㑇ఏᏊࢆྵࡴDNA᩿∦ࡢࢧࣈ
ࢡ࣮ࣟࢽࣥࢢ
1.3.2 㡯, 1.3.3 㡯, ࠾ࡼࡧ 1.3.4 㡯࡛ᚓࡽࢀࡓ VHࡲࡓࡣ VL㑇ఏᏊࢆྵࡴ DNA ᩿∦
(#3VH; 7 mg, #3VL; 1 mg, #10VH࠾ࡼࡧ#10VL; ྛ10 mg) , ไ㝈㓝⣲Xma I (#3VH, VLࡢሙ
ྜࡣ 25 U, #10VH, VLࡢሙྜࡣ 50 U) ࢆຍ࠼, BSA (100 mg/mL) ࢆྵࡴᛂ⏝⦆⾪ᾮ୰
(#3VH, VLࡢሙྜࡣ100 mL, #10VH, VLࡢሙྜࡣ200 mL), 37Υ୍࡛ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ.
ᛂᾮ୰ࡢቑᖜ⏘≀ࢆ DNA ⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ, ࡑࡢ㔞, ไ㝈㓝⣲
Sal I (#3VH, VLࡢሙྜࡣ25 U, #10VH, VLࡢሙྜࡣ50 U) ࢆຍ࠼, ᛂ⏝⦆⾪ᾮ୰ (100 mL), 37Υ୍࡛ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ᛂᾮࢆࡧ DNA ⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ,
ࡑࡢ୍㒊 (#3VH, VLࡢሙྜࡣ250 ng, #10VH, VLࡢሙྜࡣ125 ng) , ྠᵝXma I࠾ࡼࡧ Sal I࡛ᾘࡋࡓpBluescript II࣋ࢡࢱ࣮ (#3VH, VLࡢሙྜࡣ500 ng, #10VH, VLࡢሙྜࡣ250 ng) ࢆ⁛⳦Ỉ⁐ゎࡋ (#3VHࡢᛂ࡛ࡣ43 mL, #3VL࠾ࡼࡧ#10VH, VLࡢᛂ࡛ࡣ16 mL), 42Υ࡛ 5ศ㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࡢࡕ┤ࡕị෭ࡋࡓ. ࡇࡢ⁐ᾮ 10ಸ⃰ᗘࡢ
ᛂ⏝⦆⾪ᾮ (#3VHࡢᛂ࡛ࡣ 5 mL, #3VL࠾ࡼࡧ#10VH, VLࡢᛂ࡛ࡣ 2 mL) T4 DNA࣮ࣜ࢞ࢮ (ྛ800 U, 2 mL) ࢆຍ࠼࡚16Υ୍࡛ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ࡇࢀࡽࡢ
ᛂᾮࢆ, ࣇ࢙ࣀ࣮ࣝ-CHCl3-ࢯ࣑ࣝࣝࢥ࣮ࣝᢳฟࡋ, ỿẊ࣮࢟ࣕࣜࣖࡋ࡚ࢢࣜ
ࢥ࣮ࢤࣥ (20 mg) ࢆῧຍࡋࡓࡢࡕ EtOH ỿẊࢆ⾜࠸, ᚓࡽࢀࡿỿẊࢆࡑࢀࡒࢀ⁛⳦Ỉ
(10 mL) ࡛⁐ゎࡋࡓ.
ࡇࢀࡽࡢ DNA Ỉ⁐ᾮࢆ࢚ࣞࢡࢺࣟࢥࣥࣆࢸࣥࢺ⣽⬊ (#3VHࡢᛂ⏕ᡂ≀ࡣ XL1-Blueࢆ, #3VL࠾ࡼࡧ#10VH, VLࡢᛂ⏕ᡂ≀ࡣXLOLRࢆ⏝) ࡢᠱ⃮ᾮ (100 mL)
ຍ࠼࡚ΰྜࡋ, ࡑࡢ㔞ࢆ࢟ࣗ࣋ࢵࢺ㟁ᴟ⛣ࡋ࡚ịୖ࡛ 20 ศ㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ࡇࢀࢆ㑇ఏᏊᑟධ⨨╔ࡋ, 50 mF, ༳ຍ㟁ᅽ1800 V, ෆ㒊ᢠ125 Wࡢ᮲௳࡛㟁Ẽ
ࣃࣝࢫࢆຍ࠼ࡓ. ࡑࡢ┤ᚋ࢟ࣗ࣋ࢵࢺෆᐜ≀SOCᇵᆅ (900 mL) ࢆῧຍࡋ, 37Υ࡛
1㛫࠺ᇵ㣴 (~200 rpm) ࡋࡓ. ᇵ㣴ᾮࡢ୍㒊ࢆẁ㝵ᕼ㔘ࡋࡓࡢࡕ, 2YT-ATG (1%)
࣮࢞ࣉ࣮ࣞࢺሬᕸࡋ࡚37Υ୍࡛ᬌᇵ㣴ࡋࡓ.
ࡇࡢࣉ࣮ࣞࢺୖࡽࢥࣟࢽ࣮ࢆࣛࣥࢲ࣒㑅ᢥࡋ, 2YTᇵᆅ (5 mL) ᠱ⃮ࡉࡏࡓ. ࡇࡢᠱ⃮ᾮ (1 mL) , 5’࠾ࡼࡧ3’ࣉ࣐࣮ࣛ [#3VHࡢቑᖜࡣMHV-2࠾ࡼࡧMHC (ྛ 5 pmol), #3VLࡢቑᖜࡣAUAP࠾ࡼࡧmk-GSP-3 (ྛ4 pmol), #10VHࡲࡓࡣ#10VLࡢቑᖜ
ࡣKS-back࠾ࡼࡧKS-for (ྛ5 pmol)], dNTPΰྜ≀ (ྛ4 nmol), ࠾ࡼࡧAmpliTaq DNA
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࣏࣓࣮ࣜࣛࢮ (0.5 U) ࢆຍ࠼, ᑓ⏝⦆⾪ᾮ ୰ (#3VH; 20 mL, #3VL; 40 mL, #10VH࠾ࡼࡧ
#10VL; 20 mL) ࡛PCR (ࢥࣟࢽ࣮PCR) ࢆ⾜ࡗࡓ. PCR᮲௳ࡣ⇕ኚᛶ95Υ, 1ศ, ࢽ࣮ࣜ
ࣥࢢ64Υ, 1ศ, ఙ㛗72Υ, 2ศࡋ, ࡇࢀࢆ35ᅇ⧞ࡾ㏉ࡋࡓࡢࡕ, 72Υ࡛10ศࡢఙ㛗
ᛂࢆຍ࠼ࡓ. ᛂᾮࢆ ࣮࢞ࣟࢫࢤࣝ (2%) 㟁ẼὋືࡋ࡚┠ⓗ ࡢ㑇ఏᏊࡢ᭷↓
ࢆㄪ, VH㑇ఏᏊࡲࡓࡣVL㑇ఏᏊࢆಖᣢࡍࡿᙧ㉁㌿⳦ࢆ≉ᐃࡋࡓ.
1.3.6㡯 Ab-CS#3࠾ࡼࡧAb-CS#10ᢠయࡢVH࠾ࡼࡧVL㑇ఏᏊࡢሷᇶ㓄ิࡢỴᐃ
๓㡯࡛VHࡲࡓࡣVL㑇ఏᏊࡀㄆࡵࡽࢀࡓᙧ㉁㌿⳦ࡘ࠸࡚, ࡑࡢࢢࣜࢭ࣮ࣟࣝ⤖
ಖᏑᾮࢆᑡ㔞ࡾ, 2YT-ATG (1%) ᇵᆅ (10 mL) ᥋✀ࡋ࡚, 37Υ୍࡛ᬌ࠺ᇵ㣴 (~200 rpm) ࡋࡓ. ᇵ㣴ᾮࢆ㐲ᚰศ㞳 (1800g, 20 ศ, ᐊ ) ࡋ࡚㞟⳦ࡋࡓࡢࡕ, ࣉࣛࢫ࣑
ࢻDNA ᢳฟ࢟ࢵࢺࢆ⏝࠸࡚⤌࠼ࣉࣛࢫ࣑ࢻࢆㄪ〇ࡋࡓ. ࡇࢀࡽࡢࣉࣛࢫ࣑ࢻࡢ୍㒊
(600 ng) ࢆ⏝࠸, ᤄධࡉࢀࡓ㑇ఏᏊࡢሷᇶ㓄ิࢆゎᯒࡋࡓ.
1.3.7㡯 scFv㑇ఏᏊᵓ⠏ࡢࡓࡵࡢVH-DNA࠾ࡼࡧVL-DNA᩿∦ࡢㄪ〇
๓㡯࡛ㄪ〇ࡋࡓ⤌࠼ࣉࣛࢫ࣑ࢻ (50 ng) , 5’࠾ࡼࡧ3’ࣉ࣐࣮ࣛ (#3VHࡢቑᖜ
ࡣ CS#3VH-Rev ࠾ࡼࡧ CS#3VH-For, #3VLࡢቑᖜࡣ CS#3VL-Rev ࠾ࡼࡧ CS#3VL-For,
#10VHࡢቑᖜࡣCS#10VH-Rev࠾ࡼࡧCS#10VH-For, #10VLࡢቑᖜࡣCS#10VL-Rev࠾
ࡼࡧCS#10VL-For) (ྛ50 pmol), dNTPΰྜ≀ (ྛ20 nmol), ࠾ࡼࡧEx Taq DNA࣏࣓ࣜࣛ
࣮ࢮ (2.5 U) ࢆຍ࠼, ᑓ⏝⦆⾪ᾮ୰ (100 mL) ࡛PCRࢆ⾜ࡗࡓ. PCR᮲௳ࡣ⇕ኚᛶ95Υ, 1ศ, ࢽ࣮ࣜࣥࢢ64Υ, 1 ศ, ఙ㛗72Υ, 2ศࡋ, ࡇࢀࢆ35ᅇ⧞ࡾ㏉ࡋࡓࡢࡕ, 72Υ
࡛10ศࡢఙ㛗ᛂࢆຍ࠼ࡓ. ᛂᾮ୰ࡢቑᖜ⏘≀ࢆ DNA⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭
〇ࡋ, VH-DNA࠾ࡼࡧVL-DNA᩿∦ࢆᚓࡓ.
1.3.8㡯 Ab-CS#3ᢠయscFv㑇ఏᏊࡢࢡ࣮ࣟࢽࣥࢢ
pEXmide 7࣋ࢡࢱ࣮, ࡲࡎVH㑇ఏᏊࢆ⤌ࡳ㎸ࢇࡔ. ๓㡯࡛ㄪ〇ࡋࡓ#3VH-DNA᩿∦
(5 mg) , ไ㝈㓝⣲Nco I࠾ࡼࡧXho I (ྛ25 U) ࢆຍ࠼, ᛂ⏝⦆⾪ᾮ୰ (100 mL), 37Υ
୍࡛ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ᛂᾮ୰ࡢቑᖜ⏘≀ࢆDNA⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇 ࡋ, ࡑࡢ୍㒊 (125 ng) ࢆ, ྠᵝ Nco I࠾ࡼࡧ Xho I ࡛ᾘࡋࡓpEXmide 7 ࣋ࢡࢱ࣮
(250 ng) ࡢỈ⁐ᾮ (16 mL) ΰྜࡋ࡚, 1.3.5㡯‽ࡌ࡚ࣛࢤ࣮ࢩࣙࣥᛂࡋ, ࡑ
ࡢ⏘≀ࢆ1.3.5㡯‽ࡌࡓ᪉ἲ࡛XL1-Blue࢚ࣞࢡࢺࣟࢥࣥࣆࢸࣥࢺ⣽⬊ᑟධࡋࡓ. ᙧ
㉁㌿⳦ࢆ 2YT-ATG (1%) ࣮࢞ࣉ࣮ࣞࢺሬᕸࡋ࡚ 37Υ୍࡛ᬌᇵ㣴ࡋࡓ. ฟ⌧ࡋ ࡓࢥࣟࢽ࣮ࢆࣛࣥࢲ࣒㑅ᢥࡋ, ࢥࣟࢽ࣮PCR ࢆ⾜ࡗࡓ. ࡞࠾, ࣉ࣐࣮ࣛࡣ CS#3VH -Rev࠾ࡼࡧCS#3VH-For (ྛ2 pmol) ࢆ⏝࠸ࡓ. ᛂᾮࢆ࣮࢞ࣟࢫࢤࣝ (2%) 㟁ẼὋື
ࡋ࡚┠ⓗࡢ㑇ఏᏊࡢ᭷↓ࢆㄪ, VH㑇ఏᏊࢆಖᣢࡍࡿᙧ㉁㌿⳦ࢆ≉ᐃࡋࡓ. ࡇࢀ
ࡽ ࡢ ࢥ ࣟ ࢽ ࣮ ࢆ 2YT-ATG (1%) ᇵ ᆅ (10 mL) ᥋ ✀ ࡋ ࡚ 37Υ ࡛ ୍ ᬌ ࠺ ᇵ 㣴 (~200 rpm) ࡋ, 1.3.6㡯‽ࡌ࡚ࣉࣛࢫ࣑ࢻࢆᢳฟࡋࡓ.
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ḟ࠸࡛, ࡇࡢࣉࣛࢫ࣑ࢻVL㑇ఏᏊࢆ⤌ࡳ㎸ࢇࡔ. 1.3.7㡯࡛ㄪ〇ࡋࡓ#3VL-DNA᩿∦
(5 mg) ࢆᛂ⏝⦆⾪ᾮ (100 mL) ⁐ࡋ, ไ㝈㓝⣲Sal I࠾ࡼࡧNot I (ྛ25 U) ࢆຍ࠼
࡚ 37Υ୍࡛ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ᛂᾮ୰ࡢቑᖜ⏘≀ࢆ DNA ⢭〇࢟ࢵࢺ (A) ࡼ
ࡾ⢭〇ࡋ, ࡑࡢ୍㒊 (125 ng) , ྠᵝ Sal I࠾ࡼࡧ Not I࡛ᾘࡋࡓୖグࣉࣛࢫ࣑ࢻ
(250 ng) ࢆΰྜࡋ࡚ୖグྠᵝࣛࢤ࣮ࢩࣙࣥᛂࡋ, ࡑࡢ⏘≀ࢆ 1.3.5 㡯‽
ࡌ࡚XL1-Blue࢚ࣞࢡࢺࣟࢥࣥࣆࢸࣥࢺ⣽⬊ᑟධࡋࡓ. ᙧ㉁㌿⳦ࢆ2YT-ATG (1%)
࣮࢞ࣉ࣮ࣞࢺሬᕸࡋ࡚ 37Υ୍࡛ᬌᇵ㣴ࡋࡓ. ฟ⌧ࡋࡓࢥࣟࢽ࣮ࢆࢥࣟࢽ࣮PCR
ࡋ࡚VL㑇ఏᏊࢆಖᣢࡍࡿᙧ㉁㌿⳦ࢆྠᐃࡋࡓ. ࡞࠾, ࣉ࣐࣮ࣛࡣCS#3VL-Rev࠾
ࡼࡧ CS#3VL-For (ྛ 2 pmol) ࢆ⏝࠸ࡓ. ≉ᐃࡉࢀࡓࢡ࣮ࣟࣥࢆ 2YT-ATG (1%) ᇵᆅ (10 mL) ᥋✀ࡋ࡚37Υ୍࡛ᬌ࠺ᇵ㣴 (~200 rpm) ࡋ, 1.3.6㡯‽ࡌ࡚ࣉࣛࢫ࣑ࢻ
ࢆᢳฟࡋ࡚scFv㑇ఏᏊࡢሷᇶ㓄ิࢆゎᯒࡋࡓ.
1.3.9㡯 CS#10-scFv㑇ఏᏊࡢస〇⭠⳦ࡢᑟධ
1.3.7 㡯࡛స〇ࡋࡓ#10VH-DNA ࠾ࡼࡧ#10VL-DNA ᩿∦ (ྛ 200 ng) , dNTP ΰྜ≀
(ྛ10 nmol) ࠾ࡼࡧEx Taq DNA࣏࣓࣮ࣜࣛࢮ (0.65 U) ࢆຍ࠼, ᑓ⏝⦆⾪ᾮ୰ (25 mL)
࡛PCRࢆ⾜ࡗࡓ. PCR᮲௳ࡣ, ⇕ኚᛶ95Υ, 1ศ, ࢽ࣮ࣜࣥࢢ55Υ, 1ศ, ఙ㛗72Υ, 3 ศࡋ, ࡇࡢࢧࢡࣝࢆ 10 ᅇ⧞ࡾ㏉ࡋࡓࡢࡕ, 72Υ࡛ 10 ศࡢఙ㛗ᛂࢆ㏣ຍࡋࡓ. ࡇ ࡢᛂᾮࡢ୍㒊 (10 mL) , CS#10VH-Rev࠾ࡼࡧCS#10VL-Forࣉ࣐࣮ࣛ (ྛ100 pmol), dNTPΰྜ≀ (ྛ40 nmol), ࠾ࡼࡧEx Taq DNA࣏࣓࣮ࣜࣛࢮ (2.5 U) ࢆຍ࠼, ᑓ⏝⦆⾪
ᾮ୰ (100 mL), ୖグࡢ᮲௳࡛ (ࡓࡔࡋ15ࢧࢡࣝ) PCRࢆ⾜ࡗࡓ. ᛂᾮ୰ࡢቑᖜ⏘≀
ࢆDNA⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ, CS#10-scFv㑇ఏᏊ (20 mg) ࢆᚓࡓ. ࡑࡢ୍㒊 (10 mg) ไ㝈㓝⣲Nco ISal I (ྛ50 U) ࢆຍ࠼, ᛂ⏝⦆⾪ᾮ୰ (100 mL) ࡛37Υ,
୍ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ᛂᾮ୰ࡢቑᖜ⏘≀ࢆDNA⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ, ไ㝈㓝⣲ᾘ῭ࡳࡢDNA᩿∦ࢆᚓࡓ.
ࡇࡢDNA᩿∦ (250 ng) , ྠᵝไ㝈㓝⣲Nco ISal I࡛ᾘࡋ, ࣮࢞ࣟࢫࢤࣝ
(1%) 㟁ẼὋືࡼࡾ⢭〇ࡋࡓpEXmide 5 (500 ng) ࢆΰࡋ, 1.3.5㡯ྠᵝࡢࣛࢤ࣮
ࢩࣙࣥᛂࡋ࡚, ࡑࡢ⏘≀ࢆ1.3.5㡯ྠᵝXL1-Blue࢚ࣞࢡࢺࣟࢥࣥࣆࢸࣥࢺ⣽
⬊ᑟධࡋࡓ. ᙧ㉁㌿⳦ࢆ 2YT-ATG (1%) ࣮࢞ࣉ࣮ࣞࢺୖሬᕸࡋ, 37Υ୍࡛ᬌ ᇵ㣴ࡋࡓ. ࡇࡢࣉ࣮ࣞࢺࡽࣛࣥࢲ࣒㑅ᢥࡋࡓࢥࣟࢽ࣮ࢆࢥࣟࢽ࣮PCR ࡋ, scFv 㑇ఏᏊࢆಖᣢࡍࡿࢡ࣮ࣟࣥࢆྠᐃࡋࡓ. ࡞࠾, ࣉ࣐࣮ࣛࡣVHH-cPCR-back࠾ࡼࡧVHH -cPCR-for (ྛ 2 pmol) ࢆ⏝࠸ࡓ. ≉ᐃࡉࢀࡓࢡ࣮ࣟࣥࢆ 2YT-ATG (1%) ᇵᆅ (10 mL)
᥋✀ࡋ࡚37Υ୍࡛ᬌ࠺ᇵ㣴 (~200 rpm) ࡋ, 1.3.6㡯‽ࡌ࡚ࣉࣛࢫ࣑ࢻࢆᢳฟࡋ
࡚scFv㑇ఏᏊࡢሷᇶ㓄ิࢆゎᯒࡋࡓ.
1.3.10㡯 ྍ⁐ᆺCS#3-scFv㑇ఏᏊࡢస〇⭠⳦ࡢᑟධ
1.3.8㡯࡛స〇ࡋࡓࣉࣛࢫ࣑ࢻ (10 mg) , ไ㝈㓝⣲Nco I࠾ࡼࡧNot I (ྛ20 U) ࢆຍ
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࠼, ᛂ⏝⦆⾪ᾮ୰ (100 mL), 37Υ୍࡛ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ᛂᾮ୰ࡢቑᖜ⏘≀ࢆ
DNA⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋࡓࡢࡕ, ࣮࢞ࣟࢫࢤࣝ (2%) ࢆ⏝࠸ࡿ㟁ẼὋື
ࢆ⾜ࡗ࡚, ศ㞳ࡉࢀࡓ scFv 㑇ఏᏊࢆྵࡴࢤࣝࢆษࡾฟࡋࡓࡢࡕ, DNA ⢭〇࢟ࢵࢺ (B)
ࢆ⏝࠸࡚DNA᩿∦ࢆᚓࡓ. ࡇࢀࢆ, ྠᵝไ㝈㓝⣲ฎ⌮ࡋࡓpEXmide 7’࣋ࢡࢱ࣮ΰ
ྜࡋ, 1.3.5 㡯ྠᵝࡢࣛࢤ࣮ࢩࣙࣥᛂࡋ࡚, ࡑࡢ⏘≀ࢆ XL1-Blue ࢚ࣞࢡࢺࣟ
ࢥࣥࣆࢸࣥࢺ⣽⬊ᑟධࡋࡓ. ᙧ㉁㌿⳦ࢆ2YT-ATG (1%) ࣮࢞ࣉ࣮ࣞࢺሬᕸࡋ
࡚37Υ୍࡛ᬌᇵ㣴ࡋ, ฟ⌧ࡍࡿࢥࣟࢽ࣮ࢆࢥࣟࢽ࣮PCR ࡋ࡚, CS#3-scFv 㑇ఏᏊࢆ
ಖᣢࡍࡿᙧ㉁㌿⳦ࢆྠᐃࡋࡓ.
1.3.11㡯 ྍ⁐ᆺCS#10-scFv㑇ఏᏊࡢస〇⭠⳦ࡢᑟධ
1.3.9㡯࡛స〇ࡋࡓࣉࣛࢫ࣑ࢻ (10 ng) , CS#10VH-Rev࠾ࡼࡧFLAG-TAA2ࣉ࣐ࣛ
࣮ (ྛ50 pmol), dNTPΰྜ≀ (ྛ20 nmol), ࠾ࡼࡧEx Taq DNA࣏࣓࣮ࣜࣛࢮ (2.5 U) ࢆ ຍ࠼, ᑓ⏝⦆⾪ᾮ୰ (100 mL) ࡛PCRࢆ⾜ࡗࡓ. PCR᮲௳ࡣ, ⇕ኚᛶ95Υ, 1ศ, ࢽ࣮
ࣜࣥࢢ64Υ, 1ศ, ఙ㛗72Υ, 2ศࡋ, ࡇࡢࢧࢡࣝࢆ35ᅇ⧞ࡾ㏉ࡋࡓࡢࡕ, 72Υ࡛10 ศࡢఙ㛗ᛂࢆ㏣ຍࡋࡓ. ᛂᾮ୰ࡢቑᖜ⏘≀ࢆDNA⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ, ࡑࡢ㔞 (⣙3 mg) ไ㝈㓝⣲Nco ISal I (ྛ50 U) ࢆຍ࠼, ᛂ⏝⦆⾪ᾮ୰ (200 mL)
࡛37Υ, ୍ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ᛂᾮࢆDNA⢭〇࢟ࢵࢺ (A) ࡋ࡚, ไ㝈㓝⣲
ᾘ῭ࡳࡢDNA᩿∦ࢆᚓࡓ.
ࡇࡢDNA᩿∦ (250 ng) ࢆ๓㡯ྠᵝpEXmide 5࣋ࢡࢱ࣮⤌ࡳ㎸ࡳ, 1.3.5㡯ྠ
ᵝXL1-Blue࢚ࣞࢡࢺࣟࢥࣥࣆࢸࣥࢺ⣽⬊ᑟධࡋࡓ. ᙧ㉁㌿⳦ࢆ2YT-ATG (1%)
࣮࢞ࣉ࣮ࣞࢺୖሬᕸࡋ, 37Υ୍࡛ᬌᇵ㣴ࡋࡓ. ࡇࡢࣉ࣮ࣞࢺࡽࣛࣥࢲ࣒㑅ᢥࡋ ࡓࢥࣟࢽ࣮ࢆࢥࣟࢽ࣮PCRࡋ, scFv㑇ఏᏊࢆಖᣢࡍࡿࢡ࣮ࣟࣥࢆྠᐃࡋࡓ.
1.3.12㡯 ྍ⁐ᆺscFvࡢస〇⢭〇
1.3.10㡯࠾ࡼࡧ1.3.11㡯࡛స〇ࡋࡓᙧ㉁㌿⳦ࢆ2YT-AG (1%) ᇵᆅ (20 mL) ຍ
࠼, 600 nm࠾ࡅࡿ྾ගᗘࡀ0.8㐩ࡍࡿࡲ࡛37Υ࡛࠺ᇵ㣴 (~200 rpm) ࡋࡓ. ᇵ 㣴ᾮࢆ㐲ᚰศ㞳 (1800g, 30ศ, ᐊ ) ࡋ, ỿẊࢆࢱࣥࣃࢡ㉁Ⓨ⌧ㄏᑟᇵᆅ (20 mL) ᠱ
⃮ࡋ࡚, 25Υ୍࡛ᬌ࠺ᇵ㣴 (120 rpm) ࡋࡓ. ࡇࢀࢆࡧ㐲ᚰศ㞳 (1800g, 30 ศ, ᐊ ) ࡋ, ỿẊࢆᾐ㏱ᅽࢩࣙࢵࢡ⏝⦆⾪ᾮ (1 mL) ࡛ᠱ⃮ࡋࡓࡢࡕ, ịୖ࡛ 1 㛫ࣥ࢟
࣮ࣗ࣋ࢺࡋࡓ. ࡇࡢΰྜᾮࢆ㐲ᚰศ㞳 (19300g, 30ศ, 4Υ) ᚋ, ୖΎ (࣌ࣜࣉࣛࢬ࣒ᢳฟ ᾮ) ࢆᅇࡋ, -20Υ࡛ಖᏑࡋࡓ. ࡇࡢ୍㒊ࢆTBSᑐࡋ࡚4Υ୍࡛ᬌ㏱ᯒࡋࡓࡢࡕ, ᢠ
FLAG-M2ᢠయᅛᐃ࣮࢞ࣟࢫࢤࣝ (⣙1 mL) ࢆሸࡋࡓࣇࢽࢸ࣮࣒࢝ࣛ
ࡋࡓ. ࣒࢝ࣛࢆTBS࡛Ὑίᚋ, 0.10 M ࢢࣜࢩࣥሷ㓟⦆⾪ᾮ (pH 3.5) ࡛ྍ⁐ᆺscFvࢆ⁐
ฟࡋࡓ. ࡇࡢ⏬ศࢆPBSᑐࡋ࡚4Υ୍࡛ᬌ㏱ᯒࡋࡓࡢࡕ, 㐲ᚰᘧ㝈እࢁ㐣⭷ (Amicon Ultra) ࢆ⏝࠸࡚⃰⦰ࡋࡓ.
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1.3.13㡯 Scatchardἲࡼࡿ⤖ྜᐃᩘࡢ⟬ฟ
1.2.4 㡯‽ࡌ࡚ᐇ㦂ࢆ⾜࠸, Scatchard ἲࡼࡾ๓㡯࡛ᚓࡽࢀࡓ CS#10-scFv ࠾ࡼࡧ
CS#3-scFvࡢCSᑐࡍࡿKaࢆ⟬ฟࡋࡓ.
1.3.14㡯 ྍ⁐ᆺCS#3-scFvࢆ⏝࠸ࡿELISA
1.2.5 㡯‽ࡌ࡚ CS-BSA ࢆᅛᐃࡋࡓ࣐ࢡࣟࣉ࣮ࣞࢺ, 1.0%ࣈࣟࢵࢡ࢚࣮ࢫ⁐
ᾮ (300 mL/࢙࢘ࣝ) ࢆῧຍࡋ࡚, 37Υ࡛2㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ⁐ᾮࢆ྾ᘬ㝖ཤࡋ, PBS࡛3ᅇὙίࡋ࡚ࣉ࣮ࣞࢺࢆࣈࣟࢵ࢟ࣥࢢࡋࡓ. ࡇࡢࣉ࣮ࣞࢺ, G-PBS࡛ᕼ㔘ࡋࡓ CS#3-scFv (1.3.12㡯࡛స〇ࡋࡓࡶࡢ) (100 mL) , ྛ✀⃰ᗘࡢCS ᶆ‽ရࢆྵࡴG-PBS
⁐ᾮ (50 mL) ࢆຍ࠼࡚ΰࡋ, 4Υ࡛2㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ⁐ᾮࢆ྾ᘬ㝖ཤࡋ, T-PBS࡛3ᅇὙίࡋࡓࡢࡕ, PVG-PBS࡛5000ಸᕼ㔘ࡋࡓPODᶆ㆑ᢠFLAGᢠయ (100 mL)
ࢆຍ࠼, 37Υ࡛30ศ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ࡧ⁐ᾮࢆ྾ᘬ㝖ཤࡋ, T-PBS࡛3ᅇὙίࡋ
ࡓࡢࡕ, 1.2.5㡯‽ࡌ࡚ᅛ┦ୖࡢPODάᛶࢆẚⰍ ᐃࡋࡓ.
➨⠇ᒓᐇ㦂
1.4.1㡯 Error-prone PCRࡼࡿኚ␗scFv㑇ఏᏊࡢㄪ〇
1.3.8 㡯࡛ᚓࡽࢀࡓ wt-scFv 㑇ఏᏊᤄධࣉࣛࢫ࣑ࢻ (1 ng) , pEX7-VH-CP5 ࠾ࡼࡧ pEX7-VL-CP3 ࣉ ࣛ ࣐࣮ (ྛ 100 pmol), dNTP ΰ ྜ ≀ [deoxyguanosine 5’-triphosphate (dGTP), dCTP, ࠾ࡼࡧdeoxythymidine 5’-triphosphate (dTTP) ྛ0.1 mmoldATP 0.02 mmol
ࢆྵࡴ)], ࠾ࡼࡧ AmpliTaq DNA ࣏࣓࣮ࣜࣛࢮ (5 U) ࢆຍ࠼, error-prone PCR ⏝⦆⾪ᾮ [16.6 mM (NH4)2SO4, 6.1 mM MgCl2, 6.7 mM EDTA, 10 mM 2-࣓ࣝ࢝ࣉࢺ࢚ࢱࣀ࣮ࣝ, 10%
(v/v) DMSO, 0.17 g/L BSA, ࠾ࡼࡧ0.10 mM MnCl2ࢆྵࡴ67 mM Tris-HCl⦆⾪ᾮ (pH 8.8)]
୰ (100 mL) ࡛PCR ࢆ⾜ࡗࡓ. PCR᮲௳ࡣ⇕ኚᛶ 95Υ, 1ศ, ࢽ࣮ࣜࣥࢢ50Υ, 1ศ, ఙ㛗72Υ, 3ศࡋࡓ. ࡇࡢࢧࢡࣝࢆ35ᅇ⧞ࡾ㏉ࡋࡓࡢࡕ, 72Υ࡛10ศࡢఙ㛗ᛂࢆ
⾜ࡗࡓ. ᛂᾮ୰ࡢቑᖜ⏘≀ࢆ DNA ⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ, ኚ␗ scFv 㑇ఏ Ꮚࡢࣛࣈ࣮ࣛࣜࢆᚓࡓ.
1.4.2㡯 ኚ␗scFv㑇ఏᏊࡢࢡ࣮ࣟࢽࣥࢢDNAሷᇶ㓄ิࡢỴᐃ
๓㡯࡛స〇ࡋࡓኚ␗scFv㑇ఏᏊ (4 mg) , ไ㝈㓝⣲Nco I࠾ࡼࡧNot I (20 U) ࢆຍ
࠼, ᛂ⏝⦆⾪ᾮ୰ (100 mL), 37Υ୍࡛ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ᛂᾮ୰ࡢቑᖜ⏘≀ࢆ
DNA ⢭〇࢟ࢵࢺ (A) ࡼࡾ⢭〇ࡋ, ࡑࡢ୍㒊 (125 ng) ࢆྠᵝไ㝈㓝⣲࡛ᾘࡋࡓ
pEXmide 7 (250 ng) ΰྜࡋ࡚ 1.3.5 㡯‽ࡌ࡚ࣛࢤ࣮ࢩࣙࣥᛂࡋ, ࡑࡢ⏘≀
ࢆ1.3.5㡯‽ࡌࡓ᪉ἲ࡛TG1࢚ࣞࢡࢺࣟࢥࣥࣆࢸࣥࢺ⣽⬊ᑟධࡋࡓ. ᙧ㉁㌿⳦ࢆ
2YT-AG (1%) ࣮࢞ࣉ࣮ࣞࢺୖሬᕸࡋ, 37Υ୍࡛ᬌᇵ㣴ࡋࡓ. ࡇࡢࣉ࣮ࣞࢺࡽࣛ
ࣥࢲ࣒㑅ᢥࡋࡓࢥࣟࢽ࣮ࢆࢥࣟࢽ࣮PCRࡋ, scFv㑇ఏᏊࡀᑟධࡉࢀࡓ⳦ࢆྠᐃࡋ
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ࡓ. ࡞࠾, ࣉ࣐࣮ࣛࡣpEX7-VH-CP5࠾ࡼࡧpEX7-VL-CP3 (ྛ2 pmol) ࢆ⏝࠸ࡓ. ≉ᐃ ࡉ ࢀ ࡓ ⳦ ࡢ ᠱ ⃮ ᾮ ࢆ 2YT-AG (1%) ᇵ ᆅ (5 mL) ᥋ ✀ ࡋ 37Υ ࡛ ୍ ᬌ ࠺ ᇵ 㣴 (~200 rpm) ࡋࡓ. ࡑࡢ୍㒊 (1 mL) ᑐࡋ࡚, ⤊⃰ᗘࡀ15%࡞ࡿࡼ࠺ࢢࣜࢭ࣮ࣟࣝ
ࢆῧຍࡋ࡚-80Υ࡛ಖᏑࡋࡓ. ṧࡾࡢᇵ㣴ᾮࡽ㐲ᚰศ㞳 (1800g, 20 ศ, ᐊ ) ࡼࡾ
㞟⳦ࡋࡓࡢࡕ, 1.3.6㡯ྠᵝࣉࣛࢫ࣑ࢻࢆᢳฟࡋ, scFv㑇ఏᏊࡢሷᇶ㓄ิࢆゎᯒࡋࡓ.
1.4.3㡯 KM13࣊ࣝࣃ࣮ࣇ࣮ࢪࡢㄪ〇
⭠⳦TG1-Tr⣽⬊ࡢࢢࣜࢭ࣮ࣟࣝ⤖ಖᏑᾮ (200 mL) ࢆ, 2YTᇵᆅ (20 mL) ᥋✀ࡋ࡚, ᑐᩘቑṪᮇ㐩ࡍࡿࡲ࡛ 37Υ࡛࠺ᇵ㣴 (~200 rpm) ࡋࡓ. ࡇࡢᇵ㣴ᾮࡢ
୍㒊 (200 mL) PBS ࡛ẁ㝵ᕼ㔘 (1102~1012 ಸᕼ㔘) ࡋࡓ KM13 ࣊ࣝࣃ࣮ࣇ࣮ࢪ
ᾮ (10 mL) ࢆࡑࢀࡒࢀῧຍࡋ, 37Υ࡛30ศ㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ࡇࡢࣇ࣮ࢪᠱ⃮ᾮ
, 42Υಖ ࡋࡓࢺࢵࣉ࣮࢞ (3.0 mL) ࢆຍ࠼࡚ΰࡋࡓࡢࡕ, TYE࣮࢞ࣉ࣮ࣞࢺ
ሬᕸࡋ࡚37Υ୍࡛ᬌᇵ㣴ࡋࡓ. ࡇࡢࣉ࣮ࣞࢺࡽࢩࣥࢢࣝࣉ࣮ࣛࢡࢆྲྀࡾ, ࡇࢀࢆᑐ
ᩘቑṪᮇࡢTG1-Trᇵ㣴ᾮ (5.0 mL) ῧຍࡋ࡚, 37Υ࡛⣙2㛫࠺ᇵ㣴 (~200 rpm) ࡋࡓ. ࡇࡢᇵ㣴ᾮࡢ㔞ࢆ 2YT ᇵᆅ (500 mL) ⛣ࡋ࡚, 37Υ࡛ 1 㛫࠺ᇵ㣴 (~200 rpm) ࡋࡓࡢࡕ, ◲㓟࢝ࢼ࣐ࢩࣥ (50 mg/L; 500 mL) ࢆῧຍࡋ࡚, 30Υ࡛ࡉࡽ୍
ᬌ࠺ᇵ㣴 (~200 rpm) ࡋࡓ. 㐲ᚰศ㞳 (10800g, 15 ศ, 4Υ) ᚋ, ୖΎ (480 mL)
PEG/NaClᾮ (120 mL) ࢆຍ࠼࡚ΰྜࡋ, ịୖ࡛1㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ࡇࡢΰྜᾮ
ࢆ㐲ᚰศ㞳 (10800g, 30ศ, 4Υ) ࡋ࡚ୖΎࢆ㝖ཤࡋ, ỿẊࡋࡓࣇ࣮ࢪࢆ PBS (9.6 mL)
⁐ゎࡋ࡚ᗘPEG/NaClᾮ (2.4 mL) ࢆຍ࠼࡚ΰྜࡋ, ịୖ࡛20ศ㛫࣮ࣥ࢟ࣗ࣋ࢺ ࡋࡓ. 㐲ᚰศ㞳 (3300g, 30ศ, 4Υ) ᚋ, ỿẊࢆPBS (6.0 mL) ᠱ⃮ࡋ, ṧᏑࡍࡿ⭠⳦
⏤᮶ࡢᅛᙧ≀ࢆ㝖ࡃࡓࡵࡧ㐲ᚰศ㞳 (11600g, 10ศ, ᐊ ) ࡋ࡚, KM13࣊ࣝࣃ࣮ࣇ
࣮ࢪࢆྵࡴୖΎࢆ4Υ࡛ಖᏑࡋࡓ.
1.4.4㡯 KM13࣊ࣝࣃ࣮ࣇ࣮ࢪࡢຊ౯⟬ᐃ
๓㡯࡛ㄪ〇ࡋࡓ࣊ࣝࣃ࣮ࣇ࣮ࢪᾮ (45 mL) , PBS࡛ᕼ㔘ࡋࡓࢺࣜࣉࢩࣥ⁐ᾮ (1.0 g/L; 5.0 mL) ࡲࡓࡣPBS (5.0 mL) ࢆῧຍࡋ, 37Υ࡛30ศ㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ࢺࣜࣉ
ࢩࣥฎ⌮ࡲࡓࡣᮍฎ⌮ࡢࣇ࣮ࢪࢆ PBS ࡛ẁ㝵ᕼ㔘 (1103~1013 ಸᕼ㔘) ࡋ࡚, ྛᕼ 㔘ᾮ (50 mL) ࢆ, ᑐᩘቑṪᮇࡢTG1-Trᇵ㣴ᾮ (1 mL) ῧຍࡋࡓ. ┤ᚋ, 42Υಖ ࡋࡓࢺࢵࣉ࣮࢞ (3 mL) ࢆຍ࠼࡚⣽⬊ࢆᠱ⃮ࡋ, ࡇࢀࢆ TYE ࣮࢞ࣉ࣮ࣞࢺሬᕸ ࡋ࡚37Υ୍࡛ᬌᇵ㣴ࡋࡓ. ฟ⌧ࡋࡓࣉ࣮ࣛࢡࢆィᩘࡍࡿࡇࡼࡾ, KM13 ࣊ࣝࣃ࣮ࣇ
࣮ࢪࡢຊ౯ࢆpfu/mLࡋ࡚⟬ᐃࡋࡓ.
1.4.5㡯 ኚ␗scFvᥦ♧ࣇ࣮ࢪࣛࣈ࣮ࣛࣜࡢㄪ〇ࣇ࣮ࢪࡢຊ౯⟬ᐃ
1.4.2 㡯࡛స〇ࡋࡓኚ␗ scFv 㑇ఏᏊࢆಖᣢࡍࡿ⭠⳦⩌ࡢࢢࣜࢭ࣮ࣟࣝ⤖ಖᏑᾮ
(50 mL) ࢆ2YT-AG (2%) ᇵᆅ (50 mL) ຍ࠼, ᑐᩘቑṪᮇ㐩ࡍࡿࡲ࡛37Υ࡛
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࠺ᇵ㣴 (~200 rpm) ࡋࡓ. ࡇࡢᇵ㣴ᾮࡢ୍㒊 (20 mL2) 1.4.3㡯࡛స〇ࡋࡓKM13࣊
ࣝࣃ࣮ࣇ࣮ࢪࢆῧຍࡋ, 37Υ࡛30ศ㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ࡇࡢࡁ, ࣊ࣝࣃ࣮ࣇ
࣮ࢪࡢMOI (multiplicity of infection; ࣇ࣮ࢪ pfu/⭠⳦⣽⬊ᩘ) ࡣ20ࡋࡓ. ᇵ㣴ᾮ
ࢆ㐲ᚰศ㞳 (1800g, 20ศ, ᐊ ) ࡋ, ỿẊࡋࡓ⣽⬊ࢆ 2YT-AKᇵᆅ (20 mL2) ᠱ
⃮ࡋࡓࡢࡕ, 25Υ୍࡛ᬌ࠺ᇵ㣴 (120 rpm) ࡋࡓ. ᇵ㣴ᾮࢆ㐲ᚰศ㞳 (1800g, 20 ศ, ᐊ ) ࡋ, ୖΎ PEG/NaClᾮ (5 mL2) ࢆຍ࠼࡚ΰྜࡋ, ịୖ࡛1㛫࣮ࣥ࢟ࣗ࣋
ࢺࡋࡓ. ࡇࡢΰྜᾮࢆ㐲ᚰศ㞳 (10000g, 30 ศ, 4Υ) ࡋ࡚ୖΎࢆ㝖ཤࡋ, ỿẊࡋࡓࣇ
࣮ࢪࢆ PBS-2 (200 mL2) ᠱ⃮ࡋࡓ. ṧᏑࡍࡿ⭠⳦⏤᮶ࡢᅛᙧ≀ࢆ㝖ࡃࡓࡵ,
ࡧ㐲ᚰศ㞳 (15700g, 10ศ, 4Υ) ࡋ࡚, ୖΎࡋ࡚ᚓࡽࢀࡿscFvᥦ♧ࣇ࣮ࢪᾮࢆ1ࡘ
ࡲࡵ (400 mL), 4Υ࡛ಖᏑࡋࡓ. ࡑࡢ୍㒊 (5.0 mL) ࢆPBS-2࡛ẁ㝵ᕼ㔘 (1107~109 ಸᕼ㔘) ࡋ, ྛᕼ㔘ᾮ (10 mL) ࢆ, ᑐᩘቑṪᮇࡢXL1-Blueᇵ㣴ᾮ (90 mL) ῧຍࡋ࡚, 37Υ࡛30ศ㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ࡇࡢᇵ㣴ᾮࡢ୍㒊 (10 mL) ࢆ2YT-ATG (1%)
࣮࢞ࣉ࣮ࣞࢺୗࡋ࡚ 37Υ୍࡛ᬌᇵ㣴ࡋࡓ. ฟ⌧ࡋࡓࢥࣟࢽ࣮ࢆィᩘࡍࡿࡇࡼ
ࡾ, ࣇ࣮ࢪᾮ1 mL࠶ࡓࡾࡢࢥࣟࢽ࣮ᩘ (cfu/mL) ⟬ࡋ, scFvᥦ♧ࣇ࣮ࢪࡢຊ౯
ࢆ♧ࡍᣦᶆࡋ࡚⏝࠸ࡓ.
1.4.6㡯 ᢠCSάᛶࢆᣢࡘኚ␗scFvᥦ♧ࣇ࣮ࢪࡢࣃࣥࢽࣥࢢࡼࡿ㑅ᢥ
࣒ࣀࢳ࣮ࣗࣈCS-BSA (5.0 mg/mL) ࢆྵࡴCB (2.0 mL) ࢆῧຍࡋ, ᐊ ୍࡛ᬌᨺ
⨨ࡋࡓ. ⁐ᾮࢆ྾ᘬ㝖ཤࡋ, PBS-2࡛3ᅇὙίࡋࡓࡢࡕ, M-PBS-2 (4 mL) ࢆῧຍࡋ, 37Υ
࡛2㛫ᨺ⨨ࡋࡓ. ⁐ᾮࢆ྾ᘬ㝖ཤࡋ, T-PBS࡛3ᅇὙίࡋ࡚CS-BSAᅛᐃࢳ࣮ࣗࣈ
ࢆస〇ࡋࡓ. ࡇࡢࢳ࣮ࣗࣈ, PVG-PBS ࡛ᕼ㔘ㄪ〇ࡋࡓኚ␗ scFv ᥦ♧ࣇ࣮ࢪࣛࣈ
࣮ࣛࣜ (11011 cfu/mL) (1 mL) ࢆῧຍࡋ, 37Υ࡛1㛫, ࣒ࣀ࣮ࣟࢱ࣮࡛ಽ❧ΰࡋ ࡓ. ⁐ᾮࢆ྾ᘬ㝖ཤᚋ, T-PBS-2࡛3ᅇὙίࡋ, 0.10 M ࢢࣜࢩࣥሷ㓟⦆⾪ᾮ (pH 2.2, 1.0 mL) ࠶ࡿ࠸ࡣ 0.10 M ࢺ࢚ࣜࢳ࣑ࣝࣥ (pH 12, 1.0 mL) ࢆῧຍࡋ, ᐊ ࡛10ศ㛫᧠ᢾ
ࡍ ࡿ ࡇ ࡛, ࢳ ࣮ࣗ ࣈ⤖ ྜ ࡋࡓ ࣇ ࣮ ࢪ ࢆ⁐ฟ ࡋ ࡓ. ࡇࡢ ࣇ ࣮ ࢪ⁐ᾮ ࡑࢀ ࡒ ࢀ 2.0 M Tris (35 mL) ࠶ࡿ࠸ࡣ1.0 M Tris-HCl (pH 7.4) ⦆⾪ᾮ (0.50 mL) ࢆຍ࠼࡚୰ࡋࡓ
ࡢࡕ, 㔞ࢆᑐᩘቑṪᮇࡢTG1ᇵ㣴ᾮ (9 mL) ῧຍࡋ࡚, 37Υ࡛30ศ㛫࣮ࣥ࢟ࣗ࣋
ࢺ ࡋࡓ. ࡇ ࡢᇵ㣴 ᾮࡢ ୍㒊 ࢆẁ 㝵ᕼ 㔘 (1101~104 ಸ ᕼ 㔘) ࡋࡓ ࡢࡕ, ྛ ᕼ㔘 ᾮ (100 mL) ࢆ2YT-AG (1%) ࣮࢞ࣉ࣮ࣞࢺሬᕸࡋ࡚37Υ୍࡛ᬌᇵ㣴ࡋ, ຊ౯ (cfu) ࢆ⟬
ᐃࡋ࡚, ࣇ࣮ࢪࡢᅇ⋡ࢆồࡵࡓ. ୍᪉, ṧࡾࡢឤᰁ⳦ࢆ㐲ᚰศ㞳 (10000g, 10ศ, ᐊ ) ࡋ, ỿẊࢆ2YTᇵᆅ (100 mL) ᠱ⃮ࡋࡓࡢࡕ, 2YT-AG (1%) ࣮࢞ࣉ࣮ࣞࢺ
ሬᕸࡋ࡚ 37Υ୍࡛ᬌᇵ㣴ࡋࡓ. ⩣᪥, 15%ࢢࣜࢭ࣮ࣟࣝࢆྵࡴ 2YT ᇵᆅ (1.5 mL)
ࢆࣉ࣮ࣞࢺୖῧຍࡋ, ࢫࢡࣞࣃ࣮ࢆ⏝࠸࡚⏕ᡂࡋࡓࢥࣟࢽ࣮ࢆᠱ⃮ࡉࡏࡓࡢࡕ,
⤖ಖᏑ⏝ࢳ࣮ࣗࣈ⛣ࡋ࡚-80Υ࡛⤖ಖᏑࡋࡓ. ࡲࡓ, ࡇࡢឤᰁ⳦ᠱ⃮ᾮࢆ⏝࠸࡚๓ 㡯ྠᵝ scFv ᥦ♧ࣇ࣮ࢪࡢㄪ〇ࢆ⾜࠸, ྠᵝࡢ᧯సࢆ⾜ࡗࡓ. ࡇࢀࡽ୍㐃ࡢ᧯స
ࢆ3ࢧࢡࣝ⧞ࡾ㏉ࡋࡓ. ࡞࠾, ࡑࢀࡒࢀࡢ⁐ฟ᮲௳ᑐࡋ, ྠᵝࡢヨ⾜ࢆ3ᅇ⾜ࡗࡓ.
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1.4.7㡯 ࢫࢡ࣮ࣜࢽࣥࢢ⏝࠸ࡿࣔࣀࢡ࣮ࣟࢼࣝኚ␗scFvᥦ♧ࣇ࣮ࢪࡢㄪ〇
๓㡯ࡢ᧯స࡛ᅇࡋࡓࣇ࣮ࢪࡢឤᰁ⳦ࢆሬᕸࡋࡓ࣮࢞ࣉ࣮ࣞࢺࡽ 1 ヨ⾜ࡘ ࡁ50ࢥࣟࢽ࣮ࢆࣛࣥࢲ࣒㑅ᢥ (503 ヨ⾜2 ᮲௳; ィ300ࢥࣟࢽ࣮) ࡋ, ྛࢥࣟࢽ
࣮ࢆ2YT-AG (2%) ᇵᆅ (1 mL) ᠱ⃮ࡋ࡚ᇵ㣴ࡋ, 1.4.5㡯‽ࡌ࡚KM13࣊ࣝࣃ࣮
ࣇ࣮ࢪࢆឤᰁࡉࡏࡓ. 㐲ᚰศ㞳 (1800g, 10 ศ, ᐊ ) ᚋ, ỿẊࢆ 2YT-AK ᇵᆅ (1 mL) ࡛ᠱ⃮ࡋ, 25Υ୍࡛ᬌ࠺ᇵ㣴 (~200 rpm) ࡋࡓ. ᇵ㣴ᾮࢆ㐲ᚰศ㞳 (1800g, 10
ศ, ᐊ ) ࡋ, ୖΎࢆ 4Υ࡛ಖᏑࡋࡓ. ࡞࠾, ࣇ࣮ࢪࡢቑᖜࡸ scFv 㑇ఏᏊࡢゎᯒ
ഛ࠼࡚, ᑐᩘቑṪᮇࡲ࡛ቑṪࡋࡓྛࢡ࣮ࣟࣥࡢᇵ㣴ᾮࡢ୍㒊 (10 mL) ࢆ4Υ࡛ಖᏑࡋࡓ.
1.4.8㡯 ࣔࣀࢡ࣮ࣟࢼࣝኚ␗scFvᥦ♧ࣇ࣮ࢪࡢࢫࢡ࣮ࣜࢽࣥࢢ
1.2.5 㡯‽ࡌ࡚స〇ࡋࡓ CS-BSA ᅛᐃ࣐ࢡࣟࣉ࣮ࣞࢺ, ๓㡯࡛ㄪ〇ࡋࡓࣇ
࣮ࢪࢆྵࡴୖΎ (100 mL) ࢆຍ࠼, 37Υ࡛1㛫ᛂࡉࡏࡓ. ⁐ᾮࢆ྾ᘬ㝖ཤࡋ, T-PBS-2࡛3ᅇὙίࡋࡓࡢࡕ, M-PBS࡛5000ಸᕼ㔘ࡋࡓPODᶆ㆑ᢠM13ࣔࣀࢡ࣮ࣟࢼࣝᢠయ
(100 mL) ࢆຍ࠼, 37Υ࡛ 30 ศ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ྠᵝὙίࡋࡓࡢࡕ, ࢙࢘ࣝୖࡢ
PODάᛶࢆ1.2.5 㡯ྠᵝẚⰍ ᐃࡋࡓ. ྾ගᗘࡀ 0.3௨ୖㄆࡵࡽࢀࡓࢡ࣮ࣟࣥࡘ
࠸࡚, 1.4.7㡯࡛ಖᏑࡋࡓ⭠⳦ᾮࡢ୍㒊 (2 mL) ࢆ2YT-AG (1%) ᇵᆅ (1 mL) ᥋✀
ࡋ࡚37Υ୍࡛ᬌᇵ㣴ࡋ, ⤊⃰ᗘࡀ15%࡞ࡿࡼ࠺ࢢࣜࢭ࣮ࣟࣝࢆຍ࠼࡚-80Υ࡛⤖
ಖᏑࡋࡓ.
1.4.9㡯 ࣔࣀࢡ࣮ࣟࢼࣝኚ␗scFvᥦ♧ࣇ࣮ࢪࡢㄪ〇
๓㡯࡛ಖᏑࡋࡓ⭠⳦ࡢ⤖ಖᏑᾮ (200 mL) ࢆ2YT-AG (1%) ᇵᆅ (20 mL) ᥋
✀ࡋ࡚, 1.4.5㡯‽ࡌ࡚ࣔࣀࢡ࣮ࣟࢼࣝscFvᥦ♧ࣇ࣮ࢪࢆྵࡴPBS-2⁐ᾮ (200 mL)
ࢆᚓࡓ.
1.4.10㡯 ࣔࣀࢡ࣮ࣟࢼࣝኚ␗scFvᥦ♧ࣇ࣮ࢪࡢᢠCSάᛶࡢホ౯
1.4.8 㡯‽ࡌ࡚ CS-BSA ࢆᅛᐃࡋࡓ࣐ࢡࣟࣉ࣮ࣞࢺ, PVG-PBS ࡛ᕼ㔘ࡋࡓ
scFvᥦ♧ࣇ࣮ࢪ (๓㡯࡛స〇ࡋࡓࡶࡢ) (100 mL) ࠾ࡼࡧྛ✀⃰ᗘࡢCSᶆ‽ရࢆྵࡴ
PVG-PBS⁐ᾮ (50 mL) ࢆຍ࠼࡚ΰࡋ, 37Υ࡛1㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ⁐ᾮࢆ྾ᘬ
㝖ཤࡋ, TPBS-2࡛3ᅇὙίࡋࡓࡢࡕ, M-PBS࡛5000ಸᕼ㔘ࡋࡓPODᶆ㆑ᢠM13ࣔࣀ
ࢡ࣮ࣟࢼࣝᢠయ (100 mL) ࢆຍ࠼ 37Υ࡛ 30 ศ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ྠᵝὙίࡋࡓࡢ ࡕ, ࢙࢘ࣝୖࡢPODάᛶࢆ1.3.14㡯ྠᵝẚⰍ ᐃࡋࡓ.
1.4.11㡯 ྍ⁐ᆺኚ␗scFvࡢㄪ〇
๓㡯࡛wt-scFvࡼࡾࡶ㧗ぶຊ࡛࠶ࡿ᥎ᐃࡉࢀࡓࢡ࣮ࣟࣥࡘ࠸࡚, 1.3.6㡯‽ࡌ
࡚ࣉࣛࢫ࣑ࢻࢆᢳฟࡋࡓ. ࡇࡢ୍㒊 (100 ng) , pEX7-VH-CP5࠾ࡼࡧpEX7-VL-CP3 ࣉ
࣐࣮ࣛ (ྛ20 pmol), dNTPΰྜ≀ (ྛ40 nmol), ࠾ࡼࡧEx Taq DNA࣏࣓࣮ࣜࣛࢮ (2.5
70
U) ࢆຍ࠼, ᑓ⏝⦆⾪ᾮ୰ (100 mL) ࡛PCRࢆ⾜ࡗࡓ. PCR᮲௳ࡣ⇕ኚᛶ95Υ, 30⛊,
ࢽ࣮ࣜࣥࢢ55Υ, 30 ⛊, ఙ㛗 72Υ, 1ศࡋࡓ. ࡇࡢࢧࢡࣝࢆ 35ᅇ⧞ࡾ㏉ࡋࡓࡢࡕ, 72Υ࡛5ศࡢఙ㛗ᛂࢆ⾜ࡗࡓ. ᛂᾮ୰ࡢቑᖜ⏘≀ࢆDNA⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚
⢭〇ࡋࡓࡢࡕ, 1.4.2 㡯ྠᵝไ㝈㓝⣲ Nco I࠾ࡼࡧ Not I࡛ᾘࡋ, ࣮࢞ࣟࢫࢤࣝ
(2%) ࢆ⏝࠸ࡿ㟁ẼὋືࢆ⾜ࡗ࡚, ไ㝈㓝⣲ฎ⌮῭ࡳDNA᩿∦ࢆᚓࡓ. ࡇࢀࢆ, 1.3.5㡯
‽ࡌ࡚, ྠᵝไ㝈㓝⣲࡛ᾘࡋࡓ pEXmide 7’㐃⤖ࡋ, ࡑࡢ⏘≀ࢆ 1.3.5 㡯‽ࡌ
ࡓ᪉ἲ࡛XL1-Blue࢚ࣞࢡࢺࣟࢥࣥࣆࢸࣥࢺ⣽⬊ᑟධࡋࡓ. ᙧ㉁㌿⳦ࢆ2YT-ATG
(1%) ࣮࢞ࣉ࣮ࣞࢺ ሬᕸࡋ࡚ 37Υ୍࡛ᬌᇵ 㣴ࡋࡓ. ฟ⌧ࡋࡓࢥࣟ ࢽ ࣮ࢆࢥࣟࢽ࣮
PCRࡋ, ≉ᐃࡋࡓ㑇ఏᏊಖᣢ⳦ࢆ2YT-AG (1%) ᇵᆅ (20 mL) ᥋✀ࡋ, 1.3.12㡯
‽ࡌ࡚࣌ࣜࣉࣛࢬ࣒ᢳฟᾮࢆᅇࡋࡓ.
1.4.12㡯 Scatchardἲࡼࡿ⤖ྜᐃᩘࡢ⟬ฟ
1.2.4㡯‽ࡌ࡚ᐇ㦂ࢆ⾜࠸, Scatchardἲࡼࡾྍ⁐ᆺኚ␗scFvࡢCSᑐࡍࡿKaࢆ
⟬ฟࡋࡓ.
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➨❶ᒓᐇ㦂
➨⠇ᒓᐇ㦂
2.2.1㡯 VCSM13࣊ࣝࣃ࣮ࣇ࣮ࢪࡢㄪ〇
⭠⳦XL1-Blueࡢࢢࣜࢭ࣮ࣟࣝ⤖ಖᏑᾮࢆᑡ㔞ࡾ, 2YT-Tᇵᆅ (10 mL) ᥋
✀ࡋ࡚ 37Υ୍࡛ᬌ࠺ᇵ㣴 (~200 rpm) ࡋࡓ. ࡑࡢ୍㒊 (800 mL) ࢆ 2YT-T ᇵᆅ
(80 mL) ຍ࠼࡚ 600 nm ࠾ࡅࡿ྾ගᗘࡀ 0.3 ࡞ࡿࡲ࡛ 37Υ࡛࠺ᇵ㣴 (~200
rpm) ࡋࡓ. ࡇࡇ, VCSM13࣊ࣝࣃ࣮ࣇ࣮ࢪ (௨ୗ, VCSM13) ࢆMOIࡀ20࡞ࡿࡼ
࠺ຍ࠼, 37Υ࡛ 30ศ࠺ᇵ㣴 (120 rpm) ࡋࡓࡢࡕ, ◲㓟࢝ࢼ࣐ࢩࣥ (50 mg/mL;
32 mL) ࢆຍ࠼࡚ࡉࡽ37Υ࡛8㛫࠺ᇵ㣴 (120 rpm) ࡋࡓ. ᇵ㣴ᾮࢆ65Υ࡛15ศ
࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࡢࡕ㐲ᚰ (1800g, 20 ศ, ᐊ ) ࡋ࡚ୖΎࢆศ㞳ࡋ, ࢁ㐣 (0.45 mm) ࡍࡿࡇ࡛⭠⳦ࢆ㝖࠸ࡓ. ࡇࡇ, PEG/NaClᾮ (20 mL) ࢆຍ࠼, ịୖ࡛1㛫ࣥ࢟
࣮ࣗ࣋ࢺࡋ࡚㐲ᚰศ㞳 (10000g, 30 ศ, 4Υ) ࡋ, ୖΎࢆ㝖ཤࡋࡓࡢࡕỿẊࡋࡓࣇ࣮ࢪ
ࢆ PBS-2 (800 mL) ⁐ ゎ ࡋ ࡓ. ṧ Ꮡ ࡍ ࡿ ⭠ ⳦ ⏤ ᮶ ࡢ ᅛ ᙧ ≀ ࢆ 㝖 ࡃ ࡓ ࡵ ࡧ 㐲 ᚰ (15700g, 10ศ, 4Υ) ࡋ࡚VCSM13ࢆྵࡴୖΎࢆศ㞳ࡋ, ⏝ࡲ࡛-20Υ࡛ಖᏑࡋࡓ.
2.2.2㡯 VCSM13࣊ࣝࣃ࣮ࣇ࣮ࢪࡢຊ౯⟬ᐃ
๓㡯࡛స〇ࡋࡓ VCSM13 ᾮ (5 mL) ࢆPBS-2 ࡛ẁ㝵ᕼ㔘 (1107~1010ಸᕼ㔘) ࡋ࡚,
ྛᕼ㔘ᾮࡢ୍㒊 (10 mL) ࢆᑐᩘቑṪᮇࡢ XL1-Blue ᇵ㣴ᾮ (100 mL) ῧຍࡋࡓ. ┤ࡕ
42Υಖ ࡋࡓࢺࢵࣉ࣮࢞ (3.5 mL) ࢆຍ࠼࡚⣽⬊ࢆᠱ⃮ࡋ, ࡇࢀࢆ2YT-T࢞
࣮ࣉ࣮ࣞࢺሬᕸࡋ࡚37Υ୍࡛ᬌᇵ㣴ࡋࡓ. ฟ⌧ࡋࡓࣉ࣮ࣛࢡࢆィᩘࡋ࡚ࣇ࣮ࢪᾮ1 mL࠶ࡓࡾࡢࣉ࣮ࣛࢡᩘ (pfu/mL) ⟬ࡋ, VCSM13ࡢຊ౯ࢆ♧ࡍᣦᶆࡋ࡚⏝࠸ࡓ.
2.2.3㡯 ᢠVCSM13ᢠయ⏘⏕ࣁࣈࣜࢻ࣮࣐ᰴࡢᶞ❧࠾ࡼࡧᢠVCSM13ࣔࣀࢡ࣮ࣟࢼ
ࣝᢠయࡢㄪ〇
2.2.1㡯࡛స〇ࡋࡓVCSM13 (11010 pfu) ࡢ⏕⌮㣗ሷỈ⁐ᾮ (160 mL) ࢆAbISCO-100
ࢪࣗࣂࣥࢺ (40 mL) ΰྜࡋࡓࡢࡕ, 8㐌㱋ࡢBALB/c࣐࢘ࢫ (㞤) ࡢ⫼୰⓶ୗᢞ
ࡋࡓ. 2㐌㛫㝸࡛3ᅇ㏣ຍචࢆ⾜࠸, ᭱⤊චࡽ7᪥ᚋ, VCSM13 (11010 pfu) ࢆྵ
ࡴ⏕⌮㣗ሷỈࢆ࣐࢘ࢫࡢ⭡⭍ෆ (500 mL) ⭁⮚ (200 mL) ᢞࡋ, 3᪥ᚋ⭁⮚ࢆ
ฟࡋࡓ. ᚓࡽࢀࡿ⭁⣽⬊ (⣙1108 ಶ) ࢆ, 1.2.2㡯‽ࡌ࡚࣑࢚࣮࣐ࣟ P3/NS1/1-Ag4-1 ⣽⬊⼥ྜࡉࡏࡓ. ⼥ྜᚋࡢ⣽⬊ࢆ, 96 ࢙࢘ࣝࢡࣛࢫࢱ࣮ࢹࢵࢩࣗ୰, HAT ᇵᆅ࡛
⣙2㐌㛫ᇵ㣴ࡍࡿࡇࡼࡾࣁࣈࣜࢻ࣮࣐ࡢ㑅ᢥࢆ⾜ࡗࡓ. ࣁࣈࣜࢻ࣮࣐ࡢቑṪࡀ
☜ㄆࡉࢀࡓ࣐ࢡ࢙ࣟ࢘ࣝࡢᇵ㣴ୖΎࢆ, ୗグࡢ ELISAࡋ࡚, ᢠVCSM13 ᢠయࡢ ศἪࡢ᭷↓ࢆㄪࡓ. 㝧ᛶࢆ♧ࡋࡓࣁࣈࣜࢻ࣮࣐⣽⬊ࢆHTᇵᆅ࡛ᣑᇵ㣴ࡋࡓࡢࡕ, 㝈⏺ᕼ㔘ἲࡼࡾࢡ࣮ࣟࣥࡋࡓ.
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2.2.4㡯 ELISAࡼࡿᇵ㣴ୖΎ୰ᢠVCSM13ᢠయࡢ᳨ฟ
VCSM13ࡢPBS⁐ᾮ (11011 pfu/mL) ࢆELISA⏝96࢙࣐࢘ࣝࢡࣟࣉ࣮ࣞࢺศ
ὀ (100 mL/࢙࢘ࣝ) ࡋ, 4Υ୍࡛ᬌᨺ⨨ࡋࡓ. ⁐ᾮࢆ྾ᘬ㝖ཤࡋ, PBS (300 mL/࢙࢘ࣝ) ࡛ 3ᅇὙίࡋࡓࡢࡕ, M-PBS (300 mL/࢙࢘ࣝ) ࢆῧຍࡋ, 37Υ࡛1㛫ᨺ⨨ࡋ࡚ࣈࣟࢵ࢟ࣥ
ࢢࢆ⾜ࡗࡓ. ࢙࢘ࣝࢆT-PBS࡛3ᅇὙίࡋࡓࡢࡕ, ᳨య࡛࠶ࡿࣁࣈࣜࢻ࣮࣐ᇵ㣴ୖΎ
(G-PBS࡛5ಸ௨ୖᕼ㔘ࡋࡓࡶࡢ; 100 mL) ࢆຍ࠼࡚37Υ࡛1㛫ᛂࡉࡏࡓ. ྠᵝ
⁐ᾮࢆ㝖ཤ, ࢙࢘ࣝࢆὙίࡋࡓࡢࡕ, PODᶆ㆑ࣖࢠᢠ࣐࢘ࢫIgGᢠయࡢG-PBS⁐ᾮ (160 ng/mL) ࢆศὀ (100 mL/࢙࢘ࣝ) ࡋ, 37Υ࡛ 1 㛫ᨺ⨨ࡋࡓ. ⁐ᾮࢆ㝖ཤ, ࢙࢘ࣝࢆ T-PBS࡛3ᅇὙίࡋࡓࡢࡕ, PODάᛶࢆ1.2.3㡯‽ࡌ࡚ẚⰍ ᐃࡋࡓ.
2.2.5㡯 Ab-M13#71ࢆ⏝࠸ࡿ➇ྜELISA
๓㡯‽ࡌ࡚ VCSM13 ࢆᅛᐃࡋࡓ࣐ࢡࣟࣉ࣮ࣞࢺ, PVG-PBS ࡛㐺ษᕼ㔘ࡋ ࡓAb-M13#71 (2.2.3㡯࡛స〇) , ྛ✀⃰ᗘࡢVCSM13ࢆྵࡴPVG-PBS⁐ᾮ (50 mL)
ࢆຍ࠼࡚ΰࡋ, 37Υ࡛1㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ⁐ᾮࢆ྾ᘬ㝖ཤࡋ, T-PBS࡛3ᅇὙ ίࡋࡓࡢࡕ, PODᶆ㆑ࣖࢠᢠ࣐࢘ࢫIgGࡢG-PBS⁐ᾮ (160 ng/mL) ࢆศὀ (100 mL/࢘
࢙ࣝ) ࡋ, 37Υ࡛30 ศ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ࡧ⁐ᾮࢆ྾ᘬ㝖ཤࡋ, T-PBS࡛3 ᅇὙί
ࡋࡓࡢࡕ, PODάᛶࢆ1.2.5㡯‽ࡌ࡚ẚⰍ ᐃࡋࡓ.
2.2.6㡯 ࣒ࣀࣈࣟࢵࢺἲࡼࡿAb-M13#71ࡢ࢚ࣆࢺ࣮ࣉࡢ᥎ᐃ
2.2.1 㡯࡛స〇ࡋࡓ VCSM13 (11010ࡲࡓࡣ 1109 pfu) ࠾ࡼࡧࢪࢳ࢜ࢫࣞࢺ࣮ࣝ
(0.8 mmol) ࢆ, ᑓ⏝⦆⾪ᾮ (ࡑࢀࡒࢀ16ࡲࡓࡣ10 mL) ΰࡋ, 70Υ࡛10ศࣥ࢟ࣗ
࣮࣋ࢺࡋࡓ. ࡇࡢΰྜᾮࢆ, ࣏ࣜࢡ࣑ࣜࣝࢻࢤࣝ (4~12%) 㟁ẼὋື (200 V, 35 ศ)
ࡋ࡚ࢱࣥࣃࢡ㉁ࢆศ㞳ࡋࡓࡢࡕ, PVDF⭷㌿ࡋࡓ. ࡇࡢ⭷ࢆ1.0%ࣈࣟࢵࢡ࢚࣮
ࢫ⁐ᾮ (~30 mL) ᾐࡋ࡚ᐊ ࡛ 2 㛫࠺ (~60 rpm) ࡍࡿࡇ࡛ࣈࣟࢵ࢟ࣥࢢࡋ
ࡓ. ⁐ᾮࢆ㝖ཤࡋ࡚T-PBS (~30 mL) ᾐࡋ, ྠᵝ10ศ㛫࠺ࡍࡿ᧯సࢆ3ᅇ⧞ࡾ
㏉ࡍࡇ࡛⭷ࢆὙίࡋࡓࡢࡕ, Ab-M13#71ࢆG-PBS࡛1000ಸᕼ㔘ࡋࡓ⁐ᾮ (~30 mL)
ᾐࡋ࡚ᐊ ࡛1㛫࠺ (~60 rpm) ࡋࡓ. ྠᵝὙίࡋࡓࡢࡕ, PODᶆ㆑ࣖࢠᢠ࣐
࢘ࢫᢠయࢆG-PBS࡛2000ಸᕼ㔘ࡋࡓ⁐ᾮࢆຍ࠼࡚ᐊ ࡛1㛫࠺ (~60 rpm) ࡋ ࡓ. Ὑίᚋ, ⢭〇Ỉ࡛ 10 ಸᕼ㔘ࡋࡓ TMB ⁐ᾮ୰ (~30 mL), ᐊ ࡛ 30 ศ࠺ (~60 rpm) ࡋ, PODάᛶࢆྍどࡋࡓ.
➨⠇ᒓᐇ㦂
2.3.1㡯 Ab-M13#71ᢠయྍኚ㒊㑇ఏᏊࢆྵࡴcDNAࡢㄪ〇
2.2.3 㡯࡛ᶞ❧ࡋࡓ Ab-M13#71 ศἪࣁࣈࣜࢻ࣮࣐⣽⬊ (~1107 ಶ) ࡽ, 1.3.1 㡯
‽ࡌ࡚⥲RNAࢆᢳฟࡋ, VH-cDNA, VL-cDNAࢆᚓࡓ. ࡞࠾, ࣉ࣐࣮ࣛࡣ, H㙐ᑐࡋ
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࡚ࡣmg2b-GSP-1, L㙐ᑐࡋ࡚ࡣmk-GSP-1ࢆ⏝࠸ࡓ.
2.3.2㡯 Ab-M13#71ᢠయH㙐ྍኚ㒊㑇ఏᏊ (VH) ࢆྵࡴDNA᩿∦ࡢㄪ〇
๓㡯࡛ㄪ〇ࡋࡓVH-cDNAࢆ1.3.3㡯‽ࡌ࡚5’RACEἲࡋ, VH㑇ఏᏊࢆྵࡴDNA
᩿∦ࢆㄪ〇ࡋࡓ. ࡞࠾, 3’ࣉ࣐࣮ࣛࡣmg2b-GSP-2࠾ࡼࡧmg2b-GSP-3ࢆ⏝࠸ࡓ.
2.3.3㡯 Ab-M13#71ᢠయL㙐ྍኚ㒊㑇ఏᏊ (VL) ࢆྵࡴDNA᩿∦ࡢㄪ〇
2.3.1㡯࡛ㄪ〇ࡋࡓVL-cDNAࡢ⁐ᾮࢆ10ಸᕼ㔘ࡋ, ࡑࡢ୍㒊 (2 mL) , Nichollsࡽ
ࡼࡾሗ࿌ࡉࢀࡓࣘࢽࣂ࣮ࢧࣝࣉ࣐࣮ࣛ (VL-I/III, -IIa, IIb, -IV/VI, -Va, -Vbࡢ6✀ࡢ
࠺ࡕ࠸ࡎࢀ 1 ✀)69) ࠾ࡼࡧ mk-GSP-3 ࣉ࣐࣮ࣛ (ྛ 0.50 mM), dNTP ΰྜ≀ (0.40 mM), ࠾ࡼࡧ Pfu DNA ࣏࣓࣮ࣜࣛࢮ (26 U/mL) ࢆῧຍࡋ, ᑓ⏝⦆⾪ᾮ୰ (20 mL) ࡛ PCRࢆ⾜ࡗࡓ. PCR᮲௳ࡣ, ⇕ኚᛶ95Υ, 1ศ, ࢽ࣮ࣜࣥࢢ50Υ, 1ศ, ఙ㛗72Υ, 2ศ
ࡋ, ࡇࢀࢆ35ᅇ⧞ࡾ㏉ࡋࡓࡢࡕ, 72Υ࡛10ศࡢఙ㛗ᛂࢆຍ࠼ࡓ. ᛂᾮ୰ࡢDNA
ࢆ࣮࢞ࣟࢫࢤࣝ (2%) ࢆ⏝࠸ࡿ㟁ẼὋື࡛ศᯒࡋࡓࡇࢁ, VL-I/III ࣉ࣐࣮ࣛࢆ⏝
࠸ࡿࡁቑᖜࡀㄆࡵࡽࢀࡓ. ࡑࡇ࡛, VL-I/III ࣉ࣐࣮ࣛࢆ⏝࠸ࡿ PCR ࢆ, ୖグྠ
ࡌ᮲௳࡛100 mL ࢫࢣ࣮ࣝࢵࣉࡋ࡚⾜࠸, ᛂᾮࢆDNA⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚
⢭〇ࡋࡓ.
2.3.4㡯 Ab-M13#71ᢠయࡢVHࡲࡓࡣVL㑇ఏᏊࢆྵࡴDNA᩿∦ࡢࢧࣈࢡ࣮ࣟࢽࣥࢢ
2.3.2㡯࠾ࡼࡧ2.3.3㡯࡛ᚓࡽࢀࡓVHࡲࡓࡣVL㑇ఏᏊࢆྵࡴDNA᩿∦ (VH; 2.5 mg, VL; 1 mg) , ไ㝈㓝⣲Xma I (25 U) ࢆຍ࠼, BSA (100 mg/mL) ࢆྵࡴᛂ⏝⦆⾪ᾮ୰ (100 mL), 37Υ୍࡛ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ᛂᾮ୰ࡢቑᖜ⏘≀ࢆDNA⢭〇࢟ࢵࢺ (A) ࢆ⏝
࠸࡚⢭〇ࡋ, ࡑࡢ㔞ไ㝈㓝⣲Sal I (25 U) ࢆຍ࠼, ᛂ⏝⦆⾪ᾮ୰ (100 mL), 37Υ࡛
୍ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ቑᖜ⏘≀ࢆࡧDNA⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ, ࡑࡢ
୍㒊 (200 ng) , ྠᵝXma I࠾ࡼࡧSal I࡛ᾘࡋࡓpBluescript II࣋ࢡࢱ࣮ (400 ng)
ࢆΰྜࡋ࡚1.3.5㡯‽ࡌ࡚ࣛࢤ࣮ࢩࣙࣥᛂࡋ, ࡑࡢ⏘≀ࢆXL1-Blue࢚ࣞࢡࢺ
ࣟࢥࣥࣆࢸࣥࢺ⣽⬊ᑟධࡋࡓ. ᙧ㉁㌿⳦ࢆ2YT-ATG (1%) ࣮࢞ࣉ࣮ࣞࢺሬᕸ ࡋ࡚37Υ୍࡛ᬌᇵ㣴ࡋࡓ.
ࡇࡢࣉ࣮ࣞࢺୖࡽࢥࣟࢽ࣮ࢆࣛࣥࢲ࣒㑅ᢥࡋ, 2YTᇵᆅ (10 mL) ᠱ⃮ࡉࡏࡓ. ࡇࡢᠱ⃮ᾮ (2 mL) , 5’࠾ࡼࡧ3’ࣉ࣐࣮ࣛ, dNTPΰྜ≀ (ྛ8 nmol), ࠾ࡼࡧEx Taq DNA࣏࣓࣮ࣜࣛࢮ (0.5 U) ࢆຍ࠼, ᑓ⏝⦆⾪ᾮ୰ (20 mL) ࡛PCR ࢆ⾜ࡗࡓ. ࡞࠾, VH
ࡢቑᖜࡣAUAP࠾ࡼࡧmg2b-GSP-3ࣉ࣐࣮ࣛ (ྛ5 pmol), VLࡢቑᖜࡣVL-I/III࠾
ࡼࡧmk-GSP-3ࣉ࣐࣮ࣛ (ྛ2 pmol) ࢆࡑࢀࡒࢀ⏝࠸ࡓ. PCR᮲௳ࡣ⇕ኚᛶ95Υ, 1ศ,
ࢽ࣮ࣜࣥࢢ64Υ, 1ศ, ఙ㛗72Υ, 2ศࡋ, ࡇࢀࢆ35ᅇ⧞ࡾ㏉ࡋࡓࡢࡕ, 72Υ࡛10ศ ࡢఙ㛗ᛂࢆຍ࠼ࡓ. ᛂᾮࢆ࣮࢞ࣟࢫࢤࣝ (2%) 㟁ẼὋືࡋ࡚┠ⓗࡢ㑇ఏᏊ ࡢ᭷↓ࢆㄪ, VH㑇ఏᏊࡲࡓࡣVL㑇ఏᏊࢆಖᣢࡍࡿᙧ㉁㌿⳦ࢆ≉ᐃࡋࡓ.
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2.3.5㡯 Ab-M13#71ᢠయࡢVH࠾ࡼࡧVL㑇ఏᏊࡢሷᇶ㓄ิࡢỴᐃ
๓㡯࡛Ab-M13#71ᢠయࡢVHࡲࡓࡣVL㑇ఏᏊࡀㄆࡵࡽࢀࡓᙧ㉁㌿⳦ࡘ࠸࡚, 1.3.6
㡯‽ࡌ࡚ࣉࣛࢫ࣑ࢻࢆᢳฟࡋ, ᤄධࡉࢀࡓ㑇ఏᏊࡢሷᇶ㓄ิࢆゎᯒࡋࡓ.
2.3.6㡯 M13-scFv㑇ఏᏊᵓ⠏ࡢࡓࡵࡢVH-DNA࠾ࡼࡧVL-DNA᩿∦ࡢㄪ〇
๓㡯࡛ㄪ〇ࡋࡓ⤌࠼ࣉࣛࢫ࣑ࢻ (50 ng) , ๓㡯࡛ゎᯒࡋࡓሷᇶ㓄ิࡽタィࡋࡓ
ࣉ ࣛ ࣐ ࣮ (VH ࡢ ቑ ᖜ ࡣ M13#71VH-Rev ࠾ ࡼ ࡧ M13#71VH-For, VL ࡢ ቑ ᖜ ࡣ M13#71VL-Rev࠾ࡼࡧM13#71VL-For) (ྛ50 pmol), dNTPΰྜ≀ (ྛ20 nmol), ࠾ࡼࡧEx Taq DNA࣏࣓࣮ࣜࣛࢮ (2.5 U) ࢆຍ࠼, ᑓ⏝⦆⾪ᾮ୰ (100 mL) ࡛PCRࢆ⾜ࡗࡓ. PCR
᮲௳ࡣ⇕ኚᛶ95Υ, 1ศ, ࢽ࣮ࣜࣥࢢ64Υ, 1ศ, ఙ㛗72Υ, 2ศࡋ, ࡇࢀࢆ35ᅇ⧞
ࡾ㏉ࡋࡓࡢࡕ, 72Υ࡛10ศࡢఙ㛗ᛂࢆຍ࠼ࡓ. ᛂᾮ୰ࡢቑᖜ⏘≀ࢆDNA⢭〇࢟ࢵ
ࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ, VH-DNA࠾ࡼࡧVL-DNA᩿∦ࢆᚓࡓ.
2.3.7㡯 Overlap extension PCRࡼࡿM13-scFv㑇ఏᏊࡢᵓ⠏ࢧࣈࢡ࣮ࣟࢽࣥࢢ
๓㡯࡛ㄪ〇ࡋࡓ VH-DNA ࠾ࡼࡧ VL-DNA ᩿∦ (ྛ 200 ng) , dNTP ΰྜ≀ (ྛ 10 nmol) ࠾ࡼࡧEx Taq DNA࣏࣓࣮ࣜࣛࢮ (0.65 U) ࢆຍ࠼, ᑓ⏝⦆⾪ᾮ୰ (25 mL) ࡛PCR
ࢆ⾜ࡗࡓ. PCR᮲௳ࡣ, ⇕ኚᛶ95Υ, 1ศ, ࢽ࣮ࣜࣥࢢ55Υ, 1ศ, ఙ㛗72Υ, 3ศࡋ, ࡇࡢࢧࢡࣝࢆ10ᅇ⧞ࡾ㏉ࡋࡓࡢࡕ, 72Υ࡛10 ศࡢఙ㛗ᛂࢆ㏣ຍࡋࡓ. ࡇࡢᛂᾮ ࡢ୍㒊 (10 mL) , M13#71VH-Rev࠾ࡼࡧFLAG-TAA2ࣉ࣐࣮ࣛ (ྛ100 pmol), dNTP ΰྜ≀ (ྛ 40 nmol), ࠾ࡼࡧ Ex Taq DNA ࣏࣓࣮ࣜࣛࢮ (2.5 U) ࢆຍ࠼, ᑓ⏝⦆⾪ᾮ୰
(100 mL), ୖグࡢ᮲௳࡛ (ࡓࡔࡋ 15 ࢧࢡࣝ) PCR ࢆ⾜ࡗࡓ. ᛂᾮ୰ࡢቑᖜ⏘≀ࢆ
DNA⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ, ࡑࡢ୍㒊 (3 mg) ไ㝈㓝⣲Nco ISal I (ྛ25 U) ࢆຍ࠼, ᛂ⏝⦆⾪ᾮ୰ (100 mL) ࡛37Υ, ୍ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ᛂᾮ୰ࡢቑ
ᖜ⏘≀ࢆDNA⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ, ไ㝈㓝⣲ᾘ῭ࡳࡢDNA᩿∦ࢆᚓࡓ. ࡇࡢDNA᩿∦ (200 ng) , ྠᵝไ㝈㓝⣲Nco ISal I࡛ᾘࡋ, ࣮࢞ࣟࢫࢤࣝ
(1%) 㟁ẼὋືࡼࡾ⢭〇ࡋࡓpEXmide 5 (400 ng) ࢆΰࡋ࡚, 1.3.5㡯ྠᵝࡢࣛࢤ
࣮ࢩࣙࣥᛂࡋ. ࡑࡢ⏘≀ࢆ XL1-Blue ࢚ࣞࢡࢺࣟࢥࣥࣆࢸࣥࢺ⣽⬊ᑟධࡋࡓ. ᙧ㉁㌿⳦ࢆ 2YT-ATG (1%) ࣮࢞ࣉ࣮ࣞࢺୖሬᕸࡋ, 37Υ୍࡛ᬌᇵ㣴ࡋࡓ. ࡇࡢ
ࣉ࣮ࣞࢺࡽࣛࣥࢲ࣒㑅ᢥࡋࡓࢥࣟࢽ࣮ࢆࢥࣟࢽ࣮PCRࡋ, scFv㑇ఏᏊࢆಖᣢࡍ
ࡿࢡ࣮ࣟࣥࢆྠᐃࡋ࡚, 1.3.6 㡯‽ࡌ࡚ࣉࣛࢫ࣑ࢻࢆᢳฟࡋࡓ. ࡇࡢ୍㒊 (600 ng) ࢆ
⏝࠸࡚scFv㑇ఏᏊࡢሷᇶ㓄ิࢆゎᯒࡋࡓ.
2.3.8㡯 ྍ⁐ᆺM13-scFvࡢⓎ⌧㟁ẼὋື
2.3.7 㡯࡛స〇ࡋࡓ M13-scFv 㑇ఏᏊಖᣢ⳦ࡽ, 1.3.12 㡯‽ࡌ࡚࣌ࣜࣉࣛࢬ࣒ᢳฟ
ᾮ ࢆᅇ ࡋ, ᢠ FLAG ᢠ యᅛ ᐃ ࢞ ࣮ࣟ ࢫࢆ ⏝࠸ ࡚⢭ 〇ࡋ ࡓ. ࡇ ࡢ୍ 㒊 (1 mg) ࢆ
2.2.6 㡯‽ࡌ࡚ SDS-PAGE ࡋ, Ὃືᚋࡢࢤࣝࢆࢡ࣐ࢩ࣮ࣈࣜࣜࣥࢺࣈ࣮ࣝᾐ
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ࡋ࡚ᐊ ࡛1㛫࠺ (~60 rpm) ࡍࡿࡇ࡛ࢱࣥࣃࢡ㉁ࢆᰁⰍࡋࡓ. ⁐ᾮࢆ㝖ཤࡋ࡚
⢭〇Ỉᾐࡋ, ྠᵝᐊ ୍࡛ᬌ࠺ࡍࡿࡇ࡛ࢤࣝࢆ⬺Ⰽࡋࡓ.
2.3.9㡯 ྍ⁐ᆺM13-scFvࢆ⏝࠸ࡿ➇ྜELISA
2.2.4㡯‽ࡌ࡚VCSM13ࢆᅛᐃࡋࡓ࣐ࢡࣟࣉ࣮ࣞࢺ, PVG-PBS࡛㐺ษᕼ㔘
ࡋࡓM13-scFv (๓㡯࡛స〇) , ྛ✀⃰ᗘࡢVCSM13ࢆྵࡴPVG-PBS⁐ᾮ (50 mL) ࢆ ຍ࠼࡚ΰࡋ, 37Υ࡛1㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ⁐ᾮࢆ྾ᘬ㝖ཤࡋ, T-PBS-2࡛3ᅇὙ ίࡋࡓࡢࡕ, G-PBS⁐ᾮ࡛1000ಸᕼ㔘ࡋࡓPODᶆ㆑ᢠFLAGᢠయࢆศὀ (100 mL/࢙࢘
ࣝ) ࡋ, 37Υ࡛30ศ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ࡧ⁐ᾮࢆ྾ᘬ㝖ཤࡋ, T-PBS-2࡛3ᅇὙίࡋ
ࡓࡢࡕ, PODάᛶࢆ1.2.5㡯‽ࡌ࡚ẚⰍ ᐃࡋࡓ.
2.3.10㡯 ࣅ࢜ࢳࣥVCSM13ࣇ࣮ࢪࡢస〇
VCSM13 (6.51011 pfu) NHS-LC-Biotin (⣙5 ng) ࢆPBS୰ (1 mL) ΰࡋ, ᧠ᢾ ࡋ࡞ࡀࡽᐊ ࡛2㛫ᛂࡉࡏࡓ. ࡇࢀࢆPBSᑐࡋ࡚4Υ୍࡛ᬌ㏱ᯒࡋࡓࡢࡕ㏱ᯒ⭷
ෆᐜ≀ࢆᅇࡋ, ┠ⓗࡢࣅ࢜ࢳࣥVCSM13ࢆPBS⁐ᾮࡋ࡚ᚓࡓ.
2.3.11㡯 ࣂ࣮࢜ࣞࣖᖸ΅ἲࡼࡿ⤖ྜᐃᩘࡢ⟬ฟ
ࢫࢺࣞࣉࢺࣅࢪࣥࡀᅛᐃࡉࢀࡓᑓ⏝ࢳࢵࣉࡢඛ➃ࢆ, PBS࡛10000ಸᕼ㔘ࡋࡓࣅ
࢜ࢳࣥVCSM13 (๓㡯࡛స〇) (4.0 mL) ᾐࡋ, 25Υ࡛2ศ࠺ (1000 rpm) ࡋࡓ. ࡇ
ࢀࢆPBS (250 mL) ᾐࡋ࡚, 25Υ࡛1ศ࠺ (1000 rpm) ࡋ, ࡇࡢࡁࡢࢩࢢࢼࣝࢆ
ᇶ‽ࡋࡓ. ᘬࡁ⥆ࡁ, ࢳࢵࣉࡢඛ➃ࢆ2.3.9㡯࡛స〇ࡋࡓM13-scFv (0~5.0 mM) ࢆྵࡴ
PBS (4 mL) ᾐࡋ࡚25Υ࡛2ศ࠺ (1000 rpm) ࡋࡓ. ࡑࡢᚋ, PBS (250 mL) ᾐࡋ
࡚25Υ࡛ 2ศ࠺ (1000 rpm) ࡋࡓ. ᚓࡽࢀࡓࢭࣥࢧ࣮ࢢ࣒ࣛࡽ, ᑓ⏝ࢯࣇࢺ࢙࢘
ࢆ⏝࠸࡚Kaࢆ⟬ฟࡋࡓ.
➨⠇ᒓᐇ㦂
2.4.1 㡯 CS#10-scFv-Gaussia ࣝࢩࣇ࢙࣮ࣛࢮ⼥ྜࢱࣥࣃࢡ㉁స〇ࡢࡓࡵࡢ GLuc 㑇ఏ
ᏊࢆྵࡴDNA᩿∦ࡢㄪ〇
pMCS-Gaussia Luc࣋ࢡࢱ࣮ (50 ng) , GLuc-Rev࠾ࡼࡧGLuc-For (ྛ50 pmol), dNTP
ΰྜ≀ (ྛ 20 nmol), ࠾ࡼࡧ Ex Taq DNA ࣏࣓࣮ࣜࣛࢮ (2.5 U) ࢆຍ࠼, ᑓ⏝⦆⾪ᾮ୰
(100 mL) ࡛PCRࢆ⾜ࡗࡓ. PCR᮲௳ࡣ⇕ኚᛶ95Υ, 1ศ, ࢽ࣮ࣜࣥࢢ55Υ, 1ศ, ఙ㛗 72Υ, 2ศࡋ, ࡇࡢࢧࢡࣝࢆ35ᅇ⧞ࡾ㏉ࡋࡓࡢࡕ, 72Υ࡛10ศࡢఙ㛗ᛂࢆ㏣ຍࡋ
ࡓ. ᛂᾮ୰ࡢቑᖜ⏘≀ࢆDNA⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ, CS#10-scFv㐃⤖ࡍ
ࡿࡓࡵࡢlinker2ࡢ୍㒊࠾ࡼࡧGLuc㑇ఏᏊࢆྵࡴDNA᩿∦ࢆᚓࡓ.
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2.4.2㡯 CS#10-scFv-Gaussiaࣝࢩࣇ࢙࣮ࣛࢮ⼥ྜࢱࣥࣃࢡ㉁స〇ࡢࡓࡵࡢ CS#10-scFv
㑇ఏᏊࢆྵࡴDNA᩿∦ࡢㄪ〇
1.3.9㡯࡛ᵓ⠏ࡋࡓCS#10-scFv㑇ఏᏊࡢ୍㒊 (100 ng) , CS#10VH-Rev࠾ࡼࡧVL -For-2ࣉ࣐࣮ࣛ (ྛ50 pmol), dNTPΰྜ≀ (ྛ20 nmol), ࠾ࡼࡧEx Taq DNA࣏࣓࣮ࣜࣛࢮ (2.5 U) ࢆຍ࠼, ᑓ⏝⦆⾪ᾮ୰ (100 mL) ࡛PCR ࢆ⾜ࡗࡓ. PCR ᮲௳ࡣ, ⇕ኚᛶ95Υ, 1
ศ, ࢽ࣮ࣜࣥࢢ55Υ, 1ศ, ఙ㛗72Υ, 2ศࡋ, ࡇࡢࢧࢡࣝࢆ35ᅇ⧞ࡾ㏉ࡋࡓࡢࡕ, 72Υ࡛ 10ศࡢఙ㛗ᛂࢆ㏣ຍࡋࡓ. ᛂᾮ୰ࡢቑᖜ⏘≀ࢆDNA⢭〇࢟ࢵࢺ (A) ࢆ⏝
࠸࡚⢭〇ࡋ, GLucࢆ㐃⤖ࡍࡿࡓࡵࡢlinker2ࡢ୍㒊CS#10-scFv㑇ఏᏊࢆྵࡴDNA᩿
∦ࢆᚓࡓ.
2.4.3㡯 Overlap extension PCRࡼࡿCS#10-scFv-GLuc㑇ఏᏊࡢᵓ⠏ࢧࣈࢡ࣮ࣟࢽ
ࣥࢢ
2.4.1㡯࠾ࡼࡧ2.4.2㡯࡛స〇ࡋࡓDNA᩿∦ (ྛ200 ng) , dNTPΰྜ≀ (ྛ10 nmol)
࠾ࡼࡧKOD DNA࣏࣓࣮ࣜࣛࢮ (1.5 U) ࢆຍ࠼, ᑓ⏝⦆⾪ᾮ୰ (26 mL) ࡛PCRࢆ⾜ࡗ ࡓ. PCR᮲௳ࡣ, ⇕ኚᛶ98Υ, 30⛊, ࢽ࣮ࣜࣥࢢ64Υ, 30⛊, ఙ㛗74Υ, 1ศࡢࢧࢡ
ࣝࡋ, ࡇࢀࢆ10ᅇ⧞ࡾ㏉ࡋࡓࡢࡕ74Υ࡛10ศࡢఙ㛗ᛂࢆ㏣ຍࡋࡓ. ࡇࡢᛂᾮࡢ
୍㒊 (10 mL) , CS#10VH-Rev࠾ࡼࡧGLuc-Forࣉ࣐࣮ࣛ (ྛ100 pmol), dNTPΰྜ≀
(ྛ40 nmol), ࠾ࡼࡧKOD DNA࣏࣓࣮ࣜࣛࢮ (5 U) ࢆຍ࠼, ᑓ⏝⦆⾪ᾮ୰ (100 mL), ୖ
グࡢ᮲௳࡛ (ࡓࡔࡋ15ࢧࢡࣝ) PCRࢆ⾜ࡗࡓ. ᛂᾮ୰ࡢቑᖜ⏘≀ࢆDNA⢭〇࢟ࢵ
ࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ, ࡑࡢ୍㒊 (1 mg) ไ㝈㓝⣲Not I (50 U) ࢆຍ࠼, ᛂ⏝⦆⾪
ᾮ୰ (100 mL) ࡛37Υ, ୍ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ቑᖜ⏘≀ࢆDNA⢭〇࢟ࢵࢺ (A) ࢆ
⏝࠸࡚⢭〇ࡋ, ࡑࡢ㔞ไ㝈㓝⣲ Sfi I (50 U) ࢆຍ࠼, ᛂ⏝⦆⾪ᾮ୰ (100 mL) ࡛ 50Υ, ୍ ᬌ ࣥ ࢟ ࣗ ࣋ ࣮ ࢺ ࡋ ࡓ. ቑ ᖜ ⏘ ≀ ࢆ DNA ⢭ 〇 ࢟ ࢵ ࢺ (A) ࢆ ⏝ ࠸ ࡚ ⢭ 〇 ࡋ, CS#10-scFv-GLuc 㑇ఏᏊ ࢆ ྵ ࡴ ไ 㝈 㓝 ⣲ ᾘ῭ ࡳ ࡢ DNA ᩿ ∦ (#10VH-linker1-#10VL -linker2-GLuc-FLAG-His6-TAATGA; CS#10-scFv-GLuc) ࢆᚓࡓ.
ࡇࡢDNA᩿∦ (125 ng) , ྠᵝไ㝈㓝⣲Not ISfi I࡛ᾘࡋ, ࣮࢞ࣟࢫࢤࣝ
(1%) 㟁ẼὋືࡼࡾ⢭〇ࡋࡓpEXmide 5 (250 ng) ࢆΰࡋ࡚, 1.3.5㡯ྠᵝࡢࣛࢤ
࣮ࢩࣙࣥᛂࡋ, ࡑࡢ⏘≀ࢆ XL1-Blue ࢚ࣞࢡࢺࣟࢥࣥࣆࢸࣥࢺ⣽⬊ᑟධࡋࡓ. ᙧ㉁㌿⳦ࢆ 2YT-ATG (1%) ࣮࢞ࣉ࣮ࣞࢺୖሬᕸࡋ, 37Υ୍࡛ᬌᇵ㣴ࡋࡓ. ࡇࡢ
ࣉ࣮ࣞࢺࡽࣛࣥࢲ࣒㑅ᢥࡋࡓࢥࣟࢽ࣮ࢆࢥࣟࢽ࣮PCRࡋ, scFv㑇ఏᏊࢆಖᣢࡍ
ࡿࢡ࣮ࣟࣥࢆྠᐃࡋࡓ. ࡞࠾, ࣉ࣐࣮ࣛࡣ VHH-cPCR-back ࠾ࡼࡧ VHH-cPCR-for (ྛ 2 pmol) ࢆ⏝࠸ࡓ.
2.4.4㡯 CS/ࣅ࢜ࢳࣥ2㔜ᶆ㆑࣏ࣜ-L-ࣜࢩࣥࡢస〇
1.2.1㡯࡛స〇ࡋࡓCS-3CMO-NHS (53 mg/mL) ࢆྵࡴ1,4-ࢪ࢜࢟ࢧࣥ (200 mL), biotin N-succinimidyl ester (25 mg/mL) ࢆྵࡴN,N-dimethylformamide (88 mL), ࠾ࡼࡧ3.0 M HCl
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(40 mL) ࢆpoly-L-lysine (1.3 mg/mL) ࢆྵࡴPB (3.4 mL) ΰࡋ, ᧠ᢾࡋ࡞ࡀࡽᐊ ࡛ 3㛫ᛂࡉࡏࡓࡢࡕ, ࡉࡽ4Υ୍࡛ᬌᛂࡉࡏࡓ. ࡇࡢᾮࢆPBᑐࡋ࡚4Υ࡛2᪥
㛫㏱ᯒࡋ, ㏱ᯒ⭷ෆᐜ≀ࢆᅇࡋ࡚⏝ࡲ࡛4Υ࡛ಖᏑࡋࡓ.
2.4.5㡯 CS#10-scFv-Gaussiaࣝࢩࣇ࢙࣮ࣛࢮ⼥ྜࢱࣥࣃࢡ㉁ࡢⓎ⌧㟁ẼὋື
2.4.3㡯࡛స〇ࡋࡓM13-scFv-GLuc㑇ఏᏊಖᣢ⳦ࡽ, 1.3.12㡯‽ࡌ࡚࣌ࣜࣉࣛࢬ࣒
ᢳฟᾮࢆㄪ〇ࡋ, ᢠ FLAG ᢠయᅛᐃ࣮࢞ࣟࢫࢆ⏝࠸࡚⢭〇ࡋࡓ. ࡇࡢ୍㒊 (1 mg)
ࢆ2.2.6㡯‽ࡌ࡚ SDS-PAGEࡋ, Ὃືᚋࡢࢤࣝࢆࢡ࣐ࢩ࣮ࣈࣜࣜࣥࢺࣈ࣮ࣝ
ࡼࡾᰁⰍࡋࡓ. ࡲࡓ, ྠ᮲௳ࡢ㟁ẼὋືࡼࡾศ㞳ࡋࡓࢱࣥࣃࢡ㉁ࢆPVDF⭷㌿ࡋ ࡓ. ࡇࡢ⭷ࢆ1.0%ࣈࣟࢵࢡ࢚࣮ࢫ⁐ᾮ (30 mL) ᾐࡋ࡚ᐊ ࡛2㛫࠺ (~60 rpm) ࡍࡿࡇ࡛ࣈࣟࢵ࢟ࣥࢢࡋ, 2.2.6㡯‽ࡌ࡚Ὑίࡋࡓ. ࡇࡢ⭷G-PBS࡛10000ಸᕼ㔘 ࡋࡓCS/ࣅ࢜ࢳࣥ2 㔜ᶆ㆑࣏ࣜ-L-ࣜࢩࣥ (๓㡯࡛స〇) (30 mL) ࢆຍ࠼࡚ᐊ ࡛1㛫
࠺ (~60 rpm) ࡋ, Ὑίࡋࡓࡢࡕ G-PBS࡛1000ಸᕼ㔘ࡋࡓPODᶆ㆑ࢫࢺࣞࣉࢺ
ࣅࢪࣥ (~30 mL) ࢆຍ࠼࡚ྠᵝᐊ ࡛ 1 㛫࠺ࡋࡓ. ู㏵, ࣈࣟࢵ࢟ࣥࢢᚋࡢ⭷
ᑐࡋ࡚G-PBS ࡛10000ಸᕼ㔘ࡋࡓPODᶆ㆑ᢠFLAGᢠయ (30 mL) ࢆຍ࠼࡚ᐊ ࡛
1㛫࠺ (~60 rpm) ࡋ, ྠᵝὙίࡋࡓ. ࡇࢀࡽࡢ⭷ࡢPODάᛶࢆ, 2.2.6㡯‽ࡌ
࡚ྍどࡋࡓ.
2.4.6㡯 CS#10-scFv-Gaussiaࣝࢩࣇ࢙࣮ࣛࢮ⼥ྜࢱࣥࣃࢡ㉁ࡢⓎග ᐃ
๓㡯࡛స〇ࡋࡓCS#10-scFv-GLuc (20 fmol) ࢆྵࡴG-PBSࢆELISA⏝ⓑⰍ96࢙࢘ࣝ
࣐ࢡࣟࣉ࣮ࣞࢺศὀࡋ (100 mL/࢙࢘ࣝ), ࢭࣞࣥࢸࣛࢪࣥ (20 mM) ࢆྵࡴPBS (100 mL/࢙࢘ࣝ) ࢆຍ࠼, ࡑࡢ30⛊ᚋࡽ900⛊ᚋࡲ࡛ࡣ60⛊ࡈ, 900⛊ࡽ1800⛊ࡲ࡛
ࡣ300⛊ࡈ475 nm࠾ࡅࡿⓎගᙉᗘࢆFLUOstar OPTIMAࢆ⏝࠸࡚ ᐃࡋࡓ.
2.4.7㡯 CS#10-scFv-Gaussiaࣝࢩࣇ࢙࣮ࣛࢮ⼥ྜࢱࣥࣃࢡ㉁ࡢ᳨ฟឤᗘࡢ᳨ウ
2.4.5㡯࡛స〇ࡋࡓCS#10-scFv-GLuc (0~1.0 fmol) ࢆྵࡴG-PBS ⁐ᾮ (20 mL) ࢭࣞ
ࣥࢸࣛࢪࣥ (20 mM) ࢆྵࡴPBS (100 mL) ࢆຍ࠼, 15⛊ᚋࡢⓎගᙉᗘࢆ๓㡯‽ࡌ࡚
ᐃࡋࡓ. ᚓࡽࢀࡓⓎගࢩࢢࢼ್ࣝࢆCS#10-scFv-GLuc㔞ᑐࡋ࡚ࣉࣟࢵࢺࡋ, ⏝㔞స⏝
᭤⥺ࢆసᡂࡋࡓ.
2.4.8 㡯 M13-scFv-Gaussia ࣝࢩࣇ࢙࣮ࣛࢮ⼥ྜࢱࣥࣃࢡ㉁స〇ࡢࡓࡵࡢ M13-scFv 㑇
ఏᏊࢆྵࡴDNA᩿∦ࡢㄪ〇
2.3.7㡯࡛స〇ࡋࡓࣉࣛࢫ࣑ࢻ (50 ng) , M13#71VH-Rev࠾ࡼࡧVL-For-2ࣉ࣐࣮ࣛ
(ྛ50 pmol), dNTPΰྜ≀ (ྛ40 nmol), ࠾ࡼࡧEx Taq DNA࣏࣓࣮ࣜࣛࢮ (2.5 U) ࢆຍ
࠼, ᑓ⏝⦆⾪ᾮ୰ (100 mL) ࡛PCRࢆ⾜ࡗࡓ. PCR᮲௳ࡣ, ⇕ኚᛶ95Υ, 1ศ, ࢽ࣮ࣜ
ࣥࢢ64Υ, 1ศ, ఙ㛗72Υ, 1ศࡋ, ࡇࡢࢧࢡࣝࢆ35ᅇ⧞ࡾ㏉ࡋࡓࡢࡕ, 72Υ࡛10ศ
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ࡢఙ㛗ᛂࢆ㏣ຍࡋࡓ. ᛂᾮ୰ࡢቑᖜ⏘≀ࢆ DNA ⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ, M13-scFv࠾ࡼࡧGLuc㐃⤖ࡍࡿࡓࡵࡢlinker2ࡢ୍㒊ࢆྵࡴDNA᩿∦ࢆᚓࡓ.
2.4.9㡯 M13-scFv-Gaussia ࣝࢩࣇ࢙࣮ࣛࢮ⼥ྜࢱࣥࣃࢡ㉁స〇ࡢࡓࡵࡢ GLuc㑇ఏᏊ
ࢆྵࡴDNA᩿∦ࡢㄪ〇
2.4.3㡯࡛స〇ࡋࡓࣉࣛࢫ࣑ࢻ (50 ng), GLuc-Rev࠾ࡼࡧGLuc-Forࣉ࣐࣮ࣛ (ྛ 50 pmol), dNTPΰྜ≀ (ྛ40 nmol), ࠾ࡼࡧKOD Fx DNA࣏࣓࣮ࣜࣛࢮ (5 U) ࢆຍ࠼,
ᑓ⏝⦆⾪ᾮ୰ (100 mL) ࡛PCRࢆ⾜ࡗࡓ. PCR᮲௳ࡣ, ⇕ኚᛶ94Υ, 2ศࡢࡢࡕ, ⇕ኚ ᛶ98Υ, 10⛊, ࢽ࣮ࣜࣥࢢ55Υ, 30⛊, ఙ㛗68Υ, 1ศࡢࢧࢡࣝࢆ35ᅇ⧞ࡾ㏉ࡋࡓ.
ᛂᾮ୰ࡢቑᖜ⏘≀ࢆDNA⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ, linker2ࡢ୍㒊GLuc㑇 ఏᏊࢆྵࡴDNA᩿∦ࢆᚓࡓ.
2.4.10㡯 Overlap extension PCRࡼࡿM13-scFv-GLuc㑇ఏᏊࡢᵓ⠏ࢧࣈࢡ࣮ࣟࢽࣥ
ࢢ
2.4.8㡯࠾ࡼࡧ2.4.9㡯࡛స〇ࡋࡓDNA᩿∦ (ྛ200 ng) , dNTPΰྜ≀ (ྛ10 nmol)
࠾ࡼࡧKOD Fx DNA࣏࣓࣮ࣜࣛࢮ (5 U) ࢆຍ࠼, ᑓ⏝⦆⾪ᾮ୰ (60 mL) ࡛PCRࢆ⾜ࡗ ࡓ. PCR᮲௳ࡣ, ⇕ኚᛶ98Υ, 30⛊, ࢽ࣮ࣜࣥࢢ64Υ, 30⛊, ఙ㛗74Υ, 1ศࡋ, ࡇ ࡢࢧࢡࣝࢆ10ᅇ⧞ࡾ㏉ࡋࡓࡢࡕ, 74Υ࡛ 10ศࡢఙ㛗ᛂࢆ㏣ຍࡋࡓ. ࡇࡢᛂᾮࡢ
୍㒊 (10 mL) , M13#71VH-Rev࠾ࡼࡧGLuc-Forࣉ࣐࣮ࣛ (ྛ100 pmol), dNTPΰྜ
≀ (ྛ40 nmol), ࠾ࡼࡧKOD Fx DNA࣏࣓࣮ࣜࣛࢮ (5 U) ࢆຍ࠼, ᑓ⏝⦆⾪ᾮ୰ (100 mL), ୖグࡢ᮲௳࡛ (ࡓࡔࡋ25ࢧࢡࣝ) PCRࢆ⾜ࡗࡓ. ᛂᾮࡢቑᖜ⏘≀ࢆDNA⢭〇
࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ, ࡑࡢ୍㒊 (5 mg) ไ㝈㓝⣲Not I (40 U) ࢆຍ࠼, ᛂ⏝
⦆⾪ᾮ୰ (100 mL) ࡛37Υ, ୍ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ቑᖜ⏘≀ࢆDNA⢭〇࢟ࢵࢺ (A)
ࢆ⏝࠸࡚⢭〇ࡋ, ࡑࡢ㔞ไ㝈㓝⣲Sfi I (50 U) ࢆຍ࠼, ᛂ⏝⦆⾪ᾮ୰ (100 mL) ࡛ 50Υ, ୍ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ቑᖜ⏘≀ࢆ DNA ⢭〇࢟ࢵࢺ (A) ࢆ⏝࠸࡚⢭〇ࡋ, ไ
㝈㓝⣲ ᾘ῭ࡳ ࡢ DNA ᩿∦ (VH-linker1-VL-linker2-GLuc-FLAG-His6-TAATGA;
scFv-GLuc) ࢆᚓࡓ.
ࡇࡢDNA᩿∦ࢆ 2.4.3㡯ྠᵝpEXmide 5⤌ࡳ㎸ࡳ, ⭠⳦XL1-Blueᑟධࡋ,
1.3.6㡯‽ࡌ࡚ࣉࣛࢫ࣑ࢻࢆᢳฟࡋ࡚, scFv-GLuc㑇ఏᏊࡢሷᇶ㓄ิࢆゎᯒࡋࡓ.
2.4.11㡯 ྍ⁐ᆺM13-scFv-GLucࡢⓎ⌧㟁ẼὋື
2.4.3㡯࡛స〇ࡋࡓM13-scFv-GLuc㑇ఏᏊಖᣢ⳦ࡽ, 1.3.12㡯‽ࡌ࡚࣌ࣜࣉࣛࢬ࣒
ᢳฟᾮࢆᅇࡋ, ᢠ FLAG ᢠయᅛᐃ࣮࢞ࣟࢫࢆ⏝࠸࡚⢭〇ࡋࡓ. ࡇࡢ୍㒊 (1 mg)
ࢆ2.2.6㡯‽ࡌ࡚ SDS-PAGEࡋ, Ὃືᚋࡢࢤࣝࢆࢡ࣐ࢩ࣮ࣈࣜࣜࣥࢺࣈ࣮ࣝ
ࡼࡾᰁⰍࡋࡓ. ࡲࡓ, ྠ᮲௳ࡢ㟁ẼὋືࡼࡾศ㞳ࡋࡓࢱࣥࣃࢡ㉁ࢆPVDF⭷㌿ࡋ
ࡓ. ࡇࡢ⭷ࢆM-PBS (~30 mL) ᾐࡋ࡚ᐊ ୍࡛ᬌ࠺ (~60 rpm) ࡍࡿࡇ࡛ࣈࣟࢵ
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࢟ࣥࢢࡋ, 2.2.6㡯‽ࡌ࡚Ὑίࡋࡓࡢࡕ, VCSM13 (11012 pfu) ࢆྵࡴPVG-PBS (30 mL)
ࢆຍ࠼࡚ᐊ ࡛1㛫࠺ (~60 rpm) ࡋࡓ. ⁐ᾮࢆ㝖ཤࡋ࡚Ὑίࡋࡓࡢࡕ, M-PBS࡛ 2000 ಸᕼ㔘ࡋࡓ POD ᶆ㆑ᢠ M13 ᢠయ (30 mL) ࢆຍ࠼࡚ᐊ ࡛ 30 ศ࠺ (~60
rpm) ࡋࡓ. ྠᵝὙίࡋࡓࡢࡕ, ⭷ୖࡢPODάᛶࢆ2.2.6㡯‽ࡌ࡚ྍどࡋࡓ.
2.4.12㡯 ྍ⁐ᆺM13-scFv-GLucࡢ⤖ྜ⬟ホ౯⏝࠸ࡿCS#10-scFvᥦ♧ࣇ࣮ࢪࡢస
〇
1.3.9 㡯࡛స〇ࡋࡓࣉࣛࢫ࣑ࢻ (⣙ 10 ng) TG1 ࢚ࣞࢡࢺࣟࢥࣥࣆࢸࣥࢺ⣽⬊ (100
mL) ࢆΰࡋ, 1.3.5 㡯‽ࡌ࡚㟁Ẽࣃࣝࢫࢆ࠼ࡓࡢࡕ SOC ᇵᆅ୰࡛ᇵ㣴ࡋࡓ. ࡇࡢ
⳦ᾮࢆ2YT-AG (1%) ࣮࢞ࣉ࣮ࣞࢺሬᕸࡋ࡚37Υ୍࡛ᬌᇵ㣴ࡋ࡚ᙧᡂࡉࢀࡿࢥࣟ
ࢽ࣮ࢆࢥࣟࢽ࣮PCRࡋ, CS#10-scFv㑇ఏᏊࢆಖᣢࡍࡿTG1⣽⬊ࢆྠᐃࡋࡓ. ࡇࡢᙧ
㉁㌿⳦ࢆ2YT-AG (2%) ᇵᆅ (20 mL) ࡛ᇵ㣴ࡋ, 1.4.5㡯‽ࡌ࡚scFv ᥦ♧ࣇ࣮
ࢪ ࢆ ྵ ࡴ PBS-2 ⁐ ᾮ (200 mL) ࢆ ᚓ ࡓ ࡢ ࡕ, ୍ 㒊 (5 mL) ࢆ ⏝ ࠸ ࡚. ࣇ ࣮ ࢪ ࡢ ຊ ౯ (cfu) ࢆ⟬ᐃࡋࡓ.
2.4.13㡯 ྍ⁐ᆺM13-scFv-GLucࢆ⏝࠸ࡿࣇ࣮ࢪELISA
CS-BSA (1.0 mg/mL) ࢆྵࡴCBࢆ, ELISA⏝ⓑⰍ96࢙࣐࢘ࣝࢡࣟࣉ࣮ࣞࢺศὀ
ࡋ (100 mL/࢙࢘ࣝ), ᐊ ୍࡛ᬌᨺ⨨ࡋࡓ. ⁐ᾮࢆ㝖ཤࡋ࡚ PBS ࡛ 3 ᅇὙίࡋࡓࡢࡕ,
1.0%ࣈࣟࢵࢡ࢚࣮ࢫ⁐ᾮ (300 mL/࢙࢘ࣝ) ࢆຍ࠼࡚ 37Υ࡛ 2 㛫ᨺ⨨ࡋࡓ. ⁐ᾮࢆ㝖
ཤࡋ࡚T-PBS࡛3ᅇὙίࡋࡓࡢࡕ, ๓㡯࡛ㄪ〇ࡋࡓCS#10-scFvᥦ♧ࣇ࣮ࢪ (1105~1
109 cfu) ࢆྵࡴ2YTᇵᆅ (200 mL/࢙࢘ࣝ) ࢆຍ࠼, 37Υ࡛1㛫࣮ࣥ࢟ࣗ࣋ࢺࡋ ࡓ. ࡧ⁐ᾮࢆ㝖ཤࡋ, T-PBS-2࡛3ᅇὙίࡋࡓࡢࡕ, M-PBS࡛500ಸᕼ㔘ࡋࡓ M13-scFv-GLuc (2.4.11㡯࡛స〇ࡋࡓ࣌ࣜࣉࣛࢬ࣒ᢳฟᾮ) ࢆຍ࠼ (100 mL/࢙࢘ࣝ), 37Υ࡛30
ศ ࣥ ࢟ ࣗ ࣋ ࣮ ࢺ ࡋ ࡓ. ྠ ᵝ Ὑ ί ᚋ, ࢭ ࣞ ࣥ ࢸ ࣛ ࢪ ࣥ (5 mM) ࢆ ྵ ࡴ PBS ࢆ ຍ ࠼ (100 mL/࢙࢘ࣝ), ┤ᚋ300~700 nm࠾ࡅࡿⓎගᙉᗘࢆSynergy HTXࢆ⏝࠸࡚ ᐃࡋ
ࡓ.
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➨❶ᒓᐇ㦂
➨⠇ᒓᐇ㦂
3.2.1㡯 ࣇ࣮ࢪస〇᮲௳ࡢホ౯⏝࠸ࡿscFvᥦ♧ࣇ࣮ࢪࡢస〇
CS#10-scFv㑇ఏᏊࢆಖᣢࡍࡿ⭠⳦XL1-Blue (1.3.9㡯࡛స〇) ࠾ࡼࡧTG1 (2.4.12㡯
࡛స〇) ࡢ⤖ಖᏑᾮࡢ୍㒊 (ྛ 200 mL) ࢆᾮయᇵᆅ [ࡑࢀࡒࢀ 2YT-ATG (2%), 2 YT-AG (2%)] (ྛ 20 mL) ῧຍࡋ, ᑐᩘቑṪᮇ㐩ࡍࡿࡲ࡛ 37Υ࡛࠺ᇵ㣴 (~200 rpm) ࡋࡓ. ࡇࢀࡽࡢᇵ㣴ᾮ, VCSM13ࡲࡓࡣKM13 (ࡑࢀࡒࢀ2.2.1㡯, 1.4.3㡯࡛స〇)
ࢆ, MOI ࡀ20࡞ࡿࡼ࠺ῧຍࡋ, 37Υ࡛30ศ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ࡇࢀࡽࡢΰྜ≀
ࢆ㐲ᚰศ㞳 (1800g, 20ศ, ᐊ ) ࡋࡓࡢࡕ, ỿẊࢆ2YT-AKᇵᆅ (20 mL) ࡛ᠱ⃮ࡋ࡚, 25Υ୍࡛ᬌ࠺ᇵ㣴 (120 rpm) ࡋࡓ. ࡇࢀࡽࡢᇵ㣴ᾮࢆ㐲ᚰศ㞳 (1800g, 20 ศ, ᐊ
) ࡋ, ୖΎྵࡲࢀࡿCS#10-scFvᥦ♧ࣇ࣮ࢪࢆ1.4.5 㡯‽ࡌ࡚PEG/NaCl ࡼࡾ
⢭〇, ⃰⦰ࡋࡓ. ᚓࡽࢀࡓࣇ࣮ࢪᾮ (200 mL) ࡣ⏝ࡲ࡛ 4Υ࡛ಖᏑࡋࡓ. ୍᪉࡛, ࡇ ࡢ୍㒊 (5 mL) ࢆ⏝࠸࡚, ࣇ࣮ࢪࡢຊ౯ (cfu) ࢆ1.4.5㡯‽ࡌ࡚⟬ᐃࡋࡓ.
3.2.2㡯 ELISAࡼࡿࣇ࣮ࢪస〇᮲௳ࡢホ౯
CS-BSAࢆ1.3.14㡯‽ࡌ࡚ᅛᐃࡋࡓ࣐ࢡࣟࣉ࣮ࣞࢺ, ๓㡯࡛స〇ࡋࡓ
CS#10-scFvᥦ♧ࣇ࣮ࢪ (ྛ1109 cfu) ࢆྵࡴPVG-PBSࢆຍ࠼ (100 mL/࢙࢘ࣝ), 37Υ࡛1
㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ⁐ᾮࢆ㝖ཤࡋ࡚T-PBS-2࡛3ᅇὙίࡋࡓࡢࡕ, M-PBS࡛2000ಸ
ᕼ㔘ࡋࡓPODᶆ㆑ᢠM13ᢠయ (100 mL/࢙࢘ࣝ) ࢆຍ࠼, 37Υ࡛30ศ࣮ࣥ࢟ࣗ࣋ࢺ ࡋࡓ. ྠᵝὙίᚋ, ᅛ┦ୖࡢPODάᛶࢆ1.2.3㡯‽ࡌ࡚ẚⰍ ᐃࡋࡓ.
3.2.3㡯 ࣞᆺ㑅ᢥἲ⏝࠸ࡿᇵᆅ⤌ᡂ࠾ࡼࡧᇵ㣴㛫ࡢホ౯
CS-BSA (1.0 mg/mL) ࢆྵࡴCBࢆ, ELISA⏝ⓑⰍ96࢙࣐࢘ࣝࢡࣟࣉ࣮ࣞࢺศὀ ࡋ (100 mL/࢙࢘ࣝ), ᐊ ୍࡛ᬌᨺ⨨ࡋࡓ. ⁐ᾮࢆ㝖ཤࡋ࡚PBS࡛3ᅇὙίࡋࡓࡢࡕ, ࢁ 㐣⁛⳦ࡋࡓ1.0%ࣈࣟࢵࢡ࢚࣮ࢫ⁐ᾮ (300 mL/࢙࢘ࣝ) ࢆຍ࠼࡚37Υ࡛2㛫ᨺ⨨ࡋࡓ.
⁐ᾮࢆ㝖ཤࡋࡓࡢࡕ, T-PBS࡛3ᅇὙίࡋ, ࣥࣆࢩࣜࣥࢼࢺ࣒ࣜ࢘ (100 mg/mL), ◲㓟
࢝ࢼ࣐ࢩࣥ (5.0ࡲࡓࡣ25 mg/mL), ࠾ࡼࡧKM13 (5108ࡲࡓࡣ5109 pfu/mL) ࢆྵ
ࡴ2YTᇵᆅࢆศὀࡋࡓ (200 mL/࢙࢘ࣝ). ୍᪉࡛, 2.4.12㡯࡛స〇ࡋࡓCS#10-scFv㑇 ఏᏊࢆಖᣢࡍࡿ⭠⳦TG1ࡢ⤖ಖᏑᾮࢆ2YTᇵᆅ࡛ᕼ㔘ࡋ (1105~108ಸᕼ㔘), 2
YT-AG (1%) ࣮࢞ࣉ࣮ࣞࢺሬᕸࡋ࡚ 37Υ୍࡛ᬌᇵ㣴ࡍࡿࡇࡼࡾࢥࣟࢽ࣮ࢆ
ᙧᡂࡉࡏࡓ. ࡇࢀࢆ, ⁛⳦ࡋࡓ∎ᯞࢆ⏝࠸࡚ୖグࡢࡼ࠺࣐ࢡࣟࣉ࣮ࣞࢺศὀࡋ ࡓᇵᆅ୰ᠱ⃮ࡋ, 25Υ࡛16, 24, ࡲࡓࡣ45㛫࠺ᇵ㣴 (800 rpm) ࡋࡓ. ᇵ㣴ᾮࢆ
㝖ཤࡋ࡚T-PBS-2࡛3ᅇὙίࡋࡓࡢࡕ, 2.4.13㡯‽ࡌ࡚ M13-scFv-GLucࢆᛂࡉࡏ, ࢭࣞࣥࢸࣛࢪࣥࢆຍ࠼࡚Ⓨගᙉᗘࢆ ᐃࡋࡓ.
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➨⠇ᒓᐇ㦂
3.3.1㡯 ኚ␗scFv㑇ఏᏊࣛࣈ࣮ࣛࣜࡼࡿ⭠⳦ࡢᙧ㉁㌿ࢥࣟࢽ࣮ศ㞳
1.4.1㡯࡛స〇ࡋࡓኚ␗scFvࣛࣈ࣮ࣛࣜࢆ1.4.2㡯‽ࡌ࡚pEXmide 7⤌ࡳ㎸ࡳ,
⭠⳦TG1࢚ࣞࢡࢺࣟࢥࣥࣆࢸࣥࢺ⣽⬊ΰࡋࡓ. ࡇࡢΰྜ≀1.3.5㡯‽ࡌ࡚㟁 Ẽ ࣃ ࣝ ࢫ ࢆ ࠼ ࡓ ࡢ ࡕ SOC ᇵ ᆅ ୰ ࡛ ᇵ 㣴 ࡋ ࡓ. ᇵ 㣴 ᾮ ࢆ SOC ᇵ ᆅ ࡛ ẁ 㝵 ᕼ 㔘 ࡋ (10~1000 ಸ ᕼ㔘), 2YT-AG (1%) ࣮࢞ ࣉ࣮ࣞ ࢺ (ゅ 2 ྕࢩࣕ ࣮࡛ࣞ స〇) ሬᕸ (200 mL/ࣉ࣮ࣞࢺ) ࡋ࡚, 37Υ୍࡛ᬌᇵ㣴ࡋࡓ.
3.3.2㡯 M13-scFv-GLucࢆ⏝࠸ࡓⓎගᙉᗘࡢࣉࣟࣇࣜࣥࢢ
3.2.3 㡯‽ࡌ࡚స〇ࡋࡓ CS-BSA ᅛᐃ࣐ࢡࣟࣉ࣮ࣞࢺ, ࣥࣆࢩࣜࣥࢼࢺࣜ
࣒࢘ (100 mg/mL), ◲㓟࢝ࢼ࣐ࢩࣥ (5.0 mg/mL), ࠾ࡼࡧKM13 (5108 pfu/mL) ࢆྵࡴ
2YTᇵᆅࢆศὀ (200 mL/࢙࢘ࣝ) ࡋࡓࡢࡕ, ࡑࡢ࡞๓㡯࡛స〇ࡋࡓࢥࣟࢽ࣮ࢆ⁛
⳦ࡋࡓ∎ᯞࢆ⏝࠸࡚ᠱ⃮ࡋࡓ. ࣉ࣮ࣞࢺࢆ25Υ, 800 rpm࡛45㛫ᇵ㣴ࡋࡓࡢࡕ, ᇵ 㣴ᾮࢆ㝖ཤࡋ࡚T-PBS-2࡛3ᅇὙίࡋ, M-PBS࡛500ಸᕼ㔘ࡋࡓM13-scFv-GLuc (2.4.11 㡯࡛స〇ࡋࡓ࣌ࣜࣉࣛࢬ࣒ᢳฟᾮ) (100 mL) ࢆຍ࠼, 37Υ࡛ 30 ศ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ.
ྠᵝὙίࡋࡓࡢࡕ, ࢭࣞࣥࢸࣛࢪࣥ (5 mM) ࢆྵࡴ PBS ࢆຍ࠼ (100 mL/࢙࢘ࣝ), ┤
ࡕ 300~700 nm࠾ࡅࡿⓎගᙉᗘࢆSynergy HTXࢆ⏝࠸࡚ ᐃࡋࡓ.
3.3.3㡯 ࣇ࣮ࢪࡢᅇࢧࣈࢡ࣮ࣟࢽࣥࢢ
๓㡯࡛ ᚓࡽࢀ ࡓⓎගᙉ ᗘࡽ ≉ᐃࡋ ࡓ࢙࢘ࣝ ṧᏑ ࡍࡿ⁐ ᾮࢆ㝖࠸ ࡓࡢࡕ, 0.10 M ࢢࣜࢩࣥሷ㓟⦆⾪ᾮ (pH 2.2, 100 mL) ࢆຍ࠼, ᐊ ࡛10ศ࠺ (600 rpm) ࡋࡓ. 2.0 M Tris (⣙ 4 mL) ࢆຍ࠼࡚୰ࡋࡓࡢࡕ, ࡑࡢ୍㒊 (50 mL) ࢆ, ᑐᩘቑṪᮇࡢ⭠⳦ TG1 (450 mL) ຍ࠼, 37Υ࡛30ศ࣮ࣥ࢟ࣗ࣋ࢩࣙࣥࡋࡓ. ࡇࡢ୍㒊 (15 mL) ࢆ2YTᇵ
ᆅ࡛ẁ㝵ᕼ㔘 (10~1000ಸᕼ㔘) ࡋ, 2YT-AG (1%) ࣮࢞ࣉ࣮ࣞࢺሬᕸࡋ࡚37Υ࡛
୍ᬌᇵ㣴ࡋࡓ. ᚓࡽࢀࡿࢥࣟࢽ࣮ࢆࢥࣟࢽ࣮PCRࡋ࡚, scFv㑇ఏᏊࢆಖᣢࡍࡿ⭠
⳦ࢆ≉ᐃࡋࡓ.
3.3.4㡯 ࣔࣀࢡ࣮ࣟࢼࣝࣇ࣮ࢪࡢㄪ〇ᢠCSάᛶࡢホ౯
๓ 㡯࡛ స〇 ࡋࡓ ᙧ㉁㌿ ⳦ࡢ ⤖ ಖᏑ ᾮࡢ୍ 㒊 (200 mL) ࢆ, 2YT-AG (2%) ᇵ ᆅ (20 mL) ࡛ᇵ㣴ࡋ, 1.4.5㡯‽ࡌ࡚scFvᥦ♧ࣇ࣮ࢪࢆྵࡴPBS-2⁐ᾮ (200 mL) ࢆᚓ
ࡓ. ࡇࡢ⁐ᾮࢆ㐺ษᕼ㔘ࡋ࡚1.4.10㡯‽ࡌ࡚ELISAࡋࡓ.
3.3.5㡯 ྍ⁐ᆺኚ␗scFvࡢㄪ〇
๓㡯࡛wt-scFvࡼࡾࡶ㧗ぶຊ࡛࠶ࡿ᥎ᐃࡉࢀࡓࢡ࣮ࣟࣥࡘ࠸࡚, scFv㑇ఏᏊࢆ
1.4.11 㡯‽ࡌ࡚ pEXmide 7’࣋ࢡࢱ࣮⤌ࡳ㎸ࡳ, ྍ⁐ᆺ scFv ࢆྵࡴ࣌ࣜࣉࣛࢬ࣒ᢳ
82 ฟᾮࢆᚓࡓ.
3.3.6㡯 Scatchardἲࡼࡿ⤖ྜᐃᩘࡢ⟬ฟ
1.2.4㡯‽ࡌ࡚ᐇ㦂ࢆ⾜࠸, Scatchardἲ
43)
ࡼࡾྍ⁐ᆺኚ␗scFvࡢCSᑐࡍࡿKa
ࢆ⟬ฟࡋࡓ.
3.3.7㡯 ྍ⁐ᆺኚ␗scFvࡢ㔞ㄪ〇⢭〇
3.3.5㡯࡛స〇ࡋࡓྍ⁐ᆺኚ␗scFv㑇ఏᏊࢆಖᣢࡍࡿ⭠⳦ࡢ⤖ಖᏑᾮࢆᑡ㔞ࡾ,
2YT-AG (1%) ᇵᆅ (20 mL) ᥋✀ࡋ࡚, 37Υ୍࡛ᬌ࠺ᇵ㣴 (~200 rpm) ࡋࡓ. ࡇ
ࡢᇵ㣴ᾮࢆ㔞2YT-AG (1%) ᇵᆅ (2 L) ຍ࠼, 600 nm࠾ࡅࡿ྾ගᗘࡀ0.8㐩 ࡍࡿࡲ࡛37Υ࡛࠺ᇵ㣴 (~200 rpm) ࡋࡓ. ࡇࢀࢆ㐲ᚰศ㞳 (1800g, 30ศ, ᐊ ) ࡋ ࡓࡢࡕ, ỿẊࢆࢱࣥࣃࢡ㉁Ⓨ⌧ㄏᑟᇵᆅ (2 L) ᠱ⃮ࡋ࡚ 25Υ୍࡛ᬌ࠺ᇵ㣴 (120 rpm) ࡋࡓ. ᇵ㣴ᾮࢆ㐲ᚰศ㞳 (6000g, 20ศ, ᐊ ) ᚋ, ỿẊࢆᾐ㏱ᅽࢩࣙࢵࢡ⏝⦆⾪ᾮ (100 mL) ᠱ⃮ࡋ, ịୖ࡛ 1 㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. 㐲ᚰศ㞳 (19300g, 30 ศ, 4Υ)
ᚋ, ୖΎࢆ࣌ࣜࣉࣛࢬ࣒ᢳฟᾮࡋ࡚ᅇࡋࡓ. ࡇࢀࢆTBSᑐࡋ࡚4Υ୍࡛ᬌ㏱ᯒࡋ ࡓࡢࡕ, ᢠ FLAG ᢠయᅛᐃ࣮࢞ࣟࢫ (⣙ 1 mL) ࢆሸࡋࡓࣇࢽࢸ࣮࣒࢝ࣛ
ࡋࡓ. ࣒࢝ࣛࢆ TBS ࡛Ὑίᚋ, 0.10 M ࢢࣜࢩࣥሷ㓟⦆⾪ᾮ (pH 3.5) ࡛⁐ฟࡋࡓ. ࡇࡢ⏬ศࢆPBSᑐࡋ࡚4Υ୍࡛ᬌ㏱ᯒࡋࡓࡢࡕ, 㐲ᚰᘧ㝈እࢁ㐣⭷ (Amicon Ultra) ࢆ
⏝࠸࡚⃰⦰ࡋࡓ.
3.3.8㡯 ⤊ṆࢥࢻࣥࡀᏑᅾࡍࡿྍ⁐ᆺኚ␗scFvࡢ࣑ࣀ㓟ࡢྠᐃ
๓㡯࡛⢭〇ࡋࡓscFvࡢ࠺ࡕ, ⤊ṆࢥࢻࣥࡀᏑᅾࡍࡿࡶࡢ (scFv#m1-2࠾ࡼࡧ1-10) ࡘ࠸࡚࣑ࣀ㓟ศᯒࢆ⾜ࡗࡓ. scFv#m1-2ࡣ⢭〇Ỉᑐࡋ࡚4Υ୍࡛ᬌ㏱ᯒࡋ, 㐲ᚰᘧ㝈 እࢁ㐣⭷ (Amicon Ultra) ࢆ⏝࠸࡚⃰⦰ࡋࡓࡶࡢ (5.6 mM, ⣙ 100 mL) ࢆN ᮎ➃࣑ࣀ
㓟㓄ิศᯒࡋࡓ. ୍᪉, scFv#m1-10ࡣ୍㒊 (5 mg) ࢆ2.2.6㡯‽ࡌ࡚㟁ẼὋື
ࡋ, ࢤࣝࢆࢡ࣐ࢩ࣮ࣈࣜࣜࣥࢺࣈ࣮ࣝᾐࡋ࡚ᐊ ࡛ 30 ศ࠺ (~60 rpm) ࡋࡓ.
⁐ᾮࢆ㝖ཤࡋ࡚ࢤࣝࢆ⁛⳦Ỉᾐࡋ, ྠᵝ୍ᬌ࠺ࡋࡓ. ᰁⰍࡉࢀࡓ scFv ࢱࣥࣃ
ࢡ㉁ࢆྵࡴࢤࣝࢆษࡾฟࡋ, ࢪࢳ࢜ࢫࣞࢺ࣮ࣝ (0.15 mg) ࢆྵࡴ 0.10 M Ⅳ㓟Ỉ⣲
ࣥࣔࢽ࣒࢘⁐ᾮ (100 mL) ᾐࡋ࡚ᐊ ࡛30ศ㟼⨨ࡋࡓ. ⁐ᾮࢆ㝖࠸ࡓࡢࡕ, ࣮ࣚࢻ
ࢭࢺ࣑ࢻ (1.0 mg) ࢆྵࡴ0.10 MⅣ㓟Ỉ⣲ࣥࣔࢽ࣒࢘⁐ᾮ (100 mL) ᾐࡋ࡚ᐊ
࡛30ศ㟼⨨ࡋࡓ. ࡧ⁐ᾮࢆ㝖࠸ࡓࡢࡕ, ㉁㔞ศᯒ⏝ࢤࣝෆᾘ࢟ࢵࢺࡼࡾ, ࢺࣜࣉ
ࢩࣥᾘࡉࢀࡓ࣌ࣉࢳࢻ᩿∦ࢆᚓࡓ. ࡇࢀࢆ࣌ࣉࢳࢻ࣐ࢫࣇࣥ ࣮࢞ࣉࣜࣥࢺἲ
ࡋ࡚࣑ࣀ㓟ࢆྠᐃࡋࡓ.
83
➨⠇ᒓᐇ㦂
3.4.1㡯 CS#10-scFv-GLuc⼥ྜࢱࣥࣃࢡ㉁ࢆ⏝࠸ࡓoff-rate㑅ᢥ᮲௳ࡢ᳨ウ
3.2.3 㡯 ‽ ࡌ ࡚ CS-BSA ࢆ ᅛ ᐃ ࡋ ࡓ ࣐ ࢡ ࣟ ࣉ ࣞ ࣮ ࢺ , 2.4.5 㡯 ࡛ స 〇 ࡋ ࡓ CS#10-scFv-GLuc⼥ྜࢱࣥࣃࢡ㉁ (⣙1 pmol) ࢆྵࡴG-PBSࢆศὀ (100 mL/࢙࢘ࣝ) ࡋ, 37Υ࡛1㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ⁐ᾮࢆ㝖ཤࡋ࡚T-PBS࡛3ᅇὙίࡋࡓࡢࡕ, ࢭࣞࣥ
ࢸࣛࢪࣥ (5 mM) ࢆྵࡴPBSࢆຍ࠼ (100 mL), 300~700 nm࠾ࡅࡿⓎගᙉᗘࢆ ᐃࡋ ࡓ. ྠᵝὙίࡋࡓࡢࡕྛ✀⃰ᗘࡢCSࢆྵࡴG-PBSࢆศὀࡋ (200 mL), 25Υ୍࡛ᐃ
㛫 (1~6 㛫) ࠺ (600 rpm) ࡋࡓ. ྠᵝὙίࡋࡓࡢࡕࢭࣞࣥࢸࣛࢪࣥࢆຍ࠼࡚Ⓨ
ගᙉᗘࢆ ᐃࡋࡓ.
3.4.2㡯 M13-scFv-GLucࢆ⏝࠸ࡓⓎගᙉᗘࡢࣉࣟࣇࣜࣥࢢ
3.3.1 㡯‽ࡌ࡚ࢥࣟࢽ࣮ศ㞳ࢆ⾜࠸, 3.3.2 㡯ྠᵝྛࢥࣟࢽ࣮ࡢⓎගᙉᗘࢆࣉࣟ
ࣇࣜࣥࢢࡋࡓ.
3.4.3㡯 ࣇ࣮ࢪࡢቑᖜM13-scFv-GLucࢆ⏝࠸ࡓoff-rateࡢほ
๓㡯࡛≉ᐃࡋࡓ࢙࢘ࣝṧᏑࡍࡿ⁐ᾮࢆ㝖ཤࡋࡓࡢࡕ, 0.10 M ࢢࣜࢩࣥሷ㓟⦆⾪ᾮ (pH 2.2, 100 mL) ࢆຍ࠼, ᐊ ࡛10ศ࠺ (600 rpm) ࡋࡓ. ࡇࡢ㔞2.0 M Tris (⣙ 4 mL) ࢆຍ࠼࡚୰ࡋࡓࡢࡕ, ࡑࡢ୍㒊 (10 mL) ࢆ, 3.2.3㡯‽ࡌ࡚స〇ࡋࡓCS-BSA
ᅛᐃⓑⰍ࣐ࢡࣟࣉ࣮ࣞࢺศὀࡋࡓᑐᩘቑṪᮇࡢTG1 (90 mL) ຍ࠼, 37Υ࡛30ศ
࣮ࣥ࢟ࣗ࣋ࢩࣙࣥࡋࡓ. ࡇࡇ, ࣥࣆࢩࣜࣥࢼࢺ࣒ࣜ࢘ (200 mg/mL), ◲㓟࢝ࢼ࣐
ࢩࣥ (10 mg/mL), ࠾ࡼࡧKM13 (1109 pfu/mL) ࢆྵࡴ2YTᇵᆅࢆຍ࠼ (100 mL), 25Υ࡛45㛫࠺ᇵ㣴 (800 rpm) ࡋࡓ. ᇵ㣴ᾮࢆ㝖ཤࡋ࡚T-PBS-2࡛3 ᅇὙίࡋࡓ
ࡢࡕ, M-PBS࡛500ಸᕼ㔘ࡋࡓM13-scFv-GLuc (2.4.11㡯࡛స〇) ࢆຍ࠼ (100 mL), 37Υ
࡛30ศ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ྠᵝὙίࡋࡓࡢࡕ, ࢭࣞࣥࢸࣛࢪࣥ (5 mM) ࢆྵࡴPBS
ࢆຍ࠼࡚ (100 mL) Ⓨගᙉᗘࢆ ᐃࡋࡓ. ⁐ᾮࢆ྾ᘬ㝖ཤࡋ࡚T-PBS࡛3ᅇὙίࡋࡓࡢ
ࡕ, CSᶆ‽ရ (5.0 mg/mL) ࢆྵࡴG-PBSࢆຍ࠼ (200 mL), 25Υ࡛4㛫࠺ (600 rpm) ࡋ, ྠᵝὙίࡋࡓࡢࡕࢭࣞࣥࢸࣛࢪࣥࢆຍ࠼࡚Ⓨගᙉᗘࢆ ᐃࡍࡿ᧯సࢆ3ᅇ⧞ࡾ㏉
ࡋࡓ.
3.4.4㡯 ࣔࣀࢡ࣮ࣟࢼࣝࣇ࣮ࢪࡢㄪ〇ELISA
๓㡯࡛ప off-rate ᥎ᐃࡉࢀࡿࢡ࣮ࣟࣥࡘ࠸࡚, ๓㡯࡛ࢢࣜࢩࣥሷ㓟⦆⾪ᾮࡼࡾ
⁐ฟࡋࡓࣇ࣮ࢪᾮ (50 mL) ࢆ⏝࠸࡚3.3.3㡯‽ࡌ࡚⭠⳦TG1ࢆᙧ㉁㌿ࡋࡓ. ࡑ ࡢ⤖ಖᏑᾮࡢ୍㒊 (200 mL) ࢆ⏝࠸࡚1.4.5㡯‽ࡌ࡚ࣔࣀࢡ࣮ࣟࢼࣝኚ␗scFvᥦ♧
ࣇ࣮ࢪࢆస〇ࡋࡓ. ࡇࡢࣇ࣮ࢪᾮࢆ㐺ษᕼ㔘ࡋ, 1.4.10㡯ྠᵝࡢELISAࡋ
࡚, ᢠCSάᛶࢆホ౯ࡋࡓ.
84
3.4.5㡯 ྍ⁐ᆺኚ␗scFvࡢㄪ〇
๓㡯ࡢᐇ㦂ࡼࡾwt-scFvࡼࡾࡶ㧗ឤᗘ࡛࠶ࡿ᥎ᐃࡉࢀࡓࢡ࣮ࣟࣥࡘ࠸࡚, 1.4.11 㡯‽ࡌ࡚ྍ⁐ᆺኚ␗scFvࢆㄪ〇ࡋࡓ.
3.4.6㡯 Scatchardἲࡼࡿ⤖ྜᐃᩘࡢ⟬ฟ
1.2.4㡯‽ࡌ࡚ᐇ㦂ࢆ⾜࠸, Scatchardἲ
43)
ࡼࡾྍ⁐ᆺኚ␗scFvࡢCSᑐࡍࡿKa
ࢆ⟬ฟࡋࡓ.
3.4.7㡯 ྍ⁐ᆺኚ␗scFvࡢ㔞ㄪ〇⢭〇
3.4.5 㡯࡛స〇ࡋࡓᙧ㉁㌿⳦ࡢ⤖ಖᏑᾮࢆᑡ㔞ࡾ, 3.3.8 㡯‽ࡌ࡚࣌ࣜࣉࣛࢬ
࣒ᢳฟᾮࢆᅇࡋ, ᢠFLAGᢠయᅛᐃ࣮࢞ࣟࢫࢆ⏝࠸࡚⢭〇ࡋࡓࡢࡕ, PBS୍࡛ᬌ
㏱ᯒᚋ, ⃰⦰ࡋࡓ.
3.4.8㡯 ࣅ࢜ࢳࣥCS-BSAࡢస〇
NHS-LC-Biotin (1.4 mg) ࢆN,N-dimethylformamide (810 mL) ⁐ゎࡋ, ࡑࡢ୍㒊 (2 mL)
ࢆCB (498 mL) ΰࡋࡓ. ࡇࡢ㔞ࢆ, 1.2.1 㡯࡛స〇ࡋࡓ CS-BSA ࡢ୍㒊 (1.0 mL)
ΰࡋ, ᧠ᢾࡋ࡞ࡀࡽᐊ ࡛2㛫ᛂࡉࡏࡓ. ࡇࡢΰྜᾮ, ࢚ࢱࣀ࣮࣑ࣝࣥ (5 mL) ࢆຍ࠼, ᐊ ࡛30ศ᧠ᢾࡋࡓ. ࡇࡢᾮࢆPBSᑐࡋ࡚4Υ୍࡛ᬌ㏱ᯒࡋ, ㏱ᯒ⭷ෆ
ᐜ≀ࢆᅇࡋ࡚⏝ࡲ࡛-20Υ࡛ಖᏑࡋࡓ.
3.4.9㡯 ࣂ࣮࢜ࣞࣖᖸ΅ἲࡼࡿゎ㞳㏿ᗘᐃᩘࡢ ᐃ
ࢫࢺࣞࣉࢺࣅࢪࣥࡀᅛᐃࡉࢀࡓᑓ⏝ࢳࢵࣉࡢඛ➃ࢆ, G-PBS࡛8ಸᕼ㔘ࡋࡓࣅ࢜
ࢳࣥCS-BSA (๓㡯࡛స〇) (4 mL) ᾐࡋ, 25Υ࡛2ศ࠺ (1000 rpm) ࡋࡓ. ࡇࢀ
ࢆG-PBS (250 mL) ᾐࡋ, 25Υ࡛30⛊࠺ (1000 rpm) ࡋ, ࡇࡢࡁࡢࢩࢢࢼࣝࢆᇶ
‽ࡋࡓ. ᘬࡁ⥆ࡁ, ࢳࢵࣉࡢඛ➃ࢆ 3.4.8 㡯࡛స〇ࡋࡓ⢭〇῭ࡳࡢኚ␗ scFv (0~5544 nM) ࢆྵࡴ G-PBS (4.0 mL) ᾐࡋ࡚ 25Υ࡛ 2 ศ࠺ (1000 rpm) ࡋࡓ. ࡑࡢᚋ, G-PBS (250 mL) ᾐࡋ࡚25Υ࡛10ศ࠺ (1000 rpm) ࡋࡓ. ᚓࡽࢀࡓࢭࣥࢧ࣮ࢢ࣒ࣛ
ࡽ, ᑓ⏝ࢯࣇࢺ࢙࢘ࢆ⏝࠸࡚ゎ㞳㏿ᗘᐃᩘ (kd) ࢆ⟬ฟࡋࡓ.
➨⠇ᒓᐇ㦂
3.5.1㡯 ྍ⁐ᆺኚ␗scFvࢆ⏝࠸ࡿELISA
CS-BSAࢆ1.3.14㡯‽ࡌ࡚ᅛᐃࡋࡓ࣐ࢡࣟࣉ࣮ࣞࢺ, G-PBS࡛㐺ษᕼ㔘ࡋ
ࡓ࣌ࣜࣉࣛࢬ࣒ᢳฟᾮ (3.3.5㡯࠾ࡼࡧ3.4.5㡯࡛స〇) (100 mL) ࠾ࡼࡧྛ✀⃰ᗘࡢCSᶆ
‽ရࢆྵࡴG-PBS ⁐ᾮ (50 mL) ࢆຍ࠼࡚ΰྜࡋ, 4Υ࡛ 2㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ⁐ ᾮࢆ㝖ཤࡋ࡚T-PBS࡛3ᅇὙίࡋࡓࡢࡕ, G-PBS࡛5000ಸᕼ㔘ࡋࡓPODᶆ㆑ᢠFLAG
85
ᢠయ (100 mL) ࢆຍ࠼࡚37Υ࡛30ศ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ. ࡧὙίࡋ࡚, ᅛ┦ୖࡢPOD
άᛶࢆ1.2.5㡯ྠᵝẚⰍ ᐃࡋࡓ.
86 ᘬ⏝ᩥ⊩
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