重组腺相关病毒(rAAV)介导的人凝血因子IX高活性突变衍生

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(C)316SCIENCEINCHINA(SeriesC)200112(rAAV) B!* **(,200433;!#$%,&’100050;()*+,-./01,200025), R338A ,AAV,rHSV/AAV!#$%&'rAAV-hFIX()!,*+,,-./012B3456789:,;=34?@ABCDEFG1=HIJ@KLMNO78PQ.RQST:(U)7834?VWXYhFIX-R338AHIJ@Z,[\15];(^)HIJhFIX-R338A34_`@DEB(34.2±5.23)%,SabcIdDE((14.27±3.4)%);(e)7834?fWXYgHIJg@Z;(h)fijO78kl@mnFopE!qr.sT:AAV$%bDEHIJhFIX-R338A78VtuvwxcId562B78@yz{v|P@}~.B  Stafford, 338  ,,!#$% &3'[1].()*+,,-./012345678 9:$; ,=?AAV@AB,C,D0*EFAAVGHIJKKrHSV/AAVLMNOGHIJ,PQRSAAV-hFIXR338ATUGH.VW,X0)*RSNOYZNB[\PQ8 ]^A_`a,bcde 9:$;X0f9ZNB ]^gh,ijZNB]^kl*mn op#$% PQ]^Fqr.2001-02-2723,2001-04-202453*67$.(89::39880019);863=?@AB(89::Z20-02-01);/CDE$.;FGHCD$.(89::98024620);FGHIJCDKL$.(89::199925);MNO$.(89::71019)PQRIJSTFUVWAB(89::JRAPOYT)XYZ[**\]^,E-mail:jlxue@fudan.ac.cn524(C)3111.1C57BL/6JB(FIXknockout)UniversityofNorthCarolida  D.W.Stafford,!#$%&B'$(),*+,-[2].rHSV/AAV.&/0123456781 9:11;=?@ABCDE[3].hFIXR338APCRFGHFG1:5I-AC-CAGAAGTCCTGTGAACC-3IFG2:5I-CATCTTCTCCACCAACAACC-3I,JKhFIX;HL2MNO,PQRSB;TUVW,XGYZ201bp.DotblotSouthernblot[\]^_`abcdKAmershamef.WesterndotblotELISAab:ghijklgm3A6nopq68YoshioKar,sghigtuvwx]y)zH{gsIgG2Calbiiochem-BerhingefX|.hFIXR338A}~ab:ST4Bio€,:APTTkit‚FIX;ƒ„dK`DiagnosticaSTAGOef.1.21.2.1hFIXR338A …†AlteredSite‡mMˆ‰23,ŠR338Aˆ‰JAF‹hFIX;.Œ_)zH,Ž,hFIX;HL1015JCˆ‰ZG,L1016JGˆ‰ZC,hFIX;;‘’;HL338J“‘ˆ‰Z”‘,*•–AAV-hFIX2—˜m.™š,›œBHKž,Ÿ ¡#¢žkl,–£+,#¢Hjklž2.1.2.2hFIXR338AhFIX-wtAAV pAAV-hFIXR338ApAAV-hFIX-wt¤¥BHKž¦,10§¨t:RPMI1640©ª.«ž90§¬­®,¯°©ª,±‹rHSV/AAV-RepCap.&/01²,³´µ¶[3]H·`¸¹@T1Hº».¥148hš,¼½ž,3¾¿ÀHÁÂÃÄ,ÅÆ1².™š,2ÇÈ]ÉÊËÌ,PBSÍÎwÏÐ]1,*Ñdotblot·`[4]1ÒÓ.1.2.3!#$%i;VWD.W.StaffordDE,ÔÕLÖ×Ø67YBÙGÚª.BÙGÛ3T,ÜT6Ý.L1TZˆ‰ÞßT,L2TZàáÞßT,L3TZPBSâ´T.㊚äåæçè0.75%BPVC4ÃÄ,™š,éê7.5×1011@T1KëìíJåæçè.Üîïðñ ¸¹òóÎ.1.2.4&'()*vôµ¶[5]H·`,õ†ELISA·`Westernblot[\ö÷`¸¹higøHÎ.1.2.5'hFIX')*ùõ†WesterndotblotBethesda·`ghFIXúgm.WesterndotblotZSnyderûh[6]9üýH·`,þÙ.Bethesda·`[7]:㊄ⴄ&: ,® (37,1h)š,,iH}~.Ñ}~50%Z ¾Bethesdaº}~jJ.)ÔU„|âìHº}~ÒÓ.1.2.6+%,-./01234 ®ÃÄ,FÃ.£ åæ::!::#:$%&'()ûT*+T*,Q¸¹ÄÎ[\T*]Î.6  rAAV!#$%&'(5251.2.7DNA56 ®ÃT*)o,D ;TDNA,¸¹PCRSouthernblotÎ.-H1DNA.´Hirt[8]H·`¸¹Ì/W,H¡T*;TDNAHD .´µ¶[9,10]H·`¸¹.Southernblotõ†GeneImagesRandomPrimeLabelingModuleandCDP-starDetectionModule(Amersham)abc,.´0123·`¸¹.1.2.8789:;=?=@A)*34Þß456H7896:;=w?4® (@A`úBCD®`)5minE™FG¸¹HI,®,ÑAPTT`¸¹i}~.4® FH,³´µ¶[7].i}~õ†ST4Bio€, `APTThFIX}~[2].22.1 B+CDEŠJKOL1R338AAˆ‰HhiM;•–KAAVNOP•,ÑCMVZQÙ•–ÛRSƒ²˜m(T1).OLH†SKUVhFIXR338A#¢®mRNAH+,~.ÙGBW¸¹XYóž2(C2C12,BHK,HeLa)HÌm#¢.™š,Ñ ¾ZH[2œ@TAAVY¡ÒÓº»H/01º»23\\rHSV/AAV.&/01232œº»rAAV-hFIXR338A@T1.Ñdotblot·`,1ÒÓ,9]H@T1ÒÓZ1.25×1012.2.2hFIXR338AF9:GHIåæ^_éêC57BL/6jB7.5×1011rAAV-hFIXR338A@T1`²çèÞߚ,Üîõ,ÑELISA·`¸¹igøHÎ(T2).T2[a,iˆ‰báG[SrAAV-hFIXR338AÞßmO½¡cd#¢,ef15îÑÔ.gL3îhiigøH#¢¢j¡k,kl¢(445.57±24.34)ng/mL.5îš„iLmn,opqe ,cd(100ng/mL),ì†rAAV-hFIX-wtÞßHmO#¢rs t.,uTÞßSL3L10î®H#¢cdùZ:hFIXR338A:(445.57±24.34)(202.92±20.29)ng/mL.hFIX-wt:(426.37±27.95)(198.45±28.28)ng/mL.uTv wNx~yz(P0.05).D{hiˆ‰báGàá$;|[@TAAV2œ,SÞßåæT*#¢,*}ÅƋ,¢j ,H„~Ó.®,€âÞß„|¸¹Xhiˆ‰báGàá$XGHWesternblotÎ,P‚N{,SÞß„|[jƒZ56kuH„…†J,›‡jˆ‘‰Š‹Œš¸¹[\ö÷ÎZhi„…(T3).2.3./01234ZhiH#¢GEçèíJå杞,€âÞßçèíåæT*:!T*¸¹X[\T*],P‚N{,SçèíJ削T* Ž[j1pAAV-hFIXR338AITRAAV,CMV,In1,hFIXR338A338!#$%&#$,P(A)’40()*+$526(C)31hi„…HS, :!T*ÑçèPBSâ´TòT*‘’,“^”P„…H#¢,D{iH#¢GEçèíJå杞.2 hFIXR338A ()2.4DNA34 Þßåæ:!T*,D ;TDNA,¸¹hi;HPCRSouthernblotÎ,P‚N{,åæT*;TDNA[•U4hiDNA†J,*Southernblot4hi–z~DNA—. :!T*PBSÞßâ´TëìT*—whiDNA†J4”(T4),D{AAV˜m2œ,M˜~hiˆ‰mbáG;[›œ¸‹™mž,*Ñ óëâ+,Hš›SKÞßåæT*žœ.2.58JKL N{,éêrAAV-hFIXR338A@T1Þߚ,Þß456]j.žï4® ŸN ¡,ÞßWYK30min ¡g5¢12minhi((7.5±3.56)min),;oC£((3.17±1.11)min);5minF¤Nx¥¦,ÞßWH(428.33±51.69)µL¥¦j(48.33±15.46)µL,íKÞßL3î®,žï4® :Fëì4();o¢j'C£.APTT® §ŸN ¡,¨i}~D©ŸN,ªªÊw ˜m92œHàá$hi;ÞßT99¢jH}~cd,ƒ¡42.4«(P0.01)(#1).3!#$%Westernblot&'(1,-./0;21234;3hFIXR338A567-890;4wt-hFIX567-890:6  rAAV!#$%&'(5272.678MN OP'CQRS)*ÑWesterndotblotÑBethe-sda·`ÞßmOgiúgmHSrs,Ñ=wÄ,Q?T*:‘’Hš:‰.P‚N{:(¬)SéêrAAV-hFIXR-338A@TAAV1Þߚ,Þßt|We

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