LV10506B - Method for preparation of polypeptides - Google Patents

Abstract

The new peptides with partial or complete primary structure and one or many animal erythropoietin (ERO) biological properties are described. These peptides are a product of exogenous DNA sequence expression in the prokaryote or eukaryote host cells. Self-replicating plasmids or virus vectors are introduced into the DNA genome, kDNA and sequences of constructed DNA, which partially or completely code amino acid residual sequence in ERO or its analogues. Bacteria, yeast or vertebrate cells are applied as the host cells. Human or simian ERO, separated from a culture cellular lysate or DNA sequence expression product fragments, displays, for example, immunological properties and biological activity in vitro and in vivo. Chemically synthesized polypeptides with biological and immunological ERO properties are also described. Complete methods of specific single-stranded polynucleotide verification in the heterological cell or virus sample obtained from e.g. DNA of plasmids, kDNA of a virus-carrier or DNA genome library are described.

Description

LV 10506

CnOCOB ΠΟΠΥΗΕΗΜΑ ΠΟΠΗΠΕΠΤΜΑΟΒ OnncaHue flaHHoe ηβπηθτοη HacTMHHbiM προηοπχβΗΜθΜ Haxoflamnxca b npoqecce o / ļHOBpeMeHHoro paccMOTpeHna 3anBOK Ha naieHT CLUA NN561024, nonaHHOM 13 fleKaSpa 1983 ROAA, 582,185, noflaHHoii 21 cfieBpanfl 1984 Rofl, m 655 841, noflaHHoii 28 ceHTadpa 1984 Rhone. nPEOnOCbinKM M3Q5PETEHMfl

HacToamee Μ3ο6ρθτθημθ othocmtch κ MaHMnynaunM γθηθτκηθοκμμμ MaTepManaMM Μ, B HaCTHOCTM, K peKOM6MHaHTHblM ΜΘΤ0 / 1ΜΚ3Μ, Π03Β0ΠΠΙ0ΙΑΚΜ nonyHMTb ΠΟΠΜΠΘΠΤΜΑΝ, oānaflaioLUMe ηβοτμηηο μπμ nonHOCTbio nepBMHHOii CTpyKTypHoū κοΗφορΜβρΜθΜ μ / μπμ oahmm μπμ 6-one 6nonornHecKMx M3, CBoiicTB cboSoaho BCTpeHatomerocn epMTponoaTMHa.

A. MaHHnvnaLiHH reHenmecKUMU MaiepnanaMU Γθηθτμηθοκμθ MaTepnanbi μοχηο b tunpoKOM cMbīcne onpeflemiTb XAX τθ χμμμηθοκμθ BeuļecTBa, KOTopbie nporpaMMnpyioT m ynpaBrmioT npon3BOflCTBOM κομποηθητοβ κπθτοκ μ M BMpyCOB HanpaBnaroT OTBeTbl ΚΠΘΤΟΚ μ BHpyCOB. ΠπΜΗΗΟΚΘΠΗΟΘ ΠΟΠΜΜβρΗΟΘ BemeCTBO, μ3βθοτηοθ κθκ Ae30KCMpn6oHyKneMHOBan KucnoTa (flHK) coflepxnT ΓβΗβτΜΗθοκΜϋ MaTepnan Bcex xnBbix κπθτοκ m BnpycoB, 3a μοκπιοηθημθμ Ηβκοτορωχ BMpyCOB, nporpaMMnpyeMbix pn6oHyKnenHOBOPi κΜοηοτοίί (PHK). CTpyKTypHbiMn 3BeHbHMM b flHK-nonnMepax πβππιοτοπ 2

HeTbīpe pa3nMHHbix HyKneoTiifla, KajKflbiii 113 KOTopbix coctomt M3 nypnHa (afleHMH m ryaHMH) ink Ti3 ntiptiMtiflMHa (TtiMtiH ie utiT03iiH) (CBn3aHHbix b fle30KCtipti6030-caxap0M, K κοτοροΜγ nptiCoefltiHeHa cjjoccļiaTHafl rpynna. RipiicoeflMHeHiie HyKneoTnpoB b φορΜβ ημηθμηογο nonuMepa προοχοριιτ nyrēM cnunHiin 5 '^ oc0aTa οαηογο HyKneoMfla c 3'-niflpoKciinbHoii rpynnoti npyroro HyxneoTtifla. OyHKutioHanbHan flHK BCTpenaeTcn b φορΜβ ycrroiiHiiBbix HByHtiTeBbtx cooSujecTB οαιιηοηηηχ HtiTeii HyKneoTtiflOB (ti3BecTHbix Kax fle30KCtioniiro-HyKneoTtiAbi) HBN coočtuecTBa o6pa3ytoTcn nyrēM BOflopoflHoii cbasii Mexny nyptiHOBbiMti m ntiptiMtifltiHOBbiMti ocHOBaHtinMii, Te " KOMnneMeHTapHbie "coočmecTBa, cymecTBytomtie tinu Mexfly afleHtiHOM Ifi Team Team TI, ink Mex < Hy ryaHtiHOM / 17 μ miito31ihom / 14 / /ρΠητο ynoMMHaTb HyKneoTtiflbi x ocHOBaHtiti, a KOMnneMeHTapHbie coočinecTBa HyicneoTiiflOB b AByHtueBOM flHK (τ.θ. A-T μ Γ-14) ynoMMHaTb Kax " shortcut a30TticTbix ocHOBamiii ". Pti6HyKnenHOBan KncnTa npeflciaBnneT co6on nonnHyKJieoTtiflp coflepxaiqiii afleHtiH, ryaHtiH, miO3iHiHiHi (Y), CKopee, μθμ tmmmh, CBfl3aHHbit c pti6o30M tin φοοφ3ΤΗθΐί rpynnoii.

Kopone roBopn, nporpaMMtipyHDmafl φyHK ^ l1fl flHK ocymecTBnneTcn rnaBHbiM 0Čpa30M nocpeflCTBOM npouecca, β κοτοροΜ οπβψιφιΐΗβοκΜβ nocneflOBaTenbHOCTti HyKneoTtiflOB flHK (reHbi) "TpaHCKpn6npyK > TCfl" b OTHoctiTenbHO HeycToiimiBbie nontiMepbi ΜΗφορΜθφιοΗΗοίί PHK (nPHK). B CBOto onepeflb, ιιΡΗΚ cnyxoiT b KanecTBe MaTptiBlue flnn o6pa30BaHtin CTpyicTypHbix, perynnTopHbix's KaTannTMHecKtix δβηκοΒ m θμιιηοκμοποτ. 3tot προμβοο "TpaHcnniJ | wi" mPHK BKniOHaeT φyHK ^ MOHMpoBaHMe μθπκμχ ημτθμ PHK (τΡΗΚ) KOTopbie TpaHcnopTnpyioT m BbipaBHMBatoT tiHAMBiiflyanbHbie aMUHOKncnoTbi Bflonb ηιιτθμ mPHK, pa3peujan o6pa30BaHne HyKneoTMflOB b Haflnexauļnx nocneflOBaTenbHOcmx aMMHOKMcnoT. “ΉηΦορμθαμη” mPHK, iicxoflflman H K η 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 op op n op op op op op op op op op op op ue ue ue af af af af af af af af af af af af af af af af af af af af af af af af af af nonunenTiifla, HaxoflMTcn β φορΜθ TpnnneTHbix "kooohob" - nocneflOBaTenbHbix rpynnnpoBaHHM Tpex HyKneoTiiflHbix οοηοβθημμ. 3 μ3βθοτηομ CMbice, o6pa30BaHtie 6enKa ηΒπηβτση OKOHHaTenbHon φορΜοίί ”3Kcnpeccnn” nporpaMMtipoBaHHoPi γθηθτμηθοκομ μηΦορμθι4μιι, ο6θοπθμμβ3θμομ HyKneoTHflHon nocne / leobaTenbHOCTbio reHa. "TlpoMOTopHbie" nocneflOBaTenbHOCTM flHK o6biHHO "npeflUjecTByioT" 'reHy b flHK-nontiMepe ti o6ecneHtiBaioT ynacTOK nnn μημρμμροβθημα TpaHCKpyntin b PHK. ”PerynflTopHbie” nocneflOBaTenbHocTH flHK o6dihho τθιοκθ ”pacnono > KeHbi BbiLue of ϊθηθημιο " (τ.θ. npe / lyiijecTByioT) reHa β αθηηομ flHK-noniiMepe, CBn3bBan δβπκιι, onpeflenniomie nacTOTy (ink cKopocTb) TpaHCKptinuiOHanbHoro μημαμμροβθημα. ΚοππβκτΜΒΗΟ ynOMHHaeMbie xax ”npOMOTOpHan / perynnTopHan ink” ynpaBnnKDman " nocneflOBaTenbHOCTb flHK, θτμ nocneflOBaTenbHocTti, KOTopbie npeflmecTByiOT ie OTo6paHHOMy reHy (Tinu pnfly γθηοβ) b ^ φyHκ ι10HaπbH0M flHK-nontiMepe, cnocočcTByeT onpeneneHiiio τογο, προΜ3θΜηβτ either TpaHCKptinqtin Μ, b Kohll kohaob, aKcnpecctin Reha. riocneAOBaTenbHOCTii flHK, KOTopbie " conpoBO) Kflai " reH b flHK-nontiMepe 11 oSecnentiBaiOT noflasy ctimana nnn OKOHHaHtin TpaHCKptinMiiii β 11PHK, ytMtHatTcn Kax " TepMtiHaTOpHbie " 3 EN 10506 B $ OKyce μμκρο6μοπογμηθοκομ τθχηοπογμμ nocneflHero αθοητιιπθτμη ηθχοαμτοπ nonbīTKa Μ3γοτοβπθημρ MHflycTpnanbHO m 0apMaLļeBTMHecKM BaxHbix BetiļecTB c ncnonb30BaHneM MMKpoopraHM3MOB, KOTopbie μπμ nepBOHananbHO Ηβ ΜΜΘΙΟΤ ΓβΗΘΤΜΗβΟΚΜ KOflMpOBaHHOM ΜΗφθρΜ3Ι4ΜΜ, OTHOCfllfleMCH K XenaeMOMy npOflyKTy, BKniOHeHHOMy B MX flHK, μπμ (b cnynae c MneKonmaioiuMMu κπθτιομμ b KynbType ) ηθ BbipaxaioT o6bNHbiM nyT§M xpOMOCOMHbiM γθη Ha 33μθτηομ ypoBHe. npoine ΓΟΒορπ, γθη, ycTaHaBnMBatoiflMM CTpyicrypy xenaeMoro nonunenTMflHoro npoflyKTa, μπμ n3onnpoBaH ot 'αοηορηογο "MHKpoopraHM3Ma, μπμ ΧΜΜΜΜΘΟΚΜ CMHTe3MpOBaH Μ 3ΘΤΘΜ yCTOMMMBO MHTpOflyflMpOBaH B flpyrOM ΜΜΙφ00ρΓ3ΗΜ3Μ, npeflnOMTMTenbHO, CaMOpennMUMpyiOU4Miica OflHOKHeTOHHblM MMKpOOpraHH3M, TaKOpi CEC čaKrepuM, flpoaoKM μπμ MneKonmatoinne κπθτκμ b KynbType. CyiuecTByiomee μθαιμηηοθ o6opyflOBaHne ansR aKcnpeccMM γθηοβ b "TpaHC0opMnpoBaHHbix" μπμ "TpaHC0eKTnpoBaHHbix" MMKpo6Hbix κπθτκθχ xo3BMHa paGoTaeT ηππ co3flaHMn xenaeMoro npoflyicra c ncnonb30BaHneM 3Κ30γθηηομ flHK b «anecTBe MaTpuLļbi flna TpaHCKpnriL | MM mPHK, KOTopan 3aTeM TpaHcnMpyeTcn b HenpepbiBHyio nocneflOBaienbHocTb aMMHOKMcnoTHbix ociaTKOB. flaHHan oOnacTb τθχημκμ npeflCTaeneHa lUMpoico b naieHTHbix m nnTepaTypHbix ny6nMKaqMnx, οτηοοπιαμχοπ κ μθτοαοπογμπμ "ρθκομ6μη3ητηοΡι PHK" αππ M3onMpoBaHMH, CHHTe3a, 04MCTKM Μ 3ΜΠΠΜφΜΚ3ΑΜΜ ΓΘΗβΤΜΗβΟΚΜΧ ΜΘΤθρΜΘΠΟΒ, MCnonb3yeMblX ΠρΜ τρ3ΗθφορΜ3ΑΜΜ OTo6paHHbix MMKpoopraHM30B xo3AHHa. naTeHTHan rpaMOTa CIUA N4237224, BbipaHHaa Cohen et al., Further preferably, othocmtcb κτρθΗΟφορΜΒΑΜΜ οαηοκπθτοηημχ MMKp00praHM3M0B X03flMHa C "rM6pMflHOM" BMpyCHOM ΜΠΜ KOnbfleBOM nna3MMflH0M flHK, BKAK5HaiOLneii OTOčpaHHbie nocneflOBaTenbHOCTM θιοογθηηομ flHK. Μθτοαμκμ β πατθητθ Cohen et al., SnepBbie βκπκ > η3ιοτ M3roTOBneHMe eetcropa τρ3ΗφορΜ3ΐ1ΜΜ nocpeflCTBOM φβρΜβΗΤ3ΤΜΒΗ0 oTinennniomeMcn BtipycHOii μπμ KOnbfleBOM nna3MMflHOM flHK flnn o6pa30BaHnn ημτθμ πμηθμηομ flHK. OTočpaHHbie MHopoflHbie (> i3K3oreHHbie " μπμ " reTeponorunecKue ") hmtm flHK, o6bNHO BK / nonatomne nocneflOBaTenbHOCTM, KOflMpytoiflMe xenaTenbHbiM npoflyKT, nonyHatOT b πμηθμηομ φορΜβ nyTēM Mcnonb30BaHMn cxoflHbix φβρΜβΗΤΟΒ. Πμηθμη3π BMpycHan ΜΠΜ nna3MMflHan flHK BbipaiiļMBaioTcn c mhopoahom flHK b npMcyrcTBMM cujMBatomMX φβρΜβΗΤΟΒ, cnoco6Hbix ocymecTBn "Tb npoflecc BoccTaHOBneHMB, a" rn6pMflHbie "BeKTOpbi o6pa3yfOTcn coflepxaiiļMMM OTočpaHHbiPi cerMeHT θιοογθηηομ flHK, " cnneTeHHbiM" b nna3MMfly BMpycHoii μπμ KonbfleBoii flHK. ΤρβΗΟφορΜΘΑΜΒ COBMeCTMMblX OflHOKneTOHHblX ΜΜΙφ00ρΓ3ΗΜ3Μ0Β X03flMHa C TM6pMflHblM ΒβΚΤΟρΟΜ 33Κ3ΗΗΜΒ3βΤ0Π 06pa30BaHMeM MHOFOKpaTHblX ΚΟΠΜΜ 3Κ30ΓβΗΗθίί flHK B nonynniļMM κηβτοκ xo3HMHa. Β ΗβκοτορΝχ cnynanx xenaeMbiM pe3ynbTaT0M πβππθτοπ npocTO βΜΠΠΜφΜκβΜΜη MHopoflHOM flHK, m BbipaujeHHbiM " npoflyKT '' npeflCTaBnneT co6om flHK. Bone NACTIS uenbK) τρ3ΗΰφορΜ3ΐ | ΜΜ flBnneTcn 3KcnpeccMn, ocymecTBnneMan πρΜ πομοιμμ κπβτοκ X03nnHa 3K3oreHHoPi flHK β φορΜβ KpynHOMacujTa6Horo cMHTe3a BbiflenneMbix κοπμηθοτβ npoMbiujneHHo BaxHbix Φρθγμθητοβ 6enxa μπμ nonMnenTMfla, KOflMpyeMbix μηοροαηομ flHK. Okeμοτρμτθ Taioke, HanpMMep, πθτθητη CIUA NN 4264731 (BbiflaHHbiPi Shine), 4273875 (BbiflaHHbiPi Manis), 4293652 (BbiflaHHbiM Cohen) m. 4

Pa3BHTne οπθΜΜφΜΗβοκΜχ nocneflOBaTenbHOCTen flHKflnn οππθτθηηη β seicropbi flHK flocTuraeTCfl πγτΘΜ πρηΜβΗβΗηη penoro pana τθχηηηθοκηχ npneMOB, 3aBHcnmnx b 3HawMTenbHOM mepe οτ οτθπθημ " nHopoflHOCTM '" poHopa "of OTHOLLieHnKD κ npoeKTnpyeMOMy xo3AHHy, a Tao < e οτ pa3Mepa nonnnenTupa, BbipaxaeMoro β χο33μηθ. Onacancb Hpe3MepHoro ynpomeHnn, μοχηο yTOBepx < flaTb, hto cymecTByioT τρη οοηοβηνχ anbTepHaTHBHbix cnocoča: / 1 / " n3onnunn ”flByHHTeBon flHK-nocneflOBaTenbHocTn οτ γθηομηομ flHKflOHOpa; / 2 / χμμμηθοκοθ nonyMeHne flHK-nocneflOBaTenbHOCTn, co3flaioineH KOA flnn μητθρθοηογο nonnnenTnfla n / 3 / 0ΜΗΤΘ3 in vitro pByHHTeBon flHK-nocneflOBaTenbHocTn nocpeflCTBOM φβρΜβΗΤβτηΒΗοη " očpaTHon TpaHCKpunpmi "ηΡΗΚ, βμηθπθηηομ H3 ηοΗοροκιιχ κπβτοκ. BbisetynoMHHyTbie cnocočbi, KOTopbie βκπιοηθιοτ o6pa30BaHne flHK- “COMMAND” η PHK, OchbiHHO ynoMMHaioT xax “KflHK”. nonyneHne nocneflOBaTenbHOCTen flHK nacTO nNNtcn μθτοαομ BbiSopa, Corfla M3BecTHa nonHan nocneflOBaTenbHOCTb aHHOKncnoTHbix ocraTKOB acenaeMoro nonnnenTnfla. Μθτοαμκμ nonyHeHM "flHK, onncaHHbie β οαηοβρθμθηηο paccMaTpnBaeMon 3anBKe Ha πθτθητ Cujas N483451, Alton et al, noflaHHon 15 anpena 1983 ropa, η οοοτβθτοτβθηηο 3a" BKE PCT US 83/0065, ony6nw < OBaHHOM 24 Hončpn 1983 ropa nofl HOMepoM W 083/04053) , Further preferably, C03flat0T cpeflCTBa απα αοοτμχθημη τακηχ Hpe3BbmanH0 xenaHHbix pe3ynbTaTOB, XAX: npeflycMOTpeHne HannHnn wepeflyioiunxcfl koaob, očbNHO BCTpeHa »OLnnxcfl b MHKpoopraHH3Me xo3nnHa, OTo6paHHan flnn SKcnpeccnn (Further preferably, npeqycMOTpeHne" npeflnoHTnTenbHbix "KOflOHOB ΑΡΟΧΟΚΘΜ MflH E.COlfl Μ36θΧ" ΗΜΘ npncyTCTBHfl HeTpaHcnnpoBāHHbix "l1HpOHHblX "nocneflOBaTenbHOCTen (oSbiHHO Haxoflnmnxcn b MneKonmaiomnx γθηομηνχ flHK-nocneflOBaTenbHOCTflx n nx nPHK-MaTpnpax) KOTopbie ηθ 6θ3 Tpypa o6pa6aTbiBatoTcn npoKapnoTHbiMn κπθτιομη xo3anHa; n36ex < aHne He > xenaTenbHbix " Beflymnx" nonnnenTnflHbix nocneflOBaTenbHOCTeii, o6bi4HO KOflnpyeMb ix γθηομημμη flHK n KflHK-nocneflOBaTenbHOCTflMn, OflHaKO, nacTO He nerxo OTmennneMbix οτ nHTepecHoro nonnneninfla npn noMomn 6aKTepnanbHbix nnn flpoxoKeBbix κηβτοκ xo3nnHa; npeflycMOTpeHne πβτκοη BCTaBKn flHK β noflxoflnmne aKcnpeccnBHbie Beicropbi β coo6mecTBax c xenaeMbiMn npoMOTopHbiMn / perynnTopHbiMn n TepMnHaTOpHbiMn nocneflOBaTenbHOCTflMn; n npeflycMOTpeHne πθγκογο ποοτροβΗηη γθηοβ, KOflnpyiomnx nonnnenTnflHbie φρ3ΓΜβΗ ^ ι n aHanorn > xenaTenbHbix nonnnenTnflOB.

Korfla nonHan nocneflOBaTenbHocTb aMnHOKncnoTHbix οοτ3τκοβ xenaeMoro nonnnenTnfla He H3BecTHa, HenocpeflCTBeHHoe nonyneHne nocneflOBaTenbHOCTeii flHK ηθβο3μοχηο n n3onnpOBaHne nocneflOBaTenbHOCTeii flHK, KOflnpyioinnx nonnnenTnflbi npn noMomn KflHK-cnoco6a, cTaHOBHTcn μθτοαομ BbiSopa, HecMOTpn Ha B03M0 > KHbie HeflOCTaTKn b nerKocTn οβορκη Ββκτοροθ aKcnpeccnn, cnoco6Hbix o6ecnennTb BbicoKne ypoBHn ΜηκροβΗοη 3KcnpeccnBH0CTn , ynoM «HyTon Bbitue. Cpefln CTaHflapTHbix MeTOflnKflnn n3onnpoBaHnn KflHK-nocneflOBaTenbHOCTen HaxoflHTcn nonyneHne nna3MnflOHecyiqnx KflHK- "6n6nnoTeK" nponcxoflnmnx οτ oāpaTHon TpaHCKpnnpnn nPHK, NACTIS BCTpenaKDinencn β ΑΟΗοροκηχ KneTKax, OTOčpaHHbix b KanecTBe OTBeTCTBeHHbix 3a SKcnpeccnio γθηοβ BbicoKoro ypoBHn (HanpnMep, δηδπηοτβκη "flHK, nponcxoflnmne οτ ΓΗΠ0φΗ38ρΗΝχ κηβτοκ, KOTopbie Bbipa > xaioT OTHOcnTenbHO 5 EN 10506 KO KO KO n n n n n n n n n n n n n n n n n n n n n n n n Γββ M3BeCTHbl 3HaHMTenbHbie HaCTM aMMHOKMcnoTHoii nocneflOBaTenbHocTM ποπμπθπτμαοβ, it MeHeHbie, 30Hfl0Bbie nocneflOBaienbHoc ™ οαηοημτθβομ flHK, flynnnunpyioineM nocneflOBaTenbHocTb, npepnono) KMTenbHO HaxoflflinyKxa b " nnaHOBoii "KflHK, mojkho ncnonb30BaTb b ΜΘΤορκκβχ flHK / flHK-m6pnflM3aLimi, npOBOflHMbix b KTiOHanbHbix κοπιιηχ KflHK, KOTOpan 6bina fleHaiypnpoBaHa b oflHOHMTeByto φopMy. [CMOTpme, rnaBHbiM o6pa30M, pacKpbiTiie n o6cyxfleHMFi flaHHOM očnacTM τθχημκμ, npeflCTaBneHHbie b naieHTe CIUA 4,394,443, BbiflaHHOM Ha mmh Weissman et al., N nocneflHne noKa3bi ncnonb30BaHMH jqnnHHbix 30hpob onnrc > HyKneoTMflHOM rnOpupnsaMnn, coočmeHHbte b pa6oiax VVallace et al., Nuc. Acids Res., 6. CTp 3543-3557 (1979), Reyes et al. P.N.A.S. (U.S.A.), 79, CTp 322-3274 (1982), by Jaye et al. Nuc Acids Res, 11, CTp 2325-2335 (1983). CMOTpme TaioKe πθτθημτ CLLIA N4358535, BbiflaHHbiii Ha MMjl Falkow et al., Kacaiomiiiicii Μβτοριικ flHK / flHK-rn6pn1M3aMMM npw προΒβββΗΜΜ flMarHOCTMKM; ony6rMKOBaHHbie EBponeCiMeNHHbie 3aaBKM N0070685 m N0070687, OTHOCHinnecH κ CBeioncnycKaioutMM MencaM Ha 0flH0HmeBbix nonHHYKJieoTMflHbix 30Hflax; p6oTy Dāvis et al. " Manual for Genetic Engineering, Advanced Bacterial Genetics ", Cold Spring Harbor Laboratory, Cold Spring Harbor. Ν.Υ. (1980) Ha στρ.55-58 m 174-176, KacaioinyK) ca MeTOflMK rn6pMflM3aunM κοποΗΜΜ μ θΠΜββΜΜΗβοκιιχ 3a6oneBaHnii; m New England Nuclear (Boston, Mass) Spoiuopbi from " Gene Screen " Hybridization Transfer Membrane Materials, NaHyHoPHOBOflCTBa nna nepeHoca m rn6pMflH3aunM flHK w PHK. KaTanor N EF-972]

Cpejqn Han6onee 3HannTenbHbix nocneflHHx αοοτμχθημμ b MeTOfļHKax rn6pnflH3aunn PNA SKpaHMpoBaHMH peKOM6MHaHTHbix κποηοβ aBnaeTca ncnonb30BaHne MeneHbix CMeiuaHHbix OnnrOHyKneOTMflHblX 30Hfl0B, KajKflblii M3 KOTOpblX HBnaeTCH nOHHblM ΚΟΜΠΠβΜβΗΤΟΜ οπβμΜφΜΗβοκοίί nocneflOBaTenbHocTM flHK b BbičopKe rn6pnflM3aL (MM, BKmoHaromeii reTeporeHHyio mixture of οαηοημτθβνχ flHK m PHK. 3τμ μθτοημκμ npM3HatOTca οοο6θηηο none3HbiMM NPM BbineneHHH KflHK -κποΗΟΒ, nonyHaeMbix H3 mctohhmkob, κοτορΗβ co3flaioT KpaPrne Manbij KonnnecTBa Nocni / ļOBaTenbHocTePi κΡΗΚηππ npeflCTaBnaioLuero HHTepec nominenTHfla. Kopone roBOpa, ncnonb30BaHne CTpornx ycnoBnPi rn6pnflM3ai4mi, HanpaBneHHbix Ha M36exaHne ηθοπμμμΦμμθοκογο CBH3biBaHMH, μοχθτ no3Bonmb, Further preferably, 3ΒΤ0ρ3ΑΜ0Γρ3φΜΗβ0ΚνΚ) BH3yanH3amiK > ΟΠβΙ4ΜφΜΜβΟΚΟΓΟ KJOHOH KflHK Ha MCXOfle m6pMflM3aMM nnaHOBoPi flHK TOMy eflMHCTBeHHOMy 30Hfly b CMecii, KOTopbii ηβπηθτοη ee nonHbiM ΚΟΜΠΠβΜβΗΤΟΜ. CMOTpiI, rnaBHbiM6pa30M, Wallace et al., Nuc.Acids Res., 9, στρ.879-897 (1981); Suggs et al, P.N.A.S. (USA), CTp61313-6617 (1981); Choo et al., 299, στρ.178-180 (1982); Kurachi et al, P.N.A.S. (USA), 79, crrp.6461-6464 (1982); Ohkubo et al, P.N.A.S. (USA), 80, CTp1919-2200; Komblihtt et al, P.N.A.S. (USA), 80, CTp 322-3222 (1983). Boo6me, MeTOflMKM CMeuiaHHbix 30haob b paOoTe Wallace et al (1981) supra 6binn pacmnpeHbi pa3nnHHbiMM pa3pa6oTHMKaMM flo μομθητθ, Korfla coo6manocb o nonyneHMn nocTOBepHbix pe3ynbTaTOB b M3onnpoBaHMM κΑΗΚ-κποηοβ c ncnonb30BaHneM 32-ηπθηηογο CMeujaHHoro "Nynas" onnroHyKneoTMflHbix 30haob nmiHoPi b 16 ocHOBaHHH (16- Mer) paBHOMepHO M3MeHHiomMxcfl nocneflOBaTenbHOCTiIt BMecte c OflMHOHHbiM ΙΙ-μθγαππ βμποπηθημπ 6 flByxno3m4HOHHoro " CMOTpme pa6oTy Singer-Sam et al, P.N.A.S. (USA), 80, στρ.802-806 (1983).

Hcn0nb30BaHMe Μ30ΠΗΤ0Β ΓβΗΟΜΗΟΜ flHK ΗΒΠΗβΤΟΗ ΗΘΜΜβΗββ paCnpOCTpaHeHHblM M3 Tpex BbiuueinMaHyTbix cnococob pa3BMTna οπθμμΦμηθοκμχ nocneflOBaTenbHocieP απα MCn0nb30BaHMa B pe OM OM6HHaHTHblX MeTOflMKaX. 3t0 OCOČeHHO ΒβρΗΟ B oOnaCTM peKOM6nHaHTHbix μθτορμκ, HanpaBneHHbix Ha oSecneneHMe mmkpočhom aKcnpeccnBHOCTM nonunenTMflOB μπθκοπμτ3Κ) ιι1μχ, m o6ycnoBneHO, maBHbiM o6pa30M, CNO) KHocTbto MneKonMTatomePi γθηομηομ JļHK. Τθκμμ o6pa30M, χοτα m cymecTByioT HaflexHbie μθτοαηκμ απα P33bmtma cf) ar-Hecymnx 6μ6πμοτθκ γθηομηομ flHK Going to the end of the bmaob MneKonMTaiomMXCA [Ομοτρμτθ, HanpMMep, par6oi Lawn et al,, 15, dp. 1157-1174 (1978), OTHOcamyioca κ μθτοαμιομ απα reHepMpoBaHMA γθηομηομ 6μ6πμοτθκμ nenoBeKa, OchbiHHO ynoMMHaeMoti xax " 5μ6πμοτθι «Maniatis "; pa6oTy Kam et al, P.N.A.S. (USA), 77, c.5172-5176 (1980), KacaK < t > myK) Cfl < / RTI > pa6oTy BLATTNER et al, Science, 196 * crp.161-169 (1977) onncbiBaioinyfO ποοτροβΗΜβ SbiHbeii γθηομηομ βΜδπΜΟτβΚΜ] npeAnpMHATO HecKonbKo OTHocMTenbHO ycneuiHbix nonbīTOK Mcnorib30BaHMA μθτοαμκ rM6pMflM3aqnn b Μ30ΠΜρ0Β3ΗΜΜ ΓΘΗΟΜΗΟΜ flHK ΠρΜ OTCyTCTBMM Flaneks MflyilļerO npeflBMfleHMA aMMHOKMCnOTHblX μπμ flHK-nocneflOBaienbHOCTeM. B KanecTBe oflHoro npMMepa μοχηο npMBecTM FoDes et al, I. Mol. and App. Genetics. 1 crp.3-18 (1981), RPE coo6uj | AETC 06 ycneujHOM Μ30ΠΑ14ΜΜ REHAU, KOflnpyiomero af ^ a-cy6-beAMHMiiy ΓMΠoφM3apHblx γπμκοπροτθμαημχ γορμοηοβ nenoBeKa M3 ΒΜβηΜοτβκΜ Maniatis nyreM Mcnonb30BaHMA " npeflenbHO απμηηογο " 30Hfia, BK / iKDHaiomero ποπημμ Φρθγμθητ 621 napb a30TMCTbix ocHOBaHMM npeflBapMTenbHO M3onnpoBaHHOM KflHK-nocneAOBaienbHOCTM απα 3ΠΒφ3-ον6τ > θΑΜΗΜΜΒΐ. B «asecTBe flpyroro npMMepa, b pačOTe Das et al, P.N.A.S. (USA), 80, στρ. M, HaKOHeij, b6 and Anderson et al., P.N.A.S. (USA), οτρ. 6838-6842 (1983) coo6maeTCA 06 Μ30ΠΑΑμμ γθηομηογο κποη3 απα 6biwbero naHKpeaTMHecicoro τρμπομηοβογο MHrM6mopa (BPTI) c Mcnonb30BaHMeM OflMHOHHOrO 30Hfla, ΜΜβΙΟΙΑβΓΟ 86 nap a30TMCTblX OCHOBaHMM B flnMHy Μ ΠΟΟΤρΟβΗΗΟΓΟ B COOTBeTCTBMM C Μ3ΒβΟΤΗΟΜ 3ΜΜΗΟΚΜΟΠΟΤΗΟΜ nOCneflOBaTenbHOCTbKD BPTI. ABTOpbl OTMenaiOT onpeneneHMe πποχμχ nepcneKTMB απα μ3οπμροβ3ημα mPHK, npuroflHOM απα cnHTe3a KflHK-6M6nMOTeKM, BBMAy οηθβμαηβιχ hm3kmx ypoBHeii mPHK b nepBOHaHa / ibHO Hai4eneHHbix MCTOHHMKax OKonoyujHoPi > xene3bi μ τκθηθμ nerxoro. BbipaxaA 3aieM HaAex < Both Ha ycnex b 30HAMp0BaHMM ΓβΗΟΜΗΟΜ δΜβΠΜΟΤβΚΜ C MCn0flb30BaHMeM CMeCM ΜΘΗβΗΗΧ 30ΗΑΟΒ, OHM KOHCTaTMpyraT " BonbmeM MacTbio, onnroAe30KCMHyi < neoTMAHbie 30HAbi CMeujaHHbix nocneAOBaTenbHOCTeM npMMeHAriMCb απα βηαθπθημα 6θπκοβμχ γθηοβ HeM3BecTHOM nocneAOBaienbHOCTM m 6μ6πμοτθκ KflHK. Ta 'Me 30HAbi npeACiaBnAiOT M3 Ceca oSbmHo mixture of 8-32 ΟΠΜΠ ^ ΚΠβΟΤΜΑΟΒ Μ ΜΜβΙΟΤ AHMHy 14-17 HyKJieOTMAOB, flBnaaCb TMnMHHblMM npeAdaBMTenaMM κ3 > καομ βο3μο > κηομ κομ6μη3αμμ κοαοηοβ απη HeSonbīuoro προτηχβΗΜΑ (5-6 odaTKOs) 3μμηοκμοποτηομ nocneAOBaTenbHOCTM. B χθοτκμχ ycnoBMfix rM6pMAM3apMM 7 EN 10506 KOTopbie CTaBHT b xyfliuee ποποχθημθ 30Hflbi c HenpaBuribHO cnapeHHbiMii a30TMCTbiMM OCHOBaHMBMM, 3TM ΟΜΘΟΜ CnOCOČHbl n0KanH30BblBaTb ΟΠΘΜΜφΜΜΘΟΚΜΘ nOCneflOBaTe / lbHOCTM ΓΘΗΟΒ B KHOHanbHblX 6μ6ΠΜΟΤΘΚ3Χ Manoii ΟΠΟΧΗΟΟΤΜ. ΤθΜ ΗΘ ΜΘΗΘΘ, BBMfly ΜΧ ΗΘ3Η3ΜΜΤΘΠΒΗ0Μ fl / n iMHbi HeoflHopo / ļHOCTM, CMemaHHbie 30Hjļbi NACTIS nmueHbi οπθμμΦμηηοοτμ, ηθοΟχοαμμομ Αππ 30HflnpoBaHna τθκμχ οποχημχ nocneflOBaTenbHOCTeM, XAX γθηομ μπθκοπμτβιοιαμχ. 3to AerieT READY cnoco6 HenpatcruHHbiM πonnπ M M3onnpOBaHnn 6enKOBbix γθηοβ μπθκοπμτθιοιαμχ, Corfla cooTBeTCTBeHHbie nPHK HeAOCTynHbi. &Quot; (CcbinKU onymeHbi).

TaKMM o6pa30M, b αθηηομ o6nacTM τθχημκμ npOAonxaeT ocTaBaTbcn Heo6xoflnMocTb b COBepilieHCTBOBaHUM Cn0C060S ΒΗΠΟΠΗΘΗΜΠ OblCTpOM Μ 3φφθΚΤΜΒΗθΡί Μ30ΠΠΜΜΜ KflHK-ΚΠΟΗΟΒ b τθχ cny4anx, Korfla Mano ητο Μ3βθοτηο o6 θμμηοκμοποτηομ nocneflOBaTenbHOCTM m κογαθ " očorameHHbie "hctohhmkm τκθημ μΡΗΚ ηθ OHeHb-το AOCTynHbi, HT06bi μχ μοχηο 6bino ncnonb30BaTb b ποοτροθΗΜΜ 6μ6πμοτθκ KflHK. Tacna ycoBepiueHCTBOBaHHbie cnocočbi 6binn 6bi οοοΟθηηο none3Hbi, ecnn 6bi ohm npuMeH ^ nncb αnn M3onnpoBaHna γθηομηηχ κποηοβ ΜΠΘΚΟΠΜΤΘΙΟΙΑΜΧ, ΓβΘ flOCTynHa HennOTHaa ΜΗφορΜΘΜΜΠ OTHOCMTenbHO aMMHOKMCnOTHblX nocneflOBaieribHOCTeiĪ nonunenTMfla, KOflMpyeMoro mckomum γθηομ. B. 3pnTpono3THH Also < npencTaBnatoiiiHM MHTepec ποπμπθπτμπ 3pmpono33, npon3BOflCTBO KpacHbix κροΒΒΗΝΧ Teneu, ocymecTBnaeTCB HenpepbiBHO b npoflonxeHne BcePi ηθποβθ4θοκομ xm3hm αππ KOMneHcaumi pa3pyuieHHbix κπβτοκ. 3pmpono33 npeflCTaBnaeT co6om 4pe3BbmaMH0 tohho perynnpyeMbiii MexaHM3M, πο3ΒΟπ * ηοιαμμ BbipaSaTbieaTbCii b κροΒΜ AOCTaTOHHOMy KonnHecTBy xpacHbix KpoBHHbix τθπθμ αππ npaBMnbHoii OKCUreHaLļMM the fabrics, HO ΗΘ CTOnb 6onbtIJOMy, HTOObl ΚΠΘΤΚΜ BOCnpenaTCTBOBa / IM KpoBooāpameHMio. 06pa30BaHne KpacHbix κροΒΠΗωχ τθπθμ προμΟχοαμτ b kocthom M03re ποα ΚΟΗΤρΟΠΘΜ ropMOHa, 3ρΐ1Τρ0Π03ΤΜΗ3. 3ρΜΤρ0Π03ΤΜΗ, ΚΜΟΠΟΤΗΗΜ ΓΠΜΚΟΠρΟΤΘΜΑ C ΜΟΠΘ ^ ΠΠρΗΗΜ ΒΘΟΟΜ npn6nH3MTenbHO 34000 flanbTOH, μοχθτ BCTpenaTbcn β τρβχ φορΜ8χ: α, β μ acnano. (DopMbi α μ β οτπμηθιοτοη He3HaHMTenbH0 β yrneBOAHbix κομποηθητθχ, οαηθκο μμθιοτ OAMHaKOByio ποτθημμιο, 6nonomHecKy! O aKTMBHOCTb μ MoneKynnpHbiii βθο. OopMa ackiano πρθαοτθβππθτ cočoii α μπμ β -4 > opMy c OTinenneHHbiM κοημθβνμ ymeBOAOM (ciianoBan κμοποτθ). SpMTponoaTMH npncyTCTByeT b oneHb ημ3Κμχ κοημθητρθμμπχ b nna3Me, κογαθ τθπο ηθχοαμτοπ b 3A0p0B0M coctohhmm, puck κοτοροΜ τκθημ nonyHaiOT AOCTaTOMHyK) oKCMreHaMMio ot cymecTByKDiuero KonMnecTBa apMTPOMMTOB. 3The HopManbHan HM3Kan κοημθητρθμμη α ° οτ3τομη3 αππ CTMMynMpOBaHMH 3aMeHbl KpaCHblX KpOBBHblX ΤΘΠΘΜ, KOTopbie OČblMHO TepntOTCB Β npOMecce CTapeHMn. Κοπμηθοτβο spMTponoaTMHa β κροθθο6ρ3ΐΐ1θΗΜΜ B03pacTaeT β ycnoBnax ΓΜΠΟΚΟΜΜ, κογαθ CHMxaeTcn non-genomic KMcnopona KpoBHHbiMM κπθτκ3μμ npM KpOBOOcpAeHMM. Γμποκομπ μοχθτ Bbi3biBaTbcn ποτβρθΜ Oonbujoro κοπμηθοτβθ κροΒΜ NPM κροΒοτθΗθΗΜΜ, pa3pymeHneM KpacHbix KpoBBHbix τθπθμ βοπθαοτβμθ paAMoaKTMBHoro nepeo6nyHeHna, οημχθημθμ ΠΟΤρθδΠΘΗΜΠ KMCHOpOAa M3-3a 6onbUJMX BbICOT ΜΠΜ npOAOnXMTenbHOrO 6eCC03HaTenbH0r0 8 cocTOHHMfl, a TaiOKe pa3nMHHbiMM (popMaMii θηθμμμ. Πορ βπμλημθμ τκ & Ηβίί, nopsep > KeHHbix ranoKCMHecKOMy CTpeccy , apuTponoaTMH ηβμμηβθτ yBennHMBaTb npon3BopcTBo KpacHbix KpOBBHblX ΤΘΠΘΙ4 nyTeM CTMMynΜρθΒ3ΗΜfl KOHBepCMM nepBMHHblX npemueCTBy (OlHMX ΚΠΘΤΟΚ b KOCTHOM Μ03ΓΘ b np03pMTpo6naCTbl, KOTOpbie BnOCneflCTBMM POBOPAT FLO C03peBaHMfl, CMHTe3npyK) T γθμογπο6μη m peanu3yioTCfl b KpoBooSpameHMU b KanecTBe KpacHbix "ροβαηνχ tubing. Korpa κοπμηθοτβο KpacHbix" ροβηηηχ hose b KpoBoodpaipeHMti 6onbtue, ηθμ ΗΘΟβΧΟρίΙΜΟ flna HOpManbHOM ΠΟΤρββΗΟΟΤΜ Τ ΙΟΗΘΜ B ΚΜΟΠΟρορβ, 3ρΜΤρ0Π03ΤΙΊΗ B KpoBoočpameHMM CHM > KaeTCB. Ομοτρμτθ, rnaBHbiMo6pa30M, Tbi Testa et al, Exp. Hematol., 8 (Supp.8), 144-152 (1980), Tony et al., J.Biol.Chem. 256 (24): 12666-12672 (1981); Goldvvasser, J. Cell.Physiol., 110 (Supp.1), 133-135 (1982); Finch, Blood., 60 (6), 1241-1246 (1982); Sytowski et al., Expt. Hemat., 8 (Supp.8), 52-64 (1980); Naughton, Ann, Clin.Lab. Sci., 13 (5): 432-438 (1983); Viss et al., Am.J.Vet.Res., 44 (10), 1832-1835 (1983); Lappin et al., Exp. Hematol., 11 (7), 661-666 (1983); Murphy et al., Acta Haematologica Japonica 46 (7), 1380-1396 (1983); Dessupris et al., Brit. J. Haematol., 56, 295-306 (1984); m Emmanouel et al., Am.Physiol., 247 (I Pt 2), F168-76 (1984).

Tax XAX apmponosTMH BaxeH b npopecce o6pa30BaHMfl xpacHbix KpoB "Hbix hose ropMOH nMeeT noTeHunanbHoe none3Hoe npMMeHemie XAX b pnamocinKe, Tax μ β πθηθημμ HapyUjeHHM ΙφΟΒΜ, XapaiCTepM3yKDipMXCn HM3KMM ΜΠΜ ΗβΟΟΒβρίυβΗΗΗΜ np0M3B0pCTB0M KpaCHblX KpoBBHbix tubing. ΟΜΟτρκτβ, rnaBHbiM o6pa30M, Ptech Pennathur-Das et al. Blood 63 (5), 1168-71 (1984) u Haddy, Am.Jour.Ped.Hamatol. ., J.Clin.lnvest. 74/2 /, οτρ.434-441, (1984), β κοτοροίί onncaH TepaneBTunecKUM peaoiM PNN ypeMMHecKoPi oeubi Ha οοηοβ3ημμ peaKpmi in vivo Ha BnuBaHue nna3Mbi, očoraipeHHoii apuTponoaTMHOM, n npepnoxeHa po3npoBKa 10 v ΕΡΟ / κγ β peHb β τβΜβΗΐιβ 15-40 pHeii Kax nonpaBKaHeMnu TaKoro Twia, KOTOpbifi accouunpyeTcn c χροΗΜΗβοκοίί ποηθηηομ HepocTaTOHHOCTbio. CMOTpuTe TaiOKe, Ty Kranc, Henry Ford Hosp.Med.J., 31/3 / 177-181 (1983).

HepaBHO 6bino oueHeHO, ητο ηθπμμμθ HelpTonHa b 6onbuiOM KonunecTBe ποθβοπμτ nenuTb Kax < pbiPi rop aHeMMM 1600000 ηθποβθκ TonbKo b CLUA. ΟΜΟτρκτβ, HanpuMep, Morrison, " Bioprocessing in Space - an Overview ", οτρ.557-571 in The World Biotech. Report 1984

Volume 2: USA, (Online Publications, New York, N.Y., 1984). nocnepHne nccneposaHun co3panu ocHOBaHMe PNN npepcKa3aHun θφφβκτΜΒΗοοτΜ apMTponoeTUHOBOM Tepannu b pa3HOo6pa3uu cocTonHMii 6one3HM, paccTpoPicTB u cocTonHkm reMaTononmecKoro HapyuieHWfl: Vedavato et al., Acta Haematol., 71.211 to 213 (1984) (6eTa-TanacceMun); Vichinsky et al., J.Pediatr., 105 / 15-21 (1984) (KncT03Hbiii φΜδρο3); Cotes et al., Brit.J.Obstet. Gyneacol., 90/4 / 304-311 (1983) (SepeMeHHOCTb, paccTpoiicTBa MeHCTpyauuPi); Haga et al., Acta et al. Acta Pediatr.Scand., 72, 827-831 (1983) (pHHnn aHeMun npex <peBpeMeHHOCTu); Claus-Walker et al., Arch.Phys. Med. Rehabil., 65,370-374 (1984) (noBpeacpeHMe οπμηηογο M03ra); Dunn et al., Eur.J. Appl. Physiol., 52,178-182 (1984) (κοοΜΜΗβοκΜίί ποπθτ); Milleret al., Brit. 3 Haematol., 52, 545-590 (1982), LV 10506 (ocrpafl ποτβρη kpobm); Udupa et al., J. Lab. Cl.Med., 103/4 / 574-580 n. n Lipschitz et al., Blood 63/3 / 502-509 (1983) (CTapeHne); m. Daniak et al., Cancer, 51/6 /, 1101-1106 (1983) m Schwartz et al., Otolaringol. npeflbiflymne ποπητκμ nonyHMTb apMTponoaTMH c χοροιιΐΜΜ βμχοαομ BbpasoTKu M3 nna3Mbl ΚρΟΒΜΟΒΜ M3 MOHM flOKa3aHbl KaK OTHOCMTenbHO HeyflaHHbie ΥοΠΟΧΗβΗΗβΠ m yTOHHeHHlHNHOPTOPHAN τθχημκθ, ΗβΜ3ββχΗ0 μ, KaK npaBuno, npuBOflm κ c6opy oneHb Manbix KommecTB 3KnHHbix m HeycTOMHMBbix aKCTpaicroB, coflepxai4MX apMTponoaTMH. B naTeHoMe rpaMOTe CUJA N 3033753 onMcaH cnocoS HacTMHHOM ommctkm apMToNoTMHa ot nN3Mbi KpOBM OBLI, hto oAcEHMBeT nonyHeHne Manbix βμχοαοβ o @ mero 6ecnpnMecHoro aKCTpaicra, co, qepxamero apMTponoaTMH. nepBOHananbHbie ποπμτκμ BbiflenMTb apMTponoaTMH m mohm πρμβθπμ κ HeycTOMHMBbiM, 6μοπογμηθοκμ HeaKTMBHbiM npenapaTaM ropMOHa. B π3τθητηομ rpaMOTe C1UA N 3865801 oncicaH ccnocoā cra6MnM3aLLMM 6μοπογμηθοκομ βκτμβηοοτμ o6mero BemecTBa, copepxamero apMTponoaTMH, BoccTaHOBneHHbiM M3 mohm. flonyHaeMbiM nippaT, coflepxaiHMf apMTponoTMH, cpepxMBaeT 90% 3κτμβηοοτμ apMTponoaTMHa μ nenneTcn ycTOMHMBbiM. flpyroii cnocoč ommctkm nenoBenecKoro apMTponoaTMHa ot mohm napMemOB, cTpapaiomMX annacTMnecKOM θηθμμθμ, onMcaH b pa6oTe Miyake et al., J.Biol. Chem., Tom 252; N 15 (10 aerycTa 1977), οτρ.5558-5564. 3Ta ceMMaTanHaa MeTopMKa BKnioHaeT MOHOo6MeHHyio χροΜ3τοτρ3φΜΐο, ocaxneHMe aTaHona, renb- ^ MnbTpauMio, ajņcop6qMOHHyio χροΜ3τθφ3φΜΐο m BbipaāaTbieaeT ημοτμμ apMTponoaTMH c ποτβΗΑΜβΚ 70400 θαμημο / μγ 6enKa NPM βμχοαθ 21%. B naTeHTHOM rpaMOTe CLUA N 4397840, BbiflaaHHOM Takezawa et al., OnMcaHbi cnoco6bi nonyHeHMn " θρμτροποθτμηοβογο npoflyKTa "M3 o6pa3LļOB MOhm 3flopoBoro nenoBeKa c 6one3HeHHbiMM κ3τμοημτ3μμ m yTBepxflaeTCfl, whereby to nonyHeHHbie nponyKTbi c hm3kmm MoneKynnpHbiM Becomes" He μμθιοτ MHrM6MpyiomMx αθμοτβμμ προτΜΒ apMTponoaTMHa ". B 3aaBKe Ha naTeHT BenMKo6pMTaHMM N 2,085,887, Sugimoto et al., Ony6nMKOBaHHOM 6 I 1982 Rofl, onMcaH cnoco6 nonyneHMn rn6pwflHWX ηΜΜφοβπθοτοΜΑΗωχ κηβτοκ nenoBeKa m cooātuaeTcn, whereby to ypoBHM npoflyqMpoBaHMn HaxonnTcn b npeflenax ot 3 flo 420 eflMHMU apMTponoaTMHa Ha μπ cycneH3MM κηβτοκ (pacnpefleneHHbix b KynbTypax Nocni pa3MH0xeHMn χοβηΜΗΟΜ MneKonMTatomero c C0AepxaHMeM from ΙΟ ^ κηβτοκ Ha μπ). Ha caMbix BbicoKMX ypoBHnx npOflyuMpoBaHMn, κοτορΜΘ, Kak yTBepxAaeTcn, αοποχημ 6biTb flocTMmyTbi, CKopocTb npoflyuMpOBaHMn apMTponoaTMHa coct3bmt ot 40 ao 4000 eflMHMu / 106 κηβτοκ / 48 nacoB b KynbType in.vitro c nocneoyiomMM nepeHocoM κηβτοκ M3 ομοτθμ pa3MH0xeHMa in vivo. (ΟΜοτρΜτβ TaKxe aKBMBaneHTHyio naTeHTHyto rpaM0Ty CUJA N 4377513). Bbinn cpenaHbi MHOrOHMCneHHbie ηρβΑΠΟΧβΗΜΠ B ΟΤΗΟΙΠβΗΜΜ ΒΜΑΘΠΘΗΜΗ apMTponoaTMHa M3 TKaHeBblX MCTOHHMKOB, ΒΚΠΙΟΗΘίΟΙΑΜΧ ΗβΟΠΠβΟΤΜΗΘΟΚΜβ ΚΠβΤΚΜ, OflHaKO BblXOflbl Bbipa60TKM SblHM cOBepuieHHO HM3KMe. CMOTpMTe, HanpMMep, supra, Jekman et al., Expt.Hematol., 11/7 / I, 581-588 (1983); Tambourin et al., P.N.A.S. (USA), 80, 6269-6273 (1983); Katsuoka et al., Gann., 74, 534-541 (1983); Hagivvara et al., Blood, 63/4 / 828-835 (1984); m. Choppin et al., Blood 64/2 / 341-347 (1984). 10 flpyrne 30 M30nnp0BaHMa, ΠρΜΜβΗΗβΜΜΘ flJlfl ΠΟΠγΜΘΗΜΑ ΟΗΜΙΜβΗΗΟΓΟ apTTONOSTMHa, βκπιοηθπμ b ce6n μμμυηοπογμμθοκμθ μθτοαμκμ. rionMKnoHanbHoe, flBnniOLneecfl npoM3BOflHbiM CbisopoTKM aHTMTeno, SpongeHHoe προτΜΒ spMTponoaTMHa, pa3BMBaeTCfl nyTeM β > MHbeqnpoBaHHbiM MenoBewecKMM apnTponosTMH pacno3HaeTCfl κθκ MHopopHoe aHTureHHoe eeinecTBo μμμυηηομ ομοτθμομ χμβοτηογο h Bbi3bieaeT npow3BOflCTBO aHTMTen προτΜΒ aHTureHa. Pa3nnMHbie κπθτκμ, pearnpyioinne Ha CTMMynnpoBaHiie nocpepcTBOM aHTureHHoro eeinecTBa, npoflyunpyiOT n BbiCBo6o > Kha (OT b KpoBooOpaineHMM aHTMTena, OTnnHatomnec "cnerKa οττβχ, KOTopbie npoflyL | MpyiOTca ppyrMMM pearnpytoinnMM κπθτκθμμ. Ακτμβηοοτβ θητμτθπ ocTaeTcn b cbieopoTKe χμβοτηογο, Korpa ero KpoBb 3KCTparnpoBaHa. H χοτη HeoHMmeHHan cbieopoTKa μπμ npenapaTbi aHTMTen, OHMīpeHHbie CEC CbieopoTOHHan φρβκρΜη MMMyHorno6ynMHa G, MoryT 6biTb 3aTeM Mcnonb30BaHbi b npo6ax PNN o6HapyxeHMn KOMnneKCoo6pa30BaHMn c ηθποβθηθοκμμ apMTponosTMHOM, BeuļecTBa McnbīTbiBaioT rnaBHoe Heypo6cTBO. 3to cbiBopoTOMHoe aHTMTeno, cocTomuee M3 Bcex pa3nMHHbix aHTMTen, npoflyqnpoBaHHbix OTpenbHbiMM κπθτκθμμ of npMpope nenneTcn nonMfcnoHanbHbiM m o6pa3yeT KOMnneKCbi c κομποηθητθμμ b οδιρΜΧ wepTax MHawe, ηθμ OflMHOHHblM 3ΡΜΤΡ0Π03ΤΜΗ HHTepec pnn npepnocbinoK HacTonipero Μ3θδρβτβΗηη npepcTaBnnt OT nocnepHMe ροοτΜΧΘΗΜΛ b odnacTM pa3pačoTKM qenocTHbix xynbTyp κηβτοκ, cnoco6Hbix npopyuMpOBaTb OflMHOMHbIM BMfl 3ΗΤΜΤβΠ3, KOTOpblM ΟΠβρΜφΜΗβΟΚΜ MMMyHOnOTMHeCKM 3ΚΤΜΒβΗ C OflMHOHHOM aHTMreHHOM jqeTepMMHaHTOii OTOčpaHHoro amureHa. CMOTpMTe, rnaBHbiMo6pa30M, Chycholm, High Technology, Tom 3, N 1, 57-63 (1983). npepnpMHMManMCb ποπμτκμ ynoTpe6nTb cnnnHMe κπβτοκ μ μθτορμκμ rM6pMflM3aqMM flnn pa3BMTMn "MOHOicnoHanbHbix" aHTMTen κ 3pMTpon03TMHy M npMMeHMTb 3TM aHTMTena B Μ30ΠΜΡ0ΒΒΗΜΜ Μ ΚΟΠΜΗβΟΤΒβΗΗΟΜ ΟΠρβρβΠβΗΜΜ nenoBenecKoro spMTponoaTMHa. B KanecTBe oflHoro EXAMPLE moxcho πρμβθοτμ cooāipeHMe, nonBMBiueecn b θηηοτμροββηηομ φορΜβ b pačoTe Lee-Huang, Abstract 1463 N Fed.Proc., 41, 520 (1982), b κοτοροΜ roBopMTcn 06 ycneuuHoii pa3pa6oTKe πμημμ κηβτοκ γμ6ρμρομμ Mbiuib-Mbiiiib, BbiflennKDUļMX MOHOKnoHanbHbie aHTMTena κ MenoBeHecKOMy spMTpono3TMHy. B KanecTBe Apyroro npMMepa, noppočHoe onMcaHMe nonyneHMn m Mcnonb30BaHMn MOHOKnoHanbHoro, ηρ0ΤΜΒ03ρΜτρ0Π03ΤΜΗ0Β0Γ0 aHTMTen nonBMnocb b paTe Weiss et al., P.N.A.S. (USA) 79: 5465-5469 (1982). CMOTpMTe TaiOKe pačOTbi Sasaki, Biomed. Biochim. Acta, 42/11/12, S 202-S 206 (1983); Yanagawa et al., Blood, 64/2 /, 357-364 (1984); Yanagawa et al., J. Biol. Chem., 259/5 / 2707-2710 (1984); m naTeHTHy »o rpaM0Ty CUJA N 4465624.

flnn npepnocbinoK Μ3θ6ρβτθΗΜ "TaioKe πρβροτβΒπηιοτ MHTepec οοοδιρβΗΜη 06 MMMyHonorMwecKOM aKTMBHOCTM ομητθτμηθοκμχ πθπτμροβ, KOTopbie of cymecTBy jqynnMMMpyiOT aMMHOKMcnoTHyio nocneflOBaTenbHOCTb, coxpaHMBiuyiocfl b cbočopho BCTpeMaiomMxcn 6enKax, rnMKonpoTeMflax m HyKnenpOTeMflax. Bone KOHKpeTHo, nonMnenTMflbi c oTHOCMTenbHO HM3KMM MoneKynnpHbiM becom noxa3aHbi yHacTByramMMM b MMMyHHbix peaKL | MNX, cxopHbix of npOpon > KMTenbHCTM M πρΟΤΑΧβΗΗΟΟΤΜ C MMMyHHblMM peaKLļMnMM φΜ3ΜΟΠΟΓΜΗβΟΚΜ BSOKHblX δβπκΟΒ, t3kmx Kax BMpycHbie aHTMreHbi, nonMnenTMpHbie ropMOHbi m Tomy nofloāHoe. CpepM 11 EN 10506 MMMyHHblX HeadMaMe Τ3ΚΜΧ ΠΟΠΜΠβΠΤΗΑΟΒ ΒΚΠΚ5ΗΘΗΟ Μ ΠρΟΒΟΜΗρΟΒβΗΜβ 06pa30BaHHfl ΟΠΘΜΜφΜΗΘΟΚΜΧ 3ΗΤΗΤβΠ Β ΜΜΜγΗΟΠΟΓΜΗΘΟΚΜ ΒΚΤΜΒΗΜΧ XHBOTHblX. CMOTpme, HanpHMep, paoTbi Lemer et al., Celi., 23, 309-310 (1981); Ross et al., Nature, 294: 654-656 (1981); Vter et al., P.N.A.S. (USA), 77, 5197-5200 (1980); Lerner et al., P.N.A.S. (USA), 78, 3403-3407 (1981); Vter et al., P.N.A.S. (USA), 78, 4882-4886 (1981); Vongong, P.N.A.S. (USA), 78, 7412-7416 (1981); Green et al., Celi., 28, 477-487 (1982); Niggetal, P.N.A.S. (USA), 5322-5326 (1982); Baron et al., Celi., 28, 395-404 (1982); Dressman et al., Nature, 295, 158-160 (1982); h Lerner, Scientific American, 248, N 2, 66-74 (1983). CMOTpme τθκχθ pa6oTy Kaiser et al., Science, 223, CTp.249-255 (1984) KacaiomyiocH āMononmecKOM m HMMyHonornwecKoPi aicrMBHOcTM CMHTeTMHecKnx nenTMflOB, KOTopbie npn6nn3menbHO ρΑ3Αβπηκ) τ BTopnHHbie CTpyicrypbi nenTMflHbix γορμοηοβ, oflHaKO, Μοιγτ He pa3flennTb mx nepBMHHyio CTpyKTypHyK) KOH0opMaqmo. BbiujeyKa3aHHbie MceneAOBaHHfl KacaiOTCfl, κοηθηηο, θμηηοκηοιοτηνχ nocneflOBaTenbHOCTeii 6θπκοβ, μηνχ, ηθχθπη spHTponosTMH - BemecTBO, απη KOToporo He Obino Ony6nHKOBaHO CymeCTBeHHOH ΗΗφθρΜ814ΗΗ οβ ΘΜΗΗΟΚΗΟΠΟΤΗΟΜ nOCneAOBaTenbHOCTH. B coBMecTHoii 3a "BKE Ha naieHT CLIL N 463724, ποαθηηοη 4 φθBpann 1983 ROFL J.Egrie, ony6nnKOBaHHoPi 22 aBrycia 1984 Rhone XAX esponeRcKaa 3aflBKa Ha πθτθητ N 0116446, oriMcaHa πηηηη κπθτοκ rn6pnflOMbi Mbiujb-MbiLUb (ATCC HB8209 N) KOTopan npoflyunpyeT KpaiiHe ΟΠΘΜΜφΜΗβΟΚΟΘ MOHOKnOHanbHOe, npOTHBOepMTponOSTMHOBOe aHTMTenO, κοτοροβ ββπηθτοη TaioKe οπβρΜφΜΗβοκΜ MMMyHOHeaKTMBHbiM c ποπηπθπτηαομ, coAepxaiiļHM cneflytomyio nocneflOBaTenbHOCTb θμηηοκμοποτ: NH2-Ala-Pro-Pro-Ary-Leu-Ile-Cys-Asp-Ser-Ary-Val-Leu-Glu-Arg-Tyr Leu-Leu-Clu-Ala-Lys-COOH. rionnnenTHAHaH nocneAOBaTenbHOCTb - sto Ta nocneAOBaienbHocTb, KOTopan 3axpenneHa 3a nepsbiMH AaaAnaTbfo aMHOKMcnoTHbiMH ocTaTKaMH co3peeuiero ηθποβθηθοκογο 3pMTpono3TMHa, M3onMpoBaHHoro b cooTBeTCTBMM co cnocoāoM, onncaHHbiM b pa6oTe Miyalke et al., J.Biol.Chem. 252, 5558-5564 (1977); 256, 7990-7997 (1981). CMOpme Taicxe was prepared by Sue et al., Proc. Nat.Acad.Sci. (USA), 80, CTp.3651-3655 (1983); -186 (1984). Χοτη nonMKnoHanbHbie ηπμ MOHOKnoHanbHbie ammrena, XAX onncaHO Bbiuue, pacnonararaT BecbMa none3HbiMM MaTepnanaMM PNN Hcnonb30BaHMfl b MMMyHOHcnbiTaHHnx Ann o6HapyxeHHn m KonnwecTBeHHoro onpeAeneHMA spMTponosTHHa h Moryr 6biTb npnroAHbi b cpoACTBeHHOM oHHCTKe spHTponosTHHa, KaxeTcn ManoBeponTHbiM, hto 3th MaTepnanbi ajin KpynHOMacujTaāHoro H3onnpoBaHHn MoryT oāecnenHTb KonnHecTBa spHTponosTHHa MneKonHTaiomMx hctohhokob, AOCTaTOHHbie απη προβθαθηηη AanbHeHuiero aHanH3a, κπηηημθοκογο oScneAOBaHHH μ βο3μοχηογο ujnpoKOHanpaBBneHHoro TepaneBTHnecKoro MCn0nb30BaHHH ΒβΙΑΘΟΤΒΘ πρΗ ΠβΗβΗΗΜ, e.g. XpOHHHeCKOH 6one3HM ποηθκ, πρπ κοτοροπ 12 nopaxeHHbie τιοη ηθ b coctobhmm noflflep > KMBaTb npoflyunpoBaHMe apuTponoaTMHa. no3TOMy npeanonaraeTCH, mto HannyHmne nepcneicTMBbi απη ποπηομ xapaKTepMCTMKM apMTponosTMHa MneKonnTatomnx m co3flaHMB 6onbLUMX κοπμμθοτβ ero απβ bo3MO > Khom AMamocTMKM m κπμημμθοκογο πρΜΜΘΗΘΗΜΑ coflepjKMT ynoTpe6neHMe peKOM6nHaHTHbix MeTOflUKflna ocyu4ecTBneHMB KpynHOMacuuTa6Horo μμκρο6ηογο CMHTe3a οοθαμηθημα.

HecMOTpa Ha το, hto b nonbīTKe M3onnpoBaTb nocneflOBaTeribHocTM flHK, KOflMpy (oinne apuTponosTMH nenoBeKa nnn ppyrnx bmaob μπθκοπμτβιοιαμχ, 6binn npeflnpuHflTbi 3HaHMTenbHbie ycnnna, HM OflHO M3 ΗΜΧ ΗΘ 6blOO yCneillHblM. 3T0 06yCn0BneH0, maBHblM 06pa30M, ΑθφΜρΜΤΟΜ TKaHeBbix mctomhmkob, οοο6θηηο μθποβθμθοκμχ TKaHeBbix mctomhmkob, οΟογθιαθηημχ b mPHK τβκ, HToSbi no3BonnTb ποοτροβΗΜβ KflHK-6M6riMOTeKM, M3 κοτοροΡί nocpeflCTBOM πρμμθηθημη M3B0CTHbix MeTopnK μοχηο BbiperiMTb nocneflOBaTenbHocTb flHK, Kopnpyioinyio spMTponosTMH. ΚρΟΜΘ ΤΟΓΟ, Mano MTO Μ3ΒΘ0ΤΗ0 0 HenpepbIBHOM nOCneflOBaTenbHOCTM aMMHOKMCriOTHblX 0CT3TK0B OpMTponoaTMHa, a nOCeMy ΗΘΒ03Μ0ΧΗ0 nOCTpOMTb, HanpMMep, flmiHHbie nonHHyKneOTMflHbie 30Hflbl, ΠΟΡΧΟΡΒΙ ^ ΜΘ flHB ΗΘΑΘΧΗΟΓΟ MCn0nb30BaHMB B flHK / flHK -rn6pMflM3ai4MOHHOM ΠρΟΟΘΜΒβΗΜΜ KflH.K Μ ΟΟΟδθΗΗΟ TeHOMHblX 6μ6πΜΟΤΘ flHK. MnniocTpaTUBHO nocneflOBaTenbHocTb AsappaTM θμμηοκμοποτ, πρμμθηβθμθβ απη reHepiipoBaHMB BbiiueyKa3aHHoro MOHOKnoHanbHoro aHTMTena, npoflyqnpoBaHHoro A.T.C.C. N HB8209, ηθ ponycKaeT ποοτροθΗΜΒ of lH03HaMHoro 60-οοηοβηογο ojinroHyKneoTMflHoro 30Hfla cnocoSoM, lHHMM Anderson et al., Supra. HaPifleHo, mto μθποβθμθοκμμ γθη απη 3pHTp0n03TMHa ΜΟΧΘΤ npOHBnflTbCfl CEC "ΓΘΗ ΟΑΜΗΟΜΗΟΡί κογμμ" β γθηομθ μθποβθκθ, Μ TAK ΜΠΜ ΜΗΒΜΘ, ΓΘΗΘΤΜΜΘΟΚΜΜ MaTepiian, KOflMpyK) LAMM ΜΘΠΟΒΘΜΘΟΚΜΜ 3ρΜΤρ0Π03ΤΜΗ, ΒβρΟΑΤΗΟ, cocTaBnaeT μθηθθ 0.00005% očineii γθηομηομ flHK MenoseKa, KOTopaa αοπχηβ npncyTCTBOBaib β γθηομηοΡι 6μ6πμοτθκθ.

Ha Hb ceroAHJUiiHMti A © Haan6onee ycneiūHbie m M3BecTHbix nonbīTOK b peKOM6nHamrHbix cnoco6ax απλ co3AaHMfl nocneAOBaTenbHOCTePi flHK, npnroAHbix b Mcnorib30BaHMM β ΜΜκροβΗοΡί sKcnpeccmi M3onnpyeMbix κοπμμθοτβ MneKonuTaioiiiero spMTponoaTMHa, ρβπβκο ηθ αοοτμγπμ qenn. EXAMPLE B KaMecTBe μοχηο πρμβθοτμ pa6oTy Farber et al., Exp.Hematol., 11, Supp. 14, Abstract 101 (1983), β κοτοροίί cooāmaeTca 06 3KCTpaKAMM mPHK H3 πομθμηνχ τκθηθΡϊ 6a6ynHOB, o6pa6oTaHHbix ΦθημπγμαΡ33μηομ, m-MH beKUMpoeaHMM mPHK b oouMTbi Xenopus Ship c AOBonbHO βρθμθηηνμ pe3ynbTaT0M πογ ^ ηθημβ in vitro ομοομ 'TpaHC / iflLiMOHHbix nPOAyKTOB', KOTOpbie ΜΜΘΠΜ ΠρΟΒΒΠΗΚΠΑΜΘΟΒ 6μΟΠΟΓΜΜΘΟΚΜΘ CBOMCTBa 3pMTp0TI03TMHa.

CoBceM HepaBHO Farber et al., Blood, 62, N 5, Supp. N 1, Abstract 392, Ha CTp.122a (1983) ΑΟΠΟχμπμ o TpaHcnauMM in vitro mPHK μθποβθμθοκομ πομκμ nflryiuaHbMMM οοαμτβμμ. nonyMeHHa CMecb npoAyicra TpaHcnauMM 6bina oeLaHe xax coAepx < aaa b nopapKe 220 mU nPOAyicra TpaHcnauMM, MMeiom aKTMBHOCTb apMTponoaTMHa Ha MMKporpaMitf ββθαθηηομ mPHK. Μ χοτβ TaKMe ypoBHM TpaHcnmļMM in vitro 3Κ30γθηηομ mPHK, KOAMpyrameM apMTponoaTMH, 6binM npM3HaHbi coeepujeHHO hm3Kmmm (from cpasHeHMio aaxe c paHee coočmeHHbiMM ypoBHflMM TpaaHcnBLļMM mPHK 6a6yMHa b MCKOMbiPi npOAyKi) ποομμτθπμ, mto pe3ynbTaTbi noATBepx < AaioT 13 EN 10506 cymecTBOBaHiie μθποβθηθοκομ ποηκμ b Kaneciee ynacTKa aKcnpeccMBHocTM aidTaTaTa, no3Bon «iomePt ποοτροβΗΜθ κβΗΚ-δΜβπΜΟτβκΜ ocorameHHon ηθποβθηθοκομ ποηκμ, M3 κοτοροη μοχηο BbipenuTb xenaeMbiM γθη. [Ομοτρμτθ τθκχθ Farber, Clin.Res., 31/4 /, 769A 91983.] KflHK ΗΘΠΟΒΘΗΘΟΚΟΓΟ 3pMTp0n03TMHa Β Ε.ΟΟίί. ΚορΟΗΘ rOBOpfl, LļenblM pflfl ΚΠΟΗΟΒ KflHK 6bin ΒΗΘΟΘΗ b nna3MMflbi E.coli. m npopyicrbi οπμπημπ p-naKTaMa3bi Sbinn οτμθηθην < a < MMMyHCKMBHbie C MOHOKJlOHanbHblM ΒΗΤΜΤβΠΟΜK HeyCTaHOBneHHOMy “anHTOny” ΗβΠΟΒΘΗβΟΚΟΓΟ 3pmp0n03TMHa. CMOTpme Lee-Huang, Proc.Nat.Acad.Sci. (USA), 81, στρ.2707-2712 (1984). KPATKOE Μ3ΠΟ > ΚΕΗΜΕ npennaratoTcn enepBbie Hobbs OHmueHHbie m M3onMpoBaHHbie nonunenTMflHbie npoflyKTbi, MMeiomne ηβοτμηηο μπμ nonHocTbio nepBMHHyio CTpyicrypHyK) KOHCpopMaijMio (Te HenpepbiBHyio nocneflOBaTenbHOCTb aMMHOKMcnoTHbix οοτβτκοβ) μ οαηο μπμ 6-one m δΜΟΠΟΓΜΗΘΟΚΜΧ CBOMCTB (HanpMMep, ΜΜΜγΗΟΠΟΓΜΗΘΟΚΜΘ CBOMCTBa M ČMOnorMHeCKafl aKTMBHOdb in vitro) cbo6oaho BCTpenaiomMerocfl apMTponoaTMHa, BKntonafl Difference annenbHbie BapMaHTbi. 3tm nonunenTMflbi TaKxe οαηο3ηθηηο oinunaiOTCH τθμ, ητο πβπηκπόπ προΑγκτοΜ aKcnpeccMM npOKapnoTHoro μπμ 3yKapnoTHoro xo3AMHa (HanpMMep, 6aKTepManbHbiMM, NPO "OKeBblMM Μ ΜΠΘΚ0ΠΜΤ8Κ3111ΜΜΜ ΚΠΘΤΚβΜΜ B KynbType) noeneAOBaTenbHOCTeii 3Κ30ΓΘΗΗ0Μ flHK, nonyHeHHbix nyreM γθηομηογο μπμ κΑΗΚ-κποημροβθημπ, μπμ nyTeM γθηηογο CMHTeaa. npoflyicTbi ΜΜκροδΗοΡί sKcnpeccMM β κπβτκβχ πο3βοηομημχ (HanpMep, MneKonMTaiomMX μ πτμμ) Μοιγτ κ Tomy xe oTnMnaTbcn cboSoahom οτ cooSmecTBa c ηθποβθηθοκμμμ 6θπκ8μμ μπμ ApyrMMM 3arpn3HMTennMM, KOTopbie Moryr accouMMpoBaTbcn c spMTponosTMHOM b ero θοτθοτβθηηομ μπθκοπμτθιοιαθμ κπθτοηηομ oxpy > KaiomeM cpefle μπμ b 3KCTpaqennionflpHbix TeKyHMx cpeflax, τβκμχ XAX nna3Ma κροβμ μπμ Mona. npoflyKTbi τμπμηηνχαροχοκθβμχ (HanpMMep, Saccaromyces cerevisial) μπμ npoKapMOTHbix (HanpMMep, E.coli) κπθτοκ χο3πμηβ CBOOflHbl OT COOameCTBa c nioSbIMM ΜΠβΚ0ΠΜΤ8ΙΟ111ΜΜΜ SenKaMM. B 33BMCMM0CTM ot πρΜΜβΗπβΜΟΓο χο3ΠΜΗ3 npeflnonaraeMbie nonMnenTMflbi Moryr 6biTb KHMK03MnMpoBaHbi MNKMMMMM μπμ ApyrMMM 3yKapM0THbiMM yrneBOAaMM, μπμ MoryT 6biTb HemMK03MnMpoBaHHbiMM. npepnonaraeMbie nonMnenTMflbi MoryTTaioKe BKniOHaTb aMMHOKMCnOTHbIM OCTaTOK HaHanbHOfO MeTMOHMHa (β ΠΟΠΟ) ΚΘΗΜΜ 1). flpeflnonaraeMbie Hobbs mMKonpoieMHOBbie προηγκτω βκπιοηβιοτ τθ, KOTopbie MMeiOT nepBMMHyto CTpyicrypHyio KOHCpopMauMto, αοοτθτοηηο AynnMLļMpyiomyio KOHcfeopMaiiMio cbočorho BCTpeHatomerocs (HanpMMep, nenoBenecKoro) apMTponosTMHa ηππ τογο, ητο6ν no3BonMTb oSnaflaHMe οαημμ μπμ 6-one M3 6μοπογμηθοκμχ cbomctb nocneflHero, m Te, KOTopbie μμθκτγ cpeflHioK) yrneBOflHyto κομπο3μι * μιο, OTnMHaiomyiocn οτ κομποβμρμμ cbočoaho BCTpenaioinerocn (HanpMMep, neoBenecKoro) apMTponoaTMHa. Κπθτκμ no3BOHOHHbix (HanpMMep, COS-I m CHO), npennaraeMbie b HacTonmeM Μ3θβρβτβΗΜΜ, coflepjKaT nepBbie κπθτκμ, Bcerfla nocTynHbie, KOTopbie Moryr pa3MHO) KaTbcn 14

HenpepbiBHO in vitro m KOTopbie bo BpeMa POCT b KynbType cnocoSHbi npoAyunpoBaTb b cpepe nx POCT 6-one in 1000 U (npeflnosTmenbHO, U 6-one 5000 m HanSonee preferably the, aone 1000 U - 5,000 U) apmponoaTMHa H 106 κπβτοκ 3a nacoB 48, CEC onpeneneHO paflM0MMMyH0Mccnefl0BaHiieM. npepnararaTCfl Taxxe οιητθτμηθοκμθ nonunenTMflbi, nonHOCTbio nnn μθοτμηηο pynnnL (MpyioinMe HenpepbiBHbie nocneflOBaienbHOCTM θμμηοκιίοποτημχ οοτθτκοβ apuTponosTMHa, KOTopbie 3fļecb enepBbie paat ^ Chembe. 3tm nocnepoBaTenbHocTM nyTeM pa3fleneHna nepBHHHbix, BTopMHHbix μπμ TpeTMHHbix CTpyicrypHbix μ κοηΦορμβμμοηημχ xapaKTepncTMK co cbočoaho BCTpenatoiuMMca spmponoaTUHOM MoryT oānaflaTb ānonorunecKoPi aKTMBHOCTblO Μ / ΜΠΜ MMMyHOnOrMHeKMMl1 CBOMCTBaMM COBMeCTHO CO CBOSOflHO BCTpeHaramMMCB npoflyicroM tack, hto ohm MoryT npiiMeHflTbCfl XAX 6μοπογμηθοκμ aīcniBHbie mv \ MMMyHonorMHecKiie 3aMecTMTenn apmponoaTMHa b τθρθπθβτμμθοκμχ m HMMyHonornHecKMX npOLļeccax. Οοοτβθτοτβθηηο, npeAnaratOTCfl MOHOKnoHanbHbie m nonmcnoHanbHbie aHTMTena, BbipačaTbiBaeMbie CTaHflapTHbiMM methods:, KOTopbie MMMyHOpeaKTMBHbi c τβκμμμ nonunenTMflaMM m, preferably the, Taoce MMMyHopeaKTMBHbi co cbočo aho BCTpeMaiomuMca spmponoaTMHOM.

HacTonmee M3o6peTeHne nnniocTpMpyiOT KnoHnpoBaHHbie π OCN epoB ATENE b h octm flHK BMflOBbix Hanan o6e3bHHbi n ηθποβθιο, a τθιοκθ ποπμπθπτμαημθ nocneflOBaTenbHOCTM, ypo6HO BbiBeAeHHbie OTTyfla m npeflCTaBnfltomne, οοοτβθτογβθηηο, nepBMMHyio CTpyiaypHyio ΚΟΗφορΜΘΜΜΙΟ BMROBblX Η3Η3Π 06e3bflHbl Μ ΗΘΠΟΒΘΚβ. ripeflnaraioTca TaiOKe Hobbs 6μοπογμμθοκμ φνΗΚΑΜΟΗθΠΒΗΜΘ Beiaopbi BnpycHofi m KonbueBoii nna3MMf | HOM flHK, oxBaTb (BaioiuMe nocneflOBaTenbHOCTM flHK μ3ο6ρθτθημα, m MMKpočHbie (Further preferably, āaKrepnanbHbie, αροχοκθβμθ m MneKonmaiomne κπθτκμ) opraHM3Mbi xo3flMHa, ycT0MHMB0 Tpab ^ opMnpyeMbie μπμ Tpai ^ eicTMpyeMbie τ3κμμμ BeicropaMM. Οοοτβθτοτβθηηο, npeAnaraiOTCfl Hobbs cnocoSbi ποιήθηκα none3Hbix ποπμπθπτμαοβ, BKAionaioLUMe KynbTMBMpyeMoe BbipamMBaHMe Taxnx Tpaf ^ opMnpyeMbix mu Tpaf ^ eKTnpyeMbix MMKpo6Hbix χο3 * θβ b ycnoBnax, o6nernaioiuMX "pynHOMacLUTa6Hyio aKcnpeccMK) 3K3oreHHbix, Beicrop-Hecymnx nocneflOBaTenbHOCTePi flHK n βμαθπθημθ xenaeMbix πΟΠΜΠΘΠΤΜΑΟΒ H3 nUTaienbHOki cpeflbl, Kn6T0HHblX ΠΜ38Τ0Β ΜΠΜ φρ3ΚΑΜΜ ΚΠβΤΟΗΗΗΧ MeMČpaH. Μ30πημμιο m OMncTKy MMKpo6nanbHo Bbipa > KeHHbix ποπμπθπτμαοβ, npeflnaraeMbix HaCTOfllUMM Μ3θβρθΤΘΗΜΘΜ, ΜΟΧΗΟ OCymeCTBMTb ΠρΜ ΠΟΜΟΙΑΜ TpaflMUMOHHblX CnOCOSOB, BKfiK) HatoiAMX, Further preferably, npuroTOBmenbHbie χροΜθτοφβφΜΗθοκΜθ pa3AeneHMn m MMMyHonorMHecxne pa3AeneHna, coAepxauļne npenapaTbi Moho "noHanbHbix h / μπμ nonnKnoHanbHbix aHTmen.

Pa3bBCHMB nocneAOBaTenbHOCTb θμμηοκμοποτηνχ οοτβτκοβ apMTponoaTUHa, aBTop M30ČpeTeHMH npeAnaraeT ποπηοθ μ / μπμ HacTMHHoe ποοτροβΗΜβ flHK-nocneAOBaTenbHocTefi, KOAnpytomnx apuTponoaTMH m coAepxamnx Tacna SnaronpuBTHbie xapaKTepMCTMKM, XAX βηθαρθημθ koaohob "npeAnoHMTaeMbix" απ "aKcnpeccnn OTOČpaHHbiMM Χ03ΒΘΒ3ΜΜ ΗΘΜΠΘΚΟΠΜΤ3ΙΟΙΑΜΧ, ΟδθΟΠΘΗΘΗΜΘ yHaCTKOB ΑΠΒ ρθΟΙΑΘΠΠΘΗΜΛ ΠΟΟρθΑΟΤΒΟΜ OrpaHMMMTenbHblX 3HA0HyKnea3HblX φΘρΜΘΗΤΟΒ Μ ΟβθΟΠΘΗΘΗΜΘ AOnOnHMTenbHblX HaManbHblX, 15 GB 10506 κοηθηηβιχ μπμ npoMe & KytOHHbix JlHK-nocneflOBaTenbHOCTeii, KOTopbie cnoco6cTByioT nocTpoeHnio πθγκο BbipaxaeMbix eeicTopoB. Οοοτβθτοτβθηηο, npepnaraeicn ποοτροθημθ (m pa3Bmne nocpeflCTBOM οπθμμΦμηθοκογο MyrareHe3a kJJHK h γθηομηομ flHK) ΠHK -nocneflOBaTenbHOCTePi, KopnpyioinMX MMKpo6Hyio sKcnpeccmo ποπμπθπτμαηηχ aHanoroB mv \ npon3BOflHbix apmponoaTHHa, KOTopbie oTniinafOTcn ot οβοΘοαηο Β0ΤρΘΗ3Κ311 (ΜΧ08 φορΜ C TOHKM 3ρθΗΜΗ MfleHTMHHOCTM Μ ΜβΟΤΟΠΟΠΟΧΘΗΜΗ OflHOrO ΜΠΜ 6θΠΘΘ aMMHOKMCnOTHblX OCTaTKOB (Τ .Θ. ΘΗβΠΟΓΟΒ / 1ΘΠΘ14ΜΜ, C0flep > KaLflMX MeHbLIJee KOnHHeCTBO Bcex ocTaTKOB, onpefleneHHbix onn EPO μ / μπμ aHanoroB 3aMemeHnn, b κοτορ ^ χ oflMH μπμ 6-one yciaHOBneHHbix οοτβτκοβ 3aMeHeHbi ppyriiMM ocTaTKaMM, μ / μπμ flononHHTenbHbix aHanoroe, b KOTopbix OflMH HHM Sone aMMHOKncnoTHbix T OB flo č OB H č ο č č č OB OB OB OB OB č č nen nen flo K K flo flo flo flo flo flo OT K μ K K T ο flo flo flo raeMbie Hobbs flHK-nocneflOBaTenbHOCTM βκπιοηβιοτ βοθ nocnepoBaienbHOCTH, npnroflHbie b ο6θοπθηθημμ 3Kcnpeccnn b npOKapnoTHbix μπμ ayKapnoTHbix κπθτιοχ xo3HHHa ποπμπθπτμαηηχ npoflyicroB, MMeiomnx of MeHbUieii mepe nacTb πθρβμηηομ CTpyicrypHOM ΚΟΗφορΜΘΜΜΙΙ H OflHO ΜΠΜ 6θΠΘΘ M3 βΜΟΠΟΓΜΗΘΟΚΜΧ CBOMCTB 3pMTp0n03TMHa, KOTOpbie 0XBaTbiBai0Tcn: / a / flHK-nocneflOBaienbHOCTnMM, npMBeneHHbiMM b Ta6nmxx Vh VI, Unu mx KOMnneMeHTapHbiMM hmtbmm; / B / flHK-nocneflOBaTenbHocTBMM, KOTopbie rn6pnflM3MpyioT (b ycnoBnnx rn6pnflM3auMM, nnniocTpMpyeMbix 3flecb, μπμ 6-one χθοτκμχ ycnoBnnx) flHK κ-nocneflOBaTenbHOCTBM, KOTopbie onpefleneHbi b / a /, b μπμ μχ φρ3ΓΜθΗΤ8χ; m Ic / flHK-nocneflOBaTenbHOCTnMM, KOTopbie βοπθαοτβμθ flereHepaflMM γθηθτμηθοκογο KOflaflon > KHbi rn6pnflM3MpoBaTb? Οοο6θηηο oxBaHeHbi b nacTM / b / γθηομηνθ flHK-nocneflOBaTenbHOCTM, flοytoenen ΜΗφenenenenenHHH ixapapapapapapTOapapapapapapTOTOTO o o o o6666bbbberberber μ μ θθοθθο spθθκοοο MMMoaoaM M μ μ / / / / μ /flflflflflfl Οοο6θηηο oxBaHeHbi b nacTM / c / nocTpoeHHbie JļHK -nocnenoBaTenbHOCTM, KOjqnpyiou4Me EPO, EPO- ^ m parMeHTbi EPO-aHanom HBM flHK-nocneflOBaTenbHocTH Μοιγτ BKnioHaTb KopoHbi, cnoco6cTByiOLHMe TpaHcnnLļHM ΜΗφορΜΒΑΜΟΗΗΟΜ PHK B X03BMHaX 6ecn03B0H0HHblX.

OxBaneH npepnaraeMbiM μ3ο6ρθτθημθμ tot Knacc ποπμπθπτμαοβ, KOTopbitf KOflnpyeTcn HacTflMM ΟΗΚ-κομππθμθητβ b βθρχηιοιο nacTb nocneflOBaTenbHOCTM ΓΘΗΟΜΗΟΜ flHK, ΜΒΘ.ρΜΒΘβΘΗΗΟίί B TaSHHpe VI, Τ.Θ. " Κ0ΜΠΠΘΜΘΗ'Τ3ρΗ0 MHBepTMpOBaHHbie 6βΠΚΜ "

KaK onMcaHO b, Tramontano et al., Nucleic Acids Research, 12, οτρ.5048-5050 (1984).

TaiOKe 0XBaHeHbi μ306ρθτθημθμ Φβρμθαθβτμηθκμθ κομπο3Μαμμ, copļep > Kainne fleficTBeHHbie κοπμηθοτβα ποπμπθπτμαημχ nponyKTOB HacToniflero μ3ο6ρθτθημπ βμθοτθ c npMrofļHbiMM pa36aBMTenflMM, CTMMynnTopaMM μ / μπμ ηοομτθππμμ, no3BonniomMMM co3flaHne 3pMTp0n03TMH0B0pi TepanMM, ΟΟΟββΗΗΟ ΠρΜ ΠΘΗΘΗΜΜ 3ΗΘΜΜΗΘ0ΚΜΧ COCTOBHMM 6θΠΘ3Ηβίί M 6-one ECER, t3kmx θηθμμηθοκμχ coctobhmm, KOTopbie πρΜΒορητ κ χροημηθοκομ ποηθηηομ η eflocTaTO η η octh. npepnaraeMbie nonnnenTHflHbie npoflyKTbi mo > Kho " noMeMaTb "KOBaneHTHoii accounai4neii c o6Hapy > KMBaeMbiM MapKepHbiM BeinecTBOM (e.g. paflMOMapKHpoBaHHbie 16 125-)) flfifl ο6θοπθηθημη peareHTaMU, npuroflHbiMM npn o6HapyxeHMM μ κοπμηθοτβθηηομ onpefleneHUM apuTponoaTMHa b τβθραομ τκθημ μ χμρκοοτηβιχ o6pa3qax, τθκμχ XAX KpoBb μπμ Mona . flHK-npoflyKTbi μοχηο Taxxe noMenaTb o6HapyxMBaeMbiMM MapKepaMM (κθκ, Further preferably, papMOMeTKM m ηθμ30τοπηηθ μθτκμ, Tacna XAX 6motmh) m Mcnonb30BaTb b npoqeccax NHK-rn6pnflM3aunn nnn noKanM3aqMM ποποχθημπ apmponoaTUHOBoro Reha μ / μπμ ποποχθημα ΠΙΟβΟΓΟ pOflCTBBHHOrO ΓΘΗΗΟΓΟ CeMeMCTBa B ΧρΟΜΟΟΟΜΗΟΜ climb ΗβΠΟΒβΚΘ, 06e3bBHbl n flpyrnx I · BMjqax MneKonmaioLHMxcfl. MxTaicxe μοχηο ncnonb30Baib βππ μαθητμΦμ ^ μμμ HapyweHMM apmonoaTMHOBoro reHa Ha J] HK-ypoBHe m ncnonb30BaTb b KanecTBe reHHbix MapKepoe pnn ΜΑβΗΤΡίφΜΚθίΐΜΜ coceflHnx γθηοβ m mx HapyweHmm.

Kax noflpo6HO ormcaHO Bbime, HacTonmee M3o6peieHne κροΜΘ τογο očecneHMBaeT 3HannTenbHbie ycoBepweHCTBOBaHMn b cnoco6ax o6HapyxeHna οπβμΜφΜΗβοκΜΧ οαηοημτθβηχ nonnHyKneoTHflOB Hen3BecTHofi nocneflOBaienbHOCTH β γθτθρογθηηομ κπθτοηηομ μπμ BnpycHOM o6pa3qe, BKmonaiOLneM KpaTHbie opHOHHTeBbie nomiHyKneoTMflbi, β κοτοροΜ: / a / nonyHa (OT mixture of μθηθηνχ 30haob οαηοημτθβνχ nonMHyKnec > THflOB c paBHOMepHO Μ3ΜΘΗΠΙΟΙ11ΜΜΜΟΗ nOCneflOBaTenbHOCTflMM OCHOBaHMM, ΠρΜΗΘΜ KaXflblM H3 yKa3aHHblX BUflOB πβππθτοπ noTeHUManbHO οπβμΜφΜΗβοκΜ KOMnneMeHTapHbiM κ nocneflOBaienbHOCTM οοηοβθημμ, KOTopaa npeflnonoxmenbHO yHHKanbHa απη očHapyxnBaeMoro nonnHyKneoTHfla, / b / npnBH3biBaiOT očpa3eq κ nnoTHOMy cy6cTpaTy, / c / o6pa6aibiBaiOT cy6cTpai, μμθιοιαμμ npMBH3aHHbiM κ HeMy o6pa3eq, αππ ocnačneHMH nanbHetimero npMBfl3biBaHMn κ HeMy nonMHyKneoTMflOB, 3a μοκπιοη θημθμ κθκ c uenbīo rnāpMflH3ai4HM κ nonnHyKneoTHflaM b yKa3aHH0M o6pa3qe, / d / o6pa6oTaHHbiii cy6cTpaT, μμθιοιαμμ npMBA3aHHbiM κ HeMy o6pa3ei4, βρθμθηηο npMBOflflT b conpMKOCHOBeHMe c yKa3aHH0M CMecbio μθηθηνχ 30HR0B b ycnoBMnx, -cnoco6cTByioinMX rn6pMflM3aqMM TonbKO MexAy from / iHOCTbio κομππθμθητθρημμμ nonMHyKneoTMAaMM, m lel οπθΜΜφΜΜθοκιιίί nonMHyKneoTMfl o6HapyxMBaioT μοημτορμηγομ Ha HanMHMe peaKiļMM rn6pnflM3ai4MM Mexqy hmm m nonHOCTbio KOMnneMeHTapHbiM 30haom disposed within yKa3aHH0M ομθομ ΜβΗΘΗΜΧ 30Hfl0B, Kak Α0Κ33ΑΗ0 ΗΒΠΜΗΜβΜ BbICllieM ΠΠΟΤΗΟΟΤΜ ΜβΗΘΗΟΓΟ MaTepMana Ha cy6cīpaTe b noKyce οπθμμΦμηθοκογο nonnHyKneoTnna of cpaBHeHmo c nnoTHOCTbio φθΗ3 ΜβπβΗΟΓΟ MaTepMana, BbrreKaiomeM m ΗβοπβΜΜφΜΗβοκΟΓΟ CBn3biBaHMn MeneHbix 30haob c cy6cTpaTOM.

MeTOflMKM οοοδβΗΗΟ θΦΦθκτμβημ b CMTyaqMnx, AMicryiomMX Mcnonb30BaHMe 64,128,256, 512.1024 μπμ 6-one CMeiuaHHbix nonMHyKneoTMflHbix 30HA0B, μμθιοιαμχ nnMHy ot 17 20 ocHOBaHMM b flHK / flHK, μπμ PHK / PHK, μπμ flHK / PHK-rM6pMflM3aqMnx.

Kax onMcaHo infra, BbiujeyKa3aHHbie ycoBepiueHCTBOBaHHbie μθτοαμκμ HarnnflHO πο3βοπμπμ ΜΑβΗΤΜφΜΜΜροΒ8 ^ κ £ 1ΗΚ-κποηβι, KOAMpytoiiļMe 3ρΜτροπθ3ΤΜΗ o6e3bHH b npeAenax 6μ6πμοτθκμ, nonyneHHOM M3 mPHK noHeHHbix κπβτοκ 8ηθμμηθοκμχ o6e3bHH. Bone κοηκρθτηο mixture of 128 paBHOMepHO M3MeHniOUļMXCn 20-Mer 30Hfl0B, OCHOBaHHblX Ha ΜΗφθρΜΒΙ (ΜΜ aMMHOKMcnoTHOM nocnenoBaTenbHOCTM, BbnreKaiomeM M3 HepeflyiomMXcn φρ3κΐ | ΜΜ senoBenecKoro apMTponosTMHa, Mcnonb30BanM β ΜβτοηΜκβχ rM6pMflM3aqMM κοποημμ αππ 17 E 10506

MfleHTucuKaLiLiMi μ μ o o o o o KM T T T T T T T T pon pon pon π π π π π T T T T pon pon pon pon 000 π π π π π p p p p 000 Flax roro 6-one, npaKTMfca πρμμθηθηm ycoBepiueHCTBOBaHHbix μθτοαμκ HaoToamero μ3ο6ρθτθημα no3Bonnna 6bicipo BbipenuTb τρΜ nono > KmenbHbix KJiOHa m npepenoB 3KpaHMpoBaHMfl 1500000 CTBpnnbHbix πατθη, cocTaBrmomM γθηομηyio 6η6πηοτθκυ nenoeeKa. 3to 6bino βμποπηθηο noopepcreoM Mcnonb30BaHMfl BbiiiieynoMflHyTofi CMeen 128 20-μθγ 30haob βμθοτθ co BTopbiM HaāopOM 128 17-Mer 30Hfl0B Ha οοηοβθημμ 3μμηοκμοποτηογο aHanM3a OTAMHaiomeMCfl HenpepbiBHoPi nocneflOBaTenbHOCTM ηθποβθηθοκογο apMTponoaTMHa.

BbiujeynoMaHyTbie nnniocTpaTMBHbie μθτοαμκμ οοοτθβπβιοτ nepBbiPt n3secTHbiPi EXAMPLE MCn0nb30BaHM51 KpaTHblX CMeiUaHHblX OnnrOHyKneOTMflHblX 30Hfl0B b npoL | eccax flHK / flHK-rn6pMflM3aMMM, HanpaBneHHbix Ha M3onnpoBaHne reHOMHbix κποηοβ MJieKonnTaKDmMX, m nepBbiti M3BecTHbiPi EXAMPLE ncnonb30BaHna ομθομ 6-one ηθμ 32 OniirOHyKneOTMflHblX 30HflOB B Μ30ΠΜρ0Β8ΗΜΜ "flHK-KilOHOB. MHoroHHcneHHbie acneKTbi m npeiiMymecTBa μ3ο6ρθτθημη CTaHyr οηθβμαηβιμμ priH cnei4nanncTOB wheel paccMOTpeHMM cnejoiyK3inero noppoSHoro οπμοθημη Μ3ο6ρβτθΗΜΗ, co3flaioijnero EXAMPLE npaKTMHecKoro πρμμθηθημα μ3ο6ρθτθημη b npeflnoHTMTenbHbix BapnaHTax ero ocymecTBneHMfl. Π0ΡΡ05Η0Ε ΟΠΙ / ICAHME 1 / 1305PETEHM91 B COOTBBTCTBMH C Η30Τ0ΗΙΙ1ΜΜ Μ3θ6ρθΤΘΗΜΘΜ ČblJlM M30JlMp0BaHbl M oxapaicrepM30BaHbi flHK-nocneflOBaienbHOCTM, KOjqnpyiomMe ηβοτμηηο μπμ nonHOdbio nonnnenTHflHyio nocneflOBaienbHOCTb apMTponoaTMHa ηθποβθηθοκογο m o6e3bHHbero bmaob (b AanbHeMiueM ΈΡΟ "). KpoMe τογο, flHK o6e3b? IHbi m ηθποβθιο cyanamide πρθαμθτομ ayKapMOTHOM m npoKapMOTHoii aKcnpecMM, co3AatomeM H30nnpyeMbie κοπμηθοτβθ ποπμπθπτμαοβ, προηβπαιοιαμχ 6μοπογμηθοκμθ (e.g. MMMyHonornHecKne) CBoiicTBa cbočoaho BCTpenaiomeroca EPO, a TaioKe 6μοπογμηθοκμθ θκτμβηοοτμ in vivo in vitro EPO m. flHK o6e3bflHbnx BMpoBbix Hanan BBI / ļennnn M3 ΑΗΚ-6μ6πμοτθκμ, ποοτροθΗΗοίί mPHK, KOTopaa npoM30iuna οτ ποηθμηομ the fabrics o6e3bHHbi b χμμμηθοκμ MHflyunpoBaHHOM aHeMHHeCKOM COCTOHHMM M Hbfl CbIBOpOTKa, Kak yCTaHOBMnM MMMyH0n0rH4eCKMM nyreM, copepacana 6-one βηοοκμθ ypoBHM EPO from cpaBHeHmo c CbiBopOTKoii HopManbHOM o6e3bflHbi. H3onnpoBaHMe Tpe6yeMbix κΑΗΚ-κποηοβ, coflepxamnx EPO-KOflHpyiOLqyio flHK, ocymecTBnfl / iocb nyreM rn6pnflM3aLļMH κοποημμ flHK / flHK c πρμμθηθημθμ Nynas 128 cMeuuaHHbix, paflMOMe4eHHbix, 20-μθγ onnroHyKneoTHflHbix 30Hfl0B, μ βκπιοηθπο SbiCTpoe aKpaHMpoBaHHe 200000 κοποημμ. PacneT onnroHyKneoTMflHbix 30haob 6bin οοηοβαη Ha nH0opMaqnn θμμηοκμοποτηομ nocneAOBaienbHocTM, πρθαοτθβπθηηομ ΦθρμθητθτμβηοΡι ΦΡθγμθητβαμθμ h οπρθαθπθημθμ ποοπθαοβθτθπβηοοτμ μθπομ npoābi ηθποβθηθοκογο EPO. flHK HenoBenecKaa cbiAa BbiAeneHa M3 6μ6πμοτθκμ γθηομηομ flHK ηθποβθκθ. l / l30finpoBaHne kaohob, οοΑβρχοίΑΜΧ EPO-KOAnpytoinyio flHK, ocymecTBnflnn nyTeM ncnonb30BaHna rn6pMAH3aLļnn ānatueK flHK / flHK c πρμμθηθημθμ BbiLueynoMHHyTbix 128 CMewaHHbix 20-Mer onnroHyi < neoTMflHbix 30haob, a TaiOKe BTOporo Nynas 128 panel-MapKnpoBaHHbix 17-μθγ 30Hfl0B, HBN nocnenoBaTenbHOCTM ocHOBbiBanncb Ha MH0opMaumi aMMHOKucnoTHoii nocneAOBaTenbHOCTM, nonyHeHHon M3 omonaioineroca? Rion) KMTenbHbie κοποημμ μ βπηιηκΜ 6binn npoeepeHbi npn πομοιαμ amaookcm-onpefleneHneM nocneAOBaTenbHOCTM KnoHanbHOM JļHKc ncnonb30BaHneM naāopa 16 nocnenoBaTenbHOCTefi b npepenax Nynas 20-μθ 30haob, m OToāpaHHbie KnoHbi 6binn noflBep > KeHbi aHann3y Ha HyicneoTMAHyio nocneflOBaTenbHOCTb c OKOHHaTenbHofi AeAyKi4Men nepBMHHOM CTpyicrypHOM κοΗφορΜβίΐΜΜ ΕΡΟ-ποπμπθπτμποβ, KOflnpyeMbix eio. fleflyqnpoBaHHbie nomineMflHbie nocneAOBaTenbHOCTM nponenM BbicGicyio CTeneHb γομοπογμμ npyr Kflpyry μ k ηθοτμηηομ nocnenoBaTenbHOCTM, reHepMpOBaHHOM nyTeM aHHOKMCnOTHOrO aHanH3a ΗΘΠΟΒΘΗΘΟΚΜΧ ΕΡΟ-φρβΓΜΘΗΤΟΒ. ĪočpapapaHHbiM nonoxnTenbHbifi o6e3b «HMM kJHHK-κποη m OTOčpaHHbifi nonoxcmenbHbiii bl ΓΘΗΟΜΗΝΜ bl SblJlM BHeceHbl B " HenHOHHblM 'flHK-ΒΘΚΤθρ, KOTOpblM 3ΜΜΠΜφΜΙ4Μρ0Β3 E. B E.COli ΜρΜΜΘΗΜΠΜ flflB ΤρβΗΟφθΚΜΜΜ ΚΠΘΤΟΚ K B KynbType. KynbTypHbiii pocT τρθΗΟφθκτΜροΒθΗΗ ^ χ κπθτοκ χο3πμη3 Bbipa3Mncn b npenapaTax, nnaBaiomMx Ha noBepxHocTn KynbTypanbHoPi cpeflbi, copep KainMX no ομθηκθ no 3000 U EPO Ha μπ KynbTypanbHOH jkmakoctm. ·

CneAyiomne npHMepbi npencTaBneHbi c Lļenbīo nnniocTpauMM Μ3θ6ρβτθΗΜΠ μ οοο6θηηο HanpaBneHbi Ha μθτοαμκμ, ocyinecTBneHHbie paHee ρπη μαθητμΦμκθαμμ EPO, KOAMpyiomero "flHK-KnoHbi o6e3bnHbi h ηθποβθηθοκμθ γθηομηνθ KnoHbi Ha μθτοαμκμ, nonyHeHHbie B pe3ynbTaje stom μαθητμΦμκθαμμ, a τθκχθ na nocnenoBaTenbHOCTM, pa3Bmne ομοτθμ 3KCnpeCCHM M MMMyHOnorMHeCKOe ΠΟΑΤΒθρ > ΚΑβΗΜΘ SKCnpeCCMM EPO β τβκμχ ομοτθμθχ.

Eonee κοηκρθτηο. ΠρηΜβρ 1 HanpaBneH Ha ispeneneHMe aMMHOKMcnoTHoPt nocneAOBaTenbHOCTM ΕΡΟ-Φρ3γμθητοβ neoBeKa h nocTpoeHne CMeceii panno-MapKMpOBaHHblX 30HAOB Ha OCHOBaHHH pe3ynbTaT0B 3Τ0Γ0 ene noeneAOBaTenbHOCTeM. (2 IpHMep HanpaBneH rnaBHbiM o6pa30M Ha μθτοαμκμ, BKmoneHHbie β μαθητμΦμκθιιμκ) nono > KMTenbHbix 06e3bnHbMx κΑΗΚ-κποηοβ, m T3Kmm o6pa30M o6ecnennBaeT nonyweHMe ΜΗφορΜ3ΐ4ΜΜ, KacaiotneMcn πθηθημπ XMBOTHbix μ npeABapMTenbHoro aHanM3a panel-MMMyH0MccneA0BaHMn (RIA) cbiBopoTOK XMB0THbix. ΠρΜΜθρ 3 HanpaBneH Ha nonyneHMe κΑΗΚ-6μ6πμοτθκμ, aKpaHMpoBaHMe rM6pMAM3auMM κοποημΡι m npoBepKy nonox < MTenbHbix ΚΠΟΗΟΒ, ΟηρβΑβΠΘΗΜΘ nocnenoBaTenbHOCTM flHK ποποχμτθπβηογο KflHK-KnoHaj a TaiOKe reHepMpOBaHMe ΜΗφορΜΘΚΜΜ nepBMHHOM CTpyKTypHOM ΚΟΗφορΜΘΜΜΜ (aMMHOKMCAOTHOM nocneAOBaTenbHOCTM) ποπμπθπτμαοβ o6e3bHHbero EPO.TlpMMep 4 HanpaBneH Ha μθτοαμκμ, BKmoneHHbie β μαθητμΦμαμροβ3ημθ nono > KMTenbHbix ηθποβθηθοκμχ reHOMHbix kaohob, m očecneHMBaeT, τθκμμ očpa30M, μηΦορμθμμθμ, oTHocmueMcn κ ncTOHHMKy γθηομηομ 6μ6πμοτθκμ, μθτοαμκθμ rM6pMAM3apMM 6nnmeK μ προΒβρκθ nono > KMTenbHbix κποηοβ. ΠρΜΜθρ 5 HanpaeneH Ha onpeneneHMn nocneAOBaTenbHOCTM flHK.nono > KMTenbHoro γθηομηογο KnoHa m reHepiipoBaHMe ΜΗφορΜθρΜΜ βμμηοκμοποτηομ nocneAOBaTenbHOCTM ποπμπθπτμαοβ nenoBesecKoro EPO, BKAtonan cpaBHeHMe c ΜΗφορΜβαηβΜ nocneAOBaTenbHOCTM EPO 19 EN 10506 oāe3bflHbi. EXAMPLE 6 HanpaBneH Ha μθτοαμκμ ηοοτροβΗΜΠ eeKiopa, BKmosaiomero EPO-Konnpyiomyio flHK, npoM30ineflUjyio ot nono) KmenbHoro KflHK-KnoHa o6e3bnHbi, ncnonb30BaHne eeKTopa nnn τρθΗοφθκτΜροΒβΗΜη κπβτοκ COS-I m KynbTypHoro POCT TpaHC0eKTnpoBaHHbix κπβτοκ. ΠρΜΜβρ 7 HanpaBneH Ha μθτοαμκμ απη ποοτροβΗΐιπ Beicropa, βκπκ5Η3Κ3ιι1θγο EPO-KOAMpyiomyK) flHK, npon30Luefliijyra ot nono > KMTenbHoro γθηομηογο KJiOHa ηθποβθκθ, ncnonb30BaHne eeKTopa αππ τρ3ΗθφθκτιιροΒ3ΗΗΗ κπθτοκ COS-I h KynbTypHoro bowl ΤρβΗΟφθΚΤΚρΟΒΘΗΗΜΧ ΚΠΘΤΟΚ. ΠρΜΜβρ 8 HanpaBneH Ha MeTOflMKH HMMyHOHCCneAOBaHHH, npoBOflMMbix Ha HaflocaflOHHbix χμακοοτπχ cpefl, nonyneHHbix M3 KynbTypHoro POCT τpaHcφeκτnpoBaHHblx κπβτοκ b οοοτβθτοτβμμ c h ΠρΜΜβρβΜΜ 6 7 9 ΠρΜΜβρ HanpaBneH Ha 6nonornHecKyKD aKTHBHOCTb in vitro m iri vivo MMKpo6nanbHO BbipaxeHHoro EPO llpHMepoB 6 m 7 ΠρΜΜβρ 10 HanpaBneH H pa3BHTne CHCTeMbi 3Kcnpeccnn xo3nnHa MneKonnTarainnx Ann KflHK EPO bmaob o6e3bHH μ γθηομηομ flHK ηθποβθκθ, BKnioHan κπθτκμ $ MHHMKa KHTaiicKoro xoMHKa (" CHO ") m Ha nMMyHonornMecKyio m 6nonornHecKyio βκτμβηοοτμ npoflyKTOB 3Tnx ομοτθμ sKcnpeccMM, a TaioKe xapaicrepMCTMKy TaKMx npOflyi < TOB. ΠρκΜθρ 11 HanpaBneH Ha nonyHeHne γθηοβ, KOflnpyiomMx EPO ΜβποΒβκβ m aHanom EPO, reHbi KOTopbix βκπιοηθιοτ pnfl npeflnoHTMTenbHbix koaohob jqnn sKcnpeccMM b E.coli h ΑροχοκβΒΗΧ KneTKax xo3BHHa, a TaioKe Ha CHCTeMbi τβκοίί sKcnpeccMM. ΠρΜΜβρ 12 οτηοομτοα k προφΜπηΜ MMMyHonornHeKoii m 6nonornHecKoii aKTMBHOCTeii BbipaxeHHbix npoflyioOB ομοτθμ, πρΜΒβΑβΗΗΗΧ b ΠρΜΜβρβ 11 ΠΡΜΜΕΡ1 A. OnpeneneHMe aMHOKMcnoTHOM nocneaoBaTenbHOCTM φρβΓΜβΗΤβ EPO EPO ηθποβθκθ nenoBeKa βμαθππιοτ M3 MOhm m nojļBepraiOT TpMnTMHecKOMy nepeeapMBaHMio, BbiTeKaioineMy b pa3BMTMe m M3onMpOBaHMe 17 pa3flenbHbix φρβΓΜβΗΤΟΒ b κοπμηθοτβθχ, ηρΜδΠΜΧΘΗΗΟ BbipSOKeHHblX B 100-150 ΠΜΚΟΜΟΠβίί.

OparMeHTaM npoM3BonbHO npMnMCbieaiOT homepa m aHanM3Mpyi0T Ha aMMHOKMcnoTHyio nocneflOBaTenbHOCTb MMKpoaHanM30M c Mcnonb30BaHMeM τβθοφββοΒΟΓΟ ceKBeHcepa (Applied Biosystems) flnn nonyHeHnn ΜΗφορΜθμΜΜ o nocneflOBaTenbHOCTM, npMBeneHHOM b TaānMue 1 ΗΜ > κβ, RNE npMMeHeHbi 6yKBeHHbie KOflbi m "X" 0č03HaMaeT ocrraTOK, He onpefleneHHHbiM 0flH03HaHHO. 20

Ta6nnqa 1

OparMeHT N Pe3ynbTar aHami3a nocnepoBaTenbHocTM 5 T4a T4b appr GKLK T9 ALGAQK T13 T16 VLER AVSGLR 10 T18 LFR T21 T25 KLFR YLLEAK T26a LlCDSR T26b LYTGEACR 15 T27 T28 TlTADTFR EAlSPPDAAMAAPLR T30 EAEXlTTGXAEHXSL- Ν-Ε-Χ-1-TMP T31 ν-Υ-SNFLR 20 T33 T35 SLTTLLR VNFYAWK T38 GQALLVXSSQPW- EPLQLHVDK B. PaoneT h nocTpoeHne CMecefi onnroHyKneoTHDHbix 30ΗΠΟΒ AMMHOKMcnoTHbie nocneflOBaTenbHOCTM, npuBefleHHbie b Ta6nnqe I, paccMaipMBaiOTCfl b CMbīcne pereHepaunu γθηθτμηθοκογο group c uenbīo BbmcHeHMfl τογο, μοχηο πμ npuMeHMTb ΜΘΤΟβΜΚΜ CMewaHHblX 30Hfl0B K ΠρΜΘΜΘΜ flHK / flHK-Ha rM6pMflM3aUMM KflHK μ / μπμ 6μ6πμοτθκ8χ γθηομηομ flHK. 3tot aHanM3 βμαβπηθτ, hto b npepenax (DparMeHTa N T-35 cymecTByeT paper H3 7 aMMH0Kncn0THbix ocTaTKOB (Val-Asn-Phe-Tyr-Ala-Trp-Lys) KOTopbifi mo > kho 0flH03Ha4H0 0XapaKTepM30BaTb ΙΟΚ KOpnpyeMblii OflHOM M3 128 B03M0 > KHblX flHK -nocneflOBaienbHOCTefi, oxBaTbiBatoipeii 20 nap a30TKCTbix ocHOBaHiiii. no3TOMy CMHTe3MpytoT nepBbiPi Ha6op M3 128 20-c μθ onnroHyKJiecmiflOB ncnorib30BaHMeM CTaHpapTHbix φοοφοΒΜΜρκτΗ ^ χ cnocofioB (CMOTpme, Further preferably, pa6oTy Beaucage, et al., Tetrahedron Letters, 22, οτρ.1859-1862 (1981 ) Ha TBeppoM HOCHTene, b οοοτβθτοτβμμ c nocnejqoBaTenbHOCTbio, npnBepeHHoii ημ > κθb Ta6nMqe II.

Ta6nnielis II

OcTaTOK - Val - Asn Phe Tyr Ala Trp Lys 3 'CAA TT AAG ATG GGA ACC TT TAAATGGCC flononHMTenbHbiM aHanH3 BbmBnaeT, hto b npeaenax Φρθγμθητθ N T38 cywecTByeT pm H3 6 aMMHOKncnoTHbix ocTaTKOB (Gln-Pro-Trp-Glu-Pro-Leu), Ha οοη.οβθ κοτοροΓΟ mo > kho nonyHMTb nyn m 128 CMewaHHbix happroHyKneoTMflHbix 17-μθγ 30haob, xax noKa3aHO b Ta6nnqe III ΗΜΧΘ.

TačmiLia III

LeatherTOK - Gln Pro Trp Gly Leu 3 'GTT GGA ACC GGA GA C T T G G C C

OnnroHyKneoTMflHbie 30Hflbi noMenaioT b 5 '-KOHiMaMe - 32ρ_ ^ χρ1 7500-8000 Ci (μμοπ / ICN), Mcnonb3ya nonMHyKneoTMflHyio KMHa3y T4 (NEN). ΠΡΜΜΕΡ2 A.MeTonuKa o6pa6oTKH o6e3bflH m PIA-aHann3

CaMOK Cynomolgus o6e3bflH Macaca fascicularias (2.5-3 κγ, 1.5-2 rona) o6pa6aTbiBaioT nOctIcHoHORA ββθαθημη pacTBopa xnoprnflpaia $ eHnnrnflpa3iiHa (pH 7.0) b fl03npoBKe 12.5 μγ / κγ Ha 1.3 m 5 fleHb. reMaTOKpm KOHTponnpyioT no / no KaxflbiM ββθαθημθμ. Ha 7 peHb, μπμ Kax TonbKO ypoeeHb reMaCe onycKaeTca ημχθ 25% οτ nepBOHanajibHoro ypoBHH, co6npaK) T cbiBopoTky μ ποηκμ nocne ββθαθημη xnoprnApia κθτ3μιίη3 b fl03npoBKe 25 μγ / κγ. CoāpaHHbie Maiepnanbi ηθμθαπθηηο 3aMopaxnBaioT b χμακομ a3crre m xpaHsiT npw TeMnepaiype -70 ° C. 22

B. EPO RIA ans npMMeHflKDT paflnonMMyHonornMeKoe ncneflOBaHue απη κοπμηθοτβθηηογο onpefleneHMB EPO b o6pa3qax β οοοτβθτοτβμμ co cneflyioiHMMM μθτοαμκ3μμ: CTaHflapT spmponosTHHa μπμ HeM3BecTHbifi očpa3eq MHKy6npytoT βμθοτθ c aHTucbiBopoTKoPi β τθηθημθ flByx nacoB npn TeMnepaiype 37 ° C. nocne flByxHacoBoro MHKy6npOBaHMB npoSnpKM c o6pa3qaMM oxnaxAaioT Ha nbAy, αοΘθβπβιοτ apmponoaTMH, noMeneHHbiM Kax 125 | Kaxqaa nccneflyeMaa npo6npKa coqep > KHT 500 mkji HHĶy6npoBaHHoii ομθομ 50 mkji pa36aBneHHoii MMMyHHOM cbiBopoTKM 10,000 cp 125 l-apmponoaTMHa 5 μκπ TpacMnona m 0-250 μκπ nnn CTaHflapTa EPO, mv \ HeM3BecTHoro o6pa3qa, m TaiOKe PBS, 0.1% BSA coAepxamnii flnfl βοοποπηθημβ ocTaBiueroca oātMa. Hcnonb3yeMoii cbiBopoTKOM ββπβθτοβ cnymeHHaa bo ΒτοροΜ προββ KpOBb KpauKa, MMyHH3HpoBaHHoro 1% -HbiM HMCTbiM npenapTOM ΗΘΠΟΒΘΗΘΟΚΟΓΟ ΜΟΜΘΒΟΓΟ 3pMTpon03TMHa. ΚΟΗΘΗΗΟΘ ρ33ΒβΑΘΗΜΘ aHTMCbIBOpOTKM retrieval τ ,κ, HTo6b coqep > KaHMe ^^ I-AidTMHa, οββ33ηηογο c amuTenoM, ηθ npeBbiiuano 10-20% OT ΟβίΙίΜΧ BXOAHblX ΚΟΠΜΗΘΟΤΒ. B o6iqeM, 3T0 COOTBeTCTByeT KOHHHOMy ρ33ΒΘΑβΗΜΙ0 aHTMCbiBopoTKM στ 1: 50000 qo 1: 100000. 125 | -3ρΜΤρ0Π03ΤΜΗ, CBB3aHHblM C ΒΗΤΜΤΘΠΟΜ, OCaXCflaiOT T M O O ne ne ne ne ne BneHMB 150 MKJI Staph A. nocne 40 MWHyTHoro MHKy6npoBaHHB očpa3qbi qempM (α) ynipyio n n aj ļ M M po po po G G G up up po a a a a po bi bi 75 75 75 75 0.75 μπ pacTBopoM 10 mM Tris-HCl, μμθιοιαμμ pH 8, 2, copep / camnM 0.15M NaCl 2 mM EDTA's 0.05% Triton 100-100. npoMbiTbie ocTaTKM OTCHMTbiBaiOT npn πομοιαμ CMeTHMKa Γ3ΜΜ3 — ΚΒ3ΗΤ0Β ΗΠΒ ΟΠρΘΑβΠΘΗΜΒ npoqeHTa CBB3M 125 | -3pMTp0nO3TMHa. KonunecTBa, CBH3aHHbie npe-MMMyHHbiMM cbiBopoCMMM, BbiHmaiOT m βοθχ KOHEHHbix 3Η3ΗΘΗΜΜ ΑΠΒ KOppeKTMpOBKM ΗΘΟΠΘΑΜφΜΗΘΟΚΟΓΟ OCaJKfleHMfl. C0flepxaHHe 3pHTp0n03TMHa ηθμ3Βθοτηβιχ o6pa3qoB onpeflennioT nyreM cpaBHeHMB co CTaHflapTHoii KpMBOvi.

BbnuenpMBefleHHyKD MeTOflMKy πρμμθηβιοτ Ha cbieopoTKe o6e3bHH, nonyseHHOM Bb, β nacTH A, a TaiOKe Ha CbieopoTKe o6pa6oTaHHbix o6e3bHH. OpροΒΗΜ HopManbHofi cbiBopoTKM, KaK onpefleneHO, cocTaBnaiOT npn6nii3MTenbHO 36 μΙΙ / μπ, Torni κάκ απη CbiBopoTKM o6pa6oiaHHbix o6e3bHH OHM cocTaBnniOT ot 1000 ao 1700 mU / mji. ΠΡΜΜΕΡ 3

A. nocTpoeHHe Kl1HK-6n6nnoTeKH o6e3bBH MH0opMaqnoHHyio flHK BbiAennioT M3 ποηθκ HopManbHbix h ηηθμμηθοκμχ o6e3bHH nocpeACTBOM MeTOAHKH c ncnonb30BaHneM TnoqnaHaia ryaHMAHHMn, onncaHHoPi b pačoTe Chirgvvin et al., Biochemistry, 18, στρ.5294 (1979), h nonu / A / + - mPHK OHHmaiOT nyiMe nponycKa ABaxAbi Mepe3.onnro / dT / -qenniono3Hyio xpoMaTorpa0mo Ha κοποηκθ, xax hapcaHO Ha 23 EN 10506 οτρ.197-198 b pa6oTe Maniatis et al., &Quot; Molecular Cloning, A Laboratory Manual " (Cold Spring Harbor Laboratory, Cold Springs, Harbor, .Υ., 1982). 5μ6πμοτθκυ kJUHK οτροπτ b οοοτβθτοτβμμ c BnpoM3MeHeHneM ocHOBHbix μθτοαμκ, M3noxeHHbix b6oTe Okayma et al, Mol. and Celi. Biol., 2, οτρ.161-170 (1982). KntoHeBbiMM μομθητ3μμ npepnoHTeHHbix Tenepb μθτοαμκ HBnHfOTca cnepyioi ^ ne: / 1 / PU C8 ncnonb3yiOT β KasecTBe noflouiBeHHoro Bektop, pa3pe3aioT Pst I m 3aieM cHa6xaiOT χβοοτομ απμηοιο β 60-80 ocHOBaHuPi npn πομοιαμ οπμγο T; / 2 / nepeBapuBaHue Hinc II Mcnonb3yioTfln5: HOW TO USE DT-XBOda οτ οαηογο KOHqa BeKTopa; / 31 ομητθ3 nepeofi hmtm m mpucoeflUHeHne xB0CTa npn πομοιαμ οπμγο ocymecTBnflioT β οοοτβθτοτβμμ c ony6nMKOBaHHOM μθτοαμκομ; / 4 / nepeBapuBaHue BamHI npuMeHatoT pna οτιαθππθημη οπμγο dG-XBOCTa οτ οαηογο KOHua BeKTopa; m / 5 / 3aMeHy PHK-hmtm If flHK npoM3BOflflT b npncyTCTBMM flByx CB83yiouMM 3BeHbeB / GATCTAAAGACCGTCCCCCCCCC ACGGTCTTTA npn TpeXKpaTHOM ΜΟΠίφΗΟΜ ΠρβΒΟΟΧΟρΟΤΒΘ Hafl ΒΘΚΤΟρΟΜ c οπμγο dG-ΧΒΟΟΤΟΜ.

B. Μθτοπμκμ rn6pnnn3aūMM κοποημμ πππ npocenBaHMa κΠΗΚ-6μ6πηοτθκμ o6e3bSH Τρ3ΗθφορΜΜροΒ3ΗΗΝβ E.coli pa36pacbiBaiOT c nnoTHOCTbio 9000 κοποημμ Ha 10x10 cm nnTaTenbHbix HauiKax, coflepxaiflMX 50 μκγ / μπ aMnMUMnnMHa. Genescreen $ nnbTpbi (New England Nuclear Catalog N NEF-972) npepBapHTenbHO yBnaxHniOT If BHI-CAM naiuKe (37 r / n 6aKToaKCTpaKTa M03r / cepflue, 2 r / n kmchot Casamino m 15 r / n arapa, coflepxamero 500 μκγ / μπ ΧΠ0ρ3ΜφΘΗΜΚ0Π3) M HCn0nb3yK) T flnn nOflHflTMB ΚΟΠΟΗΜΜ C HaiUKM. ΚΟΠΟΗΜΜ BbipaiiHBatOT B τοΡί xe cpepe β τθηθημθ 12 nacoB μπμ απη θμππμΦμμμροβθημπ ημοπθηηοοτμ nna3MMflHOM ΚΟΠΜΜ. 6ίίΜρΟΒβΗΗΜΘ ΚΟΠΟΗΜΜ (6oKa κοποημμ) o6pa6aTbiBa »OT cepMMHO, noMeiflan Φμπβτρβι Hafl flByMn KycKaMM 3 MM BaTMaHa, HacbiifleHoro cneflyioifMMM pacTBopaMM: / 1/50 mM rniOKa - 25 mM Tris-HCl / pH 8.0 / -10 mM EDTA / pH 8 / -10 mM EDTA / pH 8, 0 / β TeseHMe 5 MMH / T. / 2 / 0.5 M NaOH β τθμθημθ 10 MMHyT, m I. / 3 / 1.0 M Tris-HCl / pH 7.5 / β τθηθημθ τρβχ MMHyT.

OnnbTpb 3aTeM BbicyuMBaHDT b BaKyyMe β τθηθημθ flByx nacoB npM TeMnepaType 80 ° C. 3aTeM φMΠbτpbl noflBepraiOT nepeeapMBaHMio npoTeMHa30M K nepe3 o6pa6oTKy pacTBopoM, coflepxaiflMM 50 μκγ / μπ npoTea3Horo φβρΜβΗΤ8 b Eyφepe K [0, IM Tris-HCl / pH 8.0 / - 0.15M NaCL -10 mM EDTA / pH 8.2 / - 0 , 2% SDS. KoHKpeTHO, ηοββΒΠΠίοτ κ KaxflOMy φMΠbτpy 5 μπ pacTBopa m nepeBapMBaHMe npoflonxaraT npM TeMneparype 55 ° C β τβπβΗΜβ 30 MMHyT, nocne nero pacTBop yflannioT.

OMnbTpb 3aTeM TbBiOn 4 μπ 6yφepoM npeflBapMTenbHOM rM6pMflM3aflMM 24 (5xSSPE - 0.5% SDS -100 μκγ / mhSS E.coli flHK - 5xBFP). npeflBapnTenbHyio rn6pMflM3aunHHyK) o6pa6oTKy ocymecTBnniOT npn TeMnepaType 55 ° C, κθκ npaBuno, b τθηθημθ 4 nacoB μπμ 6onee, nocne wero pacTBop yAanntOT. (Ipopecc rn6pMflM3ai4MM προΒορητ cneflyioiyMM o6pa30M. B XAX < ABIM 0nnbTp AoSaBnntOT 3 μπ rn6pnflM3aMMOHHoro (5xSSPE - 0.5% SDS-100 μκγ / μπ αΡοχοκθβομ τΡΗΚ) copeparamero 0.025 πμκομοπθμ XAX < AOM m 128 nocneflOBaTe / ibHOCTeit 30haob, npeflCTaBneHHbix b Ta6nmie II (nonHan mixture of o6o3HaneHa κάκ mixture of ERY), μ Φμπβτρη noflflepjKMBatoT npn TeMnepaType 48 ° C for τθηθημθ 20 nacoB. 3ia TeMnepaiypa Ha 2 ° C HM > xe, ΗΘΜ ΗΜΧΗΘΘ M3 3Η8ΗβΗΜΜ TeMnepaTypbl flUCCOLlMaUMU (Td) OnpefleneHHOii flnfl ΠΙθ6θΓΟ H3 30HA0B. Πο OKOHHaHMM rn6pMflM3ai4MM 0MnbTpbl iipOMblBaiOT TPM pa3a B ΤΘΗΘΗΜΘ 10 ΜΜΗ / Γ H KananKe paeTBOpOM 6xSSC - 0.1% SDS πρκ κομηβτηομ TeMnepaType m npoMbieaioT OTflByx ao τρβχ Pa3 pacTBopoM 6xSSC and 1% SDS wheel TeMnepaType rn6pMflM3auMM (48 ° C). ABTopafliiorpacļMeM φΜΠΒτροε ΒΜηΒπηκττ ceMb & gt nono KMTenbHbix κποηοβ cpeflii npoceaHHbix 200000 κοποημμ. AHanii3 HaH anbHofi nocneflOBaTenbHocm οηηογο M3 μημμμχ κΠΗΚ-κποηοβ o6e3b "HBI (o6o3HaHeHbi κοκ κποη 83) προΒΟΑητ ρπη npoeepKM peneii nyTeM βμαομ3μθηθημλ μθτοαμκμ, onMcaHHOM VVallace et al., Gene, 16, στρ.21-26 (1981). Kopone, flHK-nna3MMfly M3 KflHK-KnoHa 83 o6e3bHHbi nnHeapn3YiTeMeNeMeMaMMM EcoRI m AeHaTypnpyioT nocpepcTBOM HarpeBaHMH b BaHHe c KMnnmeii boaom. HyKneoTMflHy (0 nocneflOBaienbHOCTb is a peptideT flHfleoKCH-cnocoα, McaHHbiM Sanger et al., PNAS (USA), 74, pp.5463-5467 (1977). peaia (M8 nocneflOBaTenbHOCTePi. C. OnpeneneHHe κΓΙΗΚ-nocnenoBaTenbHOCTeM EPO o6e3bnHbi AHann3 HyKneoTMnHbix nocneflOBaTenbHOCTePi KnoHa 83 ocymecTB / ifliOT nocpeACTBOM MeTOflMK, onncaHHbix Messing, Methods in Enzymology, 101, crp.20-78 (1983). B Ta6nnqe IV npeflCTaBneHbi pe3ynbTaTbi aHann3a KapTbi npeflBapmenbHoro orpaHMMeHMn απα Ecorl / Hind / lll κποημροβθηηογο φρβΓΜθΗΤθ KnoHa 83 c npn6nn3MTenbHO 1600 napaMna30TMCTbix ocHOBaHHM. npn6nn > KeHHbie μθοτοποποχθημβ ynacTKOB pacno3HaBaHMA orpaHMHMTenbHbix 3HA0HyKnea3Hbix φβρΜθΗτοΒ BbipaxeHbi HMcnoM ocHoeaHMM 3 'κ yHacTKy Ecorl b 5'κοηαθ φρβΓΜθΗΤΒ. OnpepeneHMe HyicneoTMAHOM nocneAOBaTenbHocTM ocymecTBnnioT ηθχοχαθημθμ nocneA OBaTenbHocTM 0TAenbHbix orpaHnmiTenbHbix Φρ3γμθητοβ, c uenbto cnapMBaHMfl nepeKpbiBatomnxcfl Φρθγμθητοβ. HanpMMep, nepexpbiBaHMe ΜΗφορΜ8ΑΜΜ nocneAOBaTenbHocTM, npeACTaBneHHoe aHanM30M HyxneoTMAOB b orpaHMHMTenbHOM φρθΓΜβΗτβ, oč03HaHeHo 0113 (SAU 3A dial ~ lll / Smal NPM -324), a οπρθαθπθημθ nocneAOBaTenbHocTM c očpambiM nopapKOM o6o3HaHeHO C73 (Alula NPM -424 / -203 Bstll NAT). 25 LV 10506

Ta6nnLia IV ynacTOK pacno3HaBaHMf npn6nn3MTenbHoe OrpaHMMMTenbHOrO φβρΜΘΗ3 Haxox < fleHne / fl / 5 Eco Rl 1 Sau3A 111 Sma I 180 BstE II 203 §m I 324 10 Cleft 371 Ra 372 Alu »424 Estl 426 Alu I 430 15 Hpa I 466 Alu I 546 EAST 601 Pvu II 604 Alu I 605 20 Alu I 782 Likes 788 Rsa I 792 Be I 807 Ael I 841 25 Alu I 927 No I 946 Jan 3A 1014 Alu I 1072 Ali I 1115 30 Alu I 1223 Be I 1301 Bsal 1343 Alu I 1384 Price III 1449 35 Alu I 1450 Price III 1585

OnpejqeneHne nocneAOBaTenbHocTM npn6nM3iiTenbHO 1342 nap a30TMCTbix οοηοβθημη (b npepenax o6nacTM ot ynacTKa 3'flo ynacTKa E corium m ynacTKa Hind III / m aHann3 Bcex B03M0) KHblX CIICTBM CHMTblBaHMfl Cn0C06CTByK) T pa3BTWO flHK Μ ΜΗφθρΜ3Ι4ΜΜ ΒΜΜΗΟΚΜΟΠΟΤΗΟίί nocnepDBaTenbHOCTM, npeflCTaBneHHofi b TaSnupe V. B TaSnuMe, MHMMbifs nepBOHaManbHbiM aMMHOKMCnOTHblii 00Τ3Τ0Κ aMMHOOKOHHaHMH co3peBwero EPO (xax npoBspeHO nyTeM Knock-on HFMHFHyrorc > aHarM3a nocneflOBaienBHOCTM flBaflLelain ocrraTKOB? npncyTCTBne onpepenaioinero μθτμοημη ATC-KOflOHa (o6o3HaMeH -27) " HanpaBneHHoro προτΜΒ τθηθημη "κοηθμηογο a / iaHUHOBoro ocTaTKa nepBOHananbHoro aMMHa, XAX nepeoro ocTaTKa, o6o3HaMeHHoro flnfl aMHHOKMCnoTHOii nocneAOBaTenbHocTM co3peBuiero 6enKa, ββπηθτοη noKa3aTeneM npaB / ļonofloSufl τογο, whereby to EPO npeBOHananbHO BbipaaceH b qnTonna3Me b φορΜΘ npefltuecTBeHHMKa, BKmonaiomfi 26 ”BeflyinyK3” o6nacTb M3 27 aMMHOKMcnoTHbix ociaTKOB, «oTopan Bbipe3aeTCfl jno βχοκαθηιία co3peBLuero EPO b KpoBoo6pameHne. Υηθοτκμ noieHMuanbHoro rnnK03nnnpoBaHna b npeflenax nonunenTupa o6o3HaHeHbi 3Be3fl04KaMn. BbiHncneHHbif MoneKynapHbiiiEcoSnacTH TpaHcnaLlMM cocTaBn ^ eT 21117 flanbTOH, a MoneKynnpHbiM Bec 165 ocraTKOB nonunemufla, cocTaBnfiHDinMx co3peBuinfi EPO6e3bBHb, paBeH 18236 flanbTOH.

Toggle V

TpaHcnaunn kDHK EPO o6e3b3Hbi

Jan 3A

GATCCCGCGCCCCCTGGACAGCCGCCCTCTCCTCCAGGCCCGTGGGGCTGGCCCTGCCC

CGCTGAACTTCCCGGGATGAGGACTCCCGGTGTGGTCACCGCGCGCCTAGGTCGCTGAG -27 -20

Met Gly Or His Glu Cys Pro Trp GGACCCCGGCCAGGCGCGGAGATG GGG GTG CAC GAA TGT CCT GCC TGG -10

Leu Trp Leu Leu Leu Ser Leu Or Ser Leu Pro Leu Gly Leu Trp CTG TGG CTT CTC CTG CTC CTG CTG CTC CCT CTG GGC CTC CCA -1 +1 10

Or Pro Gly Ala Pro Pro Arg Leu Cys Asp Ser Arg Or Leu GTC CCG GGC CCC CCA CGC CTC ATC TGT GAC AGC CGA GTC CTG 20

Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Or Thr Met GAG AGG TAC CTC TTG GAG GCC AAG GAG GCC GAG AAT GTC ACG ATG 30 * 40

Gly Cys Ser Glu Ser Cys Ser Leu Asn Glu Asnille Thr Or Pro GGC TGT TCC GAA AGC TGC AGC TTG AAT GAG AAT ATC ACC GTC CCA 50

Asp Thr Lys Or Asn Phe Tyr Field Trp Lys Arg Met Glu Or Gly GAC ACC AAA GTT AAC TTC TAT GCC TGG AAG AGG ATG GAG GTC

Gln Gln Area Or Glu Or Trp Gln Gly Leu Lower Leu Leu Ser Glu CAG CAG GCT GTA GTC GGG CAG GGC CTG GCC CTG CTC TCA GAA 80

Ala Vai Leu Arg Gly Gln Lower Or Leu Lower Asn Ser Ser Gln Pro GCT GTC CTG CGG GGC CAG GCC GTG TTG GCC AAC TCT TCC CAG CCT 90 100

Phe Glu Pro Leu Gln Leu His Met Asp Lys To Sub Ser Gly Leu TTC GAG CCC CTG CAG CTG CAC ATG GAT AAA GCC ATC AGT GGC CTT 27 EN 10506

Ta6nwua V / nponon > KeHne / 110

Arg Serle Thr Thr Leu Leu Arg Lower Leu Gly Lower Gln Glu Lower CGC AGC ATC ACC ACT CTG CTT CGG GCG CTG GGA GCC CAG GAA GCC 120 130

Ser Leu Pro Asp Ala Ala Ser Ala Ala Field Pro Leu Arg Thrle ATC.TCC CTC CCA GAT GCG GCC TCG GCT CCA CTC CGA ACC ATC 140

Thr Ala Asp Thr Phe Cys Lys Leu Phe Arg Or Tyr Ser Asn Phe ACT GCT GAC ACT TTC TGC AAA CTC TTC CGA GTC TAC TCC AAT TTC 150 160

Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Arg CTC CGG GGA AAG CTG AAG CTG TAC ACG GGG GAG GCC TGC AGG AGA 165

Gly Asp Arg OP

GGG GAC AGA TGA CCAGGTGCGTCCAGCTGGGCACATCCACCACCTCCCTCACCAACA

CTGCCTGTGCCACACCCTCCCTCACCACTCCCGAACCCCATCGAGGGGCTCTCAGCTAAG

CGCCAGCCTGTCCCATGGACACTCCAGTGCCAGCAATGACATCTCAGGGGCCAGAGGAAC

TGTCCAGAGCACAACTCTGAGATCTAAGGATGTCGCAGGGCCAACTTGAGGGCCCAGAGC

AGGAAGCATTCAGAGAGCAGCTTTAAACTCAGGAGCAGAGACAATGCAGGGAAAACACCT

GAGCTCACTCGGCCACCTGCAAAATTTGATGCAGGACACGCTTTGGAGGCAATTTACCTG

TTTTTGCACCTACCAT CAGGGACAGGAT GACTGGAGAACTTAGGTGGCAAGCT GT GACTT

CTCAAGGCCTCACGGGCACTCCCTTGGTGGCAAGAGCCCCCTTGACACTGAGAGATATT

TTGCAATCTGCAGCAGGAAAAATTACGGACAGGTTTTGGAGGTTGGAGGGTACTTGACAG

GTGTGTGGGGAAGCAGGGCGGTAGGGGTGGAGCTGGGATGCGAGTGAGAACCGTGAAGAC

AGGATGGGGGCTGGCCTCTGGTTCTCGTGGGGTCCAAGCTT

Price Hl 28

ΓΊοπμπθπτμαηθα nocneooBaienbHOCTb b Ta6nm4e V μοχθτ 6biTb 6e3 Tpyfla noflBepmyra aHann3y Ha npncyTCTBne Hpe3BbiHafiHO rnflpo0nnbHbix oānacTefi n / nnn BTOpMHHbix KOH $ OpMaUMOHHblX XapaKTepMCTMK, ΑΒΠΗΙ011 | ΜΧ0Η nOKa3aTeneM Β03Μ0ΧΗ0 OHeHb MMMyHOreHHblX o6nacTeii, Further preferably, npn πομοιιιμ cnocoSoB, npenno > KeHHbix Ηορρ βτ al., P.N.A.S. (USA), 78, CTp 3824-3828 (1981), m. Kyte et al., J. Mol.Biol., 157, cTp.105-132 (1982), μ / μπμ Chou et al., Biochem. , οτρ.222-245 (1974), Advances in Enzymology, 47, op. 45-47 (1976). BACKGROUND OF THE INVENTION BACKGROUND OF THE INVENTION BACKGROUND OF THE INVENTION c cococoOmM Ηορρ βτ al. B03M0XHa 6narojqapfl nporpaMMe, o6o3HaHeHHiii PEP Reference Section 6.7, npeflCTaBneHHoPi Intelligenetics, Inc., 124 University Avenue, Palo Alto, California. ΜΕΡ / 1ΜΕΡ 4 A. ΓeHOMHaa 6NOnNeNeNaNaNe

ΓeHOMHyio 6n6nnoTeKy HenOBesecKOM Φθτθπβηομ πθμθημ, Hecymeii φβΓ AHC 4A, nonyHeHHyto b οοοτβθτοτβμμ c μθτοαμκβμμ Lown et al., Cell., 18, οτρ.533-543 (1979) coxpaHHiOT απη ncnonb30BaHMfl b MccneflOBaHMM rn6pnflM3auMM SnaujeK B. Μθτοπμκμ rn6punn3aunn SnameKDna npocenBaHHa γθηομηομ 6μ6πμοτθκμ nenoBeica ΦaΓOBbιe HacTULļbi nopBepraioT nn3ncy m flHK φΜκα ^ ιοτ Ha φκπΒτρβχ (50000 βπΗίυβκ Ha φΜΠΒτρ) b cooTBeTCTBMM c ΜβτορΜΚθΜΜ, onMcaHHbiMH Woo, Methods In Enzymology, 68, CTp.389-395 (1979), 3a ΜΟκπιοΗβΗΜβΜ κβκρπη ncnonb30BaHMfl GeneScreen Plus φΜΠΒτροΒ (New England Nuclear Catalog N NEF-976) μ ne HarneK (NaCl, 5r; MgCl2 -6H20.2r; NZ-Amhh A, 10r; ρροχχθΒοίί aKCTpaicr, 5 r; KMcnoTbi casamino, 2 r; ManbT03a, 2 r; arap, 15 r If flHTp).

BbicyiueHHbie Ha B03pyxe φι ^ ^ τρ o6xnraioT wheel TeMnepaType 80 ° C for 1 τβΗβΗΜβ Match m 3aTeM nepeBapMBaiOT ΠροτβΜΗθβοίί K, XAX onMcano b ΠριίΜβρβ 3 nacTb B. npeflBapnTenbHyK) rn6pnflH3aunKD ocymecTBnatoT c ncnonb30BaHneM IM NaCl and 1% SDS -6yφepa wheel TeMnepaType 55 ° C b τβΗβΗΜβ 4 nacoB mvi 6onee, nocne nero 6yφep yAanaK) T. rn6pnflM3aMMOHHbie m nocTrn6pMiLH3ai4HOHHbie npoMbiBKH ocymecTBnH10T Also < OnMCaHO B npMMepe 3, HacTb B. Πρμμθηηιοτ xax CMecb 128 20-Mer 30HflOB, oO3HaHeHHbix EPQ, Tax mccb 128 17-Mer 30H, qoB Ta6nnubi III (oO3HaHeHa xax CMecb EPQ). fn6pnAH3aunKD ocymecTBniiK) T-Tile TeMnepaType 48 ° C c ncnonb30BaHneM CMecH 30heap EPV. rn6pnflH3aLmMc ncnonb30BaHneM CMecH 30HA0B EPQ ocymecTBnaK) T-Shape TeMnepaiype 46 ° C, T.e. Ha 4 rpajļyca Hnxe HaMMeHbLueH BbiHMcneHHOM Td απη ηπθηοβ CMecH. yflaneHne rn6pnAM3npoBaHHbix 30haob fl / ΙΗ ΠΟΒΤΟΡΗΟΜ rn6pMflM3aL (MM BbinOflHfltOT nyTeM ΚΜΠΗΗβΗΜΗ BI xSSC - 0.1% SDS Β ΤβΗβΗΜβ flByX MHHyT. ΑΒΤ0ρ3ΑΜ0Γρ3φΜΗ φΐΙΠΒΤρΟΒ BblflBnfleT ΤρΜ nonOXMTenbHblX ΚΠ0Η3 (c aKTMBHblX EN 10506 29 οΘθμμμ CMEC 30Hfl0B) cpeflM npoceflHHbix 1500000 $ aroBbix 6nfliueK. npOBepKa nono > KMTenbHbix κποηοβ Ha EPO-KOflupoBaHue jļocTuraeTcn nocpencTBOM onpe ^ eneHna nocnenoBaTenbHOCTM flHK μ θπθκτροηηομ MMKporpa0nMecKoii BH3yann3ai4nn I · reTepoflyrmeKCHCM (ļjopMauMM c KflHK o6e3b "HBI npur / epa 3 3Ta MeTOflUKa TatoKe naeT flOKa3aTenbCTBO npncyTCTBnn KpaTHbix mhtpohob b γθηομηομ flHK-nocneflOBaTenbHocm ΠΡ1 / ΙΜΕΡ 5

OcyujlecTBnflKDT aHanM3 HyKneoTMflHbix nocneflOBaTenbHOCThe nonoxcnTenbHbix κποηοβ (o6o3HaHeHbi λΙιΕΙ), m pe3ynbTaTbi, nonyHeHHbie Ha ceroflHflUJHMM oeHb, npMBeneHbi b Ta6nmie VI.

AAGCTT CT GGGCTT CCAGACCCAGCTACTTTGCGGAACT CAGCAACCCAGGCATCT CT GAGT CTCCGCCCA O O O Ι < 0 oo 0 OOO (5 o &o; o o o 0 < oo oo 0 < 0 0 0 ooh— 0 - 0 0 < 0 0 0 0 < o ooooo 0 I- & lt 0 0 0o < 0 < o < ooo 0S ooooo 0 < 0 < 0 0 0 OOO 0 0 0 < o oo < 0 O 0 OO o H oooo Ι Ο Ο < o 0 l · o 0 0 OO < < o 0 O 0 (- 0 0 0 < < 0 0 OOOO < 0 HOO 0 ϋ OO o 0 I- oo H 0 O 0 0 < o o o o o o o 0 o 0 0 0 oooo o o o o o o o 0 oo oo 0 < o 0 < o 0 Ι Ο I— 0 ο < 0 <: ο < ο < ο 0 ο S 0 ο 0 ο οοοο ltοοοοοοοοοο ltοοοοο οοοοΙΙΙ 0 < ο < ο < ο 0 ο < ο Ι Ο ο ο ο < 0 ο 0 0 Ι ο ο ο ο ο ο 0 0 5 ο ο ο 0 ο 0 ο ο < ο < 0 ο ο Ι Ο 0 Ι Ο Ο X 0 X ο ο ο 0 ο 0 ο ο < ο ο 0 0 0 0 ο ο 0 0 0 ο ο 0 < 0 0 ο ο ο υ ο — 0 Ο 0 ο 0 ο ο < ο 0 ο ο 0 ο 0 0 < 0 I- < 0 0 0 ο οοοοοο 0 < 0 ο 0 ο ο < ο 0 Ι Ο ο ο 0 0 Ι 0 0 0 0 Ι ο ο ο ο 0 0 ο ο ο ο Ι ο ο 0 ο ο 0 < ο < 0 0 ο

CNJ 0 0 0 Η- 0 0 0 0 0 < 0 0 0 0 0 5 0 0 ο ο ο ο < 0 0 < < ο Η 0 Η ο 0 0 ο ο ο ο 0 ο 0I < 0 0 0 0 ο 0 < 0Ε 0 ο ο 0 ο < ο 0 I - 0 0 £ > 0 0 0 ~ 0 ω Η-& < 0 < 0 0 ο 0 < < ο ο 0 0 ο ο ο ο < 0 0 0 < 0 Ι 0 ο 0 5 ο ο ο ο 0 ο 0 ο 0 0 ο ο < 0 I— 0 0 ο ο ο to >

h- CM I 0 0 0 ο ο 0 0 ο 0 0 ο υ ο Η- ο 0 ο 0 0 0 Ι 0 0 0 0 ο 0 υ ο Č 0 < 0 Η 0 ο < 0 ο 0 0 ο ο < 0 0 5 ο 1 = 0 0 0 Ι Η 0 0 0 0 0 δ 2 ο ο 0 0 1 = LV 10506 ω Ο ο h- Ο Ι — I - 0 < Ο I - 0 0 < 0 Ο < 0 0 $ 0 0 < Lt < 0 0 0

Ta6nnua VI / προποπχθημθ / 0 Ο Ι Ο 5δ ο < ο 0 Ι Ο Ι Ο 0 Ι Οδ 0 0 0 0Ε 0 0 < 0 0 < 0 0 0 0 0 0 Η < Ο 0 0 0 0 0Ε 0 < Ο Ο 0 I— 0 < 0 I— Ο Ο < Ο 0 < ο 0 0 < 0 < Ο ο 02 5 ο I- < 0 < ο < ο 0 ο < ο < ο 0δ Ι Ο Ι Ο < 0 ο Ο ο 0 ο ο ο ο ο ο ο ο Ι ί ί ί = ο ο ο < ο ο 0 < ο < ο ο ο ο I— 0 Η- ο 03 0 03 0 0 0 0 0 I— 0 ο < 0 < 0 < ο 0 0 0 0 I - ο < Ν 3 0 Φ I - 1 Ο ^ ο ω ο ω 1- 0 ΙΟ ο οΕ 3 0J δ £ -0 ι - § ο < 0 | ω > 0 ο Η < ο ΙΟ Ε? 0 < ο 2 ο 0- ο 2Ž 0L Ο ο ^ ro Ο + < 0 ν ^ 0 '0 0 3 0 ω I - 1 Ο ο φ 3 φ

Ο CM ¢ 0 3 < CHILDREN 0 < »Ο I— 0 ο < υ υ χ Ο 3 Ο Φ I - 1 Ο >, 0 ο ro > 0 0 h ο ο ο ο 0 0 Ι Ο ο 0 < ο 0 3 0 β δ ο- Ο 3 Ο ^ δ ο ω Ο V ω η 30 55 φ ο I- < Ι C (0 0 3 < 0 Ο | ο 0 3 < ο 0 0 C0 < ί * ^? ≪ ο co lt < 0 η ® < 0 0 3δ5 δ vy 0 Ε * 0 & lt < 0 2 < Ο 0 3 < 25δ _ ο to I > 0 _ < Ε? 0 < Ο L. Ο ω 0 ω < ϋϋ < η < < 0 Η < 0 < Ο Ο ο ο 0 0 0 0 < ο 0 0 0 < 3 0 0 < ο ο 3 < ο < ο ο δ < ο < ο 0 < ο ο ο ο

(Ο CN ο ο ο ο < 0 < 0 I - 0 _ 0 -C Ο I < ο Η- 0 < f < < < < ο ο 0 I— υ < ο < 0 ο I— ο 0 3 0 0 0 δ 0 < 0 0 Η 0 0 0 0 Ε 0 0 δ ο < ο 0 0 Ι ο < < 0 0δ ο

TGGTGGCCCCAAACCATACCTGAAACTAGGCAAGGAGCFFFGCCAGCAGATCCTACGCCTGTGGGCCAGGG < 5 Tačnyua VI / προποιόκθημθ / 2 ^ O § J2 o w < 0 < Λ let ^ 0 Ο H O > 0 0 0 < D O < 0 < o E < o o ΙΟ I - 0 I - o 0 0 0 o o o h - o 1 = o o o < 0 CD CD < o

CN!? »≪ I-P ω O < < _ let > 0 O >.

U.O. O H < 0.0 (Λ < < 0 2 6 to to to c c Ι — — — ο ο ο ο c (Λ < lt Ο - < 0 0 o 0 < 0 < oo Η < < 0 1 = φ 0 Ο < ι Ο g g < 0 < ζ X ο c V < ο φ 0 < 0 I— 0 0 ιο ^ < m 0 0 ~ Ο Φ Η I < 0 Ε > 0 < -I < - 0 9-0 I--ο to to 10 < 0 ο 0 0 < 0 lt < 0 < 0 < ο 0 < ο 0 < 0 0 lt < < 0 0 0 < < 0 0 0 0 0 ο I- < 0 Η $ 0 < 0 0 0 ί 0 I- < 0 0 < 0 I - ο ο 0 < 0 ο 0 Η I - ο ο ο 0 0 1 = ο 0 < 0 0 Η < 0 < 0 Ο 0 0 Η < 0 < 0 Ι 0 0 < ο ο ο ο ο 0 ο < ο 1 - ο 0 0 < ο 0 ο 0 0 0 μ_ ο ο 0 < ο ί ο Η Η <Ϊ; Ο ο ο ϊ 0 < 0 ί5 ί < Ο 0 < Ο 0 0 ß ο ο ο lt ο ο ο ο ο $ 0 < 0 I - 0 < I < ο 0 < ο 0 0 ' s < 0 < ο ο ο < 0 < ο Ε Ε 0 < 0 < 0 0 $ 0 0 < ο ο 0 < 0 ο 0 ο Η < 0 0 < 0 0 0 ο 0 0 < 0 Ι Ο 0 0 2 0 0I 0 < ο ο ο I— 0 < I— 0 0 I— 0 0 < Ε ο 0 ι— 0 0 0 3 0 I - 0 < Ο ΙΟ I— 0 Η * Ο Ο Ο 0 0 < 0 I to 0 < 0 < Lt < 0 I to 0 < Ο I— ο ο 0 < ο ο 5 < ο 0 Ι Ο < ο ο < ο < ο I- < 0 I— 0 Ι Ο 0 < 0 Η 0 < ο 0 Ι Ο 0 0 < 0 5 (3 s < 5 < ο < οΕ ο < i i 0 0 η 0 δ ο 0 0 ω 0 I - LV 10506 Ta6rmua VI / προπογρκθημθ / Ο Ο ο ο (- CD Ο £ Lt ο < ο Ι Ε Ε ο ο ο ί ί ί ί ο ι ι ι ο ο δ $ ο5 ο < ο < ο £ < ο £ < ο I - ο < ο ο Ι Ι Lt ο ο ί ο < ο ο — ο < ο < ο ο ο ο ο ο lt ο ο ο ο lt lt ο ο ο ο lt < ο < ο f—. ο < ο < ο οοοο ltοοοl ο < 3 ο οοοο lt ο ο ο ο ο οοοοοοοο £ ο I-03 03 < Ο - 5 ο§ u Ο2g ο 3 < 2 i3o η lt < π £ 23δ ο; ο ο-lt lt lt lt ο ο C3 2-0 Η- I- _ Ο > Ο 3 < ϊ Ο CD 51 ° ο - Ο «D < Ο § Ο Ο (Ο Ο ο ο g (Ο Ο (((((8 8 8 8 8 8 8 8 ο - < ο ο ο ο ίο Ο η ° Ū- Ο ^ Ο ®QU) c Ο. ι_ ί

ω W ο 00 V} Ο Η Ο _ Ο 03 h- > ο 3 Ο ω Η -J Ο Ο 03 Ο < Ο _ ο - < ο ο .ο Ο ο Ο) ^ £ ο < ο 3 Ο 03 > Ι Ο ο ί- Ο ο ο < ο οοοο οοοΗοοοοοοοοοοοο = οοοο = ο < ο < ο ο ο ο < ο ο ο < ο ο ιο ūο ο 03 03 ū < ο ο ο αο α) γ - J Ο _ Ια? Lt < Ο _ & ° 2 > ο τ- < Lt Ι Ο ο Η Ο Ο < Ο ο ο < ο ο ο < ο ο ο ί ο Η " οΕο ο £ οοοοί ο ο ο οοοΗΗο Ι Ο 5 ο ο < ο C < ο ο 3 δ ο ο Η ο ο ο 5 ο

116 120 Lys Giu Ala Ala Ser Pro Pro Asp Ala Ala Ala Ser Ala Ala GTTTTCTCCTTGGCAG AAG GAA GCC ATC TCC CCA GAT GCG GCC TCA GCT GCT < Lt < Ο ο 0 Ι Ο £ Ο 0

Ta6nnua VI / προποπ ^ θημθ / φ 0 ο σ > Ο 3 ω ω CD < ο < ο5 ο ο < ο Ι Ο ο ο Ι Ο ο < ο ο < ο ο Η Č < ο 0 0 0 J - ο ο < ο ο l · - 0 I— 0 h-0 0 < ο οδ! fl g- < < 0 ο ο < 5 < ο < 0 0 I- < ο ο ο I— 0 Ι ο 0 < ο ο 0 ο 0 < ο Ι Ο 0 < ο Ι Ο Η- ο 0 0 0 0 < 0 ο I— 0 ο ο ο ο5 0 Ι Ο ο Ι Ο < ο ο 0 ο οοοοοοο ο οοοοοl; ο ί ο I- < 0 0 3 ο Η < ο < ο Ι Ο & 2 ο 0 < 0 < 0 < ο ο Η 0Ϊ3 $ 0 0 < 0 < ο ο 0 0 0 0 < ο ο ϊ ο < 0 Η < < ο 0 < ο ο 0 Ι Ο < ο 0 I- < ο ο 0 < 0 < Lt < 0 0 0 < ο Ι Ο ο I 0 ο 0 0 < 0 0Ε ο 0 Ο < Lt < 0 0 < Ο 0 S 0 0 0 < Ο 0 < 0 < ο ο ο 0 0 0 ί 0 £ ο 5 ο ο 0 0 0 < Ο 0 Ι Ο Ο Ο < Ο 0 0 Ο Ι Ο < ο Ι Ο 0 < 0 Ι Ο ο 0 ο 03 0 0 0 Η 0 0 < Ο ο ο £ ο < 0 l · -g ο 03 0 0 h " 0 0 i = ο5 0 < 0 0 Ι Ο ο 05 0 0 < Lt < 0 0 0 < Lt Ο Ο £ Ο Ο < ο 0 Ο Ο 0 5 0 S Ο Ο 2 0 0 0 Η 0 0 I- 0 0 0 0 Ο ο < ο 0 £ ο οοοοοο 0 3 ο 0 0 Η · 0 0 5 0 δ 0 δ 0 I οS ο 0 0 0 Ι ο ο ο Ι Ο < ο 0 0 0 Η < ο 0 0 0 ο δ δ 0I ο Η 0 0 0 0 5 ο 0 0 0 Ι Ο 5 ο 0 0 Ι Ο 0 0 0 0S 0

ACACAATATGAC EN 10506 B Taānmie VI nepBOHaHanbHan HenpepbiBHan flHK-nocneflOBaTenbHOCTb o6o3HanaeT ΒΘρΧΗΙΟΚ) HMTb 620 OCHOBaHMM B TOM, HTO, ΠΟ-BMflMMOMy, ΠΒΠΠΘΤΟΠ ΗΘΤρ3Η0ΠΙφΟ83ΗΗθίί nocneflOBaienbHOCTbio, ηθμθαπθηηο npeAiuecTByiomeii τρθΗοπίφΟΒθΗΗΟΜ nacTH ROHA EPO ηθποβθκθ. Bone κοηκρθτηο, nocneflOBaTenbHOCTb coflepaoiT 5'-κοηθα Rena KOTopbiii βθαθτ k TpaHcnuposaHHoii o6nac ™ flHK, KOAMpyKDmeii nepBbie neTbīpe aMUHOKucnoTbi (ot -27 -24 flo) Nocni / ļOBaTenbHocTM-nuflepa (" NPE-nocneAOBaTenbHOCTb '). HeTbile napbi a30TMCTbix ocHOBaHuii b nocneflOBaienbHocTM, npeflLijecTBy! Ποοπθαηθμ, KOflnpyiOT Hanano slipper, OflHaKO.eme He onpepenenbi οαηο3Η8ηηο m nosTOMy o6o3Ha4eHbi " X ". 3aTeM cneoyeT mhtpoh M3 npn6nn3MTenbHO 639 nap a30TMCTbix οοηοβ3ημμ (H3 KOTopbix y 439 nap οοηοβθημμ onpefleneHa nocneAOBaTenbHOCTb, a οοτθβιημθοπ 200 nap οοηοβθημμ o6o3HaH9Hbi ηθμθαπθηηο npeAiiiecTByn KOAOHy αππ m / raMUHa, KOTopbiii o6o3HaneH XAX ociaTOK 23 TpaHcnupoBaHHoro nonunenTMfla. Gneflyioman ηθμθαπθηηο 3K30H-nocneflOBaienbHOCTb KOflnpyeT aMMHOKucnoTHbie ocTaTKM nepe3 anaHMHOBbiii οοτβτοκ (o6o3HaHeHHbiPi XAX ocTaTOK +1 aMMHOKucnoTHoii nocneAOBaTenbHOCTM co3peBiuero EPO nenoBeKa) of KOflOHa, onpeflenfltomero τρθοημη b ποποχθημμ +26, Nocni nero cnefiyeT Βτοροίί mhtpoh, οοοτοπιαμμ m3 256 ocHOBaHufi, XAX οπθαμΦμηθοκμ μ o6o3HaneHO. Cneflyioinafl 3a θτμμ mhtpohom 3K30H-nocneflOBaTenbHOCTb απη αμμηοκμοποτηηχ οοτθτκοβ 27-55 npoAonacaeTcn TpeTbMM HHTpoHOM, coflepxaiAMM 612 days a30THCTbix οοηοβθημμ. NocnenyiMMM 3K30H KOflnpyeT ocTaTKM 56-1 15 ηθποβθηθοκογο EPO, 3aTeM μαθτ MeTBepTbiPt mhtpoh M3 134 days a30TMCTbix ocHOBaHUM, KOTopbiPi nofofonxaeTcn 3K30H0M, KOflnpyKDinnM ocTaTKM 116-166 + 1 KOflOH ”οοτ3ηοβκμ " (TGA). HaKOHep, b TaSmme VI Maemo + ^ MMMpyeTcn nocneAOBaTenbHOCTb 568 nap a30TMCTbix ocHOBaHMM b Tom, HTO cocTaBnneT HeTpaHcnnpoBaHHyio očnacTb 3 'Reha EPO nenoBeKa, αβθ napbi a30TMCTbix οοηοββημμ κοτοροίί ( "X") eme ηθ onpefleneHbi 0flH03HawH0 Ha nocneflOBaTenbHOCTb.

Ta6nnqa VI cnyxcMT NRM μαθητμΦμκθμμη πθρβμηηομ CTpyicrypHOM κοΗφορΜai \ m (aMMHOKucnoTHoii nocneAOBaTenbHOCTM) co3peBiuero EPO nenoBeKa, βκπιοηθκηιιθμ 166 yCTaHOBneHHblX aMMHOKMCnOTHblX OCTaTKOB (c BblHMCneHHblM MOneKynnpHblM ΒΘΟΟΜ = 18399). B TaSnupe TaioKe BbmBneHa flHK-nocneflOBaTenbHocTb, KOAnpyiomafl nocneflOBaTenbHocTb-nnflep M3 27 OCTaTKOB βμθοτθ c flHK-nocneflOBaTenbHOCTM 5 'm3' KOTopbie MoryT 6biTb B > KHbiMM flnfl φyHK ^^ ^ 1 ί npOMOTOpa / onepaTopa, ocymecTBnneMbix reHHbiM onepoHOM. ynacTKM Ann noTeHMManbHoro.rnMK03MmipoBaHMa ποπμπθπτμαθ C03peBiuero EPO ηθποβθκθ o6o3HaneHbi B Taānupe 3ΒΘ3Α0ΗΚ3ΜΜ. ΗθΟδχΟΑΜΜΟ OTMeTMTb, HTO ΟΠΘΑΜφΜΗΘΟΚΒίΙ aMMHOKUCflOTHan nocneAOBaTenbHOCTb b Ta6nm4e VI, ΒβροητΗΟ, cocTaBnneT nocneAOBaTenbHOCTb coboooo BCTpenatomeiicfl annenbHOM φορΜ ^ ηθποβθηθοκογο assistanceTMHa. OcHOBamie αππ τθκογο ποποχθημπ Hama ^ HO b pe3ynbTaTax HenpepbiBHbix nonbīTOK οπρθαθπθημα nocneAOBaTenbHOCTeii MoneBbix μ30ππτοβ ηθποβθηθοκογο spuTponosTMHa, KOTopbie πο3βοπμπμ pacnonaraTb ΜΗφορΜθρΜθίί OTHOCMTenbHO τογο, whereby to 3HaHMTenbHoe κοπμηθοτβο MoneKyn 3pMTp0n03TMHa ΜΜΘΚ3Τ ΜΘΤΜΟΗΜΗ B 126 B 00Τ3ΤΚΘ npOTMBOnOnOJKHOCTb CepMHy, Kak n0Ka3aH0 B

Ta6nnqe. 36

Ta6nnqa VII nnmocTpnpyeT CTeneHb γομοπογμμ ποπμπθπτμαηομ nocneflOBaTenbHOCTM Mexgly EPO does not come out o6e3bHHbi. B ΒβρχΗβίί HenpepbiBHofi πμημμ Ta6nmibi 6yKBeHHbie o6o3HaHeHna πρμμθηθηη PNA npeflCTaBneHMn fleflyuMpoBaHHbix TpaHcnMpoBaHHbix nocnejqosaTenbHOCTeii ηθποβθηθοκογο 3P0, HaHMHan c ocTaTKa -27 ημ a > κηαβ HenpepbiBHan πμημβ noKa3biBaeT AeoyunpoBaHHyio nonnnenTMflHyio nocneflOBaTenbHocTb o6e3bflHbero EPO, HaHMHaa ot HMcna -27, o6o3HaHatomero οοτβτοκ. 3βθ3ΑΟηκμ πρμθηβιοτοβ flnn BbifleneHMn γομοπογμμ nocneflOBaienbHOCTeM. CnejlyeT OTMeTMTb, hto flepyL | MpoBaHHbie nocneflOBaenbHOCTM ηθποβθηθοκογο m o6e3bBHbero 3PO βμαβπβιοτ " flononHMTenbHbiM ”ocTaTOK ΠΜ3ΜΗ3 (K) b ποπωκθΗΜΜ 116 (flnn neoBeKa). CcbinKa κ TačnMue VI 03HaHaeT, hto θτοτ ocTaTOK HaxoflMTcn Ha Kpaio μημμογο μΡΗΚ-οοθαμηθημπ b γθηομηομ nocneflOBaTenbHOCTM. npMcyTCTBMe ociaTKa nM3MHa b ποπμπθπτμαηομ nocneflOBaTenbHOCTM nenoBeKa προΒβρηιοτ nyTeM onpefleneHMn nocneflOBaTenbHOCTM KnoHa nenoBenecKOM KflHK, nonyHeHHotf M3 mPHK, BbifleneHHOM M3 κηβτοκ COS-I, KOTopbie TpaHCCpopMMpoBaHbi c γθηομηομ flHK nenoBeKa b flpMMepe 7, infra. EN 10506 bd I-Q UL > h *

1 X X CO φ vo o X O 00

Lllz _i CO 0 1 LU < o 0 > CU 3X C vo cd l · - CD m o c CDX O CL LU ffl α x i- X CD cX c 0 c CDXX CDXffl CdQ 01 z LU < LU bd o < CM LU _l _1 > CC LU 4 oi CC CL CL < 0 _i > CL _l0 _1 01 _l 17 al

bd I-Q CL > z LU < LU bd < LU _i _l > - CC LU > CC COo o _l CC CL CL < CD CL > CL_J CD _i CL_J CO 5 CO

LU idσ < CD _i < CC

LU Iσ < CD _l < CC —I * Γ- · t * t o o CO CC _l CD CO >

Q > I

o CD co CC _1 o co

G X σ: o CL LU CLσ CO COz > _J o < co σ CD CC _l > < LU co

CL LU LL CL O CO COz s 5σ occ _l > < LU CO o to CC Q 0 I to CC O < LU 0 CC Q0 CC CC O < LU0t o h »

bd 0 CC O — IO LL T- Z co > - > CC LL

bd 0 CC _i LL CO

oCM < CL 0 LU 1 > 0

< CL 0 LU 1 > CD - · * 3: 3 0 * 0 Η * σ * σ § ^ * ο CC • Μ- LL * >, > Η * LU Μ LU G * > * > < * < I < Η * ο ° * σ Η * (ο Ο «σ CC * 0 * > * 0 > _ _] 1 * ο η * co 4: * LU * 111 τ- <, * Έ Μ Έ < * (Π * CC * CC bd * bd 5 * <: α * ο <: < CL * ίο > - * b " CL LL * u_ u C0! Ζ * ζ CM ~ > > τ- < * 0 Q CL _1 co <

CC LL _l bd O LL l · - Q < H H CC cd cd X Vi X ν: κ φ σ; Φ, a ω 0 ω C0 ο co ο 0) C ω c ΙΟ φ νο φ ο X Ο X cd x K (D Ό o ΙΊΡΗΜΕΡ 6 npn nepBOHaHanbHbix nonbiTKax μμκρο6μοπογμηθοκογο CMHTe3a M3onnpyeMbix KonMHecTB apuTponoaTMHa ποπμπθπτμρηογο Maiepnana, KopnpyeMoro κ-flHK o6e3bBHbi, nonynaeMoro of μθτορμκθ npiiMepa 3 BbičpaHHan cncieMa sKcnpeccmi npepcraBnneT COCON cncTeMy, copepxaipyio KneTKM-xo3miH MneKonMTaiomero (Further preferably, κπθτκιι COS-I, ATCC, N CPL -1650). YKa3aHHbie κπθτκμ ocymecTBnnioT TpaHccpeKiimo nocpepcTBOM " ηθπηομηογο "seKTopa, oānapaiomero cnocočHOCTbio κ cnoHTaHHoii pennuKaLinn b X03BHHe E.coli (6nopapn npncyTCTBW0 flHK-npou3BopHbix ot nna3Mnp pBR322) n x03nnHa MneKonmaiomnx (SnaropapB npncyrcTBino flHK-npoii3BopHbix ot enpycoB-SV-40.

Bonee κοηκρθτηο, ΒΘκτορ sKcnpeccnn οτροιιτοη b οοοτβθτοτβμμ co cnepyioipKMM MeTopnKaMM. Κποη nna3Mnp 83, nonyHeHHbiii b npuMepe 3, aMnm ^ ni4HiPYi b 6 chrepmi E.coli m npn nepeBapuBaHmi EcoRI m Hindlll M3onnpyioT npn6nn3MTenbHO 1.4 kbflHK o6e3b «Hbi -Copypyioinio HelpTMH. V nna3Mnpbi pBR322 oTpenbHO BbipeneHO 4.0kb $ parMeHTa Price LLJ / Hall. M3 flHK, copepacaipeii noBTopbi M13m p10 RFflHK (R n l na necopy) 30 days a30THdbix ocHOBaHun (bp) cpparMema " cpennaioero 3BeHa " EcoRI / Scar. YKa3aHHoe " cijlennfliotpee 3βθηο ”βκπκη3θτ nocnepoBaTenbHO EcoRI-nnnKUti κοηθμ, 3a KOTopbiM cnepyK > T yHacTKM pacno3HaBaHMH Sstl. Smal. BamHI n Xbal n nnnKnii κοηθρ Hall. BbiiueyKa3aHHbie τριι 0parMeHTa cuiMBatoTcn c ^ πογ ηθμιθμ npnMepHO 5.4xb προμθχ / γοηηοη nna3MHflbi (" perse ") EPO-flHK, κοτοροη rpammm c orhom cTopOHOii npn Mcnonb30BaHMM " 6noKa "none3Hbix yHacTKOB orpaHtmeHMfl pacno3HaBaHnn 3HR0Hyiaiea3bi. riocne θτογο nna3MHfla pERS τρθΗΟφορΜ ^ θτοη čaicrepMflMii Hind III κ Sali c πο / ψηθημθμ 3pnTpono3TMHa H3 flHK, n EcORI b " CLjennfliomee 3βθηο ”gail (MI3mp10). YKa3aHHbiii paHee kbρ3ΓΜβΗΤ flHK ΑΠΜΗοΡί 1.4 kb cujHBaeiCfl c ynacTKOM BamHI / SalI npn6nn3menbHO απμηομ 4.0 κ nna3MMpbi pBR322 n ppyrnM " cpennaioiAMM 3βθηομ ”φρ3ΓΜθΗΤ3 M.I3mp1Q Hind III / Bam Hl. cDparMem " cuennaiomero 3BeHa " MI3 npepcTaBnaeT cočom Hind III - πηπκκη κοηθμ, nocne ηθγο cnepyiOT ynacTKM y3HaBaHM5: Psil, SM, Xbal μ πμπκm κοηθο.BamHI. npopyKTOM cujmbkh βηοββ CTaHOBmcn none3Han np0MexyT0HHan nna3Mnpa (" pBR-EPO "), copepxaipafl 3PO-flHK, KOTOP orpaHMHeHa c o6enx οτοροΗ " HaKonneHHbiMn pnpaMn " orpaHMHMTenbHoro yHacn < a.

BbiSpaHHbiii pna sKcnpeccnn EPO-flHK b KneTKax COS-I (" pDSVLI ") Βθκτορ CTpomcn 3pcsHee pna bo3moxhocth ocymecTBneHHB OTČOP n ayiopynnn Clinics b E.Coli. YKa3aHHbie xapaKTepncTMKH pocTMratOTCB 3a οηθτ Hannan pennuKauinn n nopapKa cnepoBamiii γθηοβ flHK, ctohkhx KfleficTBUio aMnnpiinniiHa, Koiopbie npncyTCTByK) T b oSnacTM, ocyipecTBnfliomefi CTnrnBaHue HyKneoTnpoB 2448 po 4362 nna3Miipbi pBR322 origin. YKa3aHHbiCi nopapOK cnepoBamifl CTpy «TypHO Mop ^ nuiipyeTca nocpepcTBOM ββθρθηκα " cpennaiomero 3BeHa " Hap "py c πορβοροΜ ΒΘΚΤΟρΟΒ PNA SKCnpeCCMH EPO-flHK κ ppyrnM none3HbiM cBoncTBaM othocatcb cnocoSHOCTb κ ayiopynnnKapnn b" neTKax COS-I m HannHne nocnepoBaTenbHOCTM, peMCTByKDipeii CEC BwpycHbiPi ycnnnTenb b KneTKax MneKonnTatoipnx. YKa3aHHbie xapaicrepncTMKn pocTuraiOTca nocpepcTBOM Hannan nopapKa 39 EN 10506 cneflOBaHMfl flHK-pennuKaMmi m nocneflOBaTenbHocTM "πο3αηθγο" Reha flHK, ABnaiomerocfl BnpycHbiM ycmvneneM, κοτορωίί npncyTCTByeT b uenu, coflepxamen 342 napbi ocnoBanm, CTarMBatoū4yto nncno HyKneoTMAOB ot 5170 flo 270 Rehome SV40. yHMKanbHbiM orpaHMHmenbHbiii ynacTOK (BamHI) o6pa3yeTca Ha Ββκτορβ, cpa3y xe npMMbiKaa κ papy BnpycHoro ycnnmenfl ānaroAapa ncnonb30BaHmo CTaHflapTHoii nocneflOBaTenbHOCTM ycnnnTenfl nocpeflCTBOM Mcnonb30BaHMB noc / ieflOBaTenbHOCTM "cLļenn« iouļero 3BeHa "MMeiomeroca Ha pbiHKe (Collaborative Research). flononHmenbHo b Ββκτορ BBeneHa nocneflOBaienbHocTb 237 nap a30TMCTbix οοηοβθημμ (npon3BOflHaa CEC HyKneoTMflHoe wncno ot 2553 of 2770 Bnpyca SV40) coflep > Kainero cnmari nonnafleHMnnpoBaHMB "no3flHero Reha" BnpycHoPi w-PHK (o6bNHO n3BecTHbiii Ka < τβρΜΜΗβτορ TpaHCKpMnLļmi). yKa3aHHbifi Φρβγμθητ pacnonaraer b eeicrope b cooTBeTCTByiomeii opneThe OTHOcmenbHo BnpycHoro ycnniiTenH “πο3αηθγο reHa” nepe3 yHMKanbHbiiiHacTOK BamHI. B yKa33HH0M Beicrope TaiOKe npefldaBneH flpyroPi γθη MneKonHTaioiuero, Haxoflflmniicfl b ποποχθημμ He MaiepnanbHOM OTHOcmenbHO bo3moxhom TpaHCKpnnL (nn REHAU, KOTopbiM ποΜβϋιβΗ Ha yKa3aHHOM yHMKa / ibHOM ywacTKe Mex (fly nocneAOBaTenbHOCTaMM BnpycHoro ycnnmena m TepMMHaTopa. [YKa3aHHbiK REH MneKorurraiomero copepacan b ce6e npn6nn3MTenbHo 2500 bp αμπιαροΦοπητ peAyiaa3a (DHFR) - MHHMreHa MbiniH, BbiAeneHHoro 113 nna3MMAbi pMG, Gassem ap-, PNAS (USA) (CllJA), t.79, dp.6522-6526, 1982 r. -4362 HyKneoTMAOB nna3MKAbi pBR322 HapaAy c HynexTMAaMM 5171-270 (342 bp) v53-770 (273 bp) BMpycHoii flHK, coApxaine Bnpyc SV40. lytO flHK H30AMpy (OT M3 nna3MHAbl pBR-EPO B KaneCTBe φρ3ΘΓΜΘΗΤ3 BamHI m BUYMBaiOT b nna3MHAy pDSVL.1, Bbipe3aHHyio BamHI. flna yAOCTOse peHMB ββθαθημη EPOHa b npaBMnbHoPi opneHTaqm β ρ, βγκ m nonyHeHHbix κποημροβθηημχ ΒβκτοροΒ (AynnnqnpoBaHHbie Beicropbi H h L) προβοαητ aHanM3 c ncnonb30BaHneM orpaHMHmenbHoro φθρΜθΗΤ3 (μ. Φμγ.2, nnnK5CTpnpyiomy (o nna3MMAy pDSVL1-MKE. Βθκτορω c HenpaBMnbHoii opneHTaqneM ΕΡΟ-γθηοβ οοτθβμπμ b KanecTBe perynaTopoB OTpnqaTenbHbix βθπμημη b 3KcnepwvieHTax τρβΗΟφβκΑΜίι, npeAHa3HaHeHHbix απη οπρθαθπθημη ypOBHeii 3Kcnpeccnn EPO X03HMHaX B, KOTOpbie ΤρβΗΟφορΜΙ ^ ΙΟΤΟΗ noepeACTBOM BeKTOpOB C npaBMAbHOii OpMeHTaUMeM EPO-flHK .

Beicropbi H, L, F, X m G ο6τ > θαμημπιι c ηοομτθπθμ flHK (flHK neneHH m cene3eHKM Mbiiun). flna ocymecTBneHMfl τρβηοΦθκαμμ ncnonb3yioT AynneTHbie 60 mm nnacTMHKM, πρμμθηηη MeTOAbi MMKpoocaxgļeHMfl noepeACTBOM φοοφβτβ Kaj appropriateufl. flynnnKaTHbie 60 mm nnacTMHKM Taxxe τρ3ΗθφθκτκροΒ3ΠΜ flHK-HOCMTeneM KanecTBe " MMHTaLlMH ”ynpaBneHHfl τρ3ΗθφορΜ3ΐ4Μβίί HeraTWBHbix βθπμημη. Hepe3 5 ΑΗβίί bch KynbTypanbHafi cpepa noABepmacb McnbīTaHmo Ha npncyTCTBne ποπμπθπτμαοβ, KOTOpbie očnaAaioT MMMyHonornHecKMMM CBOMCTBaMM 3pMTp0n03TMHa ΠρίΐρΟΑΗΟΓΟ ηρΟΜΟΧΟΧΑβΗΜΒ. 40 ΠΡΜΜΕΡ 7.

. CHEAPER 3Kcnpeccnn ncxoUHoro EPO c iicnonb30BaHneM κπβτοκ COS-I

Bbi6paHHaa Ha nepBOHanaribHOM 3Tane MMKpočnonornHecKOro CMHTe3a noflBepraeMbix H30nHLļHH ΚΟΠΜΗΘΟΤΒ ΠΟΠΜΠΘΤΜ / 1ΗΟΓΟ ΜΘΤθρΜΘΠΘ ΗΘΠΟΒΘΗΘΟΚΟΓΟ 3pMTp0n03TMHa, KOfllipyeMOrO nocpeflCTBOM ΕΡΟ-KOOHa γθηομηοη flHK ηθποβθκθ, cncieMa τθιοκθ βκπιοηβθτ b ce6a sKcnpeccnto npn3HaK0B b KneTKax-xo3flHH MneKonnTaiomnx (Further preferably, κπθτκιι COS-I, ATCC N CRL-1650 ). ΕΡΟ-γθη ηθποβθκθ nepBOHananbHo cy6KnoHiipy (OT b "4enHOHHbii4 " Βθκτορ, κοτορΒίΜ oānaflaeT cnocočHocTbio κ θβτοηομηομ pennuKapnn b o6onx xo3aiiHax 6aicrepnii E.coli (Gnaropapa flHK-npon3BOflHyto ot nna3MHflbi pBR 322) h β κπθτκθ nmm COS-I MneKonmaKDinero (čnaroflapfl steward Bnpyca SV40 Bnpyc-npon3BOflHbix flHK). Ηθπηο4ηηΡι Βθκτορ, coflepacatunn ΕΡΟ-γθη 3aieM τρ3Ηθφθκτι ^ θτοΗ β κπθτκιι COS-I. PO-nonnnenTHflHbiM MaTepnan o6pa3yeTcn b τρ3ΗθφθκπιροΒ3ΗΗΒΐχ κπθτκθχ μ βμαθππθτοπ b KneT04Hyto KynbTypanbHyio cpepy. Βοπθθ ΚΟΗΚρΘΤΗΟ, ΒΘΚΤΟρ SKCnpeCCIIH πρΐΊ3Η3Κ0Β CTpOMTCfl Β ΟΟΟΤΒΘΤΘΤΒΜΜ co cnenyioinMMM μθτοαιικ3μιι flHK, BbifleneHHa n3 KnoHa ληΕ1 6thsKa, copepacamero reHOMHbiii ΕΡΟ-γθη nepeBapHBaeTcn c noMombio BamHI n Hindlll orpaHH4HTenbHbix 3HflOHyKnea3 h n3onnpyeTcn 5.6 kb flHK-KjjparMeHTa, κάκ o6 HapyxeHo, cofleparainero nonHbiii ΕΡΟ-γθη. YKa3aHHbin Φρθγμθητ flHK CMeiunBaraT h cmnBaiOT c SaKTepnanbHon nna3Miifloii pVCB (Bethesda Research Laboratories, Inc.), KOTopan aHanoni4HbiM o6pa30M nepeBapMBaeTca, co3flaBaa npoMexyT04Hyio nna3MHfly " puc8-HUE ", o6pa3yn tbkum o6pa30M, nOflXOflailļHH HCT04HHK OrpaHMHMBaiOmerO φρ3ΓΜΘΗΤ3 .

BbiSpaHHbie Beicropbi pna sKcnpeccnn EPO-flHK b xneTKax COS-I ctpohtch 3-wayHee. nna3MHfla pSV4SET, KOTopan copep > CPA flHK-nocnepoBaTenbHOCTn, PaTT B03M0XH0CTb Bbipop μ ayT0flynnnKai4iiM b E.coli. YKa3aHHbie xapaicrepncTiiKH AocTHraiOTCJi HanaooM B03HMKH0BeHHH penniiKaunn h πορπηκοΜ cnenoBaHHfl Reha flHK, ycTOH4HBoro κ aMnnuHnniiHy, KOTopbiPi npncyTCTByeT β oānacTH, οτηγμβθιοιαθμ HyKneoTHflbi ot 2448 flo 4362 6aicrepnanbHoPi nna3MHflbi pBR 322 YKa3aHHaa nocneflOBaTenbHOdb η3μθηπθτ CTpyicrypy nocpeflCTBOM ββθαθημπ " cuennaiomero "3BeHa, o6ecne4HBan ynacTOK pacno3HaBaHnn Hindlll cpa3y xe npH nptineraHMH κ HyKneoTHAy 2448. nna3MHfla pSV4SET TaioKe cnocoHa κ ayTOflynnnKauna β κπθτκβχ COS-I. YKa3aHHan xapaicrepncTHKa flOCTnraeTcn nocpeflCTBOM ncnonb30BaHna ΦΡ3γμθητ3 342 bp, conepxamero BnpycHoe Hanano pennnChannel c noMOUbBO Bnpyca SV 40 (KonnnecTBO HyKneoTHflOB ot 5171 no 270). YKa3aHHbin Φρ8γμθητ MOf ^ nunpyiOT npn ββθαθηηη " cuennaioiuero " 3BeHa, KOTopbin o6ecneHHBaeT yHacTOK pacno3HaBaHna EcoRI, npnneraioLUHH κ HyKneoTiifly 270, n " cuennnioinero "3seHa, o6ecne4HBaiomero ynacTOK pacno3HaBaHHB, npnMbiKaioiunn κ HyKneoTHfly 5171st Β αθηηομ Βθκτορθ TaioKe npncyTCTByeT ΦΡ3γμθητ 1061 bp Bnpyca SV 40 (mmcho HyioieoTHflOB ot 1711 of 2772 c flononHiiTenbHbiM & quot ciennenninHM ”3Βθηομ, κοτοροθ ο6θπθμμβ8θτ y4acT0K pacno3HaBaHnn Sal npnneran κ 41 LV 10506

HyKneormy 1772. B npeflenax yKa3aHHoro φρβΓΜβΗΤθ MMeeTca yHHKanbHaa nocne / leOBaTenbHOCTb pacno3HaBaHMA BamHI. CyMmnpya BbimecKa3aHHoe, nna3MHfla pSV4SEt copepxMT b ce6e yHHKaribHbie yHacTKM pacno3HaBaHMa BamHI m Price UJ, flaBaa 803M0XH0CTb ββθρθηιια reHa EPO MenoeeKa. yKa3aHHbie nocneflOBaienbHOCTM patch B03M0XH0CTb ocyilecTBneHMfl PenetrationMMm m36npaTenbHoro oTop b E.coli n pennMKauMM b κπθτκθχ COS-I. flrm BBBflBHna EPO-Reha b nna3MMfly pSV4SEt, p nna3MHAa C8-Hue npepesapuBaeTca nocpeflCTBOM BamHI m Hindlll the orpaHMHMBaramefi 3HA0HyKnea3bi, m M3onnpyeTca 5.6 kb EPO, KOflnpyiomero ΑΓΚ (fjparivieHTa. nna3MMfla pSV4SEt τβκχβ nepeBapuBaeTca c noMombio BamHI m Hindlll. n ocHOBHan nacTb φρ8ΓΜβΗΤ3 , 2513 copepacamaa nap οοηοβθημμ, M3onnpyeTCfl (coxpaH88 npn 3tom BCE H0o6xoflMMbie φyHκ ^^ l ^ 1). yKa3aHHbie Φρ3γμθητη CMeiuMBaroica m cujMBaioTCB, o6pa3yn κοηθηηνμ Βθκτορ " pSVg Hu EPO1 '(ομ.Φμγ.3). 3τοτ Ββκτορ pa3BOflmca b E .col. Ββκτορ a flHK n3onnpyeTCA. flna ποΑΤΒβρχηβΗΜΑ BBOAa EPO-Reha προβοαητ aHann3 OrpaHHMHTenbHOrO φβρΜβΗΤ3. flHK nna3MHAbi pSVgHuEPO ncnonb3yeicfl απη BbipaxeHMA npn3HaKOB EPO-nonnneriTMAHoro Maiepnana b ιοίβτκβχ COS-I. fleianbHO M3naraa, flHK nna3MMAbi pSVgHu EPO očteAMHfleTCfl c ΑΗΚ-ηοομτθπθμ m τρβΗοφβκτι ^ βτοΑ b TpnnnnKaTHbie 60 mm acTMHKM κπβτοκ COS-I. B KanecTBe κοΗτροπΒ flHK nocmenb oahh τρθΗΟφβκτΜρνβτοΑ b κπθτκθχ COS-I. KneTOMHaa KynbTyparibHafl cpeAa OT6npanacb cnycTa 5-7 ΑΗβίί m noABepranacb McnbrraHMto Ha npncyTCTBne ποπμπθπτιίαοβ, KOTopbie oānaAaiOT MMMyHonorMHecKMMH CBOMCTBaMH, xapaKiepHbiMM απ "cbo6oaho BCTpeMafoineroca EPO nenoeeKa. B. Btopaa ομοτθμβ 3Kcnpeccnn npH3HaK0B EPO β κπβτκβχ COS-I.

Eme OAHa CMCTeMa aKcnpeccmi πρμμθηηθτοα απη ποιήθηκα ynyHiueHHbix npoAyicroB ΗΘΠΟΒβΗβΟΚΟΓΟ 3ρΜΤρ0Π09ΤΜΗ-Π0ΠΜΠβΠΤΜΑΗ0Γ0 MaT9pnana, KOAMpyeMOrO C nOMOLlļbKD KJlOHa HenoBeKa γθηομηομ flHK, o6pa3yiomeH EPO β κπβτκθχ COS-I (A.T.C.C. CRL-1650 N). Β πρβΑΐυβσι ^ ιοΐΑβΗ ομοτθμθ npM3HaKM EPO BbipaxaioTCfl β κπθτκθχ COS-c ncnonb30BaHneM CBoero οο6οτβθηηογο ycnnnTena, KOTopbiii npncyTCTByeT b npeAenax orpaHMHMTenbHoro φρ3ΓΜβΗΤ3 BamHI OTHOCMTenbHO HindIII. μμθιοιαθγο AnnHy 5.6 kb B cneAyiomeM ποοτροβΗΜΜ ΕΡΟ-γθη HepeAyeTca c nonyHeHneM 3Kcnpeccnw npn3HaKOB, ncnonb3yfl no3AHHH ycmiwenb Bnpyca SV40.

Bonee κοΗκρβτΗΟ, orpaHMHHTenbHbiii φρβΓΜβΗτ γθηομηογο ηθποβθηθοκογο EPO, npeACTaBnaiomnti KAOHiipoBaHHbiM 5.6 kb BamHI OTHOCMTenbHO HindIII μοαμΦμα ^ ιοτ b οοοτΒβτοτΒΜΜ co cneAyiomefi μθτοαμκομ. Kax oniicbiBaeTCfl Bbime, nna3MMAa pvC8-HuE pacmennaeTCfl nocpeACTBOM BamHI m orpaHMHmenbHofi 3HA0HyKnea3bi BstE II. OrpaHMMHTenbHaa 3HA0HyKnea3a BstE II pacmennaeT b npeAenax 5.6 kb ΕΡΟ-γθη β ποποχθημμ, κοτοροβ npeACTaBnaeT 44 napbi οοηοββημμ 5'ao nHnunnpyiomiix ATG, KOAnpya npeAiuecTBeHHHK-nenTHA w πρΜΜβρΗΟ 680 nap οοηοβαημμ 3 ao orpaHMHMTenbHoro yHacTKa Hindlll. 3aTeM BbiAenneTca φρθΓΜβΗΤ, βκπιοηθκηαμμ npn6mi3MTenbH0 4900 days οοηοβθημμ. OparMeHT flHK c cMHTeTMHecKMM " cuennajoiAMM " 3ΒΘηομ, coAepxaiAnii Hall m BStEII πμπκμθ 42 Kohlich μ ΒΗγτρβΗΗΜΜ ysacTOK pacno3HaBaHnn SamHI. noflBepraeTcn CMHTe3y μ οημοτκθ. YKa3aHHbie psa Φρθγμθητθ CMeujMBaioTCH m ciuiiBaioTCfl c nna3MMFOM pBR 322, KOTOpan Bbipe3aeTcn c from MOlBi SalIs BaM Hl αππ nonyHeHMfl npoMexyTOHHoii nna3MMflbi pBRgHE. Γθηομημμ γθη EPO neoBeKho m3onnpoBaTb m Hee β βμαθ cpparMema nepeBapiiBaHMH BamHI c 4900 nap ocHOBaHiiii, Hecymero nonHyio CTpyicrypy reHa c opmhohhom ATG 44 bp 3 OTHOcnTenbHO ynacTKa BamHI. npinoidal difference κ oOnacTH OKOHsaHMfl KOfliipOBaHi aMMHorpynn. nonyHeHHbiM Φρθγμθητ ii3onnpyiOT μ βοαπτ KaK Φρ3γμθητ BamHI b nna3MMfly pDSVL I pacemakerHHoro BeKTopa SKcnpeccine BamHI (cm. npiiMep 6). riony4aiomaflCH b pe3ynbTeu aToro nna3MMfla pSVLg Hu EPO, KaK noKa3aHO Ha Φμγ.4, ncnonb3yeTcn απη BbipaxeHM ^ npn3HaK0B EPO nonuneriTMflHoro MaTepnana M3 κπθτοκ COS, KaK omicaHO b npMMepax 6 m 7a. ΠΡΙ / ΙΜΕΡ 8

KynbTypanbHayi cpejļa, BbipameHHan m 6 τρθΗοφβκτΜροΒβΗΗΒΐχ KynbTyp κπβτοκ COS-I npuMepa 6 npoaHanM3MpoBaHa c noMombio paflnonMMyHononiHecKoro aHanii3a comacHO ΜΘΤΟβΜΚΘ, 113ΠΟ > ΚΘΗΗΟΜ Β ΠρίΙΜθρθ 2 HaCTb B. KajKflbIM 06pa3eL4 aHanM3MpOBancyi πριι 3HaHeHH5ļX anuKBOTHofi npo6bi 250.125, 50, 25 μκπ. HaflcaflOHHbie cnon m pocia κπθτοκ " ΜΜΜΤΘΜΜΟΗΗΟ " Τρ3ΗθφθΚΤΜρθΒ3ΗΗΒΙΧ ΜΠΜ ΤρθΗΟφθΚΤΜρΟΒΒΗΗΜΧ C nOMOmblO ΒΘΚΤΟρΟΒ C

HenpaBunbHOM opueHTauneM γθηοβ EPO μμθπμ oThePlantBHbie 3Η8ηθηιιπ Ha MMMyHopeaKTHBHocTb. flnn Kaxfloro o6pa3qa fleyx HaflcaflOHHbix cnoes xmakoctm, nonyHeHHbix II3 bowl κπθτοκ COS-I, KOTopbie τρ3ΗθφθκτιιροΒ3Ηω Βθκτορ3Μΐι (ie H L) c npaBunbHOM opiieHTauiiefi flHK EPO% ιιηγιι6ιιμμιι ^ 251.epo, NPI / iBfl3aHHoro κ aHTMTeny, Kone6ancn b npepenax ot 72 jqo 88 %, KOTopbie noMemaioT βοθ 3HaHeHMn β βθρχηθθ ποποχθημθ CTaHfiapTHOtf ΚρΜΒΟίΐ. ri03T0My Henb3fl TOHHO paCCHMTaTb MCTMHHYO KOHLLeTeLHH) apMTpon03THHa B nnaBaK) mnx cnonx KynbTypbi. Ebin προβθαθη CKpoMHbiPi pacneT, hBHBH 300mV / Mn, of1HaKO, BbinonHeHHbiii πριι 3Ha4eHnnx caMoii 6onbujoPi aniiKBOTHOii npočbi (250 μκπ). 06pa3eq KynbTypanbHOM xhrkocth, nonyHeHHoii cornacHO npnMepy 6, 5-7 m AHeBHbie KynbTypanbHbie xiiakoctm, onncaHHbie β npiiMepe 7a, noflBepranncb paflnoiiMMyHonornMecKOMy aHann3y αππ cpaBHeHHn aKTiiBHOCTH peKOMāiiHaHTHbix ΕΡΟ-MaTepnanoB o6e3bHHbi ιι ηθποβθκθ οτηοομτθπβηο CBOčoflHO BCTpenatomerocn EPO ηθποβθκθ, OTBenatoiuero CTaHflapTy. nonyHeHHbie pe3ynbTaTbi npeflCTaBneHbi β φ3φΜΗθθκοιι φορΜθ Ha Phc.I. "Opposes rosopn, npejļCTaBneHHbie pe3ynbTaTbi ηθοχμαθηηο noKa3ann, whereby to peKOM6iiHaHTHbiH EPO o6e3bHHbi cymecTBeHHO KOHKypnpyeT M3-3a aHTMTena, οβπ33ηηογο c aHTH-EPO ηθποβθκθ, χοτπ ηθ ypanocb nonHocTbio nHrii6npoBaTb CBn3ytoinee b ycnoBnnx iīcnbrraHHn. MaKCMManbHbift% 3HaMeHMii ΜΗΠίδΜΜΙΙΙΙ flnn ρθΚΟΜδΜΗΘΗΤΗΟΓΟ 3pllTp0n03TMHa ΗΘΠΟΒΘΚβ, ΤΘΜ Ηθ ΜΘΗΘΘ ΠΟΗΤΜ npn6niixaeTcn κ CTaHflapTHbiM noKa3aTennM. riapannenbHbiii xapaepep KpMBoii β033-3φφθκτ npeflnonaraeT MMMyHonorMHecKyio HfleHTMHHOCTb nocneflOBaTenbHOCTeii (θπμτοποβ) β ο6ιμθμ. 43 EN 10506 tire βνποπηθημθμ pacneTOB KOHMeHTpaLļnn 3P0 o6e3bHHb b KynbTypanbHbix χμακοογπχ 6binn npoBefleHbi noBTopHbie ομθηκμ npM yKa3aHHbix βμοοκμχ 3HaneHMnx pa3čaBneHMn. Bbino o6napy > KeHO) hto ohm KoneOanMCb b MHTepeane ot2,91 flo 9,12 / μπ. BbiHMcneHHbie ypOBHM npoflyunpoBaHMfl EPO Heneena ycTaHOBneHbi οοοτβθτοτβθηηο npM 3HaHeHMnx 392 V / μπ αππ o6pa3iii c 5-αηθβηημ poctom m 567 itiV / μπ nnn o6pa3ua c οθμμαηθβηημ poctom. PacneTHbie ypoBHM npopyunPOBaHi EPO o6e3bHHb b npuMepe 7 B CMCTeMbi aKcnpeccMM 6binn aHanornHHbiMM Tin NyHLUMMM. ΠΡΙ / ΙΜΕΡ 9

KynbTypanbHbie χμακοοτμ, nonyHeHHbie cornacHO npuMepaM 6 m 7, nohsepram aHami3y in vitro pna onpe / enna Hna θκτμβηοοτμ EPO b οοοτβθτοτβμμ c μθτοαμκομ Goldwasser et al., Endocrinology, 97, 2, οτρ.315-313,1975 r. BbiHMCneHHbie 3HaHeHMn EPO o6e3bflHbi flnn ncnbiTyeMbix KynbTypanbHbix χμακοοτθμ Konečanncb b npejļenax ot 3,2 of 4,3 V / μπ. KynbTypanbHbie jkmakoctm EPO ηθποβθκθ TaioKe προπΒπππΜ atcmBHOcTb b nonyneHHbix pacHeTax npn anann3e in vitro μ, κρομθτογο, yKa3aHHyio aKTUBHocib μοχηο Hefrrpami30BaTb non nePiCTBueM aHTmena " amn EPO. KynbTypanbHbie χμακοοτμ ρθκομ6μη3ητηογο EPO o6e3bflHbi of npMMepy 6 Taoce noflBepranu KonnHecTBeHHOMy onpepeneHUio in vivo čHononiHecKoii aKTMBHOCTM cornacHO očmeMy MeTOfly Cotes et al., Nature, 191, οτρ.1065-1067 1967 r., M Hammond et al., Ann.NVAcad.Sci ., τ.149, οτρ.516-527,1968 r. YKa3aHHbie ypoBHM aKTMBHOCTM MachineMCb ot 0.94 flo 1.24 V μπ. ΠΡ14ΜΕΡ 10 B npeflbifiymMx npMMepax peMomMaHTHbiii EPO-MaTepMan HenoeKa μπμ o6e3bflHbi nonyHa (OT ot eeicropoB, KOTopbie Mcnonb3yK) Tcn βππ τρ3ΗϋφβκτΜροΒ3ΗΜπ κπβτοκ COS-I. YKa3aHHbie eeicTopbi ocymecTBnniOT pAnnMKauMto b κπθτκθχ COS-I Snaropapn npMcyTCTBMio Τ-aHTMreHa BMpyca SV 40 BHyrpM κπθτκμ Hanana βο3ημκηοβθημπ pennMKaijMM SV 40 Ha yKa3aHHbix Beicropax. Χοτπ nonyneHHbie ΒβκτόρΜ βοοπρομ3βοαπτ none3Hbie KanecTBa EPO b KneTKax COS-I, mx BbipaxeHHOCTb -TonbKO BpeMeHHan (b τθηθημθ 7-14 αηθμ) M3-3a B03MOXHblX nOTepb BeKTOpa. KpOMe ΤΟΓΟ, TOHbKO He3HaHMTenbHblM% BOCnpOM3BefleHMM COS-I CTaHOBMTCH npOflyiCTMBHO ΤρβΗΟφθΚΤΜρΟΒβΗΗωΜ yKa33HHblMM BeKTOpaMM. HaCTOniUMM npMMep onMCbieaeT CMCTeMbi aKcnpeccMM npM3HaK0B, Mcnonb3yiOLUMX κπθτκμ DHFR nMHHMKa KMTaiicKOR μομπηκ3 (CHO) m ceneKTMpyeMbiM MapKep, DHFR. (Flnn o6cyxfleHMn poflCTBeHHbix ομοτθμ aKcnpeccMM CMOTpMTe naTeHTHyio rpaMOTy Cujas N 4399216 μ 33πβκμ Ha BbiflaHy EsponaTeHTa N 117058.117059 m 117 060, ony6nMKOBaHHyio 29 aBrycTa 1984 r.) B κπθτκ3χ CHO DHFR (DUX-BII) CHO Kl, Urlaub et al., Proc.Nat.Acad. Sci. (Cujas), t.77, οτρ.4416,1980 r., He xeaTaeT 3Η3ΜΜ-βΜΓΜΑροφοππτ-ρβί ^ κτ33Ν (DHFR) M3-3a MyTauMM, nponcxoflniiļMx b cTpyicTypHbix reHax m, cneflOBaTenbHO, ηθο6χοαμμο HanMHMe γπμαμηθ, 44 runoKcaHTMHa m TMMMflMHa b KynbTypanbHoPi cpefle. rina3MMflbi pDSVL-MKE (npuMep 6) μπμ pDSVL-gHuEPO (npuMep 7B) TpaHC0eKTnpyiOT βμθοτθ c flHK ηοομτθπθμ b DHFR κπθτκμ CHO, KOTopbie BbipaiuMBaioT b cpefle, coflepxameM rnnoKcaHTMH, tmmmamh m γπμμμη b 60 mm KynbTypa / ibHbix nnacTMHax. i1na3MMfly pSVgHuEPO (npuMep 7a) CMeiuMBaiOT c nna3MMflofi pMG, coflepxainMii γθη Mbiiun Ha οοηοβθ flnniflpo0? CMecb nna3MMfl n flHK ηοθμτθππ TpaHC $ eicrMpyK DHFR - CHO T b κπθτκμ. (Κπθτκμ, KOTopbie 3axBaTbiBaiOT OflHy nna3MMfly, očbiMHO TaioKe 33ΧΒ3ΤΠΤ BTopyio nna3MMjqy). Hepe3 3 αηπ nonyMeHHbie κπθτκμ flMcneprMpyiOT nocpeflCTBOM TpMncMHM3MpOBaHMn b HecKonbKMx 100 mm KynbTypanbHbix nnacTMHax b cpefle, b κοτοροΜ He xeaTaeT γμποκοβητμηθ μ TMMMflMHa. TonbKO τβ κπθτκμ, KOTopbie τpaHCφopMMpyκ) τcπ b ycTOMHMBoe cocTOHHMe nocpeflCTBOM reHa DHFR, προπΒππιοτ βνχμβ3ημθ b αθηηομ cpefle reHa EPO. 4epe3 7-21 fleHb CTaHOBflTcn BMflHbi κοποημμ XMByiflMX κπβτοκ. YKa3aHHbie κοποημμ TpaHCCpopMaHTOB Nocni flMcnpempoBaHMn nyTeM TpMncMHM3MpoBaHMa MoryT HenpepbiBHO pa3MH0 > KaTbcn b cpefle c HeflocTaTOHHOCTbio γμποκοθητμηβ μ τμμμαμηθ, co3flaean Hobbs ojTaMMbi κπβτοκ (HanpMMep, CHO pDSVL-MkEPO, CHO pSVgHuEPO, CHO-p-DSGE 9HuEP0).

KynbTypanbHbie xmakoctm, nonyweHHbie M3 BbimeyKa3aHHbix κπθτοηηβιχ ιιιτθμμοβ, noflBepraKDT paflMOMMMyHonorMHecKOMy aHanM3y Ha HanMHMe ρθκομ6μη3ητηογο EPO o6e3bBHbi μπμ HenoBeKa; Cpena ηππ ιιπώμμοβ CHO p DSVL-MkEPO coflepxMT EPO c τθκμμμ xe MMMyHOflOrMHeCKMMM CBOMCTBaMM, KaK M EPO, nOnyHeHHblM M3 κπβτοκ COS-I, TpaHC (l) eKTMpoBaHHbix nna3MMfloii pD8VL-MkEPO. 06pa3eq 65 sacoBOM KynbTypanbHOM) Κμακοοτμ coflepxMT EPO o6e3bBHbi πρΜ 3Η3μθημμ 0.60 V / μπ.

KynbTypanbHbie xmakoctm, nonyHeHHbie M3 CHO pSVgHuEPO m CHO pDSVL-gHuEPO, coflepxaT peKOMČMHamrHbiM EPO nenoBeKa, o6naflaraiflMM τθκμμμ xe MMMyHonorMHecKMMM CBOMCTBaMM, Kak m EPO, nonyneHHbiM M3 κπβτοκ COS-I, τρβΗοφΘκτΜροΒβΗΗΝχ nna3MMflOM pSVgHuEPO μπμ pDSVL-gHuEPO. 06pa3eq 3-αηθβηομ KynbTypanbHOM xmakoctm, nonyneHHOM M3 CHO pSVgHuEPO, coflepxMT 2.99 V / μπ EPO Henoee, aOcpa3eu c 5,5-αηθβηομ BbiflepxKOM, nonyweHHOM M3 CHO pDSV-gHuEPO, coflepjKMT 18,2V / Mn EPO NoBeCe, CaCaBiOT pe3ynbTaTbi paflMOMMMyHonorMHecKoro aHanM3a.

KanecTBO EPO, nony &4; ^ H3MM-flMΓMflpoφoππτ-peflyκτa3a (DHFR), KOTop npeflCTaBnneT COCOM npOflyiCT, KOflMpyeMblM ΓΘΗΟΜ DHFR, ΜΟΧβΤ 6blTb ΜΗΓΜβΜρΟΒΘΗ C nOMOLLļbK)

MeTOTpeKcaTa (ΜΤΧ). B HacTHOCTM, κπθτκμ, pa3MH0xaeMbie b cpefle c HeflocTaTOHHOCTbio rMnoKcaHTMHa μ τμμμαμηθ, MHrM6npyioTCfl μπμ y6MBaraTcn MeTOTpeKcaTOM. pin cooTBeTCTByioiflMx ycnoBMnx (HanpMMep, πρΜ MMHMManbHOM MOUNTING μ) μοχηο nonynMTb ycTOiiHMBOCTb κπβτοκ m cnocodHOCTb κ pocTy β ΜΤΧ. 06HapyxeHO, hO ya 3aHHbie κπθτκμ CTaHOBflTCH ycTOMHMBbiMM κ ΜΤΧ n of of of of of of of of of of of c ne ne ne ne io io n pnfla mx DHFR γθηοβ, b pe3ynbTaTe nero noBbimaeTcn npoflyuMpoBaHMe DHFR 3H3MMa. > KMByiflMe κπθτκμ μοχηο, β 45 LV 10506 cbok) onepepb, o6pa6oTaTb noBbHHbiMM KOHqeHTpaqnnMM MeTOTpeKcaia, b pe3ynbTaTe nero Β UJTaMMaX ΚΠβΤΚΜ γΒΘΠΜΗΜΒβθΤΟΒ ΚΟΠΜΗΘΟΤΒΟ ΗΟΒβΗΟΒ DHFR. " MuMoneTHbie reHbl " (HanpMMep, EPO), nepeHOCMMbie Ha Ββκτορ 3Kcnpeccnn ΒΜβστβ c γθηομ DHFR Tinu τρβΗοφορΜί ^ βΜΒίβ c hmm, KaK nacTO o6HapyxtiBaeTcn, noBbinatoT Mice Cbomx Bocnpoti3BepeHiiM. B KanecTBe npMMep DisposalHecKOtiMiMiMiMaNtMiMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMiMaNeMaMM κπβτκΜ CHOpDSVL-MKePaAaAbiTiTi ΜβτοτρβκοβτοΜ πγτβΜ ποβηιπθηιιπ Difference Between KOHqeHTpaqi (OnM, 30nM n 100nM). 06pa3qbi, npepcTaBnaiomiie 65-MacoByio cpepy KynbTMBnpoBaHtin na KaxpoM 3Tane 3μππκΦμκ3ρμμ no paHHbiM paflnoMMMyHononiHecKoro aHami3a copepxaT 3pMTpon03THH, ΜΜβΙΟΐρβΓΟ 3Η3ΗβΗΜΗ 0.60, 2.45 H 6.10 / ΜΠ, ΟΟΟΤΒΘΤΟΤΒΘΗΗΟ. O ΚΠΘΤΟΚ CHO pDSVL-gHuEPO nopsepraioT očpaSoTKe Μβτοτρβκο3τθΜ (ΜΤΧ). c ceptideHbiM noBbiLueHneM difference in KOHqeHTpaqtiii: 30nM, 50nM, 100nM, 200nM, 1 μΜm 5 μΜ. 06pa3eq, npeflCTaBnfliomMM 3-xpHeBHyio HapocaflowHyio xiiflKOCTb Ha 3Tane ero o6pa6oTKH ΜβτοτρβκοβτοΜ (ΜΤΧ) c KOHqeHTpai4neii 10OnM οορβρχιιτ of paHHbiM papnoiiMMyHO-norHHecKoro aHann3a apMTponoaTMH nenoBeKa, MMeiomero 3HaHeHMH 3089 + -129 U / μπ. 06pa3qbi, npepcTaBnaioiuMe 48-HacoByro KynbTypanbHyra cpepy, o6pa6oTaHHbie MeTOTpeKcaTOM c KOHqeHTpauneii 100nm h 1μΜ copepxaT apMTponosTHH nenoBeKa of paHHbiM papnonMMyHO-nortmecKoro aHann3a, MMetoīpero 3Η3ηθημβ 466 ιι 1352 V / μπ, οοοτΒβτοτΒβΗΗΟ (ycpepHeHHoe HMcno, nonyHeHHoe b pe3ynbTaīe 3-x pa3oeoro McnbrraHtin očpa3qoB) . Πο yKa3aHH0M Μβτοριικβ b 60 mm HALUKM ΠθτρΜ, copepxamtie 5 μπ cpepbi, BbiceBaioT no 1x10® κπβτοκ. Hepe3 24 Match Nocni 3Toro cpepy ypann! OT, 3aMeHnn ee H 5 μπ CBexeft cpepbr, β κοτοροίί 0TcyTCTByeT cbiBopoTKa CDMEM c BbicoKMM copepxaHneM rni0K03bi, b KOTopyio flononHmenbHo poSaBnnioT 0.1 Μ Ηβ-OCHOBHb / Χ aMMHOKMCnOT H L-rnyTaMHH. 3pMTpon03THHy flatOT 803M0XH0CTb HaKanmīBaTbcn b τβπβΗΜβ 48 nacoB b cpepe, He copepxameii cbieopoTKii. 3aTeM cpepy ccupaio pnn HcnbrraHt nocpepcTBOM papnotiMMyHononiwecKoro aHann3a, κπβτκΜ TpnncMHM3npyiOT h 3aieM noflCHMTbiBaiOT. CpeflHMe 3HaweHnn o6pa3qoe b pe3ynbiaie papMO-MMMyHonornHecKoro aHami3a cocTaBnntOT spmponosTHH nopnpKa 467 m 1352 V / μπ ρππ κπβτοκ, BbipaipeHHbix nptII οδρβδοτκβ Μβτοτρβκο3τοΜ c KOHqempaqiieii 100nm ιι 1μΜ οοοτΒβτοτΒβΗΗΟ, oGecneHMBaioT φβκτΜΗβοκιΐΜ Bbixop apmponosTMHa nopnpKa 2335 m 6750 V H MawKy.

CpepHee κοπμηθοτβο κπβτοκ Ha HaujKy cocTaBnneT 1.94 x 106 h 3.T2 x 10® κπβτοκ οοοτΒβτοτΒβΗΗΟ. TaKMM o6pa30M, CKopocTb 3φφβκτΜΒΗθτο npopyqnpoBaHt npw yKa3aHHbix ycnoBnnx KynbTMBnpOBaHnn ΜΜββτ 3HaweHMfl 1264 h 2167 V / 10® κπβτοκ 3a 48 nacoB.

OnncaHHbie Bbirne KneTOHHbie KynbTypbi npepcTaBnatoT co6oii ΓβΗβΤΜΗβοκκ pa3HOpopHyio nonynnqnKD. C qenbio M30nnqmi ΓβΗβΤΜΗβοκΜ HeopHopopHbix κποηοβ, o6napatomtix HaMBbicuieM cnocoāHocTbio κ npopyqnpOBaHmo, ncnonb3yiOT CTaHpapTHbie ΜβτορΜ OT6opa (cM.pa3pen A, nacTb 2 " Points to considera in Characterization of Cell Lines Used to Produce Biology " 1 μιοηπ 1984 r., Οτρβπ of HayHHOii προθβρκβ 6nonopenTTOB, UeHTp no neKapcTBeHHbiM m 6nonpipTaM, ΥπρβΒπβΗΜβ no nmpeBbiM m neKapcTBeHHbiM npopyicraM ClUA.

OnncaHHyKD Bbime npoflyKTMBHOCTb πμημμ κπβτοκ CHO, npopyqnpyiomMX apMTponosTHH, 46 MO > KHO ynyHUJMTb npn MCn0nb30BaHHH COOTBeTCTByiOmMX MeTOflMK KynbTMBMpOBaHMB ΚΠΘΤΟΚ. Pa3MH0 > KeHMe κπθτοκ MneMax xμβοτημχ b KynbTypanbHofi cpefle o6bNHO Tpe6yeT HanuMHB cbiBopoTk b pocTOBOM cpefle. Cnoco6 nonyHeHMB apconoaTUHa m κπθτοκ CHO b SeccbiBopOTOHHOM cpepe b 3HaHMTenbHoPi οτθπθημ. očnernaeT ρβκτΜφΜΚθρΜίο spuTponoaTMHa M3 KynbTypanbHOM cpeflbi. OnncbiBaeMbiPi Hnxe cnoco6 paEt B03M0XH0CTb θκοηομηογο nOnyMeHMB apMTponoaTMHa B 6onbUJMX ΚΟΠΜΜΘΟΤΒβΧ B CeCCbIBOpOTOHHOM cpepe, flOCTaTOHHblX ΑΠΠ np0M3B0ACTBa. Κπθτκμ πμηmm CHOpDSVL-gHuEPO, BbipatfleHHbie b CTaHflapTHbix ycnoBnax KynbTMBMpOBaHMB ΚΠΘΤΟΚ, flflfl noeeBa ηρΜΜΘΗΠΙΟΤ 0naKOHbl C nepeBMBaeMOii ΚΠΘΤΟΗΗΟΜ KynbTypoii, flnn noeeBa πρμμθηπιοτ 0naKOHbi c nepeBMsaeMoii κπθτοηηομ KynbTypoM. YKa3aHHbie κπθτκμ pa3MH0xat0Tcn β βμαθ cycneH3MOHHoii κπθτοηηομ πμημμ βο Φπθκοηβχ, coflepxaiflMX nepeBMTyio KneTOHHyio KynbTypy, b cpefle, KOTopan coctomt M3 50 + 50 ομθομ DMEM c BbicoKMM οορβρχβΗΜθΜ rnK) K03bi m F 12 Ham, b KOTopyioflononHMTenbHo flčSaBneHO 5% CbiBOpOTKa 3M6pnoHa KopoBbi, L-rnyTaMMH , πθημμμππμη h οτρθπτομμμμη, 0.05 mM HeOCHOBHblX aMUHOKMCnOT, Μ ΜΘΤΟΤρβΚΟΘΤ C COTBeTCTByiOIJ4eM KOHMeHTpMMMM. KneTOMHbie KynbTypbi β βμαθ cycneH3MM flaiT B03MoxHOCTb flnn SbicTporo pa3MHOxeHMn flo 6onbWMX οβτ, ΘΜΟΒ 3pnTpono3TMHy, nonyMomomerocfl M3 κπθτοκ CHO. Κπθτκμ CHO, BbipamneaeMbie β cycneH3MH, Mcnonb3yiOT flnn mx noeeBa BpamaioiMMeca cocyflbi npii nepBOHananbHOM πποτηοαγμ 3aceBaHMB 1.5x107 XM3Hecnoco6Hbix κπθτοκ Ha 850 cm2 yKa3aHHoro cocyfla, coflepxamero 200 μπ cpeflbi. Β? ΠρΜΜθΗπθΜθπ β naHHOM φθ3θ pocTa κπθτοκ cpefla aHanorMHHa cpefle, Mcnonb3yeMOM flnn KynbTMBMpnoBaHMn κπθτοκ b cycneH3MM. K KOHfly 3-χαηθβηογο nepMOfla pocTa κπθτοκ cpefly, coflepxamyK) cbiBopoTKy, oTflenntOT μ θθμθηπιοτ ee Ha 100 μπ čccbiBopoTOHHOM cpeflbi. BeccbiBopoTOMHan cpefla coflepxMT 50-50 ομθομ DMEM c bucokhm coflepxaHMeM0K03bi m F 12 Ham, b KOTopyio αοποπημτθπβηο ββθαθηο 0.05 mM ηθοοηοβηβιχ θμμηοκμοποτ m L-rnyraMMH. Bpatnaramnecn cocyflbi c πρμγοτοβπθηηομ cpefloPi noMemaioT If 2-3 naca b MHKy6aTop m 3aTeM ee BHOBb yflannKDT, 3aMeHnn Ha 100 μπ οβθχθμ ηθ coflepxameM cbiBopoTKM cpeflOM. 3ρΜ 1-3 HaCOBOM MHKy6aLMM B CeCeBIBOpOTOHHOM Cpefle npOMCXOflMT ΟΗΜΧβΗΜβ KOHfleHTpaflMM 3arpn3HeHMn δβπκοΒ cbiBopoTKOM. 3aTeM BpamatoiUMecn eocyflbi lBBMMOTOTS If 7 αηθμ β MHKy6aTop, β TeneHMe KOTopbix npoMcxoflMT HaKONHMe apMTponosTMHa b SeccbiBopoTOHHOM KynbTypanbHoii cpefle. K KOHLly 7-αηθβηομ φθ3Βΐ npoflyuMpoBaHMH spMTponoaTMHa CTapyio cpefly yflannK3T, 3aMeHan ee Ha CBexyio, ηθ coflepxamyio CbiBopoTKy cpefly, Heo6xoflMMyio flnn npoBefleHMH BTopor UMK npoflyuMpoBaHMn. EXAMPLE B BMfle npaKTMnecKOM peanM3aflMM yKa3aHH0M CMCTeMbi npoflyuMpOBaHMn npeflCTaBneH o6pa3eu, HaxoflniflMMcn β τθηθημθ 7 αηθμ β SeccbiBopoTOHHOM cpefle, KOTopbiii of αθηημμ paflMOMMMyHonorMHecKoro aHanM3a coflepXMT 3ρΜΤρ0Π03ΤΜΗ, MMetOIflMM 3Η3ΗΘΗΜΠ 3892 ± 409 V / ΜΠ. (IpMHMMan ΒΟ ΒΗΜΜΒΗΜβ flaHHbie paCHeTiNiNg pActIon 0.9-1.8x105 κπθτοκ Ha cm2, b KaxflOM BpainaiomeMcn cocyfle θ6ΐ »ΘΜΟΜ 850 CM2 COflepXMTCfl OT 0.75 flO 1.5x108 ΚΠΘΤΟΚ, M MCXOflfl M3 3Τ0Γ0, T3KMM 06pa30M, 47 GB 10506 CKopocTb npoflyi4MpoBaHMfl apMTONoTMHa b τθηθημθ 7 αηθμ b 100 ΜΠ KynbTypbi flOCTMraeT θηθηθημμ B npepenax ot 750 of 1470 V / 106 κπθτοκ 3a 48 nacoB.

Ha / llHOHHbie χμακοοτμ m κπθτοηηομ nmm CHO pDSVL-MKEPO, aMnDMMMPOBaHHbie b MeTOTpeKcaTe c COGeTaPeTeMe 10 M; 0čpa3eu, npiiroTOBneHHbiii b COURSEHOPUPBaHHiii cpepe npn paflMOMMMyHorioriiHecKOM aHann3e copepxnr MK-apMTponosTMHa 41.2 ± 1.4 V / μπ; 41.2 ± 0.064 V / mπ, κθκ onpepeneHO npn aHann3e Ha 6nonornHecKyio callrochocTb in vitro n 42.5 ± 5 V / μπ no paHHMM aHann3a onpepeneHnn Ομοπογμηθοκομ βκτιίβηοοτι / ι in vivo. wheel onpepeneHnn θμκηοκμοποτηηχ ocraTKOB ποπμπθτμαηνχ npoflyKTOB o6HapyxMBaeicn Hanunne apnTpono3TMHOBbix nponyicroB, πρμηθμ flOMUHaHTHbiM BHA BKntOHaeT 3 ocTaTKa " nnAiipyiomeM "nocneflOBaTenbHOCTM, npuMbiKaiOLnefi κ npepnonaraeMOMy 3aBepweHH (o aMHHorpynn anaHMHOM. B HacToniiļee BpeMa ηθ acho, πβππθτοη πμ 3το pe3ynbTaT0M HenpaBunbHoti o6pa6oTKH nppa nonunenTupa b κπθτκθχ CHO, nnn 0Tpa > xaeT pa3nnHne b CTpyiaype κοηθηηογο 3aBepujeHMH aMHHorbingTontonTUHa o6e3bnHbi OTHOcmenbHO hoopHHa ηθποβθκθ.

HaflOcaflOHHyio XMAK0CTb, npnROTOBneHHyto M3 πμημμ κπθτοκ CHO pDSVL-gHuEPO nopBepraioT τρβΜ ncnbuaHnnM. Hο naHHMM paflnMMyHonornHecKoro aHanM3a 5,5-flHeBHbiM o6pa3eq copep KMT ρθκομ6μη3ητημμ apnTponosTUH ηθποβθκθ b yKa3aHH0M cpepe, MMeiomnii 3Η8ηθημθ nopnpKa 18,2 V / μπ; npn aHann3e in vitro with a 15.8 ± 4.6 V / μπ m pin anan in vivo at 16.8 ± 3.0 V / μπ. npnroTOBneHHyio m nmm κπθτοκ CHO pDSVL-gHuEPO c nocTeneHHbiM 3ΜΠΠΜφΐΊ141Ίρ0Β3ΗΜΘΜ ΜΘΤΟΤρβΚΟΘΤΟΜ C ΚΟΗΜβΗΤρθΑΜθίί 100πΜ nOfLBepraiOT TpeM MCnblTaHMnM. Hο naHHbiM paflnoMMMyHonornHecKoro aHann3a, 3-χ αηθβηημ o6pa3eu οοΑβρχιιτ peKOMČMHaHTHbm aidTMH ηθποβθκ3, nMeioylHM 3HaHeHne 3089 + 29V / mh; in vitro - 2589171.5V / Mn u npn μοπντθημμ o6pa3Li in vivo - 20401160V / Mn. nocneflOBaTenbHOCTb θμμηοκμοποτ noKa3bieaeT 3aBepineHMe aMMHorpynn, cooTBeTCTByiomee npeACTaBneHHOMy b Ta6nnqe 4.

KneTOHHyio KOHflMMMOHMpoBaHHyio cpeny, npnroTOBneHHyio m κπθτοκ CHO, KOTopan τρ3ΗθφθκτιιροΒ3Η3 nna3MMfloii pDSK MKE b MeTOTpeKcaTe c KOHMempamieii 10 nm oāteflMHnraT m MeTOTpeKcaT flnann3npyioT b τβΜβΗΜβ HecKonbKnx αηθμ, b pe3ynbTaTe nero 06pa3yeTcn KynbTypa c aicrMBHOcTbio apuTponosTMHa nopnflKa 22115.1 V / μπ (apiiTponoaTMH-κπθτομηθπ KOHflMunoHnpoBaHHan cpefla ) (EPO-CCM). flnn onpepeneHnn ΑΘμοτβμη EPO-CCM Ha noKa3aTEN reMaCe b nmm Baib / c očbHHoii Mbiuin npOBOAflT cneAyioiHMM 3KcnepnMeHT. KneTOHHyio KOHAMMnoHMpoBaHHyto cpeAy, npnroTOBneHHyio H3 ΗβΤρ3ΗθφθΚΤΜρθΒ3ΗΗΒΙΧ ΚΠΘΤΟΚ CHO (CMM) M EPO-CCM perynnpy (OT npn MCnonb30BaHMM CTaHAapTHoro 6yφepHOΓO pacTBopaAnn ρθτμ ^ ποαμτοβ (PB). KneTOHHyio KOHAHUnoHnpoBaHHyio · cpeny ηρΜΜθΗπιοτ Ha KomponbHOM rpynne xmbothux (3 MbiiuKu), a PNN 3KcnepnMeHTanbHbix rpynn ( 2 MbiLUKM Ha rpynny) ncnonb3yioT 2 ypoBHn ao3mpobkh EPO-CCM 4 eAHHmļbi Ha MHĪ.eKUHIO M 44 eAHHHLļbl Ha HHbeKUHIO. B ΤβΗΘΗΙ / ΙΘ 5-HeAenbHOrO Kypca 7 MblUJKaM πρ0Μ3Β0ΑΠΤ 48 BHyTpn6pKDUJMHHbl0 MHbeKLJMM ΠΟ 3 pa3a Β ΗΘΑΘΠΙΟ. ΠΟΟΠΘ 8-ΟΜ ΜΗΐ > ΘΚ14ΜΜ ΟρβΑΗΜΘ Π0Κ338ΤΘΠΜ reMaTOKpma y KOHTponbHoii rpynnbi, Kak yciaHOBneHO, οοοτθβππιοτ 50.5% αππ rpynnbi npn ββθαθημ'μ 4 θαμημα EPO-CCM ohm οοοτθβπηιοτ 55.1% m NPM ββθαθημμ 44 θαμημα EPO-CCM -67.9%. npOflyKTbi sKcnpeccmi κπθτοκ μπθκοπμτθιοιαμχ χμβοτηβιχ mo > kho 6e3 3aTpyflHeHMH M3BneKaTb b flOBonbHO οημιαθηηομ βμαθ M3 KynbTypanbHofi cpeflbi c ncnonb30BaHneM HPLC (C) c sTaHonbHbin rpafliieHTOM, more preferably npn 3HaneHMM pH-seventh npoBefleHa npeflBapmenbHafl nonbīTKa of onpefleneHmo cboPictb ρθκομΘμηθητηβιχ ΓΠΜΚΟΠρΟΤΘΜΗΟΒΜΧ npOflyKTOB, nOnyHeHHblX M3 KOHflMMMOHHOBaHHOfi Cpeflbl 3KCnpeCCMM ΚΠΘΤΟΚ COS-I m CHO reHa apmponoaTMHa M3 mohm neoBeKe npM Mcnonb30BaHMM KaK aHann3a Western blot (oKpatunBaHMfl), Tax m aHann3a SDS-PAGE. YKa3aHHbie μοοπθαοβθημη noKa3biBaiOT, whereby to apmponoaTMHOBbiM MaTepnan, npoflyqnpyeMbiM κπθτκ3μμ CHO, μμθθτ HecKonbKO SonbīūMM MoneKynnpHbiM βθο, ηθμ npoAyi < Tbi aKcnpeccMM κπθτοκ COS-I, κοτορωΜ, b cboio onepeflb, ηθμηογο 6onbiue μοηθβογο SKCTpaicra ηθποβθκθ nynoBoro μοτοηημκθ. Βοθ npoflyicrbi β ηθκοτορομ οτθπθημ reieporeHHbi. 06pa6oTKa cf > epMeHTOM-HefipaMMHMAa3a απη yAaneHMfl cnanoBOM κμοποτβι πρμβοαμτ κ Tomy, hto b pe3ynbTaie peKOMGnHaHTHbie npoAyKTbi κπθτοκ COS-I m CHO μμθιοτ npuMepHO OAMHaKOBbm MoneKynnpHbm βθο, KOTopbie τθμ ηθ μθηθθ, Kak οαμη, TAK m Apyroii, Sonboje MoneKynnpHoro Beca nonyHaiomerocfl μοηθβογο sKCTpaicra ηθποβθκθ , copep ^ amero a3nanorpynnbi. 06pa6oTKa F-Φθρμθητομ 3HA0rniOKO3MAa3a (EC 3.2.1) ρθκομ6μη8ητηογο npoAytcra κπθτοκ CHO m npoAyicra μοηθβογο aKCTpaicra (αππ ποπηογο yAaneHnn ymeBOAOB M3 o6omx npoAyKToe) πρμβοαμτ β pe3ynbTaTe κ nonyHeHMto πόητμ γομογθηημχ npoAyKTOB, κοτορωβ, b ochobhom, μμθιοτ OAMHaKOBbie MoneKynnpHbie EECA. Οημιαθηηνμ apuTponoaTMH, nonyHeHHbiii M3 MOhm nenoBeKa, m peKOMČMHaHTHbiii, npoAyUMpyeMbiii M3 κπθτοκ CHO, apnTponoaTMH cornacHO HacTonmeMy Μ3θ6ρβτθΗΜΐο nOABepraioT aHanM3y Ha οπρθαθπθημθ yrneBOAHoro cocTaBa of μθτοαμκθ, onMcaHHOM Ledeen m ApyrMMM. Methods in Enzymology t.83, nacTb, ατρ.139-191.1982 r., KOTOpan MOAMc) MMMpoBaHa npM Mcaonb30saHMM MeTOAa rMAponM3a, isMcaHHoro Nesser m APyrMMM, Anal.Biochem., Τ.142, οτρ.58-67.1984 r . OnpeAeneHHbie 3KcnepMMeHTanbHbiM NyiMeKa3aTenM yrneBOAHoro cocTaBa αππ μοηθβογο M3on «Ta (BbipaxeHHbie β βμαθ MonnpHbix οτηοιπθημμ β yKa3aHHbix npoAyicrax) μμθιοτ cneAyK) iAMe 3Η8ΗΘημπ: reKC03a -1.73; -aqeTMn-rntoK03aMMHH-1; -θμθτμπ ηθμρθμμηοβομ KMcnoTbi - 0.93; cpyK03a - 0; m -aqeTMnranaKT03aMMH -0. CooTBeTCTByioinMe θηθηθημη αππ peKOM6MHaHTHbix npoAyKTOB (np0M3B0AHbix ot πμημμ κπθτοκ CHO pDVSL -gHuEPO) 3 χ αηθβηομ KynbTypanbHOPi cpeAbi, ycnneHHOM μθτοτρθκο3τομ npn KOHueHTpaunM 100 Μ) μμθθτ cneAyioiiiMe noKa3aTenn: reKC03a -15.09; N-aqeTMnrnK03aMMH -1; Ν-θαθτμπ ηθμρθμμηοβομ κμοποτβι - 0.998; 0yKO3a - 0 μ N-aqeTMnranaKT03aMMH - 0. rionyHeHHbie pe3ynbTaTbi cosnapaiT c pe3ynbTaTaMM onMCbiBaeMbixBbiu aHanM30B OKpaiuMBaHMn Westernblot m SDS-PAGE. 49 EN 10506 ΓπΜκοπροτθΜΗΟΒΝθ npoflyKTbi, nonyHaeMbie cornacHO HacTO? ImeMy n3odpeTeHnio,

TaKMM o6pa30M, npepcTaBnatoT COCOM ποοτμχοιμμθ npoflyKTbi, nepBMMHan cTpyKTypHaa ΚΟΗφΟρΜΘΜΜΑ KOTOpblX B flOCTaTOM.HOM ΟΤΘΠΘΗΜ BOCnpOM3BOflMT ΚΟΗφορΜΘΜΜΚ) 3ρΜΤρ0Π03ΤΜΗ0Β ΘΟΤΘΟΤΒΘΗΗΟΓΟ ΠρΟΜΟΧΟΧΑΘΗΜΗ ΗΠ51 npMflaHMfl MM OflHOrO ΜΠΜ HeCKOilbKMX 6ΜΟΠΟΓΜΗΘΟΚΜΧ cbomctb, m KonMHecTBeHHbiM cocTaB ymeBOAOB KOTopbix omMHaeTca ot cocTaBa 3ΡΜΤΡ0Π03ΤΜΗ0Β ΘΟΤΘΟΤΒΘΗΗΟΓΟ ΠρΟΜΟΧΟΧΑβΗΜΑ. ΠΡΜΜΕΡ 11 npennaraeMbiM EXAMPLE οτηοομτοα the eceMy UMicny nonyHeHMn apMTponoaTMHOB nOCpeflCTBOM ΟβορΚΜ HyKneOTMflHblX OCHOBaHMM flByX CTpyKTypHblX ΓΘΗΟΒ, KOflMpyiOULlMX nocneflOBaTenbHOCTb apMTponosTMHa ηθποβθκθ, npeACTaBneHHOM b TadnMpe VI C0AepxamMX m, ΟΟΟΤΒΘΤΟΤΒΘΗΗΟ "FlpeAnOHTMTeribHbie" KOAOHbl ΑΠΗ 3KCnpeCCMM npM3HaK0B B E.coli M ApoxxeBbix "neTKax (S. serevisial). B yKa3aHHOM npMMepe αοποπημτθπβηο HoaHa KOHCTpyKLlnn γθηοβ, KOAMpyiouļMX aHanorM spMTponosTMHa. Copons roBopa, npoTOKonbi, KOTopbie Mcnonb30BaHbi, npeACTaBneHbi b ο6ιαθμ, K3K yKa3aH0 b PAnYMMHeMM οπμοθημμ Alton m APyrnMM. (WO 83/04053). YKa3aHHbie reHbi CKOHCTpyMpOBaHbi Ann NepBOHaHibHOM οβορκΜ οοοτθβπαιοιαμχ OnMrOHy «neOTMAOB B ΟΠΟΧΗΟΜ, KOTOpbie, B CBOK} OHepeAb, COSpaHbl B TpM AMCKpeTHbie οθκαμμ (6ποκοβ). YKa3aHHbie 6ποκμ nocTpoeHbi απα βμππμΦμκ3αμμ μ πρπ yAaneHMM Μ3 GMCTeMbi θμππμΦμκβμμμ Moryr 6brrb co6paHbi nocneAOBaTenbHO μπμ nepe3 nMraTypy Φρβγμθητβ co οποχηομ CTpytcrypoM b ποαχοαλιαμμ Βθκτορ 3KcnpeccMM.

Ha Ta6nMqax VIII through XIV noKa3aHa in Scheme m cčopKa nonyneHHoro REHAU, KOAMpyioiAero npoAyi < T TpaHcnnuMM apMTponosTMHa, b κοτοροΜ 0TcyTCTByeT KaKan-πΜβο Apyraa nMAMpy (oman μπμ npeAuuecTBytomaH nocneAOBaTenbHocTb, ho οοαθρχμτοη MCXOAHbiii οοτβτοκ μθτμοημηβ b ποποχθημμ S. EOne τογο, γθη, ββθαθηηημ b ocHOBHyio nacTb κπθτκμ E.Col, OTAaeT ΠρβΑΠΟΗΤΘΗΜΘ KOAOHaM Μ, T3KMM 06pa30M, yAa3aHHyK) KOHCtpyKllMIO Ha3blBaiOT " ECERO " .

TAENHUA VIII

OnnroHyKneoTMflbi msctm 1 ECEPO

1. AATTCTAGAAACCATGAGGGTAATAAAATA

2. CCATTATTTTATTACCCTCATGGTTTCTAG

5 3. ATG G CTCCG CCGCGTCTG ATCT GCG AC

4. CTCGAGTCGCAGATCAGACGCGGCGGAG

5. TCGAGAGTTCTGGAACGTTACCTGCTG

6. CTTCCAGCAGGTAACGTTCCAGAACT

7. GAAGCTAAAGAAGCTGAAAACATC

10 8. GTGGTGATGTTTTCAGCTTCTTTAG

9. ACCACTGGTTGTGCTGAACACTGTTC

10. CAAAGAACAGTTGAG CACAACCA

11. TTTGAACGAAAACATTACGGTACCG

15 12. GATCCGGTACCGTAATGTTTTCGTT

ΤΑΒΠΙ / IUA IX HacTb 1 ECEPO

Xbai

EcoRl 1 2 AATTCTAG AAACCATGAG GGTAATAAAA TAATGGCTCC GCCGCGTCTG GATC TTTGGTACTC CCATTATTTT ATTACCGAGG CGGCGCAGAC 20 2 4 5

ATCTGCGACT CGAGAGTTCT GGAACGTTAC CTGCTGGAAG CTAAAGAAGC TAGACGCTGA GCTCTCAAGA CCTTGCAATG GACGACCTTC GATTTCTTCG en 7 2 '11 TGAAAACATC ACCACTGGTT GTGCTGAACA CTGTTCTTTG AAACGAAAACA ACTTTTGTAG TGGTGACCAA CACGACTTGT GACAAGAAAC TTGCTTTTGT 25 2 12

Kpn1 BamHI TTACGGTACC G AATGCCATGG CCTAG IZ EN 10506 51

ΤΑΒΠΜϋΑ X

OnnroHyKneoTnnbi nacin 2 ECEPO

1 AATTCGGTACCAGACACCAAGGT 2 GTTAACCTTGGTGTCTGGTACCG 5 3 4 TAACTTCTACGCTTGGAAACGTAT TTCCATACGTTTCCAAGCGTAGAA 5 GGAAGTTGGTCAACAAGCAGTTGAAGT 6 CC AAACTT C AACTGCTTGTTG ACCAAC 10 7 8 TTGGCAGGGTGTGGCACTGCTGAGCG GCCTCGTCAGCAGTGCCAGACCCTG 9 AGGCTGTACTGCGTGGCCAGGCA 10 GCAGTGCCTGGCCAACGCAGTACA 11 CTGCTGGTAAACTCCTCCTCTCAGCCGT 12 TTCCCACGGCTGAGAGGAGTTTACCA 15 13 14 GGGAACCGCTGCAGCTGCATGTTGAC GCTTTGTCAACATGCAGCTGCAGCGG 15 AAAGCAGTATCTGGCCTGAGATCTG 16. GATCCAGATCTCAGGCCAGATACT 52

ΤΑΒΠΜϋΑ XI HacTb 2 ECEPO

EcoRl Κρπ1 1 3 A ATTCGGTACC AGACACCAAG GTTAACTTCT ACGCTTGGAA ACGTATGGAA GCCATGG TCTGTGGTTC CAATTGAAGA TGCGAACCTT TGCATACCTT 2 4

2 Z

GTTGGTCAAC AAGCAGTTGA AGTTTGGCAG GGCTTGGCAC TGCTGAGCGA CAACCAGTTG TTCGTCAACT TCAAACCGTC CCAGACCGTG ACGACTCGCT S S 2 11

GGCTGTACTG CGTGGCCAGG CACTGCTGGT AAACTCCTCT CAGCCGTGGG 10 CCGACATGAC GCACCGGTCC GTGACGACCA TTTGAGGAGA GTCGGCACCC m 12 12 12 Bgļ11 BamHl AACCGCTGCA GCTGCATGTT GACAAAGCAG TATCTGGCCT GAGATCTG TTGGCGACGT CGACGTACAA CTGTTTCGTC ATAGACCGGA CTCTAGACCTAC 14 14 E 10506 53

TASnUUA XIX HacTb 3 ECEPO 1 GATCCAGATCTCTGACTACTCTGC 5 ACGCAGCAGAGTAGTCAGAGATCTG 2 3 4 TGCGTGCTCTGGGTGCACAGAAAGAGG GATAGCCTGTTTCTGTGCACCCAGAGC 5 CTATCTCTCCGCCGGATGCTGCATCT 6 CAGCAGATGCAGGATCCTGGCGGAGA 10 7 8 GCTGCACCGCTGCGTACCATCACTG ATCAGCAGTGATGGTACGCAGCGGTG 9 CTG AT ACCTTCCG C AAACTTGTTTCG 10 AT ACG AC AAACAGTTTG CG G AAGGT 11 TGTAT ACT CTA ACTT CCTGCGTGGTA 15 12. CAGTTTACCACGCAGGAAGTAGAGT 13. AACTGAAACTGTATACTGGCGAAGC 14. GGCATGCTTCGCCAGTATACAGTTT 15. ATGCCGTACTGGTGACCGCTAATAG 16. TCGACTATTAGCGGTCACCAGTAC 20 25 54

ΤΑΕΠΙ / IUA XIII MacTb 3 ECEPO

Bam Hl Bgļ11 GA TCCAGATCTCTG GTCTAGAGAC 5 12 5 ACTACTCTGC TGCGTGCTCT GGGTGCACAG AAAGAGGCTA TCTCTCCGCC TGATGAGACG ACGCACGAGA CCCACGTGTC TTTCTCCGAT AGAGAGGCGG 2 4 Z 2 GGATGCTGCA TCTGCTGCAC CGCTGCGTAC CATCACTGCT GATACCTTCC CCTACGACGT AGACGACGTG GCGACGCATG GTAGTGACGA CTATGGAAGG 10 2 2 11 12 GCAAACTGTT TCGTGTATAC TCTAACTTCC TGCGTGGTAA ACTGAAACTG CGTTTGACAA AGCACATATG AGATTGAAGG ACGCACCATT TGACTTTGAC 1Q 12 12 Hall TATACTGGCG AAGCATGCCG TACTGGTGAC CGCTAATAG ATATGACCGC TTCGTACGGC ATGACCACTG GCGATTATC AGCT 14 lfi 15 EN

TA5f11 / ILlA XIV Γθη SCEPO -1 1

Xbal MetAla

CTAG AAACCATGAG GGTAATAAAA TAATGGCTCC GCCGCGTCTG TTTGGTACTC CCATTATTTT ATTACCGAGG CGGCGCAGAC

5 ATCTGCGACT CGAGAGTTCT GGAACGTTAC CTGCTGGAAG CTAAAGAAGC TAGACGCTGA GCTCTCAAGA CCTTGCAATG GACGACCTTC GATTTCTTCG

TGAAAACATC ACCACTGGTT GTGCTGAACA CTGTTCTTTG AACGAAAACA ACTTTTGTAG TGGTGACCAA CACGACTTGT GACAAGAAAC TTGCTTTTGT

10 TTACGGTACC AGACACCAAG GTTAACTTCT ACGCTTGGAA ACGTATGGAA AATGCCATGG TCTGTGGTTC CAATTGAAGA TGCGAACCTT TGCATACCTT

GTTGGTCAAC AAGCAGTTGA AGTTTGGCAG GGTCTGGCAC TGCTGAGCGA CAACCAGTTG TTCGTCAACT TCAAACCGTC CCAGACCGTG ACGACTCGCT GGCTGTACTG CGTGGCCAGG CACTGCTGGT AAACTCCTCT CAGCCGTGGG CCGACATGAC GCACCGGTCC GTGACGACCA TTTGAGGAGA GTCGGCACCC 15

AACCGCTGCA GCTGCATGTT GACAAAGCAG TATCTGGCCT GAGATCTCTG TTGGCGACGT CGACGTACAA CTGTTTCGTC ATAGACCGGA CTCTAGAGAC

ACTACTCTGC TGCGTGCTCT GGGTGCACAG AAAGAGGCTA TCTCTCCGCC TGATGAGACG ACGCACGAGA CCCACGTGTC TTTCTCCGAT AGAGAGGCGG

20 GGATGCTGCA TCTGCTGCAC CGCTGCGTAC CATCACTGCT GATACCTTCC CCTACGACGT AGACGACGTG GCGACGCATG GTAGTGACGA CTATGGAAGG

GCAAACTGTT TCGTGTATAC TCTAACTTCC TGCGTGGTAA ACTGAAACTG CGTTTGACAA AGCACATATG AGATTGAAGG ACGCACCATT TGACTTTGAC

Hall

TATACTGGCG AAGCATGCCG TACTGGTGAC CGCTAATAG ATATGACCGC TTCGTACGGC ATGACCACTG GCGATTATCA GCT 25

Eonee peranbHO, Tačnnua VIII nnnKDCTpnpyeTnMeReReReReReTMflOB gna reHepupoBaHMH HacTM I reHa ECEPO, KOflMpyramero COATBbie ocTaTKM θμμηο nenoBenecKOro nonMnenTMpa. OnnroHyKneoTMflbi coSnpaioT b flynneKCbi (1 m 2, 3 m 4, m T.fl.), KOTopbie eaieM cutMBaiOTflN nNeNHMn HacTH I ECEPO, KaK b TaSnMLlue IX. CneflyeT OTMBTMTb, hto co6paHHan nacTb βκπιοηβθτ cooTBeTCTBeHHbie πμπκμθ COOKING KOHEHHbix EcoRI m BamHI. πρμηθμ no ποτοκ / 'πμπκογο KOHga EcoRl HanpaBneH ynacTOK pacno3HaBaHMn OralHUHMTenbHoro φβρΜθΗτβ Xbal, a " ηροτΜΒ noTOKa πμπκογο Kornia BamHI HanpaBneH ynacTOK pacn03HaBaHMn Kpnl. HacTb I mojkho 6e3 Tpypa 3ΜΠΠΜφΜ14Μρ0Β3ΤΒ, MCn0nb3yfl ΒβΚΤΟρ φ3Γ8 MI3, ΠρΜΜβΗΠβΜΜΜ βΠΠ ΠρΟΒβρΚΜ nocneflOBaTenbHOCTM nacTM. HeKOTopbie TpyflH0CTM HeoxnflaHHO B03HMKat0T ripM BbipeneHMM MacTM b KanecTBe φρ8ΓΜΘΗΤΒ Xbal / Kpnl M3 flHK RF, reHepMpoBaHHOM b E.coli, βθροπτηο, M3-3a μθτμπμροβ3ημπ οοηοβ3ημμ ynacTKa pacno3HaBaHMa Kpnl b npepenax X03HMHa. riO3T0My M30HMpyi0T flHK φ8Γ8 M BOCnpOM3BOflflT B flByHMT6B0M φορΜΘ ΙΠ VltiO noopepcTBOM pacTnxeHMn 3aTpaBK0M, m nocne nero M30nMpytOT Tpe6yeMbiM flByHMTeBbifi φρθΓΜΘΗΤ.

HacTM 2 m 3 reHa ECEPO (TaanMalbi XI μ XIII) στροπτ nof10bHbiM o6pa30M M3 happroHyKneoTMflOB TabnMLi X μ XII, οοοτβθτοτβθηηο. Kaxpyip nacTb aMΠΠMφM ^ Mpyιoτ Ha Beicrope MI3, npMMeHneMbiM ρππ προΒθρκΜ nocneflOBaTenbHOCTM, m BbiflenniOT m flHK φθΓθ. Kax BBCTByeT m Ta6nMflbi XI, HacTb 2 ECEPO CTpomcn c πμπκμμμ KOHflaMM EcoRl m BamHI μ μοχθτ 6biTb BbipeneHa b KanecTBe Φρθγμθητθ Kpnl / Bglil. Tohho TaiOKe HacTb 3 ECEPO nonyneHa c πμπκμμμ κοημθμμ BamHI m Sali μ μοχθτ 6biTb BbipeneHa M3 flHK RF κθκ Φρθγμθητ Bglll / Sall. l < lonyHeHHbie tbkmm o6pa30M τρΜ nacTM μοχηο 6e3 Tpypa cobpaTb b HenpepbiBHyto nocneflOBaienbHOCTb flHK (TabnMLta XIV), KopMpyioinyio 'ΠΟΠΜΠΘΠΤΜΡ ΠΟΠΗΟΓΟ ΗβΠΟΒΘΗΘΟΚΟΓΟ EPO C MeTMOHMHOBbIM KOflOHOM BMMHO OKOHHaHMH (ATG) ΑΠΠ TpanennUMOHHOrO MHMUMMpOBaHMH E.COli. CneflyeT OTMeTMTb, hto " npOTMB noTOK " nepBOHananbHoro ATG HanpaBneH pnfl a30TMCTbra οοηοββημμ, αοοήτοηηο fylynnMUMpyKDi | flna nepeHOca ECEPO μοχηο Mcnonb30BaTb močoii ποαχοηπιαμμ Ββκτορ aKcnpeccMM. ΟπβρΜφΜΗβοκΜΜ ΒβκτοροΜ, BbičpaHHbiM flna aKcnpeccMM reHa ECEPO b KanecTBe ”TeMnepaTypHO — HyscTBMTenbHOM nna3MMflbi pCFM536, πβππθτοπ προμ3βοπηοθ nna3MMflbi pCFM414 / A.T.C.C.C. 40076 / - KaK οπμοθηο b οαηοβρθμθηηο paccMaTpMBaeMoii 3a «BKe Ha naTernCUJA N 636727, POPHHOM 6 aBrycia 1984 r.

Bonee κοΗκρβτΗΟ, pCFM536 nepeeapMBaiOT c Xbal m Hind III; 6onbuiOM φρ8ΓΜβΗΤ M3HOnMpyiOT Μ ΠρΜΜβΗΠΙΟΤ B flByXCTOpOHHeM CUJMBaHMM C ΓβΗΟΜ ECEPO. HaCTM I Xbal / Kpnl / r 2 / Kpnl / Bglil / m 3 / Bglll / Sall / npejlBapMTenbHO co nM Bb mnbHOM cf. 3tot ΦΡ3γμθητ BKniOHaeT nacTb nonMCBfl3yomero 3BeHa m MI3 Μρθ-φβτβ, CTnrMBaiomero b ηθμ ynacTKM Salt b Hind III. Komponb 3a 3KcnpeccMeM b nonynaeMOM nna3MMpe 57 EN 10506 SKcnpeccmi, p536, BbinonHfliOT npn πομοιαμ npOMOTopa nflMČpa Pl, KOTopbiii cam μοχθτ perynnpoBaTbCfl penpeccopHbiM γθηομ C1857 (tskmm. Κβκ tot KOTopbiii nonyHeH b KI2AHtrp hmtm E.coli). riOCTpoeHHblM ΓΘΗ ECEPO ΜΟΧβΤ ČbITb MOflMtjMUMpOBaH3mHHblM 06pa30M ΑΠΑ KOflupoBaHMfl aHanoroBTMHa, τ3κμχ KaK [ASn2, des-Pro2 no ILe8] h EPO m [HI57] h EPO, KaK happcaHO Bbirne.

A. [Asn2, des-Pro2 no IL6] h EPO nna3MMAa 536, Hecymaa noTpoEHHbiii γθη ECEPO TaSniiUbi XIV KaK βοτθβκθ Xbal b Hind III, nepeBapuBaeTcn c Hind III m Xhol. 3HflOHyKnea3a ποοπθαηθγο pa3pe3aeT γθη I · ECEPO b yHMKanbHOM ynacTKe pacno3HaBaHna, μμθιοιαθμ 6 nap a30TMCTbix οοηοβθημμ, CTiirMBaa ποοπθαηθθ οοηοβ3ημθ KOflOHa, KOflnpy »Omero ASp8 co BTopbiM οοηοβθημθμ KOflOHa Arg18. CTpomca nocneflOBaTenbHocTb ”CBH3yiomero 3BeHa” Xbal / Xhol. HMeOn cneflyioinyK3 nocneflOBaTenbHocTb: XM + l 2 7 8 9 Me Ala • Asn Cys Asp Xhol -CTAG ATG GCT AAT TGC GAC- -3 '3' - TAC CGA TTA ACG CTG AGCT -5 'CBH3yK) mee 3βθηο Xbal / Xhol m cfipaFMeHT nocneflOBaTenbHOc ™ Reha ECEPO Xhol / Hind III βοτθβπαιοτ b 6onbuuoM Φρβγμθητ, nonyHatomMiicfl b pe3.ynbTaie nepeBapnBaHM? i Xbal h Hind III nna3MMflbi pCFM526 - npon3BOflHoe nna3MMAbi pCFM414 (ATCC 40076) - CEC onncaHo b οαηοβρθμθηηο paccMaTpMBaeMoii 3aaBKe Ha naTeHT CLLIA N 636727, noAaHHOii 6 aBrycTa 1984 ropa Charles F. Morris, απα reHepnpoBaHnn nna3MMAa-Hecymei flHK-nocneAOBaTenbHocTH, KOAnpyiomePi aKcnpeccmo E.coli cfropMbi Met '" * xcenaeMoro aHanora.

B. [His7] hEPO rina3MMAy 536 nepeBapMBaioT c Price III m Xhol. KaK b nacTH A. CBfl3yiomee 3ΒΘΗΟ Xbal / Xhol nocTpoeHa co cneAyiomeM nocneAOBaTabHocTbio:

Xbal +1 2 3 4 5 6 7 8 Xhfil

Me Ala Pro Pro Arg Leu for His Asp 5 '- CTAG ATG GCT CCG CCA CGT CTG ATC CAT GAC 3' 3'- TAC CGA GGC GGT GCA GAG TAG GTA CTG AGCT-5 '58 CBH3yioinee 3βθηο μ Φρθγμθητ nocjiepoBaTenbHOCTH ECEPO Xhol / Price II! 3βτβΜ BCTaBnatoT b pCFM526 pna reHepHposaHua rma3MMfla-Hecymeii flHK-nocnenoBaTenbHocTH, ConversionM sKcnpeccmo E.coli φορΜΝ Met '- * xenaeMoro aHanora. ΠοστροθΗΜΘ μ3γοτοβπθηηογο γθη3 (" SCERO "), BKononotinero b bleed flpoxxeBbie npeflnoMTUTenbHbie KOflOHb, nponcxoflHT KaK happcaHO b cneflytomnx Ta6nnuax XV-XXI. TaK'xe XAX mb cnynae c γθηομ ECEPO, ποπηοθ ποοτροθημθ coflepxnT o6pa30BaHne Tpex HaSopoB onnroHyi < neoTMflOB (Ta6nnL (bi XV, XVII μ XIX) KOTopbie o6pa30Banncb b flynneKCbi m coāpamicb b ceKumi (Ta6nnubi XVI, XVIII μ XX). CnenyeT OTMeTMTb, mto ομητθ3 o6nernaeTCH nacTHHHO ncnonb30BaHneM HeKOTopbix cy6onTMManbHbix koaohob b o6omx EPO m ECEPO ποσφοβΗΜΑΧ, Te onnroHyKneoTMflbi 7-12 CeKLinn I o6omx γθηοβ 6binn MfleHTMMHbi, Kak 6binn MfleHTMHHbi οπηγο ^ ^ κπθοτηα 1-6 CeKLļnn 2 b KaxflOM γθηθ. EN 10506

ΤΑΕΠΙ / IUA XV

OniiirHyKneoTMUbi SCEPO nacTM 1

1. AATTCAAGCTTGGATAAAAGAGCT

2. GTGGAGCTCTTTTATCCAAGCTTG

3. CCACCAAGATTGATCTGTGACTC

4. TCTCGAGTCACAGATCAATCTTG

5. GAGAGTTTTGGAAAGATACTTGTTG

6. CTTCCAACAAGTATCTTTCCAAAAC

7. GAAG CTAAAGAAG CTGAAAACATC

8. GTGGTGATGTTTTCAGCTTCTTTAG

9. ACCACTGGTTGTGCTGAACACTGTTC

10. CAAAGAACAGTGTTCAGCACAACAA

11. TTTGAACGAAAACATTACGGTACCG

12. GATCCGGTACCGTAATGTTTTCGTT

ΤΑΕΠΜυΑ XVI Mactb 1 SCEPO

EcoRI AATTCA GT

Hindlll 1 AGCTTGGATA TCGAACCTAT 2

AAAGAGCfjcC ACCAAGATTG ATCTGTGACT GAGAGAGTTTT TTTCTCGAGG TGGGTCTAAC TAGACACTGA GCTCTjCAAAA 4 5, 1 GGAAAGATAC TTGTTGGAGAG CTAAAGAAGC TGAAAACATC |

2 11 Kpn1 BamHI

GTGCTGAACA CTGTTCflTTG AACGAAAACA TTACGGTACC G CACGACTTGT GACAAGAAACl TTGCTTTTGT AATGCCATGG CCTAG 12 60 TASNUUA χνιι

OnnroHyKneoTnnbi SCEPO nacTM 2

1 Aatto CGGT ACCAGACACCAAGGT 5 GTTAACCTTGGTGTCTGGTACCG 2 3 4 TAACTTCTACGCTTGGAAACGTAT TTCCATACGTTTCCAAGCGTAGAA 5 GGAAGTTGGTCAACAAGCAGTTGAAGT 6 AAACTT CC C G AACT CTTGTTG ACCAAC 10 7 8 TTGGCAAGGTTTGGCCTTGTTATCTG G CTT C AG AT AACAAG G G 9 CCAAACCTT AAGCTGTTTTGAGAGGTCAAGCCT 10 AACAAGGCTTGACCTCTCAAAACA eleventh TGTTGGTTAACTCTTCTCAACCATGGG 15 12. TGGTTCCCATGGTTGAGAAGAGTTAACC 13. AACCATTGCAATTGCACGTCGAT 14. CTTTATCG ACGT G CAATTGCAA 15. AAAGCCGTCTCTGGTTTGAGATCTG 16. G ATCCAG AT CT C AAACCAG AG ACGG 61 EN 10506

TABni / IL1A XVIII Hacrb 2 SCEPO

Kpnļ

EcoRI 1

A ATTCGGTACC AGACACCAAG 5 GCCATGG TCTGTGGTTC 2 GTtrAACTTCT ACGCTTGGAA ACGTAT | gGAA GTTGGTCAAC AAGCTGTTGA CAATTGjAAGA TGCGAACCTT TGCATACCTTJCAACCAGTTG TTCGACAACT 4 Z 2

AGTTTGGCAA GGTTTGGCCT TGTTATCTGGA AGCTGTTTTG AGAGGTCAAG 10 TCAAHCGGTT CCAAACCGGA ACAATAGACT TCGLACAAAAC TCTCCAGTTC a m n i n

CCTTTGTTGGT TAACTCTTCT CAACCATGGG ATTGCACGTC GGAAATCCA ATTGAGACAG GTTGGTACCC TTGGTAGAACGT TAACGTGCAG 12 14

and Egl11 BamHI

GATEL ^ AAGCCG TCTCTGGTTT GAGATCTG CTATTT ^ GGC AGAGACCAAA CTCTAGACCTA G ia

62 TABNUUA XIX

OnnroHyiaieoTMūbi SCEP nactn 6 1 G AT AT CCAG CTTT G ACT GTT ACTTT 5 TCTCAACAAAGTAGTCAAAGATCTG 2 3 4 GAGAGCTTTGGGTGCTCAAAAGGAAG ATG G AG CTTCCTTTTG CACCCAAAGC 5 CCATTTCCCCACCAGACGCTGCTT 6 GCAGAAGCAGCGTCTGGTGGGGAA 10 7 G CCG CT CTCC ATTG AG AT AACC C 8 CAGTGATGGTTCTCAATGGAGCG 9. ACTGCTGATACCTTCAGAAAGTT 10. G AATT CTG AAG GTAT CAG 11. ATT C AG AGTTT ACTCC AACTT CT 15 12. CTCAAGAAGTTGGAGTAAACTCT 13. TGAGAGGTAAATTGAAGTTGTACAC - 14. ACCG GTGT AC AACTT CAATTT ACCT 15. CGGTGAAGCCTGTAGAACTGGT 16. CTGT C ACC AGTT CTAC AG G CTT C 20 17. GACAGATAAGCCCGACTGATAA 18. GTTGTTATCAGTCGGGCTTAT 19. CAACAGTGTAGATGTAACAAAG 20. TCGACTTTGTTACATCTACACT 63 EN 10506

ΤΑΕΠΙ / IUA XX HacTb 3 SCEPO

Bam Hl Eal11 1 GATC CAGATCTTTG ACTACTTTGT t] gAGAGCTTT GTCTAGAAAC TGATGAAACA ACTCTJCGAAA 5 2 3 5

GGGTGCTCAA AAGGAAGGCCA TTTCCCCACC AGACGCTGCT TCTGCCGCTC CCGACGAGTT TTCCTTCGGT ALAGGGGGGGGGGGGGGGGGG AGGGGCGAG 4 5 I 2.. 11 CATTGAGAAC CATCALACTGCT GATACCTTCA GAAAGTWT CAGAGTTTAC GTAACTCTTG GTAGTGCGGA CTATGGAAGT CTTTCAATAA GILTCTCAAATG 10 en 1 £ 12, 15

TCCAACTTCT (TAGAGAGGT ATTGAAGTTG TACACALCGGTG AAGCCTGTAG AGGTTGAAGA TCTATT TCACTATAAC ATGTGGCCACC TTCGGACATC 14 lfi 17 1 £

AACTGGTTLSAC AGATAAGCCC GACTGATAAALC AACAGTGTAC TTGACCACTG TCfTATTCGGG CTGACTATTG TTGfTCACATC 15 ia

Hall

ATGTAACAAA G TACATTGTTT CAGCT m 20 25 64

TAEfīMLIA XXI Γθη SCEPO

Hindlll -1 + 1

ArgAla AGCTTGGATA AAAGAGCTCC ACCAAGATTG ATCTGTGACT CGAGAGTTTT ACCTAT TTTCTCGAGG TGGTTCTACC TAGACACTGA GCTCTCAAAA 5

GGAAAGATAC TTGTTGGAAG CTAAAGAAGC TGAAAACATC ACCACTGGTT CCTTTCTATG AACAACCTTC GATTTCTTCG ACTTTTGTAG TGGTGACCAA

GTGCTGAACA CTGTTCTTTG AACGAAAACA TTACGGTACC AGACACCAAG CACGACTTGT GACAAGAAAC TTGCTTTTGT AATGCCATGG TCTGTGGTTC

10 GTTAACTTCT ACGCTTGGAA ACGTATGGAA GTTGGTCAAC AAGCTGTTGA GAATTGAAGA TGCGAACCTT TGCATACCTT CAACCAGTTG TTCGACAACT

AGTTTGGCAA GGTTTGGCCT TGTTATCTGA AGCTGTTTTG AGAGGTCAAG TCAAACCGTT CCAAACCGGA ACAATAGACT TCGACAAAAC TCTCCAGTTC CCTTGTTGGT TAACTCTTCT CAACCATGGG AACCATTGCA ATTGCACGTC GGAACAACCA ATTGAGAAGT GTTGGTACCC TTGGTAACGT TAACGTGCAG 15

GATAAAGCCG TCTCTGGTTT GAGATCTTTG ACTACTTTGT TGAGAGCTTT CTATTTCGGC AGAGACCAAA CTCTAGAAAC TGATGAAACA ACTCTCGAAA

GGGTGCTCAA AAGGAAGCCA TTTCCCCACC AGACGCTGCT TCTGCCGCTC CCCTCGAGTT TTCCTTCGGT AAAGGGGTGG TCTGCGACGA AGACGGCGAG

20 CATTGAGAAC CATCACTGCT GATACCTTCA GAAAGTTATT CAGAGTTTAC GTAACTCTTG GTAGTGACGA CTATGGAAGT CTTTCATA GTCTCAAATG

TCCAACTTCT TGAGAGGTA ATTGAAGTTG TACACCGGTG AAGCCTGTAG AGGTTGAAGA ACTCTCCATT TAACTTCAAC ATGTGGCCAC TTCGGACATC

AACTGGTGAC AGATAAGCCC GACTGATAAC AACAGTGTAG TTGACCACTG TCTATTCGGG CTGACTATTG TTGTCACATC

ATGTAACAAAG TACATTGTTT CAGCT LV 10506

CoāpaHHbie οθκμμμ SCEPO cnepyeT b MI3 h CeKLlnn 1.2 n 3 BbiflenntoTcn H3 c α ar b KanecTBe Φρθγμθητοβ Hind IH / Kpnlr Kpnl / Bglll n Bglll / Sal. npeflnoMUTaeMoii cePinac ομοτθμοη sKcnpeccmd αππ προργκτοΒ Reha SCEP ηΒπηβτοη CHCTeMa ceKpegmi Ha οοηοβθ ceKpegnn a- $ aKTOpa S.cerevisial, Kak onncaHO b Haxoflnmeiicfl b npogecce οαηοβρθμθηηογο paccMOTpeHMa 3anBKe Ha naTeHT CLIL N 487753, noflaHHOM 22 anpenn 1983 Ron Grant A.Bitter, ony6nnKOBaHHOM 31 οκτηβρη 1984 ROFL CEC 3anBKa Ha eBponeHCKMM naTeHT 0,123,294th Kopone roBopn, CHCTeMa βκπιοηβθτ KOHCTpyKiļnn, RPE flHK, K0flnpy (0inaa Beflymyto nocneflOBaTenbHOCTb Reha a-0aKTopa ηροχοκθίί, pacnonaraeTCn cpa3y 5 'κ KOflnpyKDi4eii očnacTM Bbipa > KaeMoro 3Κ30γθηηογο Reha. B pe3ynbTaTe nero TpaHcnnpyeMbiii reHHbiH npoflyKT BKmonaeT Beflymyio mv \ cnmanbHyio nocneflOBaTenbHOCTb, KOTopan "o6pa6aTbiBaeTcn " sHporeHHbiM φβρΜβΗΤΟΜ flpoxxefi b HanpaBneHHH ceKpeqnn ocTaBLueiĪCH nacTM npoflyicra. Tak κθκ KOHCTpyKi4Msi flaeT B03M0XH0CTb ncnonb30BaHHjļ Kopona MHHLļHMpoBaHHjļ TpaHcn" UNN alkyl aicropa (ATG), He 6bino Heo6xopnMOCTH b C03flaHHM Takor KOflOHa b ποποχβΗΜΜ S Reha SCEP. Kak μοχηο 3aKmomiTb m Ta6nnL (bi XXI, anaHMH (+1) KOflnpyioiAMfi nocneflOBaTenbHOCTb, ctomt 3a nocneflOBaTenbHOCTbio CBfl3yromero 3BeHa, whereby to πο3Βοπηβτ ocyLflecTBMTb HenocpeflCTBeHHyio BCTaBKy b nna3MMfly, B n nK) wafOLut flHK pnn nepBbix 80 ocTaKOB npoBOflHMKa anpaicropa, cnenyiomero 3a προΜοτροΜ α-φθκτορβ. ΟπβΜΜφΜΜβοκοβ npeflnoHHTaeMoe nocTpoeHHe pnn 3Kcnpeccnn reHa CEPO copepacHT MeTbipexcTopoHHee cuHBaHHe, bkmonan BbiiueyKa3aHHbie φpaΓMeHTbl ceKLlMM CEPO m chonbuioii φρ3ΓΜβΗΤ nepeBapHBaHMn Hind Ill / Sal nna3MMfloii paC3. \ Ae nonyHaeMoii nna3MHflHoii paC3 / SCEP BbiflennioT npoMOTopHyio m npoBOflamyio nocneflOBaTenbHocTM a- ^ aKTopa, a TaioKe REH SCEP nyreM nepeeapMBaHMfl c BamHI m cmneaioT b nepeBapeHHyto nna3MHflHyro Pye BamHI c o6pa30BaHneM nna3MMflHoPt PAE / SCEP SKcnpeccmi. ΜΕΡ4ΜΕΡ 12

EXAMPLE HacTonuļMM othocmtcb k 3Kcnpeccnn peK0M6iiHāHTHbix npoflyKTOB, nocTpoeHHbix ECEPO m SCEP - γθηοβ b npepenax CHCTeMbi sKcnpeccuM npmviepa 11 ΠρΜ ncnonb30BaHHM CMCTebi 3Kcnpeccmi, paccmiTaHHoii ρπη πρΜΜβΗβΗΜη b KneTKax X03BMHa E.coli. nna3My p536 ilpHMepa 11 τpaHcφopM ^ φyιoτ β κπβτκβχ E.coli AM7, pHee τρ3ΗϋφορΜΜροΒ3ΗΗΒΐβ c npnroflHOM nna3MMflOM, pMWI, npMHBBUjeM κ ce6e reH 0 ^ 357. KynbTypbi κπβτοκ b LB 6ynbOHe (θμπμψιππμη 50 μκγ / μπ m κθηβμμρμη 5 μκγ / μπ, npeflnoHTHTenbHO c 10 μΜ MgSO ^ coxpaHnioT npw TeMnepaType 28 ° C m bo epeMn POCT κπβτοκ b KynbType po O.Dgoo = 0.1 sKcnpeccmo EPO MHflyi4npyioT njrreM noBbiineHMn I.

TeMnepaTypbi at 42 ° C. KneTKH pacTyr po npnčnn3MTenbH0 40 O.D., očecneHMBan nonyHeHMe EPO (ορβΗβΗΟ reneM) οκοπο 5 μγ / O.D π.

ΚπβτκΜ co6npaioT, noflsepratOT nM3Mcy, pa36nBaioT Φρθη ^ οκμμ npeccoM (10,000 psi) h 06pa6aTbiBaiOT πμ30ρμμομ m peTepremOM NP-40, OcaflOK, nonyHeHHbiii b pe3ynbTaTe i4eHTp ^ yrnpoBaHnn 24,000 xg, pacTBopnioT ryaHMflMHOBoii HCI m noflBepraioT panbHeMiueM 0flH0pa30B0ii OHMCTKe npn noMoīpīd C4 (výdajů) Reverse Phase HPLC / EtOH, 0-80%, 50mM 66 NH4AC, ρΗ 4.5. OnpefleneHne nocnepoBaTenbHOCTM ocHOBaHuPi 6enKa BbmBnneT, ητο προργκτ hmct 6-one ηθΜήθ 95%, a nonyMeHHbie npopytcrbi BbmBnniOTpBa pa3Hbix θμμηο-οκοηηβημπ, appr ... n ... PPR npn OTHOCMTenbHOM κοπμηθοτβθηηομ οτηοιιιθημμ, paBHOM 3: 1.3to ποοπθρηθθ HaSmoneHne hEPO n [des Alal] hEPO-npoflyKTOB 03HaHaTe, hto " o6pa6oTKa " Aktmbhoctb, onpepeneHHan papMOMMMyHO-nccneflOBaHueM, pna μ30ππτοβ HaxoflMTCB Ha ypoBHe 150000 - 160000 V / μγ; aKTMBHOCTb, BbmBneHHaa in vitro, οοοτθβπηθτ nopnpKa 30000 -62000 V / μγ; h CallmBHOCTb, BbinBneHHaa in vivo, BapbMpyeTCB b npepenax ot 120 po 720 V / μγ. (CpaBHeHO co CTaHpapTOM ηθποβθηθοκογο μοηθβογο H30PPT = 70000 V / μγ, b kedkpom μοπητθημμ). KpuBap ρθ33-3φφθκτ pnn peKOM6nHaHTHoro npopyic b McnbrraHMM in vivo, 3HannTenbH0 OTnnwaeTCH ot kpmbom daHpapTa ηθποβθμθοκογο μοηθβογο EPO. nna3Mupbi EPO-aHanora, o6pa30BaHHbie b ηθοτπχ Am B npMMepa 11, Kaxpan τpaHCφopM ^ ιpyeτcπ b ρΜννί-τρθΗοφορΜΜροΒθΗΗΗθ κπθτκμ E.coli ΑΜ7. μ κπθτκμ KynbTMBnpyiOTI xax õncaHO Bbiiue. Οημιρθηηηθ μ30ππτη McnbīTbieatoT KaK RIA, τθκ m aHanii3aMM in vitro. 3η8ηθημπ, nonyyeHHbie npn McnbrraHMnx RIA m in vitro ρππ προργκτοβ oKcnpeccm [ASN2, des-Pro from lle®] h EPO, cocTaBnnfOT npn6nn3MTenbHO HOOOV / m μγ 6000 V / μγ npoTenHa, οοοτβθτοτβθηηο, Torpa XAX 3Η3ηθημπ aHann3a PNN [His?] HEPO οοστβΒπηιοτ οκοπο 41000 V / μγ m 14000 V / μγ 6enKa, οοοτβθτοτβθηηο. 3th 03HaHeT, hto npopyKTbi aHanoroe πβππιοτοπ Ha 1 / 4-1 / 10 "aKTHBHbiMM", TaK xe KaK npopyKT aKcnpeccMM "popnTena" b ncnbiTaHnnx. B ΟΜΟΤΘΜΘ 3KCnpeCCMM, npeflHa3HaHeHHOii ΡΠΠ MCn0nb30BaHMa B ΚΠΘΤΚΘΧ X03PHHa S.Cerevisial, nna3Mnpy PYE / SCEP τρθΗΟφορΜ ^ ιοτ b pea pa3nnHHbix uiTaMMa, YSD P4 (γθηοτμπ apep4-3 trp ^ / n RK81 / γθηοτμπ ααΡθΡ4-3 throne. TDaHCCboDMHPOBaHHbie xo3aeBa YSDP4 BbipaipHBatOTcn b cpepe SD (MeTopbi γθηθτμκμ ρροχοκθίί, Cold Spring Harbor Laboratory, Cold Spring Harbor, Ν.Υ. κπθτκμ BbipacTaioT po 36 OD, copepx < aT npopyKTbi EPO Ha ypoBHe οκοπο 244 V / mh / 97 μκγ / OD n, whereby to BbineneHO nocpepcTBOM RIA /. ΤρθΗΟφορΜΜροΒθΗΗΝθ κπθτκμ RK8I, BbipaipeHHbie po 6.5 OD, o6ecneHMBaiOT C03paHMe cpep c KOHpeHTpauMBMM EPO οκοπο 80 -90 V / mh / 34 μκγ / OD n, hb BbmBneHO RIA / llpepBapMTenbHbie aHanM3bi βνπβππιοτ 3HaHMTenbHyio reTeporeHHOCTb b npopyKTax, nonyHHbix nocpepcTBOM ρμμθηθημπ CMCTeMbi sKcnpeccMM, hto from-BMpMMOMy, o6ycnoBneHO Μ3ΜΘΗΘΗΜΠΜΜ Β ΓΠΜΚ03ΜΠΜρ0Β3ΗΜΜ BbipaxeHHblX 6θΠΚΟΒ, a Τ3ΚΧΘ OTHOCMTenbHO BbICOKMM copepxaHMeM MaHH03bi πρμοοθρμηθηηογο yrneBopa. f1na3MMflbi PaC3 m Pye b KneTKax E.coli HBI0I SbinM cpaHbi PNN perMCTpapMM b ΟΟΟΤΒΘΤΟΤΒΜΜ C ΠρθΒΜΠΜΜΜ npOM3BOpCTBa ΡΘΠ Π3ΤΘΗΤΗΟΓΟ ΒβρΟΜΟΤΒΡ CLIL OT 27 ΟΘΗΤΠδρΠ 1984 ropa aMepMKaHCKOM κοππθκρμθμ τμποβνχ KynbTyp, 12301 Parklown Drive, Rockvllle, Marylond, nop peno3MTapHbiMM HOMepaMM A.T.C.C. 39882 οοοτβθτοτβθηηο. rina3MMpbi pCFM526 β κπθτκθχ AM7, pCFM536 b KneTKax MI03 m pMwl β κπθτκθχ MI03 6binM 3perMCTpMpoeaHbi nopoSHbiM o6pa30M 21 Ηοπδρπ 1984 ropa KaK A.T.C.C. 33932, 33934 m 67 EN 10506 33933 οοοτβθτοτβθηηο. UJiMMbi YSPD4 m RK8I Saccharomyces cerevisial 6binn 3aperncTpnpoBaHbi 21 ΗΟΗδρη 1984 roAa KaK A.T.C.C. 20734 m 20733 οοοτβθτοτβθηηο. £ | 0 g > ΚΗ0 CTaTb OHeBMflHblM M3 paCCMOTpeHMH npeflCTaBneHHblX Bbliue MnnK3CTpaTHBHblX npMMepoB, hacTonmee Μ3θ6ρθτθΗΜθ b θγο μηογμχ acneicrax npeflnaraeT μοκπιοημτθπβηο peHHbie nponyKTbi m cnoco6bi. npeflnaraeMbie ποπμπθπτμαη ηβπηιοτοη θθμθτηο none3HbiMM MaTepManaMM, 6yflb ohh MMKpo6nanbHO BbipaxeHHblMM npOflyKTaMM ΜΠΜ ΟΜΗΤΘΤΜΗΘΟΚΜΜΜ npOflyiCTaMM) nepBHHHaa, BTOpMHHafl ΜΠΜ ΤρθΤΜΗΗΘΗ ΚΟΗφθρΜ3Ι4ΜΗ KOTOpblX CTA / ia ΒΠθρΒΜΘ Μ3ΒΘ0ΤΗ3 ČnarOAapfl HacTO "meMy μ3ο6ρθτθημιο.

Kax oTMeHanocb paHee, ρθκομΘμηθητηο npoflyqnpyeMbie h ομητθτμηθοκμθ npoAyicTbi Μ3θ6ρθΤΘΗΜΗ pa3flen ^ K3T B ρ33ΠΜΗΗθίί ΟΤΘΠΘΗΜ βΜΟΠΟΓΜΗΘΟ ^ ΙΟ aKTHBHOCTb in Vitro Μ30ΠΗΤ0Β EPO M3 HaTypanbHbix mctohhmkob m noaTOMy npoAyMnpyiOTCfl c τθμ, HTočbi KMeTb npnroflHOCTb b KanečTBe 3aMecTMTeneii μ30πητοβ EPO b KynbTypanbHbix cpeflax, πρμμθηαθμβιχ απα POCT κπθτοκ apMTponoaTMHa b KynbType. Taoce, nocKonbKy ποπμπθπτμαηηθ npoAyKTbi HaoToaiiļero M3o6peTeHna pa3AenniOT aKTMBHOCTb in vivo HaTypajibHbix μ30πητοβ EPO ohm ηβπβιοτοη 3ΘΜΘΤΗ0 npkirOAHblMH ΑΠΑ MCn0 / lb30BaHMH B ΜΘΤΟΑΜΚΘΧ 3ρΜΤρ0Π03ΤΜΗ0Β0Μ ΤθρθΠΜΜ, πρμμθηηθμμχ Ha ΜΠθκοπΜΤθίοίΑΜΧοίΐ, BKjiKDHan HenoeeKa, απη pa3pa6oTKn niodoro μπμ βοθχ 3φφθκτοΒ, npncymnx EPO in vivo, HanpMMep, cTHMynnpoBaHiie οτβθτθ peTMKynoqMTOB, pa3BHTne φθρροκΜΗβτΜΗθοκΜχ ΘφφθκτοΒ (τθκμχ CEC 3φφθκτΜ Kpyroo6opoTa xene3a b nnasMe h 3φφθκ ™ ερβΜβΗΜ nepexoAa b μο3γοβοθ βθιαθοτβο) MaccoočMeHbi spMTpoLļMTa, CTMMynnpoBaHne CMHTe3a reMomočMHa C (cm. Esenbach et al., supra μ, κθκ οτμθηθθτοα b EXAMPLE 10 yBennMeHneγροΒΗβΡί γθμθτοκρμτθ b μπθκοπμτθιοιαμχοη, B rpynny πιοαθΡϊ, o6pa6aTbiBaeMbix npeAnaraeMbiMM npoAyKTaMM, βκπι οηθην πθμμθητμ, Kak npaBMno, HyxAaiotnMecfl B πθρθπμβθηm κροΒΜ μ βκπιοηθιοιαμθ acepTBbi TpaBM, xnpyprMwecKMe OonbHbie, OoribHbie-ΠΟΗΘΗΗΜΚΜ, ΒΚΠΚ3Η3Β AHanH3HblX 6onbHblX, H naUMeHTbl C pa3HOBMAHOCTbK3 COCTaBa κροΒΜ, Bbi3biBaiomero HapymeHHB, TaKMe CEC γθμοΦμπμη, cepnoBMAHO-κγιθτοηη3Α 6one3Hb, Φμ3μοπογμμθοκμ aHeMMaa M Tomy ΠΟΑ06ΗΟΘ . ΜΜΗΜΜΜ3ΘΑΜί1 ΗΘΟβΧΟΑΜΜΟΟΤΜ B TepariHM C ΠΘΡΘΠΜΒΘΗΜΘΜ ΚρΟΒΜ δΠΘΓΟΑθρA MCnonb30BaHMK) TepanMM 3pHTp0n03TMH0M, MO > KHO &Kt; npeAnaraeMbie npoAyKTbi, Onaropapa mx nonyHeHmo πρΜ πομοιαμ ρθκομΟμηθητηηχ οποοοΟοβ, ηθαο nonaraTb, ηθ copepjKaT nkiporeHbi, HaTypanbHbie ymeTatomne BemecTBa h Tomy ποαο6ηοθ, m no3TOMy ηθοομηθηηο AOJDKHbi oOecnenMBaTb noBbiiueHHyio o6myto OTAany b TepaneBTimecKMX μθτοα3χ of omoiuenmo κ npMpOAHbiM npoAyicraM. OpMTponoaTMHOBaa Tepankifl c ncnonb30BaHneM npeAnaraeMbix npOAyKTOB Taoce AonxcHa CTaTb ποπθ3Ηομ b nOBblUieHMM KMCnopOAOnpOnyCKaiOmeii CnOCOČHOCTM MHAMBMAyyMOB, CTanKHBaKDlAHXCB c ΓΜΠΟΚΟΜΗΘΟΚΜΜΜ ycnOBkinMM BHeuJHeii CpeAbl M, B03M0JKH0, B ΟδθΟΠΘΗΘΗΜΜ 6naronpnaTHblX ΟΘΡΑΘΗΗΟ - COCyAMCTblX 3φφθΚΤ0Β. npeAnoHTMTenbHbiM cnocočoM ββθαθημη npeAnaraeMbix ποπμπτμαηβιχ npoAyKTOB HBnatOTCB SolarHPPHHBie (HanpMMep, BHyTpMBeHHbie, BHyTpMMbimeHHbie, ποακοχηηθ 68

HilM BHyTpn6ptOUJMHHbie) nyTM Irt BBepeHHbie Κ0ΜΠ03ΜΜΜΜ flOn > KHb !, KaK 06blHH0, COflepaOTb repaneBTunecKu a00eKTHBHbie KonnHecTBa npopyicra b coneraHun c npneMneMbiMM pa36aBMTenRMM, ηοομτθπημμ μ / μπμ CTMMynflTopaMM. npeflBapuTenbHbie φβρ'ΜΒκοκΜΗθΤΗΗβοκκβ MccneflOBaHMfl OTMenato 6onee flnmenbHbiii nepnop nonypacnaaa in vivonnn npoflyicroB EPO o6e3biiHbi npn ββθαθημμ mx BHyTpMMbiiueHHO, CKopee ηθχθπμ BHyTpMBeHHO. CEC npefļnonaraeīca, θφφβκτΜΒΗΝβ fl03np0BKM MoryT M3MeHirrbCfl b 3HaHHTenbH0M CTenemi b 3aBMCMM0CTM ot ycnoBMM, oflHaKO, TepaneBTunecKue fl03bi, Kak cefinac OKMflaioT, flonxHbi BapbupoBaTbca b npenenax ot 0.1 / ~ 7v / po 100 / -7000 V / μκγ / κγ Beca Tena βκτμβηογο Maiepnana . CiaHflapTHbie pa36aBmenn, τθκμθ CEC cbiBopoTOHHbifi anb6yMMH nenoBeKa, npepnonaraioTca nna ncnonb30BaHMfl b Φθρμβμθβτμμθοκμχ κομπο3μμμηχ HacTOflinero Μ3θβρβΤΘΗΜ "Hapflfly CO CTaHflapTHbIMM HOCmenaMM, TaKMMM CEC φΜ3Μ0Π0Π1ΗΘ0ΚΜίί A solution.

CtmMynmpy iolumθ MaTepnanbi, npuroflHbie pn "ncnonb30BaHna b npeflnaraeMbix Κ0ΜΠ03Μ14Μί) Χ, ΒΚΠΚ) Η3Κ) Τ ΟΟθρίΙΗΘΗΜΛ, yKa3aHHbie CaMOCTOflTejlbHO fl / LFL 3ρΜΤρ0Π03ΤΜΜΘ0ΚΜΧ CTMMynnpyiomMx ΘφφβκτοΒ, Kak Further preferably, TecTociepoHbi, CTMMynyrropbi npefluuecTBy» oinMX ΚΠΘΤΟΚ, φ3ΚΤ0ρ POCT MHCynMHOnOfl06HblX, npOCTarnaHflMHbl, CepOTOHMH, LļMKJlUHeCKMpi Ona, npNAKTMH m TpuMflOTupoHMH, a τβκχθ cpe / skc, oaabHHO npHMermeMbie npw πθηθημμ annacTMHeCeMn, τ κμθ KaK μθτθηοπθη, CTaH03on n HaHpponoH ((cm., HanpuMep, Resegotti et al., Panminerva Medica, 23, 243-248 (1981)); McGonigle et al., Kidney Int., 26/2 /, 437-444 (1984), Pavlovic-Kautera et al., Expt Hematol., 8 / Supp. 8 / 283-291 (1980); , FEBS Letters, 14a / l /, 105-108 (1982)] TaioKe npeflnonaraeTca b KanecTBe CTMMyn «TopoB ncnonb30BaTb BemecTBa, yBenMHMBaK inMe fleiicBiifl μπμ CMHeprM3MiPiOn pMTponoaTMH μπμ acuano-EPO, TaKne KaK aflp eHepruMecKue aroHMCTbi, TuponflHbie ropMOHb, aHflporeHbi μ BPA (cm. Dunn, Current Concepts in ErythiOpoiesis, John Wiley α Sons / Chichester, England, 1983 /; Weiland et al., Blut, 44/3. 173-175 (1982); Kalmanti, Kidney Int., 22, 383-391 (1982); Shanidi

New Eceg.J.Med., 289, 72-80 (1973), Fisher et al., Steroids, 30/6 /, 833-845 (1977), Urabe et al., J.Exp.Med. οτρ.1314-1325 (1979) m Billat et al., Expt.Hematol. al., Acta Haemat., 69, οτρ.171-179 (1983) & " apMTpoTponMHbi ”(KaK happcaHO Congote et al., b Abstract 364, Proceedings 7 ~ International Congress of Endocrinology /

Quebec City, Quebec, April 1-7, 1984 ropa /; Congote Biochem. Biophys.Res.Comm., 115/2 /, 447-483 (1983) n Congote, Anal. Biochem., 140, 428-433 (1984)] m "spMTporeHMHbi" (KaK luckHoBeTe Rothman et al., J.Surg.Ocol., 20, CTp.105-108 (1982)). npeABapmenbHbie npocenBaHua, npeflHa3HaHeHHbie flrm H3MepeHMfl 3pMTponoaTHHecKMX οτβθτοβ SKC -rnnoKCMHecKnx nonnunTeMMHecKnx MbiLiiePt, npeflBapmenbHO o6pa6oTaHHbix nn6o 5 - flurnflpoTecTocTepoHOM, nn6o HaHpponoHOM, a 3aTeM npepnaraeMbiM apMTponosTMHOM, ITO ^ Bnanu coMHUTenbHbie pe3ynbTaTbi. fluaroHocTMMecKue ncnonb30BaHna npejqnaraeMbix nonunenTUflOB TaioKe o6wnpHbi m BKJIKDMaiOT ΠρΜΜΘΗβΗΜβ ΜβΗΘΗΜΧ Μ ΗβΜβΗβΗ ^ Χ φθρΜ Β pa3H006pa3MM ΜβΤΟβΜΚ MMMyHO-nccneflOBaHua, BKmoHaa RIA, ELISA m Tomy nopoČHoe, a TaioKe b pa3HOo6pa3mi aHarui30B 69 EN 10506 npoBOflMMbix flnfl BbiflBneHMfl βκτμβηοοτμ in vitro μ in vivo. Cm., HanpMMep, paoTbi Dunn et al., Expt.Hematol. II / 7 /, οτρ. 590-600 (1983); Gibson et al., Pathology, 16, οτρ.155-156 (1984); Krystal, Expt.Hematol., 11/7, pp.649-660 (1983); Saito et al., Jap.J. Med., 23/1 /, στρ.16-21 (1984); Nathan et al., New Eng.J.Med., 308/9, CTp.520-522 (1983); m pa3nMHHbie ccbinoHHbie MaTepnanbi, MMeiomne OTHouieHne κ ynoMMHaeMbiM b θτμχ pa6oTax MccneflOBaHMflM. llpeflnaraeMbie ποπμπθπτμαν, BKJiioHaioLiļMe ΟΜΗτβτΜΗβοκΜβ nenTUflbi, KOTOpbie coflepxaT nocneflOBaTenbHOCTH οοτθτκοβ BnepBbie βναβπθηηογο EPO, TaiOKe pacnonaraiOT Hpe3BbiHaMHO none3HbiMH HMCTbiMM MaTepnanaMM απη npon3BOflCTBa nonm < noHanbHbix aHTMTen μ " 6ποκοβ "M.oHOKnoHanbHbix amuTen, οπβΑΜφΜΗβοκΜΧ απη OTnnHatoinnxca HenpepbiBHbix m npepbiBMCTbix 3ΠΜΤ0Π0Β EPO. B KasecTBe οαηογο npMMepa, npeflBapmenbHbifi aHann3 aMMHOKHcnoTHbix nocneflOBaienbHocTeii Taffilbil VI b CMbit βοαοπθημμοοτμ b οοοτβθτοτβμμ c Ηορρ βτ al., P.N.A.S. (USA), 78, οτρ.3824-3828 (1981) m. , οταγμβθιοιαμχ no3MLļMM 41-57, BKHKDHMTenbHO, 116-118, BKHlOHMTenbHO, m 144-166, BKAlOHMTenbHO, ΒβρΟΛΤΗΟ, jqonXHbl npOflyiļMpOBaTb 4pe3BbiHaMH0 aHTMreHHyra peaKLļMio m BbipačaTbieaTb none3Hbie MOHOKriOHanbHbie m nonMKnoHajībHbie aHMTena, MMMyHopeaicTMBHbie XAX ομητθτμμθοκμμ πθπτμαομ c, m τθκ LienbīM 6θπκομ. TaKMe MaTepna / ibi, xax οχΜΑββτοί ?, ponxHbi 6biTb none3Hbi npn o6HapyxeHMM m CpOflCTBeHHOM ΟΗΜΟΤΚβ EPO M CBB3aHHblX C HMM npOflyKTOB.

MnntOCTpaTMBHO SblHM nOnyneHbl TpM ΟΜΗΤβΤΜΗβΟΚΜΧ nenTMfla: / 1 / h EPO 41-57, V-P-D-T-K-N-F-Y-A-W-K-R-M-E-V-G; / 2 / h EPO 116-128, K-E-A-1-S-P-D-A-A-S-A-A;

131 h EPO 144-166, VYSNFLRGKLKLYTGEACRTG-DR npeflBapMTenbHbie MCCneflOBaHMil MMMyHM3ai4MM C ΠρΜΜβΗβΗΜΘΜ BblUieyKa3aHHblX ποπΜπβΤΜβΟΒ, BbiPBMHM OTHOCMTenbHO cna6yio nonoxMTenbHyio peaKLļMK) Ha hEPO 41-57, He nopflaioinytoca oueHKe peaKUMio Ha hEPO 116-128 m CMnbHyio nonoxMTenbHyio peaKLļMK) Ha hEPO 144-166, Kax onpepeneHO cnoco6HOCTbto cbiBopoTOMHbix aHTMTen KponMKa MMMyHOOCaXflaTb Μ30ΠΗΤΗ ΜΘΠΟΒβΗΘΟΟΚΟΓΟ ΜΟΜβΒΟΓΟ EPO, Μ3ρΚΜρ0Β3ΗΗ0Γ0 I. npeflBapMTenbHbie μοοπθαοβθημβ 3κτμβηοοτμ in vivo, npoBeneHHbie Ha τρβχ nenTMflax, ηθ BblBBMJlM 3HaHMTenbHOM aKTMBHOCTM K3K ΚΘΧβΟΓΟ B OTflenbHOCTM, T3K Μ B KOM6MHaLļMM.

HecMOTpB Ha to mto BbiBepeHHbie nocneAOBaTenbHOCTM θμμηοκμοποτηνχ οοτβτκοβ EPO ΜΠβΚ0ΠΜΤ3Ι0111ΜΧ, ΠρβΑΟΤΘΒΠΘΗΗωθ B ΜΠΠΚΧΤφΒΤΜΒΗΗΧ πρΜΜβρθΧ, ΠΟ CytlļeCTBy ΟΠρβρβΠΗΙΟΤ nepBMHHyio cTpyKrypHyio κοΗφορΜθΑΜίο co3peBmero EPO, cnenyeT OH ^ Tb, mto οπβιΐΜφΜΗβοκθίΐ nocneflOBaTeribHOCTb 165 θμμηοκμοποτηβιχ οοτβτκοβ EPO o6e3bflHbi b TačnMLļe V m 166 οοτβτκοβ EPO MenoeeKa b TačnMLļe VI He orpaHMMMBeTeMaTeOn3Hbix nojiMnenTMflOB, npeflnaraeMbix Μ3θ6ρβτβΗΜβΜ. Οχβθτνβθιοτοη HacTO $ uiļMM Μ3θ6ρθτβΗΜβΜ τβ pa3nMHHbie CBOčoflHO BCTpenaioiiļMecH annenbHbie φορινίΝ EPO, KOTOpbie b npouinoM BomnM b MCCneflOBaHMB 6ΜΟΠΟΓΜΗΘΟΚΜ aKTMBHblX ΠΟΠΜΠβΤΜΑΟΒ ΜΠβΚΟΠΜΤΘΙΟΙΑΜΧ, Kak ΗθΠ-ρΜΜΘρ, ΜβΠΟΒβΜβΟΚΜΜ γ-ΜΗΤβρφθρΟΗ. (CpaBHMTe, HanpMMep, ΜβΠΟΒβΜΘΟΚΜΜ MMMyHHblM ΜΗΤβρφθρΟΗ 70 μμθιοιαμμ ocTaTOK apruHHHa b ποποχθημμ N140 b EPO, ο κοτοροΜ coočmanocb b ony6nnKOBaHHOM 33ηβκθ Ha naieHT 0,077,670, μ BMA, μμθιοιαμμ rnyraMMH b ποποχθημμ N140, Kak cooSmanocb b paOoie Gray et al., Nature, 295 , pp. 503-508 (1982) 06a BMfla οτπμηθιοτοη τθμ, hto cocTaBrmiOT nocneAOBaTenbHOCTM " co3peBiijero " nenoBenecKoro γ-ΜΗΤβρφβροΗθ). ArmeribHbie φορΜΜ ποπμπθπτμαοβ co3peBwero EPO Moryr OTnunaTbca ppyr ot Apyra n ot nocneflOBaTenbHOCTeii, npeflCTaBneHHbix b Ta6nnuax V μ VI, b οτηοιαθημμ flnuHbi m nocnepoBaTenbHOCTn μ / μπμ b οτηοιιιθημμ penepuM, 33μθιαθημμ, BCTaBOK μπμ αο63βπθημμ aMMHOKMcnoT b nocneflOBaTenbHocTb Hapafly c πογμηθοκμ β ^ τθκθιοιαμμμ BapnaMnaMM b cnocoāHOCTM flna γπμκοομπμροβθημπ. Kax OTMenanocb paHee, oflHa npeAnonoxMTenbHan annenbHaa φορΜθ μθποβθηθοκογο EPO, Kak KaxeTcn, ponxHa BKniOHaTb ocTaTOK μθτμοημηθ b ποποχθημμ 126 npeflnonoxMTenbHo, cbočoaho BCTpenaioLUMecfl annenbHbie φορΜ ^ nocneflOBaTenbHOCTeM KflHK m EPO-K0RMpyiomeM γθηομηομ flHK, ΒβροπτΗΟ, τθκχθ AonxHbi BCTpeTMTbcn, npMHeM KOflMpyiomMMM BbiujeyKa3aHHbie TMnbi annenbHbix ποπμπθπτμαοβ ΜΠΜ npOCTO MCnOnb3ytOUlMMM ΟΤΠΜΗΘΙΟΙΑΜΘΟΠ KOflOHbl ΑΠΠ θ6θ3Η3ΗΘΗΜΠ Τ3ΚΜΧ Χθ ΠΟΠΜΠΘΠΤΜΑΟΒ, KOTopbie yKa3aHbi. B βΟΠΟΠΗΘΗΜΘ K CBOČOAHO BCTpeHatOLlMMCn annenbHbIM φσρΜΘΜ 0Ο3ρθΒΐυβΓΟ EPO HacTOHLnee μ3ο6ρθτθημθ τθκχθ βκπιοηθθτ b ce6a opyrMe 'np0flyKTbi EPO, τθκμθ KaK nonMneTMflHbie aHanorM EPO μ Φρθγμθητν ”co3peBwero EPO. Cnepyn μθτοαμκθμ Bbiiue-ynoMHHyTOM ony6nMKOBaHHOM 33πβκμ Ha naTem- Alton et al., / WO / 83/04053 /, μοχηο 6β3 Tpyna paccHMTaTb μ μθγοτοβμτβ reHbi, KOAMpyK > iAne MMKpo6MajibHy (o sKcnpeccMio ποπμπθτμαοβ, ΜΜΘΙΟΙΜΜΧ nepBMHHytO ΚΟΗφορΜΘΜΜΚ) KOTOpafl OTJlMHaeTCfl OT ΚΟΗφΟρΜΘΑΜΜ, yKa3aHHOM 3flecb

Ann co3peBioero EPO, ctohkm 3peHMn μαθητμηηοοτμ μπομοποποχθημπ οαηογομπμ 6οπθθ ocTaTKOB (HanpMMep, 3aMemeHMn, κοηθηηνθ μπμ npoMexyTOHHbie fločaBneHMn μ αθπθμμμ). flononHMTenbHO, μοαμΦμκβαμμ KflHK μ γθηοβ γθηομηογο EPO MoryT 6biTb πθγκο ocymecTBneHb noopepcTBOM χοροιοο M3BecTHbix μθτοαμκ caMT-HanpaBneHHoro MyTareHe3a m Mcnonb30BaHbi ΑΠΠ an aHanOTOB Μ np0M3B0flHblX EPO. ΤθΚΜΘ npOflyiCTbl EPO flOnXHbl pa3flenaTb no KpaMHeii Mepe oaho m 6μοπογμηθοκμχ cbomctb EPO, ho MoryT OTnMHaTbca b flpyrMX. CEC npMMepbi, npoeKTMpyeMbie nponyKTbi EPO HacTonmero ΜβοβρθτβΗΜΠ βκπιοηθιοτ τθ, KOTopbie coKpameHbi nyTeM, HanpMMep, fleneuMM [ASN2, des -Pro2 of lle6] hEPO, [des- Thr163 of Arg166] hEPO μ " Δ27-55 hEPO "npMHeM nocneflHaa ΜΜββτ οοτβτκμ , KOflMpyeMbie BbiHepKHyTbiM pencils 3K30hom; μπμ KOTopbie 6onee ycTOMHMBbi κ rMflponM3y (m, cneflOBaTenbHO, MoryT MMeTb 6onee BbipaxeHHbie μπμ flonbuie npononxaiomMeca βπιίπημπ, ηθμ cbočoaho BCTpenaiomMMCfl EPO); μπμ KOTopbie 6binM μ3μθηθην απα BbmepKMBaHMn οαηογο μπμ 6one nOTeHUManbHblX ynaCTKOB ΑΠΠ ΓΠΜΚ03ΜΠΜρ0Β3ΗΜΑ (KOTOpan ΜΟΧβΤ ΠρΜΒβΟΤΜ K BbICOKOM aKTMBHOCTM ΑΠΠ npOAyKTOB, npOAyUMpyeMblX ΑΡΟΧΧΘΜΜ); ΜΠΜ KOTopbie ΜΜβΙΟΤ OAMH μπμ 6-one OCTaTKOB LļMCTeMHa, BblHepKHyTblX ΜΠΜ 3aMeiAeHHblX, HanpMMep, 0CTaTK3MM rMCTMAMHa μπμ cepMHa (κθκ HanpMMep, aHanor [His ^] hEPO / m noTeHAManbHO 6-one πβτκο ββιαθππκποπ b ΘΚΤΜΒΗΟΜ φορΜβ M3 MMKpo6ManbHblX ΟΜΟΤβΜ; ΜΠΜ KOTOpbie ΜΜβΚ) Τ OAMH ΜΠΜ 6θΠββ OCTaTKOB TMpo3MHa, 3aMeHeHHbix φβΗΜΠ3Π3ΗΜΗ0Μ (κθκ HanpMMep, aHanorM [Phe " l3] hEPO, 71 LV 10506 [Phe49] hEPO, [Phe145] hEPO) μ μογ / τ CBfl3biBaTbcn 6onee nnn μθηθθ πθγκοc peueniopMU EPO Ha nnaHOBbix κπθτκθχ. Taxxe oxBaHeHbi ποπμπθπτμαηηθ Φρθγμθητν, flynnMflMpyiomMe TonbKO nacTb HenpepbiBHOti θμμηοκμοποτηομ nocneflOBaTenbHOCTM μπμ BTOpMHHbix κοΗφορΜθρΜΜ b npepenax co3peBwero EPO, npuneM φρβΓΜβΗΤΝ Moiyr odnapaTb oahom aKTMBHOCTbio EPO (HanpMMep, pepenTopHbiM CBB3biBaHMeM) n HeppyrnMH (HanpMMep, apMTponosTUHecKafl aioruBHocTb). OcoSeHHO BaxHbi b 3T0M othoiiibhhm τθ noTeHiIngNbHbie φρ3ΓΜΘΗΤΒΙ EPO, KOTOpbie 06-bBCHeHbl npn paCCMOTpeHMM nOCneflOBaTenbHOCTM ΓβΗΟΜΗΟΜ flHK ηθποβθκθ, npeflCTaBneHHOM b Ta6nnpe VI, T.e. " φρθΓΜβΗΤΗ ”ποπηομ HenpepbiBHon EPO-nocnepoBaTenbHOCTM,« OTopan OnepneHa MmpoHHbiMM nocneflOBaTenbHOCTHMM HOME μοχθτ cocTaBmb ρθ3Κ0 BbipaxeHHbie " npnMenaTenbHO, whereby to OTcyTCTBne aKTUBHOCTM in vivop nn ntočoro οαηογο μπμ 6-one "npoflyKTOB'EPO" Haoroniiļero Μ3θδρβτβΗΜ ^ b uenoM ηθ npennTCTByeT TepaneBTMHecKoii ποπθ3ηοοτμ (cM.VVeiland et al., supra) μπμ none3HocTM b ppyrnx κοΗΤβκοτβχ, τθκμχ Kax EPO-EPO McnbnraHMfl μπμ -aHTarHH3M. AHTarHMCTbiTMHaMaTeMMa3aMiTaNMMaTeTeMMMa3a μπμ b cnynanx nepenpoM3BOflCTBa EPO (cm., HanpuMep, Adamson, Hosp.Practice, 18/12 /, στρ. 49-57 (1983) Hellman et al., Clin.Lab. ., 5, στρ.335-342 (1983)). flpyroPi ocoāeHHOCTbio HacTonmero Μ3θ6ρβτβΗΜπ nBnneTcn the hto KnoHMpoeaHHbie flHK-Nocni / ļOBaTenbHOCTM, omicaHHbie 3flecb, KOTOpbie KOflnpyraT ποπμπθπτμαμ EPO nenoBeKa M 06e3bHHbl, BeCbMa LļeHHbl ΑΠΠ ΜΗφθρΜ3ί1ΜΜ, KOTOpyKD OHM npepnaranDT B OTHOUJeHMM aMMHOKHCnOTHOM nOCneflOBaTenbHOCTM 3pMTp0n03TMHa ΜΠβΚΟΠΜΤβΚΟΙΑΜΧ, KOTOpbIM flO CHX P 6bin HeflocTyneH, HecMOTpn Ha flecnTMneTMHMM npoBOflMBUjyiocn aHanMTMHecKyio o6pa6oTKy Μ3θπητοΒ CBoSoflHO BCTpeHaiomMxcfl npoflyioroB. flHK-nocneflOBaTenbHocTM TaiOKe BecbMa MGHHbi xax npoflyKTbi, npMroflHbie b ocymecTBneHMM KpynHOMacuiTa6Horo MMKpo6ManbHoro CMHTe3a 3ΡΜΤΡ0Π03ΤΜΗ3 npM Π0Μ0111Μ pa3H006pa3Mn ρβΚΟΜβΜΗβΗΤΗΗΧ ΜβΤΟβΜΚ. ΚρΟΜΘ ΤΟΓΟ, npeflnaraeMbie flHK-nocnefloeaTenbHOCTM none3Hbi b BbipaāaTbiBaHnn ηοβηχ m npMroflHbix BeKTOpOB BMpyCHOM M KOnbfleBOM nna3MMflHOM flHK, HOBblX Μ Π0Πβ3ΗΗΧ Τρ3ΗθφθρΜΜρθΒ3ΗΗΜΧ Μ Τρ3ΗϋφβΚΤΜρθΒ3ΗΗΝΧ MMKpo6nanbHblX ΚΠβΤΟΚ ΠρΟΚβρΜΟΤΗΟΓΟ M 3yKapMOTHOrO X03? IMHA (BKnKDnan κπθτκμ 6aKTepMM μ Αροχχβίί μ κπθτκμ μπθκοπμτβιοιιιμχ, BbipameHHbix b xynbType) a TaKxe HOBbie m npMroflHbie cnoco6bi KynbTMBMpyeMoro pocTa τθκμχ MMKpo6ManbHbix κπβτοκ xo3BMHa, cnoco6Hbix o6ecneHMBaTb 3KCnpeccMio EPO m npoflyiCTOB EPO. flHK-nocneflOBaTenbHOCTM npeflnaraeMoro ΜβοβρβτβΗΜΠ TaiOKe nsnaioTCR BecbMa npnroflHbiMM MaTepwanaMM flnn Mcnonb30BaHnn b KanecTBe μθηθημχ 30ηαοβ b μ30πμροβ3ημμ EPO m CBH3aHHoro c HMM 6enKa, KOflMpytomero nocneflOBaTenbHOCTM KflHK μ γθηομηομ flHK MneKonnTaioiflMX, flpyrnx, ηθχθπμ ΗβποΒβκ μ o6e3bnHa, MnmocTpnpyeMbie 3flecb αιβρΜφΜΗβααι. CTeneHb βο3μοχηογο Mcnonb30BaHMa flHK-nocneflOBaTenbHocTeM b pa3nMHHbix anbTepHaTMBHbix cnoco6ax ββπκοΒΟΓο CMHTe3a (HanpMMep, b xneTKax HaceKOMbix) μπμ b reHeTMHecKOM TepanMM ntonePi m flpyrMX μπθκοπμτβιοιιιμχ, eme ηθβο3μοχηο onpeflenMTb. npeflnaraeMbie flHK-nocneflOBaTenbHOCTM, XAX oxMflaeTcn, αοπχηη craTb none3HbiMM b pa3pa6oT < e TpaHcreHHbix bmrob μπθκοπμτθιοιαμχ, KOTOpbie Moryr cnyxMTb b KanecTBe 72 3yKapnoTHbix " χο3ηθβ "απη nonyHeHMa apMTponosTMHa μ npoflyicroB apmponoaTMHa β SorībmoM KonunecTBe. CMOTpu, rn HbiM odpa30M, PayType Palmiteretal, Science, 222/4625 /, CTp.809-814 (1983).

PaccMOTpeHHbie b stom CBH3M KOHKpeTHbie pacKpbrmn noncHMTenbHbix npMMepoB, HecoMHeHHO, orpaHMHUBaraT oSbeMa HacToamero Μ3θ6ρθτβΗΜη, mcnepnanMCTaM 6ynyT BMflHbl MHOrOHMCneHHbie MOflMCl) HKai4Hl1 Μ Μ3ΜΘΗΘΗΜΑ. B KaHeCTBe OflHOrO EXAMPLE, ΧΟΤΗ ΠΗΚ-nocneflOBaTenbHocTM, npeflnaraeMbie noacHmenbHbiMH npuMepaMM, βκαιοηθιοτ nocneflOBaienbHOCTM KflHK μ γθηομηομ flHK, nocKonbKy AaHHan 3a "BKA odecneHMBaeT ΜΗφθρΜ3φ1βΜ aMMHOKMCAOTHOM nOCneAOBaTenbHOCTM, CymeCTBeHHOii ΑΠΡ nOCTpOeHMA JļHK-nocneAOBaTenbHocTM, M3o6peieHne TaioKe npeAnonaraei Tacna M3roTOBneHHbie JUHK-nocneAOBaTenbHOCTM, KOTopbie μοχηο nocTpomb If ocHOBaHMM 3HaHMn oč aMMHOKMcnoTHbix nocneAOBaTenbHOCTnx EPO. Ohm MoryT KOAnpoBaTb EPO (κβκ b flpuMepe 12), a TaioKe φρθΓΜβΗΤΗ EPO m nonuneTMAHbie aHanoru EPO (τ.β. " npoflyicrbi EPO "), KOTopbie Moryr pa3AenaTb oaho μπμ done M3 6nonornHecKnx cbomctb cbočoaho BCTpenaiomerocn EPO, οαη3κο, ηθ pa3Aen "Tb Apyrnx (μπμ odnanaTb flpymMM b pa3HMHHOM οτθπθημ). flHK-nocneAOBaTenbHOCTM, npeAnaraeMbie HacToamnM Μ3θβρβΤΘΗΜβΜ, τθκμμ odpa30M, AonxHbi oxBaTbiBaTb ECE flHK-nocneflOBaTenbHOCTM, npuroAHbie flnn ncnonb30BaHna b odecneneHMM sKcnpeccMM β κπβτκβ npoKapMOTHoro μπμ 3yKapnoTHoro xo3AMHa ποπμπθτμαηογο npoAyicra, MMetoiflero of KpaidHeM mepe, MacTb nepBMHHoii CTpyicrypHOM κοΗφορΜ8ΑΜΜ m oaho μπμ done M3 6μοπογμηθοκμχ cbomctb mAtTaMa, m OTdMpatoTcn m: / a / flHK-nocneAOBaTenbHOCTeii, npeACTaBneHHbix β Ta6nnuax V μ VI; / b / flHK-nocneflOBaTenbHOCTeM, KOTopbie rn6pnAM3MpyiOT κ JHH-nocneAOBaTenbHOCTflM, onpefleneHHbiM b / a /, μπμ mx .... ΦΡθγμθητβμ; / c / flHK-nocneflOBaTenbHOCTeM, KOTopbie M3-3a flereHepauMM γθηθτμηθοκογο κοαθ AonxHbi rn6pnAH3MpoBaTb κ flHK-nocneAOBaTenbHOCTAM, onpefleneHHbiM B / a / M / b /. B 3T0M cba3m 3acny > KMBaeT βημμβημπ, HanpMMep, the hto cymecTByioiflMe nocneflOBaTenbHOCTM γθηοβ annenbHoro EPO nenoeeKa m ode3bAHbi m nocneflOBaienbHOCTM γθηοβ flpyrnx bmaob ΜΠβΚΟΠΜΤ3 (ΟΙΙ1ΜΧ, K3K OXMflaeTCA, Flon > KHbl rn6pMflH3HpOBaTb K nOCneflOBaTenbHOCTAM Β Ta6nnuax V μ VI μπμ μχ φρβΓΜβΗτβΜ. ΚροΜβ τογο , M3-3a flereHepaflMM reHeTHHecKoro Kona reHbi SCEP m ECEPO m M3roTOBneHHbie μπμ MyTareHM3MpoBaHHbie nocneflOBaienbHOCTM KflHK μπμ γθηομηομ flHK, KOflMpyioiHMe pa3nMHHbie φρθΓΜβΗ ™ EPO m ero aHanoru, αοπχημ TaioKe rn6pnflM3MpoBaTb κ BbiiiieynoMHHyTbiM flHK-nocneflOBaTenbHOCTAM. TaKMe rMdpMflM3auMM μοχηο de3 Tpyna ocymecTBMTb b ycnoBnnx rn6pnflM3aumi, onMcaHHbix 3flecb b oTHomeHMM nepBOHaHanbHoii μ30ππμμμ flHK, KOflMpyioinio EPO nOnBeKaMaMe3bflHBi, MnM donee CTporMX ycnoBnnx, ecnM acenaTenbHO CHM3MTb rMdpMAM3auMio φοΗβ. MnniocTpMpyKDT Μ3θ6ρβτβΗΜβ B OTHOLUeHMM MMKpOČManbHOM SKCnpeCCMM npOflyKTOB EPO Β ΚΟΗΤβΚΟΤβ SKCnpeCCMM κπβτοκ ΜπβκοπΜΤ3Κ) ΐΐ1ΜΧ flHK, BCTaBneHHOM β rMčpMflHbiM Ββκτορ 6aKTepnanbHbix nna3MMflHblX M BnpycHbix renoMHbix BMflOB, β npeflenax paccMOTpeHMn Μ3θ6ρβτθΗΐΐη HaxoflMTcn 6onbiuoe pa3Hoodpa3Me CMCTeM aKcnpeccMM. OcodeHHO oxBaHeHbi CMCTeMbi aKcnpeccMM, BKmonaiomMe 73 EN 10506

BSKTOpbl rOMOreHHblX BMflOB, OTHOCBLLļMXCfl K pa3H006pa3M! 0 6aKTepManbHblX, ΑΡΟΧΟΚΘΒΜΧ M μπθκοπμτθιοιαμχ κπθτοκ b KynbType, a τθιοκθ k ομοτθμθμ sKcnpeccMM, ηθ BKmonaiomMM BeKTOpbi (Kax HanpiiMep, τρΒΗοφθκρΜη κπθτοκ φοοφορΗΟΚΜοη ^ Μ KanbpueM). B stom cbh3m ctshbt ποηπτηο, Ητο sKcnpeccMn, e.g. flHK o6e3bHHbi b KneTKax xo3anHa o6e3bflHbi b KynbType n KneTKax xo3nnHa ηθποβθκθ b KynbType A®MCTBMTenbHococTaBnaeT npnMep sKcnpeccMM "3Κ30γθηηομ " flHK, TaK KaK flHK EPO, Hba sKcnpeccnn BbicoKoro ypoBHa pa3biCKHBaeTcn, ηθ 6yjqeT HMeTb cbomx hctohhhkob β γθηομθ xo3HHHa. CncTeMbi SKcnpeccMM HacTOiunero μ3ο6ρθτθημπ, κροΜθ τογο, npepnonaraiOT stm αθμοτβμπ, ββιτθκθιοιαμθ b UHTonna3MaTMHecKoe o6pa30BaHne nponyKTOB EPO m ηθκθππμβθημθ rnMK03MnnpoBaHHbix m HernnK03nnnpoBaHHbix npoAyKTOB EPO b umonna3Me κπθτκμ xo3HHHa μπμ MeM6paHax (e.g. HaKannviBaHHe b 6aTepnanbHbix npocTpaHCTBax nepnnna3Mbi), Una b ηθαοοθαοηημχ χμακοοτηχ KynbTypanbHbix cpep, Kak ποποηθηο Bbiiue, μπμ b poBonbHO peflKHx ομοτθμθχ,

KaK HanpHMep, CMCTeMax 3Kcnpeccnn Piqeruginosa (luckHHixx b6oie Gray et al., Biotechnology, 2, οτρ.161-165 (1984).

YcoBepiijeHCTBOBaHHbie μθτοαοπογμμ rM6pMAM3aLļMM HacTomnero Μ3θβρθτθΗΜπ (χοτπ h 6binn HnniocTpaTMBHO npHMeHeHbi BbiLue κ npoceMBaHHHM flHK / flHK- rn6pMflM3ai4mi, b paBHoPi οτθπθημ npHMeHHMbi κ npoceMBaHmo PHK / PHK PHK m / flHK. Μθτοαμκμ CMeuiaHHbix 30HA0B, Kak ποποηθηο 3flecb, maBHbiM o6pa30M cocTaBnniOT PAA ycoBepiueHCTBOBaHMM b rnāpnflH3aukiOHHbix npoueccax , Π03Β0ΠΠΠ ocymecTBMTb 6-one 6biCTpbie m Hafle > KHbie M30rtnuMM nonnHyKneoTMAOB. 3τμ μηογμθ oTflenbHbie ycoBepiueHCTBOBaHnn o6pa6oTKM ΒκπιοΗβιοτ: ynyHiueHHbiM npenoc κοποημμ μ μθτοαμκμ οοχρθηθημπ; Mcnonb30BaHMe φΜΠΒτροΒ na οοηοβθ HaMnoHa, Τ3ΚΜΧ CEC Gene Screen m Gene Screen Plus, pa3peinaioiAMX ποβτορηοθ 30ΗΑΜρθΒ3ΗΜΘ 3TMMM ΧΘ φΜΠΒΤρ3ΜΜ Μ ΠΟΒΤΟρΗΟΘ J0Π0ΠΒ30Β3ΗΜΘ (J) MnbTpa, ΠρΜΜΘΗΘΗΜΘ HOBbix μθτοαοβ πθηθημπ np0Tea30M (cpaBHMTe, HanpMMep, c Taub et a l., Anal.Biochem., 126, οτρ.222-230 (1982)) Mcnonb30BaHMe oneHb ημ3κμχ OTAenbHbix KOHueHTpaiļMM (nopapKa 0.025 πμκομοππ) Oonbujoro HMcna CMemaHHbix 30HA0B (HanpMMep, ηθμ 6-one 32) μ προβθαθημθ 14ΜΚΠΟΒ TM6pMAM3ai4MM M nOCT-TM6pMAM3aUMM Β ΟΤρΟΓΜΧ TeMnepaTypHblX MHTepBanaX, 6ΠΜ3ΚΟ npM6nMxaioiAMXca (τ.β. b npeqenax 4 ° C, npeAnoHTMTenbHO, b npeAenax 2 ° C) κ HaMMeHbuieM BbiCMMTaHHOM TeMnepaType AMecouMauMM Nochor M3 πρμμθηπθμμχ CMeuiaHHbix 30HA0B. 3tm ycoBepmeHCTBOBaHMa ο6τ > θαμηπιοτοπ c nonyneHMeM pe3ynbTaTOB, KOTopbie Henb3n 6bino oxMpaTb. 3το b ποπηομ mepe ποποηπθτοπ τθμ, ητο μθτοαμκμ CMemaHHbix 30haob, βκπιοη3Κ31αμθ 4 pa3a κοπμηθοτβο 30ΗΑ0Β, κοτοροβ, Kak COOSmanOCb, ΠρθΧΑΘ ΗΘΜ ΒΟΘΓΑΘ c ycnexoM Mcnonb3oeanocb Aaxe b cMTax KflHK Ha βμαθχ ΜΗφορΜ3ΐ4ΜθΗΗθΜ flHK οτηοομτθπβηο Manom ΗΜΟΠΘΗΗΟΟΤΜ, ČblHM ycneillHO ηρΜΜΘΗΘΗΜ Κ Μ30ΠΠΙ4ΜΜ ΓΘΗ3 yHMKanbHOM nocneAOBaTenbHOCTM b npoceMBaHMM γθηομηομ 6μ6πμοτθκμ m 1500000 CTepMnbHbix ππτθη. 3tot ποαβμγ 6bin ocyiAecTBneH of cyiAecTBy, coBnaAan c ony6nMKOBaHMeM μηθημπ Anderson et al., Supra, whereby to cnocočbi npoceMBaHMfl c Mcnonb3oeaHMeM cMeiuaHHbix 30ηαοβ 6μπμ " ... HenpaKTMHHbl ΑΠΠ Μ30ΠΜρ0Β3ΗΜΠ βθΠΚΟΒΜΧ ΓΘΗΟΒ ΜΠΘΚΟΠΜΤ3Κ3111ΜΧ Kota ΟΟΟΤΒΘΤΟΤΒΘΗΗΜΘ flHK HeAOCTynHbi ". 74 OTnuHMTenbHbie npM3HaKM Μ3θ6ρβτβΗΜΑ, pacKpbiTbie b npefliuecTByiomeM onncannn, b cneflytouj (EII cf > opMyne Μ3ο6ρθτθημβ μ / μπμ b npnnaraeMbix Ηβρτθχοχ, MoryT oTpenbHO μ b πιοδοιί KOMČMHaumi mx ciaTb MaTepnanoM nna ocymecTB / ieHM5i μ3ο6ρθτθημβ b pa3Hoo6pa3Hbix ero cpopMax.

Claims (60)

A purified and isolated polypeptide having a partial or complete primary structure conformation and one or more free erythropoietin biological properties, characterized in that it is a prokaryotic or eukaryotic expression exogenous DNA sequence product. . A polypeptide according to claim 1, characterized in that it is free of compatibility with any mammalian protein. The polypeptide of claim 1, wherein the exogenous DNA sequence is a kDNA sequence. 4. A polypeptide according to claim 1 wherein the exogenous DNA sequence is a constructed DNA sequence. 5. A polypeptide according to claim 1, wherein the exogenous DNA sequence is a genomic DNA sequence. The polypeptide of claim 1, wherein the exogenous DNA sequence is transferred by a self-replicating plasmid DNA or a virus vector. 7. The polypeptide of claim 1, characterized in that it has a partial or complete conformation of the primary structure of human erythropoietin as shown in Table VI or any of its freely occurring allelic variants. 8. The polypeptide of claim 1, characterized in that it has a partial or complete conformation of the primary structure of the monkey erythropoietin as shown in Table V or any of its freely occurring allelic variants. 9. A polypeptide according to claim 1, characterized in that it has the free immunological properties of erythropoietin. 10. A polypeptide according to claim 1, characterized in that it exhibits free in vivo biological activity of erythropoietin. 11. A polypeptide according to claim 1, characterized in that it exhibits free in vitro biological activity of erythropoietin. 12. A polypeptide according to claim 1, wherein the covalent bond is to the labeled substance being sought. 13. The polypeptide of claim 12, wherein the labeled target is a radiolabel. 2 14. A DNA sequence expressed as a polypeptide product in a prokaryotic or eukaryotic host cell that has at least a conformational portion of the first structure and one or more biological properties that are free of erythropoietin, wherein said DNA sequence is selected from: (a) DNA - the sequences shown in Tables V and VI or complementary yarns; (b) DNA sequences which hybridize to the DNA sequences defined in (a) or fragments thereof; and (c) DNA sequences which, due to the degeneracy of the genetic code, have to hybridize to the DNA sequences defined in (a) and (b). 15. A cell of a prokaryotic or eukaryotic host transformed or transfected with a DNA sequence, after 14, allowing the host cell to express the specified polypeptide product. 16. The DNA sequence expression polypeptide product of claim 14, which is a prokaryotic or eukaryotic host. A purified and isolated DNA sequence encoding the expression of a prokaryotic or eukaryotic host polypeptide having partial or complete conformation of the primary structure and one or more of the biological properties of cytopoietin. 18th kDNA sequence after 17p. 19. Erythropoietin monkey encoded by the DNA sequence after 18p. 20. The DNA sequence of 19.p. and protein-containing coding region shown in Table V. 21. Genomic DNA Sequence after 17p. 22. Human erythropoietin encoded by the DNA sequence at 21p. 23. The DNA sequence after 22nd and the protein-containing coding region shown in Table VI. 24. Constructed DNA Sequence after 14p. 25. The engineered DNA sequence of item 24 and comprising one or more codons selected for expression in E.Coli cells. 26. A constructed DNA sequence of 25p encoding expression of human erythropoietin. 27. The constructed DNA sequence of item 26 containing the protein coding region is shown in Table XIV. 28. The engineered DNA sequence of item 24 and comprising one or more codons selected for expression in yeast cells. 29. Constructed DNA sequence after expression of 28.p. encoding human erythropoietin. 3 EN 10506 30. The construct DNA sequence of Fig. 29 containing the protein and the coding region is shown in Table XXI. 31. The DNA sequence according to item 17 is covalently linked to the labeled target substance. 32. A DNA sequence after 31st, wherein the searchable feature is a radio tag. 33. Single-stranded DNA sequence after 31p. 34. A DNA sequence encoding a free erythropoietin polypeptide fragment or polypeptide analogue. 35. The DNA sequence encoding [Phe15] hEPO, [Phe49] hEPO, [Phe145] hEPO, [His7] hEPO, [Asn2des-PRO2 to [Le [beta] HEPO, [des-1 H] 63 to Arg- * 63] hEPO or [D27-55] hEPO. 36. The DNA sequence of Paragraph 34 constructed in the sequence. 37. A biologically functional ring-plasmid or viral DNA vector containing a DNA sequence of p-ths 14,17,34 or 35. 38. The cell of a prokaryotic or eukaryotic host is stably transformed or transfected with a DNA vector after 37p. 39. The polypeptide product of the DNA sequence after 17 or 34 of the prokaryotic or eukaryotic host cell. 40. A glycoprotein product having a conformational structure that duplicates the conformation of the free erythropoietin sufficiently to allow it to exhibit one or more of its biological properties, and which has a medium carbohydrate composition different from the free erythropoietin composition. 41. A glycoprotein product having a primary structure conformation that duplicates freely human erythropoietin to exhibit one or more of its biological properties and has a medium carbohydrate composition different from the free human erythropoietin composition. 42. The cells of the vertebrate that can be reproduced continuously in vitro and which are capable of producing more than 100 units of erythropoietin per 10 6 cells per 48 hours of culture in culture, as determined by radioimmunoassay. 43. The vertebrate cells of step 42 which are capable of producing more than 500 units of erythropoietin per 106 cells in 48 hours. 44. The vertebrate cells of step 42 which are capable of producing more than 1000 units of erythropoietin per 106 cells in 48 hours. 45. The vertebrate cells at 42, which are mammalian or bird cells. 46. The vertebrate cells at 45, which are COS-I or CHO cells. 4 47. A synthetic polypeptide having a partial or complete amino acid sequence as shown in Table V and having one or more free biological activities of the monkey erythropoietin in vivo and in vitro. 48. A synthetic polypeptide having a partial or complete amino acid sequence set forth in Table VI other than a sequence of amino acid residues in the region designated 1-20 and having the biological properties of free human erythropoietin. 49. A synthetic polypeptide having a secondary conformation of part or all of the amino acid sequence shown in Table VI other than a sequence of amino acid residues in the region designated 1-20 and having the biological properties of free human erythropoietin. 50. A method for obtaining a polypeptide having partial or complete conformation of the primary structure and one or more of the free biological properties of erythropoietin, characterized in that, under useful feeding conditions, the cultivation of prokaryotic or eukaryotic host cells transformed or transfected with DNA. vector after Fig. 37 and isolation of the desired DNA sequence expression polypeptide products in the indicated vector. 51. An antibody which is distinct from immunoreactivity against erythropoietin and a synthetic polypeptide having a conformational structure, a sufficiently duplicating sequence of continuous amino acid residues, with the exception of any polypeptide comprising the amino acid residue sequence, completely contained within the sequence boundaries A-P-P-P-L-1-C-D-S-R-V-L-E-R-Y-L-L-E-A-K. 52. The antibody of claim 51, which is a monoclonal antibody. 53. The antibody of claim 51, which is a polyclonal antibody. 54. The antibody of claim 51 p-ta, that is immunoreactive to erythropoietin and a synthetic polypeptide having a sequence selected from: V-P-D-T-K-V-N-F-Y-A-W-K-R-M-E-V-G, K-E-A-L-S-P-P-D-A-A- -A-A and V-Y-S-N-F-L-R-G-K-L-K-L-Y-T-G-E-A-C-R-T-G-D-R. 55. A pharmaceutical composition comprising an effective amount of a polypeptide according to any one of claims 1, 16, 39, 40 or 41 and a pharmaceutically acceptable solvent, stimulant or carrier. 56. The polypeptide according to p.p. As pharmaceutical agent 1, 16, 39, 40 or 41. 57. The method of claim 56, wherein the therapy is to increase hematocrit levels. 5 LV 10506 58. A purified and isolated DNA sequence as shown in Table V or VI, or a fragment thereof, or a complementary thread of such a sequence or fragment. 59. The DNA sequence expression polypeptide product of 58 cell, prokaryotic or eukaryotic host cell. 60. Multiple-strand polynucleotide-specific single-stranded polynucleotide-specific single-stranded polynucleotide-specific single-stranded polynucleotide constanting technique comprising a heterogeneous cell or virus sample, characterized in that (a) produces a single-stranded polynucleotide. a mixture of labeled probes with homogeneously variable base sequences, wherein each of said probes is potentially specifically complementary to a base sequence that is possibly unique to the target polynucleotide; (b) binding the sample to a dense substrate; (c) treating the substrate to which the sample is attached to attenuate further attachment of the polynucleotide to it, except for hybridization with polynucleotides of a given sample; (d) temporarily adhering to the treated substrate to which the sample is attached, the indicated labeled probe mixture under conditions that promote hybridization only between completely complementary polynucleotides, and (e) detecting the specific polynucleotide by monitoring the presence of a hybridization reaction between their fully complementary probe labeled the probe mixture, as demonstrated by the highest density of the labeled material on the substrate-specific polynucleotide locus, compared to the background density of the labeled material resulting from the non-specific binding of the labeled probes to the substrate, wherein the indicated enhancement includes the use of more than 32 mixed probes and one or more performance of operations: (1) using dense substrate on nylon paper; (2) treating with protease in step (c); (3) Apply to individual labeled probes about 0.025 picomolar concentration; and (4) apply, at any of the applicable probes, as one of the hybridisation rules (d), strict temperature limits approaching 4 C values from the lowest calculated Td value.