JP 2013-237673 A 2013.11.28 10 (57)【要約】 （修正有）
Contents
1. 4 JP 2013 237673 A 2013 11 28 U ULU l uu U Hod IHOOUdaddaCddO IU U Ll V UB LHUBWLtl uu Hod Dg E DE J HOUdaddaCddO V UB UBWtltl U ULU U U ULU HOOUdgdgdaddO A duck Mongol i a 54 01 H5N2 9 HAD O HAJ AJ duck Hokkai do 9 99 H9N2 Pumgumu2. A duck Hokkai do 9 99 H9N2 NPO O 0 0 O 5 ACG CGT CG CGC GCT TCA AGG CAC CAA ACG ATC 3 7 7000000000 5 CCC AAG CTT CIC CIC TCC ATT GTC TCC GA A 3 000000000000 One Step RNA PCR kit AW TaKaRal O O O O PCRI H D dtl NPQ cDNA O D O JUUUUIUUIUUUUULU OHO00g0 00 Sal Hi ndl iig j Uu uluttkuWuDliuitilliiutititiitlu4t 000 pQE31 Qagen OOO00000000 DNA Escherichia coli M50 O O O NPQ pppggooo0o000000000000QGagenO O D UU IUL Pe NP 0 1 O 1 LBO O 1 0 Tryptone 0 5 Yeast extract 1 0 sod i um chl ori def anpi cillin sodi umsalt 100ntg m
3. uggggdguuuuuauuseuguuuuuuuuuuuuucgdsotn 0 0 O uggggdguusuugaugamgumgguuguiuuguuuuuugdutHutl tl U U popg00o00800 U I JHL JOUOUNUNU UDONHNWUL U U DID DD Uu t UL D O JDU DOUDNUNU IOHOUUdO IOHOUdOH B Fa E ZW lu NES ENEI Sones EZ Don Es MES Es B DN HUnggggugaguggggguauguggggguausguuuulb U D DNjUugguuamgmgggggguusguuuuuudtdtl tl 704000000 0 90 60 SSC 40 25 C U DD D D 10 SSQ 55 C H D D D D D 0000000 U U REGI iDgmbpstl t iumngiWuul lOO iDgmbpEstl t iumngiguutu 30 0 0 5000000000 800 000000 Dg O DU IUIUUIUUUIUU UUIUUIUIUU VD UNUL OHUDUDOY 0 DDB O0 D U O FASTAQ BLAST PSI BLAST SSEARCH D D DNJog00000DBpp00000000000 nd Analysis 0000 http www ddbj ni g ac j p E mail homol ogy j National Center for Biotechnol ogy Information NCBI 000 0 BLA 0 O0 O0 O0 O0 O0 O0 O0 0 O0 O O0
4. act 2 peptone 0 0000496 bi oti n yeast nitrogen base Invitrogen 100mM phos phate buffer pH 00 O O O O O D O OD D D 00 28 C 10000 160 0 0 O methan OU IUN 00000000000 60000000000 O00050 00 ol iguumgmguuuuu seocyuuuduldtl 100000 breaking buffe r 50m Tris Cl 1nM EDTA 5 ycerol pH8 0 0 D D D O glass beads 425 600 m crons Sigma O O O Bi oruptor Cosmo biol O 300 0 0 a 300 0 O D 300 30 0 O UNUI UI 0 0 0 DO 14 0009 en 300 0 0000000000 22mem BpHugmgugugumgusuuuuuuutlislttl ulu p pp0g00000000 0000 HiPrep 16 10 O XL O O GE healthcare po0000000 20nM Tris Cl pH8 0 10 0 O 20nM Tris Cl pH8 0 9 1M NaCl O U ugHnusumuuuBstiutl JUDUNUL 30kDa H 00000 20nM Tri s C pH8 0 150mM NaCl o O poo UNUI UUU O HAD O O O 10 SD S PAGE iugi 000 gt U DDHDHOUNLULNU U 10 JP 2013 237673 A 2013 11 28
5. 50 0 COSI 3T30 0 H D D O O BHK baby hanster ki dney HeLa Vero O O utu Valle et al Nature 1981 291 358 340 Sf 9 Sf 211 Tn5 000 000000060 CHO DG44 CHO DXB11 COS7 BA p0p00000000 IUUUUUUUUUU utu chog o0 000 000 00000000000000 U DEAE OOO0000000000 0 U DOTAP u dt Od O IUUUUDUUUUDUUUUUUUUUUUDUUU og U U DU 00000000 N coti ana tabacu 00000 UDUDUDUDUDUDUDUDUUUUUU Saccharomyces 0O00 S O O O O O O O O OD O OD OD D O O Saccharomyces cerevisiae O 00000000 0 O Saccharonyces ponbel Aspergillus 0000000000000000 Aspergillus ni ger IHUUUUU IH JUDUL 000000 J MLO9 DH5a HB101 DU po0000060 DNA po0000000000invitroop 0o000000000000 m O E I EI CI ELI
6. 10 30 40 50 9 JP 2013 237673 A 2013 11 28 ugagggggagugauuagugggaggagaauagumuagumuuesutl HA IO A duck Mongol i a 54 01 H5N2 0 O HA D D O OD O OD O N
7. O 0 O NP 1000000000 20mM Tris HCl pH8 O O 5M NaCl 0 O0 O0 UI O a0 NPU U U U 10 SDS PAGE DH D d 0 But 0000 00 0 0 O O0 O0 O0 O0 O0 O O0 0 O0 O0 O0 UI DO O Ceai O gt GTC GIT GIC GIT GIC GIT 3 Ameiss et al 2006 Vet Immunol Immunopathol 110 25 7 67 J O000000 D DM CpG oligo di nucl eoti def CoG ODN U Oper on IDUUUDUUUUUUUUUUUUUUUIUU 00000000D RI d CpG ODN OO D O D O D D D D 00 U PBS 137nM NaCl 8 1nM Na HPO 2 6 8nM KCI D 1 47nM KH PO D 0000000000 20 C UU LU UU HULBLuL U 4400000000 linemgopoo0000000050 Uu tl lg A duck Hokkai do Vac 1 04 H5N1 OO OO 0000 0 10 TQ D50 UU 0 2m Y 11 D 34 C 72 LV Ll 4coop000000 U 3 000 g0 200 O 0 0 O 0 05 v v 0 0 D O beta propi olactone D000 4 CO 5 Og U HA O Huit PBSI 5 120 HAU m uu light liquid paraffin 65 tetraoleic acid pol yoxye thyl ene 40 sorbitol 1 4 w v sorbitan sesqui oleate 2 096 w VI 0000 0 0000 4L mnpg 8 000r pm 0 5kPJ30 00000000 11 JP 2013 237
8. 48024 AAO BA32 CAM CA20 DNI2 EAMA GALL 40085 AAO3 A438 DN CD62 EEO FF14 44045 BAIO GAO1 DM6 EA31 FA
9. n CpG Ameiss et al 2006 Vet Immunol mmunopat hol 110 257 67 DUUNOODOZZOOUOOOOOZZOOOOOOZZUH O u umgagggggagugauuaguggguggagaamuagumuaguguuecsluct JP 2013 237673 A 2013 MoO Lj ELE EI CI LI ET Rem ngt on s mpany Easton U S Al o O ULUI
10. A D Altstein iBpggNeggguguuugguuuauaduuuuuuuuHlddtd O Altstein et al 2006 Arch Virol 151 921 931 G Le Gal Recueppgoop000oo0oo0o00000000000000000 IO O GG Le Gall Recule et al 2007 Avian Di s 51 490 494 Y Asahi aki popopo0oo000o00oo000000000000000 HA igaggggmuaaagggmguumuadaggBBl vY Asahi Czaki e t al 2006 Mcrobes Infect 8 2706 2714 3 JP 2013 237673 A 2013 11 28 iggggggaaguagumugaguggaugmgaguaaguuiub D oooga O D D 2000 2538760 D D 2003 528570 O0 O 2007 2826360 Bagchi 2008 CMA 178 1415 Wight 2008 N Engl J Med 358 2540 2543 Altstein et al 2006 Arch Virol 151 921 931 G Le Gall Recule et al 2007 Avian Dis 51 490 494 Y Asahi Ozaki et al 2006 Mcrobes Infect 8 2706 2714 Eli asson D G et al Vaccine 2008 26 1243 52 Sylte M J et al Vaccine
11. kanamycin sul f ate 25nvg m 37 C 0 0 O 0 O O O0 O IUL O0 1009 00000 LB O0 UI 0 0 O O 0D ood 0 60 0000 O0 37 cC0 0000000000000000 0000 00000 0000 I PTG 000 1m0 NEO DOO OD DODDOODODOO0X01D0 37 co 00000 15 0009 gd 200 0 D B B D D D D 0 0 O O O0 O0 0 0 0 0 U 20nM Tris HCl pH 8 0 O O O O O O O UI U lysozyrnel 0 0 B 0 4m mD 00000 0 30 D O O O O O0 O0 O0 O0 O0 O O O0 O0 0 1 8 O O U sodi um d eoxychl orate 137 CH 300 000000000000 Polytron 8 000 rpm 20 10 000 g0 300 0000000000 50m 0 5m Ni NTA Agarose Giagen U 0 D D 440 1000000 0 O U Polypropylene Colums 53m 4D 102000 0000000 A 20nM Tri s HCI 0 5M NaCl 20nM in dazol ef pH8 0 0 0000000008 20nM Tris HCI 0 5M NaCl 75nM i m dazol el pH8 OO O O O D D OD D OD D OD O D 2 571 5 OD D D 20nM Tris HCl 0 5M NaCl 250mM in dazol e pHe OD 40 000000000
12. 0 DNA O0 Hirose S et al Proc Natl Acad S gapped dupl e Methods in Enzymol 154 350 367 1987 ILU Dd g 5 g 5 6 JP 2013 237673 A 2013 11 28
13. 000000000000000 oo0ooo0oro0000r0000000 poo0000800000000000000000000E sgo00000000 pnuugugugugggaaagngdgugggugga a mgugggdgdgugonvaug d d uug I an nn uganacainugugggganangugnagnnanuunuuuna NN p000000000000000 po00000 AS JP 2013 237673 A 2013 11 28 10 20 2000 2538760 0 O O a r RE nn nn H H 2003 528570 2007 282636
14. 2007 25 3763 72 Ohba K et al Vaccine 2007 25 4291 300 Roy S et al Vaccine 2007 25 6845 51 Huleatt J W et al Vaccine 2008 26 201 14 V bster R G et al Vaccine 1991 9 303 8 J ll yi nskii P O et al Vaccine 2008 26 2177 85 Zanvit P et al Immunol Lett 2008 115 144 52 Isaka M et al Mcrobiol Immunol 2008 52 55 63 Horthongkham N et al J I mune Based Ther Vaccines 2007 oO poo o0oggg00000000000000 O 0 O O O O0 O0 0 O O O O0 O O O HAG O O NP O umgagggguggggaauaggaguguguuaggulutu O U O7PGQIOOOOOO0O0C0000000000C00D O IUUUULU BU U ceq 0000000000000000 O O Og HOOO0d0ddag D O O poo0000 UU oo O uuu Oo 10 20 30 40 50
15. 673 A 2013 11 28 D gt E z 1 024 HAU m DH DH Hr PECH HN 000 512HA O0O001000 0 5m VE IV a E I D 1000 o osm O O O O O O O O D D Na HCO 1 0 0 198 900000000040 00 wood 960 ELISA 0 O O D WIE 0000000 4c IDO 000000000 0 05 Tween20 137nM NaCl 8 1nM Na HPO 2 6 8nM KCI 1 47nM KH PO O 0000000 1000003 w000 P o LI w UJ n 4 BS T D I DOODOODODODOOOODODO00 37 c0 10000000000 10 O 0000000 PBS T li LU UULHULEULULHULl PBS TI 400000000 UU 1009 0 51 200 200000000000000 u o O 37 C 1 Od UU t iunuggmuuuuHuulg PBS TI q O O I O 12 0000 000 HRP I gG ZYMED UU 37 C 1 LU UL PBS TO 4000000060 Na HPO 14 69 L ci trate monohydrate 10 2g L ortho phenyl enedi ani ne 1g L hydrogen peroxi de 1ni L 1 O00 37 CU 150 ppo000000000000000000 5N sulfuri ABS 492nnl O0 00000000000000 Corona MP 120
16. A iDngggggganagnaggugngwmnugunanauund D OO ooo OO0Oep M0 00FFO00000000O000000000 DELE Pet Pe a DF oa na nu oa na nu oa na nu 40 12 JP 2013 237673 A 2013 11 28 load 1 A FAA X HA UR 1 B KA A NP 175 175 83 0 83 0 62 0 62 0 47 5 47 5 32 5 32 5 25 0 25 0 kDa kDa CBB TAR TOY A CBBRfS VYrx v7uyh LU D de 2008 0 4 8 12 16 192021 0 4 8 12 16 1920 21 VI A 4 ILOOFVARRORAAM DEALT ILIDFVAERMEO RAM ZZ 19384 E NED f HA NP CpGif D Bde 5 HA NPKE D B 10155 PRS Control p 0 05 p lt 0 01 RSR BE E O HE I DU lu 2013237673000001 app 13 74 74 74 74 74 74 72 72 72 72 72 JP 2013 237673 A 2013 11 28
17. JP 2013 237673 A 2013 11 28 19 BEER JP a2 Bd Fe F A 11 Sgt ib BU B 15882013 237673 P2013 237673A UJAME MmM25 11288 2013 11 28 51 Int Cl F 1 ATI 8 A61K 39 145 2006 01 A61K 39 45 ZNA 4BO24 A61K 39 39 2006 01 A61K 39 39 4C085 A61P 31 16 2006 01 A61P 31 16 4HO45 CO7K 14 11 2006 01 CO7K 14 11 C12N 15 09 2006 01 C12N 15 00 A BERK BEROROSS8S OL 13 Si 21 Hees 882013 127162 P2013 127162 71 HERA 511018642 22 HA 3 BE257E6H 18H 2013 6 18 AE RH DIF ABRAS AL 62 FORT 882010 525677 P2010 525677 HE RAAT mity TE 111 DRA TAREA 100102978 RERA 3E ER2 F8H 13A 2009 8 13 FET WK g 31 RABERBS S HER2008 210021 P2008 210021 74 KFEA 100102118 32 tH 3E 2048 A 18H 2008 8 18 FEE FH EX 33 RARER A UP 74 EBA 100160923 FET WA fx CHAI ESAS FR 1 ORB RAR KRA 74 K A 100119507 PURPA SEER MRE RS ESA DES REL ME P RENT DA FE HA FU AA INM IV RAS SERES BERE 74 REA 100142929 GKS 0 EBIRERITACKS bo BR fHI jk KE MILE 1 OR OMSL ZEE 74 RI A 100148699 FEL ER Abt FRAT CEC 64 RAOR Lutte LAR RUS CAM EMT Siva BOLI hb 47 ALAFO DFY DEMRE HE 570000 00000 ODo0O0OOOC0O0O000C000000000000000000000000 oo 0o0o0r0o000000000000000000
18. NCBIC O0 O0 O0 O 0 O0 O O0 O 0 000 U BLAST 7 JP 2013 237673 A 2013 11 28 http www ncbi nl m ni h gov BLAST Altschul S F et al J Mol Bio l 1990 215 3 403 10 Altschul S F Gish W Meth Enzynol 1996 266 46 0 480 Altschul S F et al Nucleic Acids Res 1997 25 3389 3402f 000 p00 00 0 O Advanced BLAST 2 19 90000 0000000000000000000bla stp M M O ExpectO Y 100 Filter O M OFFO M O O Metri xi BLOSUM62 O O O Gap exis tence cost Per residue gap cost 0 O0 O Lambda ratio 0 O0 0 O 110 10 0 850 0 IUUUUIUUUUUDIUUDUUI identityGO00 OO0O000D0COOOD TT Karlin S and S F Altschul 1990 Proc Natl Acad Sci USA 87 2264 68 Ka rlin S and S F Altschul 1993 Proc Natl Acad Sci USA 90 5873 7 0 Dea tik EM CHO J Exp Med 1995 108 94
19. O D OD 1700005280 00 iugggudggpenujnuuuugdtetEu t DOE 00000 5 GCC CTA CGT AGA CCA AAT TTG CAT TGG TTA C 3 5 ATA GIT TAG CGG CCG CTT A TAT TTG GTA AGT TCC 3 D D One Step RNA PCR kit AM TakaRal 0 d O PCRO D D D U cDNA 00000 IDOWUOOUUOCNONWU UNIUNII SnaBl Noth G 0 H 0 D pPPICSKI O O O O Multi Copy Pichia Expression Kit In vi trogeni O D D O O D D O O HI D D D OD D D Pichia pastoris GS115 MIti C opy Pichia Expression Kit Invitrogen UN D O DO DO D O O D O D D OD 00 0 Miti Co py Pichia Expression Ki t I UUUUUUUUI IOHOUUdO UI UNIUNI HA U 000000 O Rabbit Polyclonal anti A vian Influenza A Henagglutinin Novus M O Goat anti Rabbit IgG heavy and light chain cross adsorbed Anti body HRP Conj ugated Bethyl Laboratories O O 0 iunabBmBmuBHbBlellbit dU Dt 0 0 O HAI O 0O O O YPDU 2 gl ucose 1 yeast extract 2 peptone 000 28 C0 p00000000000000 Mai O 1 gl ycerol 0 00004 bi oti ni yeast nitrogen base Invitrogen O0O 0000 0 28c0 000000 OD o oD 1 50 200000000000 161837 D 0 00000000000000 2 0000 gl 50 0 O0 O O0 O0 O0 O0 O0 O0 O0 O O0 0 O0 O0 00 0 0 0 0 BMW U 0 5 methanol 1 yeast extr
20. uuHgBHdgEHtutu NP 000000 SDS PAGE Od Hod UU t U UU t LU UL OO HAT NP Dg E 0000 HAQ NP 0000 UDmUDUDmUImU UDUDUDUUUUUU Dg E IUN UNU UNUI iDnggBgusuuuttllu i nn i Ne DOBR itn ei AjDnuuldl u LU UULHBULEMLIULEBUtLELULl NME NORU Ge HA UU HAD D UUUUNMIUIUUUU ULUI UUUUUULU U 0 0 0 go 50 HAT 0000 iDpnggBguguuuttlulu uu U UULUYBStLuBWulBW4ultu poda n0o0000000 og uu uu H5 H7 HOOdadCddO PO UL Ll uu U JUDUNUL U U IUUUUU LU UL U Ll IO JUDUL O Uu D O O Uu 0000000 HA NP DD MI 10 20 30 40 50 5 JP 2013 237673 A 2013 11 28 z 9 gana HAT 10 20 3
21. w Q O w O Inn 20 ELISAJDODOODOOGOCOOOCOOOOCOO 0000000000190 IDOOODOHDOOONOOGODZONOOOOGOCPEGOONINOIOJONZOLOTG 1 0 D O HA 100ncgH D O NP 100neg CpG ODN 25neg D D O 11 0 DD HA 100nc 90 OO O NP 100neg 0 0 O iii PBSHOOO0037000000 400000000000 CpG ODN Peso OOO 2MO00000000000T00000000000000 IOC007000000000000000000100000 ESA0C0000E IDO CR IDO MEJU D 0 O O O O O0 O O0 O O 0 O O O O O O O O O O O O 0 0 HAI NP 30 CpG ON DOO0O00C000C0000000000000 00 0000 000 po 05 0000
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