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- ねんたろう はしかわ
- 5 years ago
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2 MHC T HLA I HLA 21 2 HLA-A2402/K b HLA-A2402 PCR H-2K b PCR H-2K b PCR HLA-A2402/K b PSA cdna HLA-A2402/K b HLA-A2402/K b HLA
3 2.3.1 HLA-A2402 PCR H-2K b PCR HLA-A2402/K b HLA-A2402/K b HLA-A2402/K b HLA-A2402/K b HLA-A24 CTL PSA CTL PSA PSA HLA-A HLA β2m HLA β2m HLA CD8 + CTL HLA HLA-A24 CTL
4 MHC Major Histocompatibility Complex HLA Human Leukocyte Antigen β2m Beta 2-Microglobulin TCR T cell receptor CTL Cytotoxic T Lymphocyte TAP transporter associated with antigen processing IFA Incomplete Freund's Adjuvant DMSO dimethyl sulfoxide APC antigen presenting cell PSA prostate specific antigen MAGE melanoma-associated antigen CEA carcinoembryonic antigen TERT telomerase reverse transcriptase WT1 Wilms' tumor suppressor gene 1 ELISA Enzyme-Linked Immunosorbent Assay IFN interferon 3
5 HLA I CD8 + T CTL CD8 + CTL HLA I HLA I in vivo HLA CD8 + CTL CD8 + CTL HLA-A24 HLA-A24 HLA-A24 HLA I CD8 HLA-A2402 α3 MHC I H-2K b HLA HLA-A2402/K b CD8 + CTL HLA CD8 + CTL PSA HLA-A24 HLA-A24 HLA-A2402 HLA-A24 HLA-A2402 N HLA-A2402 HLA-A24 HLA-A24 HLA-A2402/K b 4
6 CD8 + T HLA-A24 CD8 + T HLA-A24 5
7 1 1.1 LAK TIL BRM LAK lymphokine activated killer cells IL-2 TIL LAK IL-2 BRM Biological Response Modifiers BCG OK-432 PSK Lentinan Bestatin IL-2 IL-12 IFN TNF 6
8 1991 T. Boon CD8 + T CTL 1 CD8 + CTL CD8 + CTL HLA CD8 + CTL CD8 + CTL CD8 + CTL CD8 + CTL CD8 + CTL CD8 + CTL CD8 + CTL T T CD8 + CTL HLA TCR 2 3 7
9 CpG TLR α-galcer CD1d 4 5 T CD25 + CD4 + Treg 6 8
10 1.2 MHC T major histocompatibility complex MHC MHC MHC I II HLA human histocompatibility leukocyte antigen HLA I II MHC I CD8 + CTL TCR MHC I MHC I MHC I MHC α MHC β2 β2m α MHC I α α1 α2 α3 β2m MHC I α1 α2 MHC I MHC I 8 12 MHC TCR 9
11 MHC I CD8 + T A) HLA-A2 P1 P9 9 N C HLA-A2 P3 P7) TCR B HLA-A2 TCR α A, B, F HLA-I CHO C) HLA-I CD8 + T NK KIR killer-cell inhibitory receptor 10
12 MHC II B antigen presenting cell APC CD4 + T MHC II T APC MHC II α β α β MHC II MHC II CD4 + T TCR CD8 + CTL CD4 + T 11
13 MHC-II CD4 + T A) HLA-DR1 HA MHC-II N Tyr position 1 (PI) C MHC-II MHC-II MHC-II MHC-II B ) HA MHC-II TCR HA MHC-II Pl, P4, P6, P7, P9 ) MHC-II MHC-II HLA-DR1 C MHC-II CD4 + T α1 α2 β1 β2 MHC-II TCR α β TCR α β C V 12
14 T Th Th MHC I 20S 19S/PA700 11S/PA28 IFN-γ IFN-γ IFA-γ 20S PA28 19S-20S-19S 11S/PA28-20S-11S ATP 13
15 CD8 + T 30-80% defective ribosomal products DRiPs HSP70 TAP transporter-associated with antigen processing ATP MHC I MHC I MHC I MHC I MHC II CD4 + T MHC I CD8 + T MHC I CD8 + T 14
16 (step 1) (step 2) APC CSP (step 3) APC empty MHC I 9 empty MHC I MHC I step 4 (step 5 6) (EC) (P) step 7 (PS) TAP (ER) (step 8) empty MHC I (AP) (step 9) MHC (step 10) CD8 + T (step 11) (step 12 13) MHC I (step 14) 15
17 1.3 CD8 + CTL SYT-SSX SYT X SSX1 CD8 + CTL 8 16
18 CD8 + CTL mrna cdna cdna 100 MHC I cdna COS CD8 + CTL CD8 + CTL COS COS cdna cdna CD8 + CTL cdna cdna COS CD8 + CTL MHC CD8 + CTL 17
19 CD8 + CTL cdna cdna 9 10 MHC I in vitro CD8 + CTL CD8 + CTL cdna mrna cdna cdna 100 cdna MHC cdna COS DNA COS T T COS COS cdna cdna T cdna 18
20 1.4 HLA I HLA-A N. Holmes HLA-A2.1 CD8 + CTL 11 MHC I α1 α2 TCR α3 CD Chesnut HLA-A2.1 α3 H-2K b MHC I HLA-A2.1/K b 14 HLA-A2.1/K b HLA-A2.1 α1 α2 HLA-A2.1 HLA-A2.1/K b HLA-A2.1 CD8 + CTL CD8 HLA-A2.1/K b HLA-A2.1 CD8 + T 1996 A. Sette 38 HBV HCV HPV CD8 + CTL HLA-A2.1/K b in vivo in vitro HLA-A2.1 HLA-A2.1/K b CD8 + CTL 70% 14+13/38 HLA I CD8 + CTL 1 2 in vivo CD8 + CTL 19
21 HLA I CD8 + CTL 20
22 1.5 HLA HLA CD8 + T 1998 Altman HIV HIV HLA CD8 + CTL HLA-A HLA TCR affinity M BIAcore HLA avidity off-rate 17 HLA CD8 + T T TCR CD8 + T HLA HLA C HLA β2m in vitro HLA HLA HLA-A24 HLA molecule Avidin Core Biotinylated C-terminal tag HLA 21
23 2 HLA-A2402/K b 2.1 HLA-A HLA-A2.1 50% 45% HLA-A24 60% 20% 33% 27% 18 HLA-A2.1 HLA-A24 HLA I CD8 + CTL HLA in vivo HLA CD8 + CTL HLA-A24 HLA-A24 CD8 + CTL PSA 22
24 RERF-LC-AI HLA-A2402 EL4 H-2K b 3.5 x ml 0.7% 1,200rpm 4 C 5 15ml 10mM Tris-HCl ph µl 10mg/ml Proteinase K 150µl 10% SDS 60 C 1 37 C 16 15ml /TE 15 9,000rpm 10 15ml / 15 9,000rpm 10 15ml 15 9,000rpm ml 3M ph ml 80% 15ml TE ph µl 10mg/ml RNase 37 C 1 10ml / 5 9,000rpm 10 10ml 5 9,000rpm 10 1ml 3M ph ml 75% 80% 2ml TE ph HLA-A2402 PCR RERF-LC-AI HLA26-1F 5 -CCC AAG CTT ACT CTC TGG CAC CAA ACT CCA TGG GAT-3 A24-BglII30 5 -CGG GAG ATC TAC AGG CGA TCA GGT AGG CGC-3 Pfu-DNA Polymerase Promega PCR Hind III Bgl II 95 C C C 72 C 35 23
25 72 C 10 pbluescript II (Bgl II + ) Hind III Bgl II DNA Ligation Kit Ver.2 JM µg/ml LB 37 C Plasmid Maxi Kit ABI PRISMTM 377 DNA ABI Applied Biosystems Genbank Accession Z72422 pb-a H-2K b PCR EL4 H-2KBF3 5 -CGC AGG CTC TCA CAC TAT TCA GGT GAT CTC-3 H-2KB3R 5 -CGG AAT TCC GAG TCT CTG ATC TTT AGC CCT GGG GGC TC-3 TaKaRa LA Taq TM PCR EcoR I 95 C C C 72 C 25 pbluescript II SK+ EcoR I Kpn I DNA Ligation Kit Ver.2 JM µg/ml LB 37 C Plasmid Maxi Kit ABI PRISMTM 377 DNA ABI Applied Biosystems Genbank Accession v00746 v H-2K b PCR EL4 H-2kbF5 5 - AGG ACT TGG ACT CTG AGA GGC AGG GTC TT -3 H-2kb5R 5 - CAT AGT CCC CTC CTT TTC CAC CTG TGA GAA -3 Pfu-PCR 95 C C C 72 C C 10 24
26 pbluescript II(Bgl II + ) BamH I Bgl II DNA Ligation Kit Ver.2 JM µg/ml LB 37 C Plasmid Maxi Kit ABI PRISMTM 377 DNA Genbank Accession v HLA-A2402/K b HLA-A2402/K b ISOGEN total RNA SuperScript Choice System GIBCO BRL 10µg total RNA Oligo(dT) cdna cdna chi.pf1 5 -CCC AAG CTT CGC CGA GGA TGG CCG TCA TGG CGC CCC GAA-3 chi.pr1 5 -CCG GAA TTC TGT CTT CAC GCT AGA GAA TGA GGG TCA TGA AC-3 Pfu-PCR Hind III EcoR I 95 C C C 68 C C 10 Hind III EcoR I pcdna3.1(+) pef1/myc-his A Invitrogen HLA-A2402/K b pchla-a2402/k b pef1-a2402/k b PSA cdna cdna Library Clontech hpsa-f 5 -CGC GGA TCC ACC ATG TGG GTC CCG GTT GTC TTC CT-3 hpsa-r 5 -CCG GAA TTC TCA GGG GTT GGC CAC GAT GGT GTC-3 Pfu-PCR BamH I EcoR I pcdna3.1(+) 25
27 pef4/myc-his A PSA pc-hpsa pef4-hpsa PSA cdna (Accession M26663) HLA-A2402/K b pb-a2402 Hind III Bgl II pb-h-2 K b BamH I EcoR I 37 2 DNA Prep-A-Gene DNA purification kit (Bio-Rad ) DNA Ligation Kit Ver.2 HLA-A2402/K b pb-a2402/k b 11µg pb-a2402/k b Hind III EcoR I Dra I SeaKem GTG HLA-A2402/K b Prep-A-Gene DNA purification kit 1 10 TE 10mM Tris ph 8 0.1mM EDTA ph 8 DNA Jurkat Hela pchla-a2402/k b EL4 pef1-a2402/k b Jurkat-A2402/K b Hela-A2402/K b EL4-A2402/K b EL4-A2402/K b pef4-hpsa Hela-A2402/K b pc-hpsa E24P H24P HLA-A2402/K b C57BL/6 HLA-A2402/K b SLC 6 HLA-A2402 HLA26-1F A24-BglII30 26
28 TaKaRa LA Taq DNA PCR 1.5kbp DNA C57BL/6 5 x 10 6 FITC HLA-ABC B Immunotech HLA-A2402/K b FACScan Becton Dickinson MHC I FITC H-2D b Pharmingen HLA T2-A24 3 x 10 5 / µg/ml PSA RPMI HLA-A HA One Lambda PE Igs Dako FACscan MFI HLA 100 x MFI MFI MFI % MFI HER-2/neu gp100 CEA MAGE-1 MAGE-3 TERT FNNFTVSFWLRVPKVSASHLE DMSO 40mg/ml 20mg/ml 27.5µl 27.5µl 500µl PBS(-) IFA water-in-oil PSA DMSO 40mg/ml 27.5µl 527.5µl 27
29 Montanide ISA 51 water-in-oil 200µl ACK 0.15M NH 4 Cl, 10mM KHCO 3, 0.1mM EDTA,pH x µg/ml RPMI %FCS 10mM HEPES 20mM L- 1mM 1mM MEM 1%MEM 55µM 2- CTM x 10 6 Jurkat-A2402/K b EL4-A2402/K b 3.7MBq 51 Cr 1 100µg/ml ml 10% FCS RPMI x 10 4 /ml E T E/T=80, 40, 20, well 51 Cr 1470 WIZARD γ Wallac 100 x 1% NP40 % specific lysis HLA-A2402/K b PSA ISA x µg/ml IL-2 30U/ml 5 in vitro MACS Miltenyi Biotec CD8 + 1 x 10 5 / Hela Hela-A2402/K b H24P 1 x 10 4 / IFN-γ ELISA 28
30 R&D Systems IFN-γ HLA-A2402/K b EL4-A2402/K b FITC HLA B FITC H-2D b Hela Hela-A2402/K b H24P HLA-A PE Igs FACScan HLA-A2402/K b pb-a2402/k b HLA-A2402/K b x 10 5 E24P HLA-A2402/K b PSA 2-10 Montanide ISA µg 200µl 23 29
31 HLA-A2402 PCR HLA-A24 Accession Z72422 HLA-A2 Accession K02883 HLA-A24 273bp HLA-A2 HLA 5 Hind III HLA-A2 HLA-A26 Accession AB Hind III HLA-A HLA-A24 Hind III HLA-A2 HLA Bgl II HLA-A2 3 HLA-A24 3 HLA-A2 Hind III HLA-A24 3 Bgl II HLA-A2402 RERF-LC-AI PCR Z H-2K b PCR H-2K b Genbank 2 Accession v00746 v00747 v ,594bp v ,837bp HLA BamH I 3 BamH I v Kpn I 30
32 v EcoR I H-2K b EL4 PCR BamH I v v00747 PCR 2 H-2K b HLA-A2402/K b HLA-A2402 Bgl II H-2K b BamH I HLA-A2402/K b HLA-A2402/K b
33 CD8 + CTL HLA-A2402/K b HLA-A2402/K b HLA-A2402/K b mrna total RNA 1 cdna 5 3 PCR HLA β2m HLA-A, B, C B HLA-A2402/K b β2m HLA-A2402/K b HLA-A2402/K b 18 A HLA-A24 CTL HLA-A2402/K b in vitro HLA-A24 CD8 + CTL MAGE MAGE gp100 int HER-2/neu HER-2/neu CEA CEA TERT TERT HLA-A2402/K b CTL 32
34 2.3.8 PSA CTL PSA HLA-A24 2 HLA BIMAS SYFPEITHI PSA 2-10 PSA 6-14 PSA PSA PSA HLA-A24 T2 HLA-A24 T2 TAP-1 TAP-2 HLA HLA empty HLA HLA 26 PSA 2-10 PSA 6-14 PSA PSA HLA-A24 HLA-A2402/K b PSA 2-10 PSA 6-14 PSA PSA PSA in vivo CTL PSA 2-10 PSA 6-14 PSA PSA CTL PSA PSA 2-10 PSA 6-14 PSA PSA HLA-A24 PSA Hela Hela-A2402/K b H24P Hela MAGE-1 MAGE-3 25 MAGE-1 MAGE-3 HLA-A24 MAGE MAGE MAGE MAGE HLA-A2402/K b CD8 + CTL Hela Hela-A2402/K b IFN- ELISA Hela-A2402/K b A CD8 33
35 HIV HIV-1 Nef CD8 + CTL HIV-1 Nef Hela-A2402/K b HLA-A2402/K b Hela-ELISA PSA 2-10 PSA 6-14 PSA PSA CD8 + CTL Hela Hela-A2402/K b H24P PSA 2-10 PSA PSA CD8 + CTL H24P CD8 PSA 6-14 CD8 + CTL H24P PSA 2-10 PSA PSA HLA-A24 B PSA HLA-A2402/K b PSA 2-10 Montanide ISA 51 n=6 PSA 2-10 PSA
36 2.4 HLA CD8 + CTL CD8 + CTL HLA in vivo HLA CD8 + CTL HLA I HLA in vivo HLA CD8 + CTL TLR CD1d CTLA-4 Treg CD8 + CTL HLA-A24 HLA-A2402/K b C57BL/6 H-2b H-2K b H-2D b BALB/c H-2d H-2K d H-2L d MHC I H-2K d HLA-A24 BALB/c HLA-A24 H-2K d CD8 + CTL 21 BALB/c HLA-A2402/K b HLA-A24 H-2K d CD8 + CTL C57BL/6 HLA-A2402/K b HLA-A24 CTL 35
37 HLA CTL HLA-A24 HLA-A24 HLA-A2402/K b PSA CD8 + CTL HLA PSA 30 PSA PSA PSA 31 PSA PSA PSA PSA CD8 + CTL PSA HLA-A24 HLA PSA 2-10 PSA 6-14 PSA PSA PSA HLA-A24 HLA-A2402/K b PSA 2-10 PSA 6-14 PSA PSA HLA-A24 CD8 + CTL HLA CD8 + CTL Hela ELISA 36
38 Hela MAGE-1 MAGE-3 MAGE MAGE HLA-A2402/K b MAGE MAGE CD8 + CTL HLA-A2402/K b Hela Hela-A2402/K b MAGE MAGE HLA-A2402/K b Hela-ELISA HLA-A2402/K b PSA CD8 + CTL Hela Hela-A2402/K b H24P PSA 2-10 PSA PSA CD8 + CTL H24P PSA 6-14 CD8 + CTL CD8 + CTL HLA-A24 HLA-A24 HLA TAP HLA β2m HLA-A24 H-2K d HLA-A2402/K b CD8 + T TCR TAP HER-2/neu HLA-A2.1 HER-2/neu HER-2/neu HER-2/neu 35 HER-2/neu HLA-A2.1 37
39 CD8 + CTL HLA-A2.1/K b 36 CD8 + CTL TCR MHC I HLA-A2.1/ K b HLA-A24 HER-2/neu HER-2/neu HER-2/neu HER-2/neu 38 HLA-A2402/K b MHC I HER-2/neu CTL HLA-A2402/K b HLA-A2.1/K b TCR CD8 + HLA-A2402/K b HLA-A2.1/K b HLA-A2.1 CD8 + CTL TCR CD4 + T CD8 + T T T 39 CD8 + CTL TCR CD8 + CTL HLA I HLA TCR CD8 + CTL HLA-A2.1/K b HLA CD8 + CTL HLA 38
40 RI HLA I HLA-A2402/K b HLA-A24 39
41 HLA-A24 genome HLA-A2 genome HLA-A24 genome HLA-A2 genome HLA-A24 genome HLA-A2 genome HLA-A24 genome HLA-A2 genome HLA-A24 genome HLA-A2 genome HLA-A24 genome HLA-A2 genome HLA-A24 genome HLA-A2 genome HLA-A24 genome HLA-A2 genome HLA-A24 genome HLA-A2 genome HLA-A24 genome HLA-A2 genome HLA-A24 genome HLA-A2 genome HLA-A24 genome HLA-A2 genome HLA-A24 genome HLA-A2 genome HLA-A24 genome HLA-A2 genome HLA-A24 genome HLA-A2 genome HLA-A24 genome HLA-A2 genome Hin d III Bgl II HLA-A24 HLA-A2 Genbank HLA-A24 HLA-A2 273bp HLA-A24 HLA Bgl II AGATCT 40
42 HLA-A24 HLA-A2 HLA-A24 HLA-A2 HLA-A24 HLA-A2 HLA-A24 HLA-A2 HLA-A24 HLA-A2 HLA-A24 HLA-A2 HLA-A24 RERF-LC-AI PCR HLA-A24 HLA-A2 41
43 Bam H I H-2Kb G G A T C C T G T G T G A C A C A C C T G T A C C T T G T C C C C C A G A G T C A G G G G C T G G G A G T C A T T T T C T C T G G C T A C A C A C T T A G T G A H-2Kb T G G C T G T T C A C T T G G A C T G A C A G T T A A T G T T G G T C A G C A A G G T G A C T A C A A T G G T T G A G T C T C A A T G G T G T C A C C T T C C A H-2Kb G G A T C A T A C A G C C C T A A T T T T A A T A T G A A C T C A A A C A C A T A T T A A A T T A G T T A T T T T C C A T T C C C T C C T C C A T T C T T T G A H-2Kb C T A C C T C T C T C A T G C T A T T G A A C A T C A C A T A A G G A T G G C C A T G T T T A C C C A A T G G C T C A T G T G G A T T C C C T C T T A G C T T C H-2Kb T G A G T C C C A A A A G A A A A T G T G C A G T C C T G T G C T G A G G G G A C C A G C T C T G C T T T T G G T C A C T A G T G C G A T G A C A G T T G A A G H-2Kb T G T C A A A C A G A C A C A T A G T T C A C T G T C A T C A T T G A T T T A A C T G A G T C T T G G G T A G A T T T C A G T T T G T C T T G T T A A T T G T G H-2Kb T G A T T T C T T A A A T C T T C C A C A C A G A T T C C C C A A A G G C C C A T G T G A C C C A T C A C A G C A G A C C T G A A G A T A A A G T C A C C C T G H-2Kb A G G T G C T G G G C C C T G G G C T T C T A C C C T G C T G A C A T C A C C C T G A C C T G G C A G T T G A A T G G G G A G G A G C T G A T C C A G G A C A T H-2Kb G G A G C T T G T G G A G A C C A G G C C T G C A G G G G A T G G A A C C T T C C A G A A G T G G G C A T C T G T G G T G G T G C C T C T T G G G A A G G A G C H-2Kb A G T A T T A C A C A T G C C A T G T G T A C C A T C A G G G G C T G C C T G A G C C C C T C A C C C T G A G A T G G G G T A A G G A G A G T G T G G G T G C A H-2Kb H-2Kb G A G C T G G G G T C A G G G A A A G C T G G A G C T T T C T G C A G A C C C T G A G C T G C T C A G G G C T G A G A G C T G G G G T C A T G A C C C T C A C C T T C A T T T C T T G T A C C T G T C C T T C C C A G A G C C T C C T C C A T C C A C T G T C T C C A A C A T G G C G A C C G T T G C T G T T C T G G T T G T C H-2Kb C T T G G A G C T G C A A T A G T C A C T G G A G C T G T G G T G G C T T T T G T G A T G A A G A T G A G A A G G A G A A A C A C A G G T A G G A A A G G G C A H-2Kb G A G T C T G A G T T T T C T C T C A G C C T C C T T T A G A G T G T G C T C T G C T C A T C A A T G G G G A A C A C A G G C A C A C C C C A C A T T G C T A C H-2Kb T G T C T C T A A C T G G G T C T G C T G T C A G T T C T G G G A A C T T C C T A G T G T C A A G A T C T T C C T G G A A C T C T C A C A G C T T T T C T T C T H-2Kb C A C A G G T G G A A A A G G A G G G G A C T A T G C T C T G G C T C C A G G T T A G T G T G G G G A C A G A G T T G T C C T G G G G A C A T T G G A G T G A A H-2Kb G T T G G A G A T G A T G G G A G C T C T G G G A A T C C A T A A T A G C T C C T C C A G A G A A A T C T T C T A G G T G C C T G A G T T G T G C C A T G A A A H-2Kb T G A A T A T G T A C A T G T A C A T A T G C A T A T A C A T T T G T T T T G T T T T A C C C T A G G C T C C C A G A C C T C T G A T C T G T C T C T C C C A G H-2Kb A T T G T A A A G G T G A C A C T C T A G G G T C T G A T T G G G G A G G G G C A A T G T G G A C A T G A T T G G G T T T C A G G A A C T C C C A G A A T C C C H-2Kb C T G T G A G T G A G T G A T G G G T T G T T C G A A T G T T G T C T T C A C A G T G A T G G T T C A T G A C C C T C A T T C T C T A G C G T G A A G A C A G C H-2Kb T G C C T G G A G T G G A C T T G G T G A C A G A C A A T G T C T T C T C A T A T C T C C T G T G A C A T C C A G A G C C C T C A G T T C T C T T T A G T C A A H-2Kb H-2Kb G T G T C T G A T G T T C C C T G T G A G C C T A T G G A C T C A A T G T G A A G A A C T G T G G A G C C C A G T C C A C C C C T C T A C A C C A G G A C C C T G T C C C T G C A C T G C T C T G T C T T C C C T T C C A C A G C C A A C C T T G C T G G T T C A G C C A A A C A C T G A G G G A C A T C T G T A G C C T G T C H-2Kb A G C T C C A T G C T A C C C T G A C C T G C A A C T C C T C A C T T C C A C A C T G A G A A T A A T A A T T T G A A T G T A A C C T T G A T T G T T A T C A T H-2Kb C T T G A C C T A G G G C T G A T T T C T T G T T A A T T T C A T G G A T T G A G A A T G C T T A G A G G T T T T G T T T G T T T G T T T G A T T G A T T T G T H-2Kb T T T T T T G A A G A A A T A A A T G A T A G A T G A A T A A A C T T C C A G A A T C T G G G T C A C T A T G C T G T G T G T A T C T G T T G G G A C A G G A T H-2Kb G A G A C T G T A G C A G C T G A G T G T G A A C A G G G C T G T G C C G A G G T G G G C T C A G T T T G C T T T G A T C T G T G A T G G G G C C A C A C C T C H-2Kb C A C T G T G T C A C C T C T G G G C T C T G T T C C C T C T A T C A C T A T G A G G C A C A T G C T G A G A G T T T G T G G T C A C A A A G A C A C A G G G A H-2Kb A G G C C T G A G C C T T G C C C T G T C C C C A G G A T T A T G A G C C C C C A G G G C T A A A G A T C A G A G A C T C G G A A T T C Eco R I H-2K b H-2K b EL4 PCR Genbank 3 HLA Bam HI GGATCC 3 EcoR I 42
44 HLA-A2402_Kb HLA-A2402_Kb HLA-A2402_Kb HLA-A2402_Kb HLA-A2402_Kb HLA-A2402_Kb HLA-A2402_Kb HLA-A2402_Kb HLA-A2402_Kb HLA-A2402_Kb HLA-A2402_Kb HLA-A2402_Kb HLA-A2402_Kb HLA-A2402_Kb HLA-A2402/K b cdna HLA-A2402/K b total RNA 1 cdna 43
45 HLA-A2402/K b HLA-A2402/K b HLA-A2402/K b HLA-A, B, C C57BL/6 H-2D b MHC I 44
46 HLA-A2402/K b HLA-A2402/K b HLA-A2402/K b 18 A2 pb-a2402/k b HLA-A2402/K b H-2K b 45
47 100 MAGE MAGE gp100 int % Specific Lysis % Specific Lysis % Specific Lysis No.1 No.2 No.3 0 No.1 No.2 No.3 0 No.1 No.2 No.3 50 HER-2/neu HER-2/neu CEA % Specific Lysis % Specific Lysis % Specific Lysis No.1 No.2 No.3 0 No.1 No.2 No.3 0 No.1 No.2 No CEA TERT TERT % Specific Lysis % Specific Lysis % Specific Lysis No.1 No.2 No.3 0 No.1 No.2 No.3 0 No.1 No.2 No.3 HLA-A2402/K b CTL HLA-A24 n=3 1 5 in vitro E Jurkat-A2402/K b T 51 Cr-release assay E/T=80 X Y % Specific lysis 46
48 Flu µm PSA HLA-A24 PSA HLA-A24 Flu MHC I 47
49 % Specific Lysis % Specific Lysis % Specific Lysis No.1 No.2 No.3 0 No.1 No.2 No.3 0 No.1 No.2 No.3 % Specific Lysis No.1 No.2 No.3 % Specific Lysis No.1 No.2 No.3 PSA CTL PSA n=3 1 5 in vitro E EL4-A2402/K b T E/T=80 X Y % Specific lysis 48
50 A 200 MAGE MAGE IFN-γ(pg/ml) IFN-γ(pg/ml) B IFN-γ(pg/ml) IFN-γ(pg/ml) HLA-A24 HLA-A24 HLA-A24 ELISA HLA-A2402/K b MAGE MAGE PSA 2-10 PSA PSA CD8 + CTL Hela Hela-A2402/K b HLA-A2402/K b Hela H24P HLA-A2402/K b PSA Hela A Hela MAGE-1 MAGE-3 B PSA Hela Hela-A2402/K b H24P Hela-A2402/K b A H24P B CD4 Hela-A2402/K b A H24P B CD8 49
51 A B volume mm days after inoculation days after inoculation C 4000 volume mm * E24P * * vac vac day days after inoculation PSA 2-10 PSA 2-10 n=6 E24P 1 A PSA 2-10 B C PSA 2-10 * Wilcoxon test p<
52 3 HLA-A HLA TCR HLA CD8 + CTL HLA CD8 + CTL CD8 + CTL CD8 + CTL CD8 + T HLA CD8 + T 6 HLA-A24 HLA-A24 51
53 HLA β2m HLA-A2402 cdna PCR HLA-A2402 cdna rsa24bsp/fw 5 -CCA TGG GCT CCC ACT CCA TGA GGT ATT TCT CCA CAT CCG T-3 A24BSP/PR 5 -GGA TCC TGG CTC CCA TCT CAG GGT GAG GGG CTT GGG CAG ACC CTC-3 Nco I BamH I HLA-A2402 cdna PCR HLA-A2402 cdna mrsa24bsp/pf 5 -CCA TGG GCA GCC ATT CTA TGC GCT ATT TTT CTA CCT CCG T-3 A24BSP/PR HLA-A2.1 cdna PCR HLA-A2.1 cdna rsa0201wt P/PF 5 - CCA TGG GCT CTC ACT CCA TGA GGT ATT TCT TCA CAT CCG T-3 A2BSP/PR 5 -GGA TCC TGC CTC CCA TCT CAG GGT GAG GGG CTT GGG CAG ACC CTC-3 HLA-A2.1 cdna PCR HLA-A2.1 cdna mrsa0201al/pf 5 - CCA TGG GCA GCC ATT CTA TGC GCT ATT TTT TTA CCT CCG T-3 A2BSP/PR Pfu-PCR pet11d Nco I BamH I pet11-mrsa2402 pet11-mrsa0201 β2m cdna PCR β2m cdna h2mpet-f1 5 -CAT ATG ATC CAG CGT ACC CCG AAA ATT CAG-3 h2mpet-r1 5 -GGA TCC TTA CAT GTC TCG ATC CCA CTT AAC-3 Pfu-PCR Nde I BamH I pet11a Nde I BamH I pet11-mrsβ2m 52
54 3.2.2 pet11-mrsa2402 pet11-mrsa0201 pet11-mrsβ2m BL21(DE3) Novagen 100 µg/ml LB 37 C LB 10 O.D.600 = mM isopropyl-β-d-thiogalactosidase IPTG 4 2 x Laemmli sample buffer 100 C 2 10/20% SDS-polyacrylamide gradient multi-gel 40mA SDS-PAGE Quick-CBB HLA-ABC clone8e9.4 Antigenix America HRP Igs Dako HLA β2m pet11-mrsa2402 pet11-mrsa0201 pet11-mrsβ2m BL21(DE3) 50 µg/ml LB 100ml 50µg/ml LB LB 10 O.D.600 = mM IPTG ml suspension buffer 25% sucrose, 50mM Tris ph8.0, 1mM EDTA, 1mM DTT, 1mM PMSF 20mg 5 Triton X µl 30 30mM MgCl 2 1mM MnCl 2 2,400-3,200 unit DNase I 75 20ml Detergent buffer 20ml Wash Buffer1 0.5% Triton X-100, 50mM Tris ph 8, 100mM NaCl 53
55 20ml Wash Buffer2 100Mm Tris ph 8, 150mM NaCl, 1mM EDTA 10ml 8M Urea Denaturing Buffer HLA HLA-A2.1 HLA-A24 hβ2m 42, 43 Refolding buffer(100mm Tris-HCl ph8, 2mM EDTA, 400mM L-arginine-HCl, 5mM Glutathione, 0.5mM Glutathione, 0.5mM PMSF) HLA 35µg/ml β2m 24µg/ml SYT-SSX1 PSA 1mg 6 3 Centriprep-10 YM-10, Amicon PD-10 desalting columns Pharmacia Centricon µl 50µl Biomax A 50µl Biomax B 1.5µl BirA Avidity minicon YM µl Superdex 200HR 10/30 Pharmacia 0.4ml/min PBS PE APC Molecular Probes 100µg HLA-A2402/β2M/ 33µg CD8 + CTL PSA HLA-A24 ISA 51 HLA-A2402/K b ISA in vitro PE PSA-HLA-A FITC CD8 Pharmingen
56 Jurkat-A2402/K b 51 Cr-release assay SYT-SSX 1 HLA-A2402 SYT-SSX1 SS393 APC SYT-SSX1-HLA-A PE CD8 Becton Dickinson 4 30 PBS 2 FACScan SS393 HLA-A2402 CIR-A*2402 HLA-A2402 SYT-SSX1 HS-SYII Fuji 51 Cr-release assay SS393 HLA-A2402 CIR-A*31012 HLA-A2402 SYT-SSX1 SW982-A24 HLA-A2402 SYT-SSX1 K562 55
57 HLA HLA recombinant soluble HLA rshla β2m recombinant soluble β2m rsβ2m rshla-a2.1 rsβ2m rshla-a rshla-a2402 rshla-a2.1 N BL21(DE3) Escherichia coli B mrshla modified rshla mrshla-a HLA-A24 CTL HLA-A2402/K b PSA in vitro in vitro CD8 PSA-HLA-A24 A CD8 + T B PSA CD8 + T 34.7% HLA-A % SYT-SSX1 CD8 SYT-SSX1-HLA-A24 A CD8 + T B CD8 + T in vitro CD8 + T CD8 + T CD8 + T 56
58 57
59 3.4 CD8 + CTL CD8 + CTL HLA-A24 HLA-A24 rshla-a2.1 rshla-a2402 HLA-A24 HLA rshla-a2402 rshla-a % rshla-a2.1 N rshla-a rshla-a2402 N rshla-a2402 rshla-a2402 rshla-a2402 rshla-a2.1 N 7 AGG Homo sapiens 11.9% E. coli B 2.1% AGG AGA 44 AGG CGC rshla-a2402 HLA-A24 PSA HLA-A2402/K b 58
60 PSA-HLA-A24 CD8 HLA-A24 SYT-SSX1 in vitro SYT-SSX1-HLA-A24 CD8 PSA HLA-A24 CD8 + CTL HLA-A24 CD8 + CTL rshla-a2402 N rshla-a2402 HLA-A24 HLA-A24 CD8 + CTL CD8 + CTL CD8 + CTL HLA-A24 CD8 + CTL 59
61 rshla-a2402 5'- ATG GGC TCC CAC TCC ATG AGG TAT TTC TCC ACA amino acids Met Gly Ser His Ser Met Arg Tyr Phe Ser Thr mrshla-a2402 5'- ATG GGC AGC CAT TCT ATG CGC TAT TTT TCT ACC frequency rshla-a0201 5'- ATG GGC TCT CAC TCC ATG AGG TAT TTC TTC ACA frequency amino acids Met Gly Ser His Ser Met Arg Tyr Phe Phe Thr mrshla-a0201 5'- ATG GGC AGC CAT TCT ATG CGC TAT TTT TTT ACC frequency HLA N HLA N Escherichia coli B rs mrs 60
62 A kda B) B rshla-a2402 mrshla-a2402 rshla-a2.1 mrshla-a2.1 rshla-a2402 mrshla-a HLA HLA LB 10 O.D.600 = mM IPTG 37 10µl 2x Laemmli sample buffer SDS-PAGE (A) CBB (B) HLA-ABC clone8e9.4 rs mrs 61
63 PSA-immunized SSA-Height R2 G1 Counts M % CD8-FITC Tetramers Non-immunized SSA-Height G2 Counts 10.59% M1 CD8-FITC Tetramers A PSA-HLA-A24 PSA HLA-A2402/K b 1 5 in vitro PSA-HLA-A24 CD8 62
64 Jurkat PSA Jurkat + PSA Jurkat-A2402/Kb + PSA Jurkat-A2402/Kb % Specific Lysis B PSA CD8 + CTL PSA HLA-A2402/K b 1 5 in vitro CD8 + CTL E T PSA Jurkat-A2402/K b PSA Jurkat-A2402/K b PSA Jurkat HLA-A24 X % Specific lysis Y E/T=80 63
65 A B Tetramer CD8 Before peptide-stimulation After peptide-stimulation C D E F Tetramer CD8 Before peptide-stimulation After peptide-stimulation A SYT-SSX1-HLA-A24 HLA-A24 SYT-SSX1 in vitro SYT-SSX1-HLA-A24 CD8 A-B HLA-A24 C-D HLA-A24 E-F 64
66 CIR-A*31012 SYT-SSX1 CIR-A*31012+SS393 CIR-A*2402 CIR-A*2402+SS393 K562 SW982-A24 HS-SYII Fuji % Specific Lysis B SYT-SSX1 CD8 + CTL HLA-A24 SYT-SSX1 SS393 in vitro CD8 + CTL E T SS393 CIR-A*2402 HLA-A24 SYT-SSX HS-SYII Fuji SS393 CIR-A*2402 SS393 CIR-A*31012 HLA-A24 K562 HLA-A24 SYT-SSX SW982-A24 HLA-A24 SYT-SSX X % Specific lysis Y E/T=30 65
67 4 HLA-A24 HLA-A2402/K b HLA CTL CTL PSA HLA-A24 5 HLA-A24 CD8 + CTL 4 3 HLA-A24 in vivo HLA-A24 N HLA-A24 HLA-A24 HLA-A24 HLA-A2402/K b PSA in vivo CD8 + CTL HLA-A24 in vitro CD8 + CTL HLA-A2402/K b HLA-A
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ウイルス 感染症研究 実験特集 不可能を可能にする NanoLuc テクノロジー NanoLuc テクノロジーをご利用のウイルス 感染症研究に携わるユーザー 4 名の方に最新の 研究成果についてご寄稿頂きました プロメガが開発した NanoLuc は 19 KDa の低分子の発光酵素であり 従来のホタルルシフェラーゼの 1/3 の分子量であるにもかかわらず 発 光レベルは 1 倍にも達します この
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TP T-13i AST ALT ALP Y CTP LOH CHE CRP 8.7 id 0.4 mg/d 21 AWL 131U/L 257 LU/L 12 111/L 234 11J/L 344 IU/L 0.30 mid No K a BUN ORE UA 142 meq/1 4.3 meqa 108 meqa 15.8 met! 0.71 mg/di 5.0 mg/d CEA 5_5 n8/m1
1. Levin B, Remine WH, Hermann RE, Scheive PS and Cohn I Jr: Panel: Cancer of the pancreas. Am J Surg (1978) 135, 185-191. 2. Van Heerden JA, Remine WH, Weiland LH and Mcllrath DC: Total pancreatectomy
Application Note 2
A P P L I C A T I O N N O T E 2 CD8 CD4T I M M U N E F U N C T I O N FastImmune Cytokine System T T 3 H- 51 Cr T TMHC- 1, 2 Enzyme-Linked Immunospot ELISPOT 3, 4 5-7 in vitro 3 ELISPOT T 8, 9 T /MHC T
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T DAC8 2011 1 27 (DAC) DAC 11 DAC8 DAC8 T Leukemia. 2008, 22, 1026 1134. Clin. Cancer. Res. 2009, 15, 91 99. DAC8 1)DAC8 2) T T T-ALL 10 1 T-ALL 30~40% T CTCL 10 1 CTCL T ATLL 1 TLV-1 120 2,000 TLV-1 16~41%
研究成果報告書
Arterioscler Thromb Vasc Biol., 27, 2400 2406 (2007) J. Agri. Food Chem., 59, 4544 4552 (2011) Bioorg. Med. Chem. Lett., 21, 1786 1791 (2011) Advanced glycation end products (AGEs) AGEs AGEs AGEs HbA1c
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2303 16 2 9 1 14 5 31 (423) 1 (4), (6) C55H84ClN17O21S3 HCl (BLM-A2) 1487.47 11 2 1 4 104 2 14 5 31 [ ] ( ) ( ) 1. 15mg 30mg( ) 5 20mL 1 5mg( ) 2. 3 15mg 30mg( ) 5mL 1mg( ) 1mL 3. 5mg 15mg( ) 4. 1 2 1
1 HLA MHC 2017; 24 (2) HLA HLA 1967 HLA HLA HLA HLA 1980 HLA HLA ABO HLA 2000 HLA DSA 2010 HLA HLA HLA HLA HLA HLA HLA HLA HLA GVHD HLA HLA
Major Histocompatibility Complex 2017; 24 (2): 150 157 抄録集 1 HLA 2017 5 13 150 1 HLA MHC 2017; 24 (2) HLA 1960 1964 HLA 1967 HLA HLA HLA HLA 1980 HLA HLA ABO HLA 2000 HLA DSA 2010 HLA HLA HLA HLA HLA HLA
Agilent Microarray Total Solution 5 5 RNA-Seq 60 mer DNA in situ DNA 5 2 QC 4200 TapeStation 2100 / mirna CGHCGH+SNP ChIP-on-chip 2 mirna QC
Microarray Agilent Microarray Total Solution Agilent Microarray Total Solution 5 5 RNA-Seq 60 mer DNA in situ DNA 5 2 QC 4200 TapeStation 2100 / mirna CGHCGH+SNP ChIP-on-chip 2 mirna QC RNA / mirna total
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種子における未承認遺伝子組換えパパイヤ (PRSV-YK) の検査法について 本検査法ではパパイヤ種子を検査対象とし DNA 抽出精製は 以下の GM quicker2( ニッポン ジーン社 ) を用いる 1 検体から2 反復で DNA を抽出し 各抽出 DNA 試料液を用いてリアルタイム PCR を用いた定性 PCR 法を実施する Ⅰ. 試料の準備 検査に用いるパパイヤ種子は 破砕粒や他の混入物を取り除いた完全粒とし