LV10120B - Method of producing of new plasmides and using thereof in producing of the plasminogen activator - Google Patents
Method of producing of new plasmides and using thereof in producing of the plasminogen activator Download PDFInfo
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- LV10120B LV10120B LVP-92-204A LV920204A LV10120B LV 10120 B LV10120 B LV 10120B LV 920204 A LV920204 A LV 920204A LV 10120 B LV10120 B LV 10120B
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6456—Plasminogen activators
- C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
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- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Saccharide Compounds (AREA)
- Peptides Or Proteins (AREA)
Description
LV 10120
CnOCOB ΠΟΛΥΗΕΗΗΗ Η0ΒΗΧ nJIA3MHfl Η HX ΠΡΜΜΕΗΕΗΗΕ ΠΡΜ ΓΟΥΜΕΗΗΗ ΑΚΤΗΒΑΤΟΡΑ ΠΙΙΑ3ΜΜΗ0ΓΕΗΑ ΠρΗ ΤβρβΠΗΗ, 06yCJI0BJieHH0ft φΗβρΗΗΟΜ, 3aKynOPKM COCyflOB κβκ HanpuMep, ΗΗφερκτβ MHOKapjja, θμΟοληη λθγκηχ, apTepnaJii>HHX COCyAHCTbIX 3a(50^eBaHHK ΚΟΗβ«ΗΟΟΤβίί Η ap., βΚΤΗΒβΤΟΡΗ nJiaSMH-HoreHa nrpaioT cymecTBeHHyio pojib. C Ha«aJia 50-ux γολοβ H3BecT-ho, «το npeBpameHHe nJia3MHHoreHa odycjioBJieHO ero npeodpa30Ba-HH6M B nJia3MHH, Τ.θ. B 9H3HM, KOTOptlft 0(5yCJI0BJIHBaeT ρβΟΠΙβΠΛΘ-HH6 φΗ0ρΜΗ3 B paCTBOpHMHe ΠΘΠΤΜβΗ H, T3KHM O0pa3OM, paCTBOpe-ΗΗΘ 0(5yCJI0BJieHHUX φΗβρΗΗΟΜ ΤΡΟΜ^ΟΒ. 8ΤΟΤ βΚΤΗΒβΤΟΡ ΠΛ33ΜΗΗ0Γβ-Ha dbiJi HasBaH ypoKHHa3oft, h oita3a;iocb, «το b aeficTBHTejibHOCTH cymecTByK)T pa3Jin«Hbie Φορμη ypOKHHa3ti. Bce 3th Φορμη noJiy«aioT-cb ojiHHaKOBo, HenocpeACTBeHHo H3 oflHoK MOJieKyjiH-npeflmecTBeHHH-Iļbl, H3BeCTH0fi npH0JIH3HT6JIbHO C CepeflHHbl 70-ΒΙΧ ΓΟΛΟΒ, UHMOrena npoypoKHHa3H. Torjia tao ycTaHOBJieHO, «to 3Ta npoypoKHHa3a oOjiaflaeT oflHoueriHoft CTpyKTypofi h OHa dujia Ha3BaHa " scu-pa" (single Chain urinary plasminogen aktivator). 8Ta scu-PA COCTOHT M3 411 aMHHOKHCJIOT H rJIHK0JIH3HP0BaH B npHpOAHOft npea-uiecTByiomeM φορΜβ flo aMMHOKHCJiOTU 302. H3 pa3Jin«Hux padoT H3BecTHo reHOT6XHOJiorn«ecKoe nojiy«eHHe HerJIHK0JIH3Mp0BaHH0fi «aCTH ΠρΟΤβΗΗβ SCU-PA, KOTOpafl Ha3BaHa " rscu-PA” (peKOMOHHHpoBaHHafl scu-PA ) η ΜΜββτ Tanse TpnBnajitHoe Ha3BaHne "capynjia3a". Πρκ TepaneBTH«ecKOM Hcnojib30BaHHH rscu-PA OKa3ajiocb, «το ero HadJiroflaeMoe fleftcTBHe η οοβμθοτμμοοτβ πρβΒΟΟχοΛΗΤ ynoīpetoeMbie ΦηΟρηηολητηκη (cm. "JlaHiļeT", 1989r. ctp.863-868). ta nojiy«eHHH HerjinK03HJinpoBaHHoro aKTHBaTopa njia3MHHo-reHa rscu-PA ncnoJib30Ba;iH nponapMOTHTbi, ο «θμ yae cymecTBOBa- 2. •jih ΗθκοτορΗβ aaHHbie b jiHTepaType. Μ3βθοτηηθ flo HacTOHmero ΒρβΜβΗΜ cnocotfbi noJiyveHHH §cu-PA ocHOBaHu Ha 9Kcnpeccnn cTpy-KTypHoro reHa scu-PA BtmeJieHHoro H3 ve.noBevecKHX TKaHei*. K coaajieHHK), πρπ θτομ flocTuraeTCH JiHuib He3HavHTejibHafl οτθπθηβ 3Kcnpeccnn, KOTopan He odecnevHBaeT 3K0H0MHvecK0e nojiyveHHe uejieBoro npoayKTa b 6ojibiunx KOJiHvecTBax. TaK HanpHMep, οπη-caHHOe Hibino H flp. B "Agrie, Biol, Chem " 52, 329-336,1988, nojiyveHHe rscu-PA 6aKTepnH e.coū aaeT CTeneHb 3Kcnpeccnn jiHiiib 2%, H3MepeHHyio b oc5iu©m KOJin^ecTBe dejuca daKTepHfi. C iipy-roK ctopohh, b.eBponeftcKofi 3ΒΗΒΚΘ Ha naTeHT 92 182 A2, a TaKae Hoimec h flp. flocTHraeMyio CTeneHb aitcnpeccHH tovho He yKa3biBa-I0T. OflHaKO πρκ npOBepKe 3THX flaHHbIX B pa3JIHVHHX ΙΠΤΒΜΜΒΧ tfblJIO nojiyveHO MeHee 2% προτβΗΗβ daKTepnfi rscu-PA.
Cjie^oBajio 6u yita3aTb Ha to, vto b dojibīDHHCTBe c;iyvaeB 3KcnpeccHH 3yKapnoTHoro npoTeHHa b daKTepHHx nojiyvaeTCH Taitae rscu-PA b KJieTKe b KavecTBe fleHaTypHpoBaHHoro BKJiioveHHoro Te-jia, κοτοροθ flajiee Ha3HBaeTca "προτθηηομ npeflBapHTejibHOft οτθληη” kotopu^ noejie npoMeayTOWHoro BH^ejieHHH, λολβθη dbiTb προτθηηοχη-MHVeCKH B03BpameH K npaBHJIbHOH VeTBepTHVHOfl CTpyKType rscu-PA. nojiyqeHHbifi τβκμμ odpa30M npoflyKT mobho eaTeM πβρβΒβοτκ jņjra onpe flejieHHH o6pa30BaBiiierocH KOJiHvecTBa rscu-PA , HanpHMep, nodaB-λθηηθμ njiaeMHHa, b flByxuenHyio HerjinK03minpoBaHHyio ypoKHHa3y ("rtcu-PA^ 8Η3ΗΜΗ3Η dKTHBHOCTb KOTOpofl OnpefleJieHa Η MOaeT CJiy-anTb Mepofl o6pa30BaBUierocfl KOJiHvecTBa rscu -pa. ΕοτβοτΒβΗΗο, momho onpeaejiflTb βηχολ npoTeHHa npeflBapHTeJibHOft CTaflHH hjih rscu-PA h TaKHM o6pa30M, οτθπθηβ 3KcnpeccHH, HanpHMep, c πομο-mbio fleHCHTOMeTpHuecicofi οηθηκη γθλθβογο ΘΛβκτροφορβββ BCero ΠΡΟΤΘΗΗΒ.
HeoaH^aHHo dbiJio odHapyaeHo, ντο όβκτβρΗΗ, τρβΗΟφορΜπρο-BaHHbie nJia3MHflaMH, nOJiyveHHHMH B COOTBeTCTBHH C Η3Ο0ρβΤβΗΗβΜ, flaioT bo μηογο pa3 6ojibiuyK) CTeneHb siccnpeccHH, veM mTaMM-xo3HMH, HfleHTHVHO Τρ3ΗθφθρΜΜρθΒ3ΗΗΗΐί nJia3MHflaMH, Η3ΒβΟΤΗΜΜΗ H3 CnOCOČa ypOBHH ΤβΧΗΗΚΗ. flOJiyVeHHbie B COOTBeTCTBHH C Η3θ6ρβΤβΗΗβΜ njia3-MHflbl, B COOTBeTCTBHH C 3THM 6θΛββ ΠΡΗΓΟΛΗΗ, V6M BC6 Η3ΒβΟΤΗΗβ flo HacTonmero ΒρβΜβΗΗ n^a3MHAU, ajih nojiyveHHH rscu-PA hjih ero npoTeHHa npeAtuecTByiomeft CTaflHH. CjiejņyeT eme ynoMHHyTb, ντο H3-BecTHUM odpa30M MoaeT feb nojiyveH TaKae c noMombio y»e ynoMH- LV 10120 'HyToro Bbiuie ajih aHajiHTHtiecKHX uejiefl pacmenjieHHH rscu-PA, HanpHMep njia3MMH0M, TaK«e h rtcu-PA - b TexHHuecKHX MacuiTaOax, nOCJie uero, djiaroflapfl ΠρβΑΛΟΚΘΗΗΟΜγ H306peTeHHK),0WeHb flOCTy-ΠβΗ rscu-PA. f1Jia3MMJQbI, nOJiyWaeMbie B ΟΟΟΤΒΘΤΟΤΒΗΗ C Η300ρβΤβΗΗΘΜ, OT-jnmaioTCfl, κροΜβ τογο, τθμ, uto nx onepoH ΜΜββτ peryjiHpyeMbiK b cooTBeTCTByioiiieM cjiy*iae cHHTeTHuecKHft npoMOTOp, aKTHBHyio, b KauecTBe MecTa cbh3h phOocom, nocjieflOBaTejibHOCTb D]HHO-,najibrapHO, CTapTOBHfi KOflOH, ΟΗΗΤβΤΜ^βΟΚΗίί CTpyKTypHHfl γθη ρ,λη scu-pa h προτηβοπολοβηηΚ pacnojioaeHHio CTpyKTypHoro reHa, no Kpaitoefl μθ-pe, ο,αηη τβρΜΜΗβτορ. npeflnouTHTejibHO ΗΜβιοτ Μβοτο ολμη 3a flpy-γμμ cjieflyroiiiHe τβρΜΗΗβτορκ, b cjiytiae κοτορυχ peub Ηββτ b uacTHO-cth o trpa TepMHHaTope h/hjih o tet A/orfi TepMHHaTope H3 τη , CM. Schollmeier H flp. B cjiyyae peryjinpyeMoro npoMOTopa pe^b HfleT, b wacTH0CTn, o tac npoMOTope HJIH trp ΠΡΟΜΟΤΟΡΘ. B KOHTpOJIbHOM ρβΓΗΟΗβ nOJiyweHHbie B C00TB6TCTBHH C H30-dpeTeHHeM njia3MHflbi ημθιοτ paccTOHHHe Mew nocjieaoBaTejibHocTbio UJnHO-^aJībrapno h CTapTOBbiM ko,e(ohom 6-12, npe^no^iTHTeJibHo 8-10 HyKJieoTH,aoB. ΠρΗ KOHCTpynpOBaHHH HCn0JIb30BaHH0r0 B HOBbIX njia3MH,E(ax cTpyKTypHoro rēna npeflnouTHTejibHo BCTpaHBaioT 3aK0flnp0BaHHbie Ajih cooTBeTCTBywmnx aMHHOKHCJiOT npHBefleHHbie b cJieAjrouieii Ta6-Jiniļe 6a30Bbie nocjie/joBaTejibHOCTH. Πρκ θτομ b CTpyKTypHOM reHe He flOJIMHbl (5bITb ΒΗΠΟΛΗΘΗΗ ΟΛΗΟΒρβΜβΗΗΟ ΒΟβ 3TH ΟΤΛΗΊΗΤβΛbHUG ΠΡΗ3Η8ΚΗ. AMHHOKncjioTa: TpnnjieT:
βρΓΗΗΗΗ CGT JieftiļHij CTG BaJIHH GTT npOJIHH CCG rJIHUHH GGT C flpyroii cTopoHbi, πρκ ποοτροθηηη cTpyKTypHoro reHa nejie- C006pa3H0, ΠΟ B03M0BH0CTH, H30eBaTb ΠΡΜΜΘΗβΗΗΛ ΟΛΘ^ΙΟϋΙβΓΟ KO-flOHa flJIH COOTBeTCTByromHX Ηβ3Β8ΗΗΗΧ aMHHOKHCJiOT (npH^eM, CHOBa ΗΘ flOJIBHbl BbinOJIHHTbCH ΟΛΗΟΒρθΜβΗΗΟ 3TH OTJIH^HTeJIbHbie ΠΡΗ3Η3ΚΗ flJIH BC8X aMHHOKHCJiOT). 4.
Kohoh. κοτορογο cjienveT Amhhokmcjtota : H36eraTb:
ATA HaojiefliiHH
GTC BaJIHH
CCC npOJIHH
AAG JIH3HH AGG AGA, CGG, CGA ΑρΓΗΗΗΗ GGA, GGG ΓΛΗΙ^ΗΗ flyTeM Bufiopa KOAOHa, b οοοτβθτοτβηη c H30tfpeTeHneM, ajih οτββΛΒΗΗχ aMHHOKHCJioT b οτργκτγρΗΟΜ rene, a τβκκβ BbnneHa3BaHHbix OTJIHWHTeJIbHHX npH3HaK0B ΚΟΗΤΡΟΛΒΗΟΓΟ Γ8Η3, ΠΟΛΗΟΟΤΒΚ) ΠρβΠΗΤ-cTByioT o(5pa30BaHHio CTa0HJibHbix BTopnwHux cTpyKTyp Me*fly CTpyK-TypHUM Γ6Η0Μ H KOHTpOJIbHHM Ρ6ΓΜ0Η0Μ.
Ajih nojiytieHHH cHHTeTHuecKoro CTpyKTypHoro reHa CHanajia CHHT83HpyiOT OJIHrOHyKJieOTHJļbI B 0ΤΡΘ3ΚΘ 40-80 OCHOBaHHŪ B 0,UHy uenomcy. L(ejiecoo6pa3HO nojiyiiaTb hx b MacmTatfe 1 mkm/moji no Me-TOAy ΤΒβρΛΗΧ Φδ3 no AaaMcy h ap· c πομοπιβιο ,QHK-CHHTe3aTopa mo-AeJlH " Biosearc8600" φΗΡΜΗ "New Brunsevick Scient '·. B Ka^eCTBe ΜΟΗΟΜβρΗΟΓΟ CHHTOHa ΠΡΜΜβΗΗβΤΟΗ ΠΡΗ 3T0M HMeiOIHHfiCfl B ΠρΟΑβΚΘ β- IļHaH0-3THJI-3aMemeHHHe ΑΗΜ30ΠρθΠΜΛ8ΜΜΗΟ-φθθφθρθΜΗΑΗΤΗ οοοτβθτ-CTByiomHx Heo6xo,qnMbix fle30Kcnpn60HyKJie03Hfl0B, nocJie oTmeaneHHH ot H0CHT8JIH HaxoAflmnecH Ha KOHuax einē TpHTMJi3aMemeHHue hhth, OdeCCOJIHBaiOTCH 5 CTepHJIbHbIX yCJIOBHHX nyTeM ΓβΛΒφΗΛΒΤρβΙΙΗΙΙ M npeflBapuTejibHo otromaioTCH n, 3aTeM, β αβθ οτβΑΗΜ, noflBepraioTCfl nepBOMy pa3,qejieHHio c πομοιιιβκ) "B03BpaTH0fi φββΗ" -hplc. Οοοτβθτ-CTByromnfi οοηοβηοΑ npo^yKT fleTpnTHJinpyioT h, nocjie flByx*flonojiHH-Te;ibHbix CTaflHft o«hctkh "B03BpaTH0fi φ83Η''-ΗΡ^, nojiynaiOT uejieBofl OJIHrOHyKJieOTHfl BHCOKOft HHCTOTbl, KaK nOKa3aJI ΓβΛΒ3ΛβΚΤρθφθρβ3-Hblfl aHaJIH3 (page). H3 rpynnw 4-9 0thx οταθλβηηχ ημτθΒ nojiy«aJin 3aTeM ajihh-hoA okojio 200 HyKJieoTHflOB Φρβγμθητη αβοΑηηχ uenefi, WTo6bi He-KOHiļeBbie oTfleJibHue ojinroHyKJieoTHflH y δ/'-κοΗΐιβ φοοφορκΛκροΒβτΒ μ cooTBeTCTByK)mHe KOMnjieMeHTapHHe hhth βμθοτθ c κοηι4θβημη ojih-roHyKJieoTMflaMM AeHaTypHpoBaTb h JinrHpoBaTb. nocjie Jinrannn 00pa30BaBiiiHeci!, cnocofiHue κ κλοηηροββηηιο, flBoftHbie uenHue φρβΓ-M8HTbI HCnOJIb3yK)T 38T6M B ΠΟβΧΟΑΗΙΙΙΗΧ JIHHeapH3HpOBaHHbIX ΒΘΚΤΟ- pax. AonojiHUTejibHbie Mepbi mokho y3HaTb H3 HHKenpnBefleHHbix npn-MepoB. 5. LV 10120
Hcnojib3yeMbie b npefljīPaeHHofi 3aflBKe coitpameHHH ΚΜβιοτ cjieflyioiHHe 3HaweHna: pu - Piong untis 6n - ocHOBHbie napu ŪE 52 - aHHOHOOŪMSHHMK φΐφΜΗ E.HTK - 3THJieHflnaMHHOT0TpayKcycHafl KHCJiOTa ΜΠΤΓ - H30nponMJI- - -TH0raJiaKT0nHpaH03Hfl PAGE - Π0ΛΗβΚΡΗΛβΜΗΛΓΘΛΒ3ΛΘΚΤΡ0φ0ρβ3 HflC - HaTpHMflOfleunjicyjib$aT
DI.’H.nocjieflOBaTejibHOCTb - ūlHHe-JlajībrapHO-nocjieflOBaTejibHOCTb TpHC- fjtt - mflpoXJIOPHfl TPHCrHflpOKCHMeTHJiaMHHOMeTaHa TBHH-80 - nOJIH9THJI0HOKCHfl COpdHTaHMOHOOJieaT JļJIfl KOHCTpyHpOBaHHH, B C00TB8TCTBMH C Η3θ6ρβΤΘΗΗΘΜ, HO- bux iuia3MHA npeflno^iTHTejibHO ηοχοαητ ot HMeiomeficfl b npoflase nJia3MHfltI pBR (4363 tfn). ΠρΘΛΠΟ^ίΤΗΤβΛΒΗό H3 H66 3JIHMHHHpyK)T nic/bom O&iaCTb H/HJIH TeTpaiļHKJIHHOBblfi pe3HCTeHTHHft Γ6Η. ΠρΗ 3T0M ΗβΤ Heo6XOJD[HMOCTH nOJIHOCTbfO tfījaJIHTb ΓβΗ ρβ3Η0ΤβΗΤΗ00ΤΗ κ TeTpaqnKJiMHy, wame Bcero AOCTaTOUHO, wto6u ruia3MHfla ηθ nepe-Aajia TpaHC®opMnpoBaHHOMy t3khm otfpa30M QiTdMMy δβκτθρκβ ycTofi-IIHBOCTM K TeTpaUHKJIHHy. C nOMOmbK) 3ΤΗΧ Mep (yflaJieHHe nic/bom o^JiacTH h uacTH reHa ρβ3ΗοτβΗΤΗθοτΗ κ TeTpaijMKJiHHy) nojiyuaioT τβκ Ha3UBaeMyx) "BucoKOHaflesHyio njia3MHfly". npeflnowTHTejibHO0 CTpaTerne8 fljia KOHCTpynpoBaHMfl hoboR n^a3MHflbI, B C00TB3TCTBHH C Η300ρβΤβΗΗΘΜ, HCXOAfl H3 ΜΟΑϋφΗΑΗρΟ-BaHHoK, κβκ yriOMHHaJiocb BHme, njia3MHAti pbr322 (hjih flpyrofl Ha3-BaHHofi, H3BecTHoK paHee, njia3MWAbi) ηβληθτοη, b Ka^ecTBe nocjie-flyiomefi CTaflHH, βοτρβηββηηθ ομητθτμ^θοκογο "caflTa MyjibTHKJioHnpo-BaHHH" Meafly m@ctom pa3pe3a eco ri m Hind m#. 8tot cafiT My;ib-THKJIOHHpOBaHHH ΗΜΘβΤ yCTaHOBJieHHyiO nOCJieAOBaTeJIbHOCTb MeCT pa3pe3a, Jieiaume μθβαυ Himh ocHOBaHHH ηβληιοτοη npoH3BOJibHO bh-OpaHHHMM, 3a ΗΟωίΧΝΘΗΗΘΜ nOCJieAOBaTeJIbHOCTH Me»Ay Xbal M NdeI, KOToptie coAepiaT Μβοτβ cbh3h pmGocom (UlHHe-AajībrapHo-nocjieAOBa-TejibHOCTb) H3 xyi-onepoHa 5'-aaggag -3'(4)B.subtilis , a Tanae ycTaHOBJi6HHoro pacTOHHHH Me«fly UI.A.-nocJieflOBaTejibHOCTbio h CTap-tobum KOflOHOMatg . Cjieflyiomne 3a ID.A.-noc^eflOBaTejibHOCTbK) ocho- ΒβΗΗΗ GAAAT ΠβρβΗΘΟβΗΜ M cBfl3aHH C CATATG , ΜΘΟΤΟΜ JDa3pe3a NdeI TaKHM odpa30M, paccTOHHHe Me«fly III.A.-nocjieflOBaTejibHocTbKī h atg cocTaBJiaeT 8 οοηοβημχ nap. PaccTOHHMfl Me»fly οταθλβηημη μθοτβμη 6. •pa3pe3a caītoa Myjibthkjiοηηροββηηη hojikhh 6ητβ , H3 cootfpaaeHHi* TeXHOJIOrHH KJIOHHpOBaHHH, ΠΟ ΜβΗΒΠΙβΑ Μβρβ OKOJIO 20 HyKJieOTHflOB f b pe3yjibTaTe wero ofaeniaeTCH yflajieHHe npoMe»yTOWHbix «fcparMeHTOB. C nOMOUlbK) 3Τ0Γ0 CafiTa MyJIbTHKJIOHMpOBaHHfl B nJia3MHfly BBO-flHT MecTa paepesa, KOTopue, nocjie BCTpāHBaHHH TepMHHaTopa Tpan-cKpnniJ[nn, fleJiaioT βο3μομηημ c;ieflyiomee npyr 3a apyroM ycneuiHoe BCTpaMBaHHe «acTHtiHoft nocjienoBaTejibHOCTH scu-pa cTpyKTypHoro rēna, npe,EtnowTHTejibHO HaunHaromerocfi ot C-KOHua uejieBoro npoTeHHa, T.e. ot 3'-KOHua ηητη HHK nojijroaiomerocfl CTpyKTypHoro reHa, a TaK «e BCTpanBaHHH, HanpHMep, οηητθτηιιθοκογο, hjih TaK*e BbmejieHHoro H3 apyrofl njiasMHflH, hjih KMeiomerocfl b npoflaae trp -npOMOTOpa. nojiHaB HyKJieoTH,qHafl nocjienoBaTejibHOCTb nojiy*ieHHoro TaKHM otfpaeoM scu-pa -CTpyKTypHoro reHa npeflCTaBJieHa c yKa3amieM aMHHOKHCJioT , COOTBeTCTByiOmHX OTfleJIbHOMy KOflOHy. ,Qpyrne ηοβηθ njia3MHflbi MoryT (5ητβ nojiyyeHH,B οοοτβθτοτβηη c H3o6peTeHHeM, H3 CKOHCTpyHpoBaHHofl KaK onHcaHO Bbiine njia3MHflbi, HanpHMep, nyīeM pa3pe3a c noMombio Eco ri h Hind iii nojiywaK>T CHHTeTH^ecKHii scu-PA-CTpyKTypHHK reH co βοθμη θληηηιιβμη peryjin-poBaHHH h, eaTeM, BCTpanBaioT b flpyryio, b 9HTepo0aKTepH8HyK>, b
UaCTHOCTH,E. coli, aBTOHOMHO MHO»amyiOCH nJia3MHfly, KOTOpyiO K TO-My .«e JiHHeapn3HpyiOT hjih yKopawHBaioT c noMombio paspe3a c πομο- mbK) ECO RI H Hind III. TaKHM »β, B ΠΡΗΗΙίΗΠβ, ΟΠΟΟθ6θΜ, HO ΠΡΗ Hcnojib30BaHHH MecTa paepesa eco ri h xba mokho 3βμθηητβ Tanie, HanpHMep trp -ΠΡΟΜΟΤΟΡ tac -ΠΡΟΜΟΤΟΡΟΜ H HaO^OpOT B nOJiytieHHOfi B COOTBeTCTBHH C Η3θ6ρβΤβΗΗβΜ nJia3MHfle. AHaJIOrHHHO MOHHO HCXOflHTb ΤβΚΚβ H3 flpyrHX Η3ΒβΟΤΗΗΧ njia3-μηλ, κοτορΗβ o^JianaioT eflHHCTBeHHbiM ΜβοτοΜ pa3pe3a.
HoBbie njia3MHflbi c yBejiHweHHUM paco'TOHHHeM Meamy IIIhho-.HajībrapHo-nocjieflOBaTejibHocTbio h CTapTOBUM koaohom ATC moikho nOJiyUHTb,B COOTBeTCTBHH C Η3θ6ρβΤ6ΗΗβΜ, ΤβΜ, MTO CKOHCTpyHpO-BaHHaH^ KaK onncaHo Bbime njiasMHfla, b κοτοροβ ynoMHHyToe paccTO-HHHe cocTaBJineT, HanpHMep, 8 HyKJieoTH(qoB, pa3pe3aiOT Nde ι, ko-Topoe 3anojiHHeT nojijreeHHoe Μβοτο paepesa h, HenocpencTBeHHO nocjie 3Toro, nojiyueHHue kohiih JiHrHpyioT, b pe3yjibTaTe wero 06-pa3yeTCH KejiaeMan HOBan njia3MHfla, b κοτοροΑ paccTOHHHe Me»,Eļy Ul.il.-nocjieflOBaTejibHOCTbK) h CTapTOBbiM κολοηομ cocTaBJineT, HanpHMep, 10 HyKJieoTHflOB. LV 10120 7.
KaK yae tao 0Ka3aH0, c ΰβκτβρΗΗΜΗ, τρ3ηοΦορμηροββηηημη nJia3MHflaMH, nOJiyueHHbIMH B COOTBGTCTBHH C H306pGTGHHGM, MOKGT 6biTb j[0CTnrHyTa bo μηογο pa3 fojibinan οτθπθηβ 3KcnpeccHH, ueM b cjiyuaflx Η3Βθοτηηχ H3 ypoBHH τθχηηκη njia3MH,q. ΤρβΗοφορΜβρΗΐο nOflXOflHmHX ΚΟΜΠβΤβΗΤΗΗΧ 0ρΓ3ΗΗ3Μ0Β-Χ03ΗβΒ OCymeCTBJIflK)T Η3ΒβΟΤ-hhm o<5pa30M. Hjih aKcnpeccHH, noJiyyeHHHX b οοοτβθτοτβηη c H30-όρβτβΗΜβΜ, njia3MHfl οοο6θηηο πρηγοαηη opraHH3Mbi mTaMMa-xo3HHHa THna E.coli Η Λρ.ΠρΗΜβΗΗβΜΗβ 3ΗΤβρ00βΚΤβρΗΗ,Κ3Κ HanpHMep, UITaM-Mbl Pseudomonas, Salmonella, Enterobacteria, Klebsiella, Serratia. ΠρβΑΠΟ^ΤΗΤβΛΒΗΗΜΗ 0praHH3MaMH-X03fleBaMH HBJIflIOTCH MTaMMH THna E.coli , ΚβΚ ΗβΠΡΜΜβρ,Ε. COli GRT-1 Η, B tiaCTHOCTH, ΙΠΤ3ΜΜΗ e. coiinonrpynnbi K12, KaK HanpHMep, E.coli K12y M101/ATCC 33876/, E.coli K12 y M103/ATCC 39403/, E.coli K12 y M105/dsm4162/, E.coli K12 ATCC 31446 hjih TaKKe E.coli K12 dni/ATCC 33849/. ΒβΘΛΘΗΗΘ 3KCnpeCCHH B 3KCnpeCCHOHHyiO CHCTeMy E.coli, ko-Topan οολθρκητ tac-προΜθτορ, mokho ocymecTBJiHTb, HanpHMep, nyTaM flodaBJieHHH JiaKT03bi hjih oidopa rjnoK03bi, ho πρθλπο^τητθλβ-ho nyTeM «ņodaBJieHKH H3onponHJi-a-D -THorajiaKTonnpaH03Hfla. OflHa-ko, ecjin b njia3MMfle ημθθτοη trp-προΜθτορ, to HHflyKijHio ocymecT- BJIHiOT npeMMyiHeCTBeHHO C nOMOUlblO HHflOJiaKPHJiyKCyCHOfi HJIH HHflOJI-aKpHJi-nponnoHOBoii KHCJioTbi. HpyrHe H3BecTHbie HHflyKTopbi, caMO codo8 pa3yMeeTCH, MoryT tob TaK»e Hcnojib30BaHbi.
flocjie hhotuhh h nocTH«eHHH onpeflejieHHofi BbiiiieyKa3aHH0ft IIJIOTHOCTH KJieTKH, KJieTKH OTUeJIHIOT ^eHτpHφyΓHpoBaHHeM H OCTa-tok, nocjie ^eHτpHφyΓHpoBaHHfl, nocjie cycneH3HpoBaHHH β βοληομ COJieBOM paCTBOpe, ΠβρβΒΟΑΗΤ β Γ0Μ0ΓβΗΗ3βΤ0ρ, B KOTOPOM KJieTKH b pe3yjibTaTe pa3HHqa nasjieHHH πολβο,ιιητ k MecTy. nocjie ηοβογο ^eHτpHφyΓHpoBaHHH noJiywaiOT β οοτβτκβ προτβΗΗ npeflBapHTeJibHofi CTaflHH rscu-PA Hapflfly C BOflOHepaCTBOpHMbtMH O6JI0MK3MH KJieTOK H πρ. B pe3yjibTaTe otfpafiOTKH pacTBOpOM ryaHHflHHB προτβΗΗ npeflBa-pHTejibHoK CTaflHH πβρβχοβΗτ b pacTBop h, nocjie ηοβογο ^eHτpHφy-ΓΗρθΒ3ΗΗΗ, OTCTOHBUIHftCH paCTBOp 0<5pa(5aTbIBaK)T ρβΛΟΚΟ-ΟΗΟΤβΜΟΚ (HanpHMep, coflepiKaineii BoccTaHOBJieHHbift hjih OKHCJieHHbifi rjiyTaTH-οη), β pe3yjibTaTe wero ocyinecTBJiHeTca odpa30BaHHe βοτβοτεβΗΗοίί ΚΟΗφθρΜ3Ι1ΗΗ, Τ.β., 06pa30B3HHe IļeJieBOfi rscu-PA. OHa HaXOflHTCfl 3aTeM B peaKIJHOHHOfi CM6CH B paCTBOpeHHOii φορΜβ Η ΜΟΚβΤ 6bITb BbiflejieHa b mhctom BHfle b pe3yjibTaTe o6uuhux CTaunft o^hctkh, HanpHMep, ΧΡ0Μ3Τ0ΓΡ3φΗΗ H nOCJie^IOUierO BbICyilIHBaHHfl 3aM0pa»H- 8. Β8ΗΗ6Μ.
Mjih aHajiMTHtiecKnx uejiefi uejiecoo(5pa3Ho φβρΜβΗΤΗροΒβτΒ TpaHC$opMMpoBaHHufi noAJieKamefi HccjieAOBaHH» njiašMHAofi οιτβμμ E. coii h, nocjie αοοτημθηηη BbruieyKa3aHH0ft onTHMajībHOfi πλοτηο-CTH CycneH3HH, ΚΛβΤΚΗ HHflyiiHpOBaTb ΠΟΛΧΟΛΗΠΙΗΜ HHflyKTOpOM 3K-cnpeccHio rscu-PA - npoTeHHa npeABapHTejibHofi ctbahh. flocjie coot-BeTCTByiomeži npoAOJiKHTejibHocTH φβρΜβΗΤθρΗΗ oTtfnpaioT ajiHKBOTHbie yaCTH, H OT^eHTpHφyΓHpOBaHHMe H3 ΗΗΧ ĶJieTKH paCTBOpHIOT 38ΤΘΜ JĪH30IIHM0M (1 ΜΓ JIH30IļHMa Ηβ 50 MJI MMOJI ^Φβρ8 TpHCrHflP0XJI0PHHa c pH 8,0, 50 mmoji 3THJieHflHaMHHT©TpayKcycH0fi khcjiotoK h 15¾ caxap03bl). TOMOreHaT JIH3HpOBaHHbIX KJI6T0K CTa0HJIH3HpyiOT B 4-5 moji pacTBopa ryaHHAHHrHAPoxjiopHAa h nocjie pastfaBJieHHH ao 1,2 MOJI ryaHHflMHrHflpOXJIOpH,Iia ripu AOtfaBJieHHfl BOCCTaHOBHTeJIbHOro cpe-IlCTBa (rjiyTaTnoHa, MepKanToaTaHOJia hjih rHCTeHHa) 3a 2-5 uacoB noflBepratoT pBaic^UHH 0(5paTH08 (cm. TaKKe , HanpHMep, BHHKJiep h Ap.). nojiyyeHHyio tskum 0(5pa30M OAHoqenHyio rscu-PA npn AodaBJie-hm njia3MHflH npeBpainaioT b AByxHHTeByio rscu-PA, βκτηβηοοτβ ko-τοροΑ onpeflejiHeTCH c πομοπιβϊο cy6cTpaTa s 2444 (nHporJno-PJiH-Apr-n-HMTpoaHHJiHA; Ka0H-AHarHOCTHKa,(IlBeiJ[Hfl), KOTopuft OTiuenjineT-Cfl TOJIbKO OT AByXHHTeBOfi ypOKHH83bI. 8TO aKTHBHpOBaHHe rscu-PA njia3MHH0M ocymecTBJiHiOT b 50 mmoji Tpnc-dy®epe, 12 mmoji xjiophcto-ro HaTpHH, 0,02¾ TBHHa-80 npn pH - 7,4 h 37°C. CooTHOineHHe rscu-PA K njia3MHHy AOJIKHO COCTaBAflTb npH6AH3KTeJIbHO 100-1500 : 1, cwraH Ha MOJinpHOCTb hjih, npH0JiH3HTejibH0, 8.000-36.000:1 c^HTan Ha 3H3HMHyio @AHHHuy. TecTOByio HHKy0aiļHK) προβοαητ b 500 mmoji Tpnc<Sy®epa, 38 mmoji xjiopHCToro η8τρηη πρκ pH 8,8 b npncyT-CTBHH 0,36 MKMMOJI anpOTHHHHa (AJIH ΤΟΡΜΟΚΘΗΗΗ nJia3MHHa) H 0,27 mmoji 5 2444 npn 37°C. B 33βηοημοοτη OT coAepmaHHH npodH rscu-PA peaKUHio nocjie 5-60 MHHyTHOfl HHKy6aiiHH ocTaHaBJiHBaioT nyTeM ao-6aBJieHHH 90%-ηο8 yKcycHofi khcjioth η η3μθρηιοτ oKcnpeccnro npn 405 hm. Π^ο yKa33HHK3 H3roTOBHTejiH cydcTpaTa s 2444, b cjiyuae 3τοίί npeABapHTejibHOfi CTaAHH Η3ΜβΗβΗΗβ 3KCTHHKUHH 03HayaeT 0,05 Ha MHHyTy πρκ 405 hm 8kthbhocth 25pu /mji τθοτοβογο pacTBopa.
KaK BHAHO H3 ΗΗΚβΠρΗΒβΑβΗΗΜΧ ΠΡΗΜβρΟΒ, B W8CTH0CTH Ta6-jiHiļbi 1 npMMepa 2, πλ83μηαμ, noJiyuaeMbie b σοοτΒβτοτΒΗΗ c H3o6pe-ΤβΗΜβΜ , OCymeCTBJIHK)T npeAnOMTHTeJIbHO B IDT8MM8X E.COli00pa30Ba-ΗΗΘ rscu-PA npoTenHa npeABapHTeJibHoii ctbahh co CTeneHbH) 3Kcnpec- LV 10120 9. cm ot 10 flo 25 Bec. προυβΗΤΟΒ, b uacTHOCTH, 14-20 Beo·.* odpa-30BaBuierocH odiiiero προτβΗΗβ. Οτθπθηβ 3Kcnpeccnn, cjienoBaTejibHo, npH ΠρΗΜβΗβΗΗΗ ΗΟΒΗΧ nJia3MHfl# ΜΟΚβΤ dblTb flOCTHrHyTa, ΠΟ ΚρβίΐΗθΚ Mepe, β 10-15 pa3 Bbinie, ueM b cjiytiae η3βθοτηηχ nJia3MHA, Kax Ha-ΠρΗΜβρ,ρϋΚ 54 trp 207-1.
PeaKUHOHHHe οηοτθμη, πρημθηηθμηθ b npnMepax Ha ocyuiecT-BJieHMe cnocoda, hbjihiotch ynoTpedHTeJiBHbiMH, ημθιοπιημηοη b npona-Μβ (CM.HanpHMep Nachr.Chem.Techn.Lab. 35, 939, 1987).
Hcnojib3yeMaH b npHMepax hjih cejieKijHH kjiohob KyjiBTypHafl cpe^a coflepKHT Ha jihtp: 7,8 γ nenTOHa H3 pudu, 7,8 r nenTOHa hs Ka3eHHa, 10 r λρομθβογο aKCTpaKTa, 5,6 r, xjiopncToro HaTpHH, 10 r γλκ)Κ03η. 9Ty cpei[y cMeuiHBaioT c 10 r arapa Ha jihtp b Tenjie H BHJIHBaiOT Ha nJiaCTHHH. ΠΡΚΜΕΡ 1. K0HCTpyHp0BaHH6 3KCnp8CCH0HH0ft nJia3MHflM pBF 160 flJIH npo-ΤβΗΗΒ npe^BapHTeJIbHOft CTaflHH rscu-PA C CHHT6THU6CKHM scu-PA-reHOM npw KOHTpojie trp-npoMOTopa. a) KoHCTpyHPQBaHH8 njia3MHūbi pbf 158. I) H3 ruia3MHflHpBR 322 (SapMauefl, iP 27-4902, 4363 dn) Bbipe3ai0T nic-bom -odJiaCTb, KOTOpaH ΗβΧΟβΗΤΟΗ M8Kfly OCHOBaHHHMH h2263, y,ijajiHF0T Kan cJiefiyeT HH*e: pbr 322 y οοηοββηηη 2298 c Hfle 1 h t3khm odpa30M JiHHeapH3HpyioT. BucTynaioiHHe kohuh HanoJiHH-k)t nyīeM peaKUHH ."3anojiHeHHfl", h tbkhm odpa30M nojiytiaf0T Tynwe KOHLļbi pBR 322. nocjie 3τογο y οοηοββηηη 2063 pa3pe3aioT c πομοπιβιο Pvu 11 h oda Tynbix KOHija ocTaBmeficfl nacTH pbr 322 c πομοπιβιο OdbHJHOft ΤβΧΗΗΚΗ CH0B3 JiHrHpyioT c ΠΟΜΟΠΙΒΙΟ T4-JiHra3bi. HenocpejiCT-ΒΘΗΗΟ nocjie 3Τ0Γ0 JIHraiļHOHHafl BCTBBKa B COOTBeTCTByiOIIIHX E.coli 12 y M 103 KJieTKax (ATCC 39403) τpaHcφopMHpyeτcH. TpaHC(£opMHpo-BaHHbie KJieTKH KyjibTHBHpyioT Ha cpene npH aodaBJieHHH 150 jfr aMnn-UHJiJiHHa/MJi. H3 nojiyweHHbix kjiohob BbīdHpaioT TaKHe, κοτορΗβ coflep-ataT njia3MH,ay pbf 157, KOTopan OTJiHMaeTCH ot ηοχο,ιιηο8 njia3MHflbi pBF 322 Ha 228 HyKJieOTHflOB a) Pst 1 X BspM Iļ,d)Pst lxBal 1-, B) Pst lx Ava Ι-φρΒΓΜβΗΤΗ. Oda β,ΙΙΗΗΟΤΒβΗΗΗΧ ΜβΟΤβ pa3pe3a Pvu II h Nde l pbr 322 dojiee He coflepiaTCH b njia3MH,qe PBR 157. II) H3 pBR 157 y,aajiHioT dojibmyio uacTB TeTpauHKJiHHOBofl no-cjieflOBHTejibHOCTH ny?eM pa3pe3a c πομοπιβιο eco rv χ Nru 1 h no-cJie<iiyK)uieii Jīnraunefi ποηβηβπιηχοη Tynux kohuob. nocjie τρΒκοφορΜβ- iIHH E.coli K 12 y M 103 h Kyjiβτηβηροββηηη Ha cpene co 150>ir aMnHUHJuiHHa /mji^ nojiywaioT kjiohh, H3 κοτορυχ BbīdnpaioT τβ, κοτορυβ 10'. coflepaaT ruia3MHAy pBF 158. OHa Ha 787 HyiuieoTHAOB Kopotie, ueM pBF 157 h oTJiHuaeTca, κροΜβ τογο, OTcyTCTBHeM MecTa pa3pesa Bam HI. ΤρβΗΟφορΜβΙίΗΑ ΙϋΤΒΜΜΟΒ daKTepnfl C ĪIOMODlblO PBE 158 ΗΘ npHflaeT hm ρθ3ηοτθητηοοτη k T6TpaiļMKJiHHy. Λ b) KoHCTpyHPQBaHH6 pBF 158-01. BB&ūeHKe CHHTeTHgecKoro MyjibTMKJioHtipyK)iiiero oaflTa; H3 pBF 158 nyTeM pa3pe3a c noMOUtbio eco ri x Hind iii yflajiHioT φρβΓΜβΗΤ·, ' βθλη*ιηηοΑ 31 HyKJieoTHA h b jiiok jiHmpyioT chh-TeTiraecKHfl caftT MyjibTHKJioHnpoBaHHH. nocjie TpaHC<£opMauMH Jinra-UHOHHOfi BCTRBKH B E.coli K 12 y M 103 W KyJIbTHBHpOBaHHfl Ha epe-Αβ co 150 ja? aMnuLinjuiHHa/MJi BudHpaioT τβ kjiohh, κοτορκβ coAep-aaT njia3MHAy pbf 158-01. OHa oTJimiaeTCH ot pbf 158 AonojiHMTejib-HblMH MeCTaMH pa3pe3a Xba l,Nde 1, Sal 1, Eag 1, Kpnl,Spl I, a TaK«e βτορημ μθοτομ pa3pe3a Pst i. C) KOHCTPyHPOBaHHe PBF 158-01 T. ΒΒΘ,ΠβΗΜβ TPaHCKPHnUHOH-Horo TepMHHaTopa. M3 pBF 158-01 yAajiflK)T φρβΓΜβΗΤ cia ι χ Hind nicafiTa Myjib-THKJIOHHpOBaHHfl H 33ΜΘΗΗΚ)Τ Cla I X Hind ΙΙΙφρβΓΜβΗΤΟΜ H3PRT 61 (5), Ha KOTOPOM HaXOAHTCfl tet A/orf L TepMHHaTOp M3Tn 10(6.) flOCJie ΤΡ3Η0φ0ρΜ811ΜΜ B ΙΙΙΤβΜΜΘ E. coli K 12 y 103 H KyJIbTH-BHpOBaHHH Ha CPβΑβ CO 150 ΜΓ aMriHIiHJIJIHHa/MJI BUdHpaJIH Te KJIOHH, κοτορΗθ coAepsajiH ruia3MHAy pbf 158-01 T. OHa OTJiimaeTCfl ot pbf 158-01 φρ3ΓΜβΗΤ0Μ* cia I X Hind III tfOJIbllIMM Ha 297 HyKJieOTMAOB. d ) Ββθαθηκθ οηητθτη^θοκογο čparMeHTa M4 - M8 jjih scu-pa-reHa. KOHCTpyKUHH nJia3MHfl pbf 158-02 T - pbf 158-06 T,
Bce CHHTeTHgecKHe Φρβγμθητη omicaHH. Ohh bboahtch b coot-B6TCTByiomHe βθκτορη b KauecTBe npndJiH3WTejibHo αβοΒηηχ mivthko- ΒΗΧ φρ3ΓΜΘΗΤ0Β AJIHHOH 200 HyKJieOTHAOB. 3Τ0Γ0 ΒβΚΤΟΡΗ pa3pe- 3aK)T OCTaTOgHbIMH 3Η3ΜΜ3ΜΗ, COOTBeTCTByramHMH Η33Β3ΗΗΗΜ MeCTBM pa3pe3a, h, c noMombio 3;ΐθκτροφορθ3β Ha arapnoM rejie, 3JieKTpo-3JitonpoBaHHH h o^ihctkh, OTAeJiflioT uepe3 AE 52 ot noAAeaamero yAajieHH!o φρ3ΓΜβΗΤ3. flocjie θτογο ocymecTBJiHK>T jmmpoBBaHHe otho-CHUļHMCfl K He'My ΟΗΗΤΘΤΙΙ^ίβΟΚΗΜ φρβΓΜβΗΤΟΜ ΑΒΟΚΗΟίί Lienu C nOMOIflbK) T4-JlHTa3U, ΤΡ3ΗθφθρΜ3ΑΜΜ B E.coli K 12 y M 103 H CeJieKUHH Ha cpeAe, coAepsameft aMnnqnjuinH. LV 10120 11. I) ·ΠμγΗΡ0Β8ΗΜΘ ΦΡβΓΜΘΗΤβ Μ 8 ΜΘΒϋν BflM Η1 Μ Cla 1 Β njia3MMny pBF 158-01 Τ. noJiyqaeTCH ruia3MHfla pbf 158-02 Τ.
OjinroHyKJieoTHfl 019 KOflnpyeT fljia C-TepMHHajībHofi nocjieito-BaTejibHOCTH scu-pa. 3a οτοπ-κοαοηομ TAA HaxoflHTCH mocto pa3pe-3a Nhe 1, pacnpocpaHHiomeecH οτ λοπολημτθλβηοΚ nocJienoBaTejibHO-CTH TpaHCKpHnUHOHHOft trpa- TepMHHaTOpa (8) flO Cla l. II) JInrHPOBaHMe āpaPMeHTa M 7 Meamy spi 1 h BaM H1 b njia3MMgy pbf 158-02 T. noJiyHaeTCH nJia3MHfla pbf 158-03 T. III) JlHrnpQBaHne čparMeHTa M 6 Me«jy κρη 1 vt spi I b njia3MHiiy pbf 158-04 T. noJiywaeTCH njia3MHfla pbf 158-04 T. IV) ,)ΐΜΓκροΒ3ΗΜβ ČparMeHTa M 5 Me«gy Eag ļ η κρη I b njia3MMjiy pbf 158-04 T. nojiyyaeTCH njia3MWfla pbf 158-05 T. V) JInrannH d)parMeHTa M4 Meamy sai I h Eag I b njia3MHay pbf 158-05 T. nojiywaeTCH njia3Mw,na pbf 158-06 T. H3 nOJiyiieHHHX KJIOHOB i-V BN(5npaK)T ΟΟΟΤΒΘΤΟΤΒΘΗΗΟ Te, KO-Topue OT^HuaioTCH οτ cooTBeTCTByiomero npefluiecTBeHHHKa HamraneM BBeneHHoro ηοβογο $parMeHTa b flonojiHHTejitHO npoBepeHHofi npa-BHJIbHOfi nOCJieflOBaTeJIbHOCTH. e) BBe,qeHne οηητθτιμθοκμχ Φρβγμθητοβ M2 η M3 jjjih scu-pa--reHa. KoHCTpynpoBaHne pbf 158-08 T.
TaK κβκ fljia. BBeaeHHH Φρθγμθητοβ M2 η M3 ΗβοδχοΛΗΜ ποτ-Ι pa3pe3 h Ha ncnojib3yeMoft ajih θτογο njia3MHfle pbf 158 HaxonHTCfl eme Pst-I MecTO pa3pe3a (β aMmmHJiJiHHOBOft nocJieflOBaTejibHocTH), κοτοροθ Meuiajio 6b\ ripu nocjieflyiomeM KJiOHHpOBaHHH, nocTynaraT cjie-li;yiomnM o(5pa30M: I) M3 MMeiometo b npoflaae (HanpuMep, "Pharmacia") njiasMH-flbi ynajiHF0T cafiT MyjibTHKJiOHnpoBaHHfl h nonojiHHTeJibHO 212 Hyiuieo-thaob ββθρχ οτ Nde I x Hind iii φρ3ΓΜθΗΤ3. B nojiyueHHOM nponyc-Ke 3'βΤβΜ JIHrnpyiOT ΟΗΗΤΘΤΗίΣΘΟΚΚΑ CaŪT MyJIbTHKJIOHMpOBaHHH H3 nJia3MHflb! pBF 158-04-3 T B BHfle Nde-I X Hind III -φρβΓΜβΗΤβ. 8īy njia3MHfly pBF 158-04-3 T nojiywaiOT H3 πλβ3μηλη pbf 158-02 T (cm. npHMep īdi ) nyTeM BBeneHHH oJinroHyKJieoTHflHoro φρβΓΜθΗΤΒ M6 h cooTBeTCTBeHHO njia3MM,iīy pBF 158-04 τ 6e3 φρβΓΜβΗτβ M7, noCJie ΤΡ3ΗθφθρΜ31(ΗΜ B E.coli K 12 y M 103 KJieTOK H KyjībTHBHpO-BaHHH Ha cpene co 150 jfr aMnnqHjuinH/MJi BuduparoT τβ κλοηη,κοτο-Pbie conep*aT njia3MHfly pBF 158-03 -3 . Ohk OTJiimaiOTca HajiimH- 12. eM φρβΓΜβΗτβ Nde I χ Hind iii ajihhoK 712 HjrKjieoTHAOB. II) H3 $parM6HTa Pst I χ sac I βηλθληιοτ JiHHeapH3HpoBaHHHfi Ββκτορ h JinrnpyiOT oji«roHyKJieoTHAHHii φρβΓΜβΗΤ M3. nocjie TpaHC-φΟρΜβυΜΗ B E.coli K 12 y M 103 W KyJIbTHBHpOBaHHfl wa cpefle co 150 jav aMnHLļHJUlHHa/MJi, BuCupaiOT τβ kjiohh, κοτορΗβ coaepaaT ruia3-MHfly. Ona oTJin^aeTCH Φρθγμθητομ Pstl χ Sac I ajihhoA 189 HyKJie-oTHflOB, KOTOpufi coflepiHHT φρβΓΜΘΗΤ M3 c npaBHJībHoK nocjieflOBaTejib- HOCTblO. III) H3 BUUieonHCaHHOft nJia3MHAM yflaJIHK)T φρβΓΜβΗΤ NdeI X Pst I Η B npOnyCK JIHrnpyK)T φρ3ΓΜβΗΤ M2. nocjie ΤΡ3ΗθφθρΜΒΙ|ΗΜ B E.coli K 12 y M 103 h KyjibTMBnpoBaHHfl Ha cpene c 150 jtr aMrin-iļHJUiHHa/MJi, Bbi6npaioT τβ kjiohh, κοτορΗβ coflep*aT ruia3MHfly. OHa OTJIHiiaeTCH HaJIHWHeM φρβΓΜβΗΤβ Nde I x Pst I flJIHHOfl 246 tfn(M2) c npaBHJIbHO flOnOJIHHTeJIbHO ΠρΟΒβρβΗΗΟϋ IlOCJieflOBaTeJIbHOCTbK). IV) M3 nJiaSMHAU ynajfIHK)T φρβΓΜβΗΤΗ M2 Η M3 B BHfle NdeI X sac I $parMeHTa h JinrnpyioT b nojiyneHHyio nocjie pa3pe30B c noMOigb»
Nde I x sac I doJibiuyio «acTb 158-06 T. nocjie TpaHC®opMauHH b E.coli K 12 y M 103 η KyjibTHBnpoBaHHH Ha cpefle co 150/mr aM-nHHHJiJiHHa/Mji BHdHpaiOT Te kjiohh, KOTopne eoaepaaT njia3MHAy Nde 158-08 T. OHa OTJiH^aeTCH οτ pbf 158-06 T HajiH^HeM φρβΓΜβΗτβ Nde I X Sac I flJIHHOft 435 HyKJieOTH,T[OB. I) KoHCTpynpoBaHne njia3MHHH pbf!60. ββθβθηκθ ομητθτη^θο-κογο tro-npoMOTOpa.
OrmcaHHaH nocjieflOBaTejibHOCTb trp-npoMOTopa pacnpocTpaHH-eTCH ao Hba I Mecia pa3pe3a h yAJWHfleTCH y δ-KOHija c noMombio nocJieAOBaTejibHOCTH 5ΆΑΤΤΙΙΤΓΑΑΑΤ-3'. B pe3yjibTaTe θτογο KOHCTpy-HPyioT eco ri MeCTO pa3pe3a. OTflejibHHe hhth 021 h 021A AeHaTy-pHpyioT η jiHrHpyioT b njia3MHfly pBF 158-08 T, pa3pe3aHHyio eco ri X Xba I. nocjie τρΒΗΟφορΜΒΗΗΗ Β E.coli K 12 y M 103 H KyJIbTH-BnpoBaHHH Ha cpeae· co 150 flr aMnuiimuinHa/MJi Bu6npaioT Te kjiohh κοτορΗβ co^epaaT pBF 160. njia3MKfla pbf 160 OTJiHMaeTCH οτ Bcex BHUieonHCaHHHX npeAIIieCTBeHHHKOB ΤΘΜ, WTO DITaMMH 6aKTepnK, TaKHM Odpa30M Τρ3Η0φ0ΡΜΙφ0Β3ΗΗΗΘ E.coli, Ha A00aBJieHHe HHflOJiaKpHJIO-BOfi KHCJIOTH 3KCnpHMHpyi0T ΓφΟΤβΗΗ npeABapHTeJIbHOfi CTaAHH rscu-PA. g) OKcnpeccHOHHbift TecT:
i) iepMeHTaiļHH
Pa3JIWiHbie UJTaMMbl E.coli, ΤρβΗΟφΟΡΜΗρΟΒβΗΗΗ C nOMOUIbiO LV 10120 13. nJiaSMHflH pBF 160, HanpHMep E.coli GTR-I, E. coli K 12 y M 103, a TaK*e e. co.uk 12 ATCC31446,npn cooTBeTCTByiomnx 0AHHaK0Bbix ycjioBMHx (JepMeHTHpyK)T b cpefle, cocTOHmefi H3 38 mmoji cyJIbφaτa aMMOHHH, 56 mmoji φοοφΒΤΗΟΓΟ ^epa pH 7,0, I mmoji cyjib®aTa Mar-ηηη,Ι% αρομθβογο BKCTpaKTa, 1% rjuoK03H, coAepKameH 150 ΜΓ aw-nniļHJiJiHHa Ha jihtp, h HHAyunpyioT c πομοικβιο 62 μγ HHAOJiaKpHJioBoii khcjiuth/ji 3KcnpeccnK) npoTemia npeABapHTejibHOft οτθαηη rscu-PA. flJIfl CpaBH8HHH BbIUieHa3BaHHUe IDTaMMbl TpaHCφopMHpyK)T C nOMODIblO BbiineonncaHHOM njia3MHflbi, KOTopan coflepiHT reH ^ejioBe^ecKoK npo-ypoKHHa3u H3 6aHKa kJIHK, nojiytieHHoro H3 MPHK KJieTOK ίβτροίίτ 562 h MMerouiHM o6o3HaueHMe ρυκ 54 trp 207-1, h 3aTeM, npw oah-HaKOBbIX yCJIOBMHX, φβρΜΘΗΤ^ΙΟΤ H nOflBepraiOT HHAyKIļHH.
FlepeA MHflyKUHeK h Ka»flbif5 ^ac nocjie HHAyKiļHH, Bcero 6 wa- COB, ΟΟΟΤΒβΤΟΤΒβΗΗΟ ^eHTpnφyΓHpyK)T 1 MJI KJieTOUHOH CyCneHHHH C OnTMtieCKOR nJIOTHOCTbK) (0Π) I ΓφΗ 578 HM, H HCnOJIb3y!OT AJIfl TecTupoBaHHfi CTeneHH 3KcnpeccHH. II) OdpaTHan peaKUHH npoTeHHa npeflBapuTejibHofi CTaAHH ao rscu-PA, ero pacmenJieHHe AO rtcu-PA H Η3ΜβρβΗΗΘ aKTHBHOCTH. OTAeJieHHbie ^eHτpHφyΓHpoBaHHeM KJieTKH, KaK onncaHo Bbiine, COeAHHfliOT C JIH30UHM0M Η 33ΤΘΜ HCriOJIb3yK)T ΓΟΜΟΓβΗβΤ JIH3HpOBaHHHX KJieTOK ajih onpeAejieHHH βκτηβηοοτη, KaK onHcaHO Bbiine. CTeneHH 3KcnpeccHH, onpeAeJieHHHe TaKHM otfpaeoM, nocjie H3MepeHHfl aKTHBHOCTH rtcu-PA, 06pa30BaHHOfi H3 rscu-PA, nojiyue-Hbi H3 npoTeHHa npeABapHTejibHoK οτβαηη. OTCIOAa BHAHO, VĪTO UITaMMU E.COli, KOTOpue dHJIH ΤρβΗΟφΟΡΜΗ-poBaHbi c noMOīĶbio njia3MHAbi pBF 160, AaroT 10-15-KpaTHbiH βηχοα 3KCnpeCCHH, WeM nJia3MHA0fi pUK 54 trp 207-1 (10), Η3Ββ0ΤΗ0Η H3 jiHTepaTypbi τpaHcφopMHpyIoιneft TaKHe «e uitamm E.coli. KpoMe τογο 0Ka3aJI0Cb, ^TO BbIXOAH 3KCnpeCCHH BO Bcex DITaMMaX, B 3aBHCHM0C-TH OT HCn0JIb3yeM0il AJIH Τρ3ΗθφθρΜ3Ι4ΗΗ nJia3MHAH, Jie«aT npH6JIH3H-TeJIbHO ΠΡΗ ΟΑΗΗβΚΟΒΗΧ 3Η3Μ3ΗΗΗΧ, Τ.β., *ΙΤΟ ΙΙΙΤβΜΜΗ (ΗΘ33ΒΗ0ΗΜ0 OT OTAeJIbHbIX E.coli IDTaMMOB), ΤΡ3ΗθφθρΜΗρθΒ3ΗΗΗβ C ΠΟΜΟΙΙΙΒΚ) nJia-3μηαη pBF 160, BcerAa AaiOT bo μηογο pa3 6ojibuiyio CTeneHb 3Kcnpec CHH, yeM ΗΑΘΗΤΗηΗΜβ UITaMMbī, ΤΡ8ΗθφθρΜΗρθΒ3ΗΗΗβ H3BeCTH0ČĪ nJia3MH floft pUK 54 trp 207-1. • F1PHMEP 2.
a) KoHCTpynpoBaHHe 3KcnpeccH0HH0fi njia3MHAH pbf-161 αλη npoTeHHa npeABapMTeJIbHO^ CTaAHH rscu-PA c ΟΗΗΤβΤΗΜβΟΚΗΜ rscu-PA 14. ,-γθηομ ripu KOHTpoJie trp npoMOTopa. njia3MHfly p3R 322 pa3pe3a^H C nOMOmbIOEoo RI H Hind III f!ojiyyeHHHii φρβΓΜθΗΤ fljiKHoft 31 HyKJieoTHfl OTfleJiHJin c πομοπιμ) nofl-roTOBHTejibHoro 3Ji6KTpo$ope3a Ha arapo3HOM rene. OcTaBmyK)CH wacTb pBF 322 aJUOMpOBaJIH C ΠΟΜΟΙξΕΙΟ 3JieKTp03JIK)Hp0BaHHH H3 ΓβΛΗ H ΟΗΗ-majin c noMombK) HE 52. njia3MHfly pbf 160 (npHMep II) TaK«e pa3pe3aroT c noMombio ECO RI H Hind III Η φρβΓΜΘΗΤ Eco RI X Hind III flJIHHOfi 1684 HyK-JieOTHfla, ΚΟΤΟρυΚ COflepKHT ΟΗΗΤΒΤΗΗβΟΚΗίί scu-PA -ΓΘΗ CO BC8MM eflHHHpaMH peryjmpoBaHHfl (trp-προΜοτορ), οτ,ΐίβΛΗιοτ c noMombfo 3JieK-τροφορβ3β Ha arapo3HOM rene, sjimpym KaK onncaHo sunie h ohh-maioT. noJiy^eHHbie TaKHM o0pa30M φρβΓΜθΗΤΗ JinrapyK)T oChuhhm cno-COČOM C nOMOlUbJO T4-JIHra3U H 38ΤΘΜ TpaHC®0pMHpyiOT B E.coli K 12 M 103. flocne KyjibTHBnpoBaHHfl Ha cpene co 150 jtr aMnwinnnHHa/Mn BbīdnpaiOT Te κλοηη, κοτορηθ co^epstaT φρβΓΜβΗτ eco ri x gīnd m nnHHoft 1684 HyKJieoTH,qa h npoflypnpyioT προτβΗΗ rscu-PA npeflBapn-TejIbHOH CTaflHH nOCJie flOČaBneHHfl HHflOnaKpHnOBOfl KHCJIOTU. 8th kjiohu conepaiaT nna3MH,ņy pBF 161. b) 3KCnpeCCH0HHblft ΜΘΤΟΛ-ΤΘΟΤ. E.coli GRT-I, E.coliK 12 y M 103 H E.coli K 12 ATCC 31 446 ΤΡ8ΗθφθρΜ^Ε0Τ G ΠΟΜΟΙ«ΒϊΟ pBF 161 Η 38ΤβΜ φβρΜβΗΤ^ΙΟΤ ΚβΚ onncaHo b npHMepe Ig h HcnbīTUBaioT pe3yjibTaT BKcnpeccnH. Βηχο£η rscu-PA προτθΗΗβ npeflBapHTenbHofi CTaflHH onpenejiHioT Kan rscu-PA aKTHBHOCTb ^epe3 1-6 HaCOB nOCJie HHHyKlJHH H OHH CpaBHHMH co 3HayeHHHMH ΒΜΧΟΛΟΒ njia3MHflbi pBF 160 nojiytieHHMX πρΗΜθΗθΗΗβΜ nna-3MHflbI pBF 160 flJIH ΤΡ3ΗθφθΡΜ814ΗΗ. nojiyyeHHbiH no aHanomu c npuMepoM īgi qeHTpn®yrnpoBaHneM KJieTO^HbiH ocauoK μοκθτ 6biTb TaKffie nonyweH nyīeM κηπηηθημη b 0,25 moji tphc HCI-(5y<ī>8pe, pH 8,0 c 4% HaTpnMfloneqnncynb®aTa, 1% MepKanT03TaH0Jia m 20¾ rnmļepMHa. PacTBopeHHbie τθκημ o6pa-30M ΠΡΟΤΘΗΗΗ OTUeJIfllOT C nOMOUļblO SDS-PAGE H fleJiaiOT BHflHMblMH C noMombK) OKpamuBaHHfl KyMaccdnK> (12). OKpaineHHHe reņu oneHHBaioT HeHCHMeTptmecKM κ HHTerpnpyioT nHTHa cpenH īihkob, KonimecTBo 0dpa30BaBll!er0CH nOCJie HHHyKUHH HHflOnaKpunOBOft KHCnOTOfi rscu-PA npoTeHHa npeflBapuTenbHoft CTaflHH, onpenennioT c noMonibio cydcTpaK-IļHH ΠΗΤβΗ, ΗΗΛΘΗΤΜφΗΙΙΙφΟΒβΗΗΗΧ B KaHeCTBe ΠρΟΤΘΗΗβ npeflBapHTeJIb-hoh CTaflHH nojioc nepefl HH,ņyKiJ(neii, ot nojioc, nojiyyeHHbix nocne MHflyKLļHH. LV 10120 15. B BHnieoriHcaHHOM npHMepe KOJimiecTBO rscu-PA npoTeHHa npe^Bapn-TejibHOH cTaflHH nocJie hh^klīhm cocTaBJineT 17,9 Bec.% ČaKTepn-ajībHoro očmero npoTeHHa. Hpyrne npHMepu yKa3aHbi b Tataije I.
TABJIMJA I
KojiH^ecTBO rscu-PA npoTeHHa npeflBapHTejibHoM CTaflMM ot o6mero KOJitmecTBa npoTeHHa ( b npoqeHTax).
TpaHC$opMHpoBaHHbiii [IlTaMM E. COli pBF 161 pUK 54 trp 207 K 12 ATCC 31446 14 1,5 K12 y M 103 17,9 1,9 GRT-1 17,8 1,7 ΠΡΜΜΕΡ 3.
KoHCTpyHDQBaHMe βκοπρθοομοηηοη njia3MMjibi jjih npoTerma npeflBaPHTeJIbHOH CTaflHM rscu-PA C ΟΜΗΤΘΤΜ^ΘΟΚΗΜ scu-ρα-γ.θηομ ripu KOHTppjie trp-npoMOTOpa b pbr 322 c flejiemieft β γθηρθ3ηο ΤΘΗΤΗΟΟΤΗ K TeTPaUHKJIMHy. a) KoHCTpynpoBaHne njia3MHAbi pbr 322 dei.
njia3MHfly pBR 322 pa3pe3aioT c πομοιιιβιο arapo3ereJib8JieKTpo-φθρΘ33, OCTaTOUHOe KOJIHUeCTBO pBR 322 H3 reJIH 3JIK)HpyK)T C nOMODlblO 8JieKTpo3JiK)HpoBaHHH, o^HiiļaKT wepe3 HE 52 Tynue KOHUbi pBR 322 Eco-RV X Nru I-OCTaTOWHOK qaCTH, H Ο&ΜΗΗΜ 0dpa30M JIHrHpyiOT c nOMOlIļbK) T4-JIHra3bI. nocJie ΤΡ3ΗθφθρΜ3Ι1ΜΗ B E.coli κ 12 y M 103 h KyjibTMBHpoBaHHH Ha cpefle co 150 μγ aMnnmuuiHHa/MJi BuOHpaiOT
Te KJIOHH, H3 ΚΟΤΟΡΗΧ 3ΛΗΚΒ0ΤΗ3Η UaCTb ΠΟΟΛβ ΠβρβΗβΟΘΗΗΗ Ha cpeny c 25 μγ TeTpauHKJiHHa /mji Ha Hei* ηθ BbipacTaeT, T.e., He oOjiaflaeT TeTpauHKJiMHOBoft pe3ncTeHTHOCTbio. Kjiohh conepsaT njias-MHfly pbr 322 del, KOTopaa OTJiHuaeTCH ot pbr 322 τβΜ, uto OHa Ha 787 HyKJieoTHflOB MeHbiiie, qeM h ηθ MoseT 6biTb pa3pe3aHa 6ojiee C nOMOUļbfO ECO RV H Nru I. b) KoHCTpynpoBaHne njia3MHflw pbf 162. pBF 322 del pa3pe3atoT c noMombio eco rv h Hind III. nojiy-tieHHuii trkmm odpasoM φρ3ΓΜβΗΤ λληηοΚ 31 HyKJieoTHH, οτλθληιοτ c nOMOUlbfO npenapaTHBHOf! 3Γ8ρθ3βΓΘΛΒ8.ΠΘΚΤρθφθρθ33, OCTaTOWHyiO WaCTb pBR 322 del 8JH0HpyK)T C nOMOmblO 8JieKTp08JIK)Hp0BaHHfl H3 ΓΘΛΗ H o^HmaiOT HE 52. 16. , l
TaKHM'otfpasoM Η3 pbf 160 nojiytiaioT Φρθγμθητ eco ri x Hind III flJIHHOČi 1684 HyKJieOTHflOB, KOTOpblft COflepMT CHHT8THlieC-KHH scu-PA -ΓΘΗ CO ΒΟβΜΗ eflHHHliaMH peryJIHpOBaHHH (trp -npOMOTOp)
Oda $parMeHTa odbmHbiM odpa30M jinrnpyjoT T4-JiHra3oft h 3aTeM TpaHc$opMHpyiOT b E.coli K 12 y M 103 KJieTKH. HocJie KyjiBTHBH-poBaHHH Ha cpefle co 150 μγ aMnnitiuiJiHHa/ΜΛ BbīdHpaioT kjiohh, ko-Topbie coflepffiaT OparMeHT eco ri x Hind III αληηοη 1684 dn h npoflyunpyioT nocJie nodaBJieHHH 62 μγ HHflOJiaKpmioBOfi khcjioth/mji rscu-ΡΑ-προτθΗΗ npoMe»yTotļHoft CTaflHH. kjiohu conepsaT iuia3-MHfly pBF .162. c) 3KcnpeccH0HHbiii τβοτ. Ē. coli GRT-I TpaHC(£opMHpyiOT C nOMOIHBK) pBF 162, H 3aTeM $epMeHTHpyK)T, κβκ orracaHo b npHMepe I η μοπητηββιοτ pe3yjibTaT 3K-cnpeccHH. Βηχοαη rscu-PA -npoTeHHa npeflBapHTeJibHOfi CTaflHH on-peneJIfliOT KaK rscu-PA-aKTHBHOCTb Μβρβ3 6 tiaCOB nocJie HHflyKUMH, KOTopan cocTaBJiHeT 1300 pu /mji ΚΛβτο^ΐΗοίί cycneHEļnn c onTHtiecKoft nJIOTHOCTbEO I, H CpaBHHM3 CO 3HaWeHHHMH BbIXOflOB nocJie τρβΗΟφΟΡ-MaUHH E. coli GRT -I C nOMOHlbK) nJia3MHflb! pBF 160, ΠΡΜΜΕΡ 4.
KoHCTpywpoBaHne SKcnpeccHOHHofi njia3MHflH pbf 171 ajih npoTeHHa npeflBapHTejibHofi CTaflHH rscu-PA c CHHTeTmiecKKM scu-pa--ΓΘΗΟΜ npH KOHTpOJie tac -npOMOTOpa. n^a3MHfly pBF 161 pa3pe3ajoT c noMombro Eco ri x Xba i. no^y^eHHbiii φρ3ΓΜ8Ητ αληηοΚ 74 HyKJieoTHfla οταθαηϊοτ c noMOiiļbio npenapaTHBHOrO 3ΓΒρθ36ΓΘ.ΠΒ3.Π8ΚΤρθφθρβ33, OCTaTOWHyiO tļaCTb ΠΛβ3-MHflbl 3JIK)HpyK)T C nOMOIIļbK) 3JI(OHpOBaHHfl Η 3βΤΘΜ O^HUiaiOT C ΠΟΜΟΙΰΒΚ) ΛΕ 52. H3 nJia3MHflbI ptac SDT ( DSM 5018) BblfleJIHlOT φρ3ΓΜΘΗΤ Eco RI ,x Xba I , KOTOpblft COflepffiHT tac-πρΟΜΟΤΟρ. HjIH 3Τ0Γ0 pa3pe3aioT ptac sdt c πομοιιιβιο eco ri x xba I h οταθληιοτ $par-MeHT c noMombio npenapaTHBHoro nojiHaKpHJiaMMflrejieBoro θΛβκτροφο-pe3a. ΦρβΓΜβΗτ 3JuonpyioT c πομοικμ) HarpeBa flo 65°C b aMMOHHfiaije-TaT/SDS -^φβρβ, pH 8,0 h MexaHHtiecKH H3Mejib*ieHHoro nojinaKpmi-aMHfla, H OUHUtaiOT ΜΗΟΓΟΚρβΤΗΟΗ 3KCTpaKIJHeft φβΗΟΛΟΜ, KOTOpblft Ha-cameH I moji τρκο^φβροΜ, pH 8,0.
Oda noJiyyeHHbix TaKHM odpaeoM φρβΓΜβΗτβ odu^HUM odpa30M jinrnpyiOT T4-jinra3oii h 3aīeM TpaHC$opMHpyioT β E. coli K 12 y M 103. flocjie KyjibTHBnpoBaHHfl Ha cpefle co 150 βΜπιιΐ1ΜΛΛΗΗβ/ΜΛ 17. LV 10120
BblČHpaiOT KJIOHbl, ΚΟΤΟΡΗΘ nOCJie ΛΟββΒΛΘΗΗΗ ΜΠΤΓ npOflyqHpyK)T rscu-PA προτθΜΗ npeflBapHTejiBHOii CTaflHM. 0TM kjiohh coflepaaT ruia3MHfly pBF 171. 6) 0KcnpeccnoHHbifi TecT. Ē. coli K 12 y M 103 TpaHC®opMHpyiūT c noMombio pbf 171, h 38τβΜ φβρΜβΗΤΜργιοτ, κθκ onncaHO b npMMepe Ig ) h HCCJie,qyīOT pe3yjibTaT 3Kcnpeccnn. Mhotuhio ocyinecTBJiHJOT c noMouļbio ποΛκβκριω-aMUJirejieeBoro 3Λβκτροφορθ3Β ( KOHe^Han KOHiieHTpauHH 0,5 mmoji). Βηχοαμ rscu-PA -npoTemia npeflBapHTejibHoii ctsahh, onpe^e-JIHKJT B BMJļe rtcu-PA aKTHBHOCTH Ηθρθ3 1-6 «acOB nOCJie HHUyKIJHH. ΟΠΗ CpaBHHMbl CO 3HaUeHMHMH BbIXOflOB, nOJiygeHHbIMH ΓφΗΜβΗβΗΗβΜ nJia3MHflbI pBF 160 B OflHHaKOBbIX ΜΤ3ΜΜ3Χ E. COli. ΠΡΗΜΕΡ 5. а) KoHCTpynpoBaHHe 3KcnpeccH0HH0ft njia3MMflu pbf 172 kjih rscu-PA -npOTenHa npeflBapHTejibHof! CTaflHH c CHHTeTimecKHM scu-γθηομ npH KOHTpojie tac-npoMOTopa b pBR 322 c fl.ejieijMeft β γθηθ pe3cnTeHTH0CTH κ TeTpaunKJiHHy.
IlJia3MHfly pBR 322 del (cm. npHMep 3a) pa3pe3aioT c πομο-UļblO ECO Rī x Hind III . riOJiyHeHHblfi φρβΓΜΘΗΤ flJIHHOft 31 HyKJieOTHfl OTflejiHiOT c noMombK) npenapaTMBHoro arapo3orejieBoro θΛβκτροφορβ-3a, ocTaToyHyro gacTb pBR 322 del 9jnoHpyioT c noMouibio 9JieKTpo-3JIK)MpOBaHHH H3 ΓΘΛΗ H 3aTeM OHHUiaiOT flE 52.
TaKHM »e o(5pa30M nojiygaioT H3 pbf 171 φpaΓMβHT Eco rix Hind III, KOTOpblfi COflepMHT CHHTCTHUeCKHK scu-PA -ΓβΗ C.O BC6MH eflHHHIiaMM peryJIHpHOCTH ( tac-npOMOTOp). 06a nojiyueHHbix TaKHM o6pa30M φρ3ΓΜθΗΤ3 JiHrHpyiOT ο6ηηηημ o6pa30M T4 Jinra3oft h saTeM τpaHCφppMHpyκ)τ b E.coli K 12 y M 103 kji©tkh . Oocjie KyjibTHBHpoBaHHH Ha cpejie co 150 μγ aMnHUHJi-jiMHa/MJi BudHpaioT KJioHbi, ko. opbie npoflyqHpyFOT b npHcyTCTBHH 0,5 mmoji ΜΠΤΓ rscu-PA -προτβΗΗ npeflBapHTeJibHofi CTaflHH. Θτη kjiohh coflepmaT njia3MHfly pbf 172. б) OKcnpeccHOHHHfi τβοτ. E. coli K 12 y M 103 τpaHcφopMHpyίoτ c noMombio pbf 172 h 33τβΜ φepMeHTMpyκ)τ KaK onHcaHo b npMMepe 16 ) h HCCJieflyioT pe-3yjIbTaT 3KCnpeCCHH. HhJ^KIIHK) OCymeCTBJIHK)T C nOMOUIbK) Ι4ΠΤΓ (ko-HetļHan KOHLieHTpaijHfl 0.5 mmoji). Βηχοαη rscu -npoTeHHa npenBapn-TeJIbHOfl CTaflHH OnpefleJIHlOT KaK rtcu-PA -aKTHBHOCTb uepe3 1-4 18. tiacoB nocjie MHflyKLiHH h OHa cocTaBJineT πρκ<5λη3ητθλβηο 1100 /mji KJieTCmHOfl CycneH3HH ΟΠΤΗ«ΘΟΚθβ IUIOTHOCTH I H CpaBHHMH CO 3Ha-MeHHBMH ΒΗΧΟΛΟΒ, ΠΟΛ^βΗΗΗΧ ΠΡΗΜΘΗΘΗΗΘΜ nJia3MHflbl pBF 160 C ΤβΜ Κθ ΙΙΙΤβΜΜΟΜ E.coli. ΠΡΜΜΕΡ 6 Μ3μθηθηηθ paccTOHHHH MejRAy Hl.fl.-nocjieflOBaTejibHOCTbio h CTapTOBHM KOflOHOM B 3KCnpeCCH0HH0fi nJia3MHfle flJIfl rscu-PA -ΠΡΟ-TeHHa npeflBapHTejibHO^ CTannn. CTapTOBfeIH KOflOH, ATG , OriHCaHHblfi BO Bcex KOHCTpyKUHHX npuMepoB 1-5, ABJineTCH cocTaBHofi wacTtio Nde-I-MecTa pa3pe3a -CATAT - Nde -I, pa3pe3aeT 3a πθρβημ A reKcaHyKJieoTMHHyK> nocjie-AOBaTejibHOCTb h jņaeT 5-kohph, κοτορκβ nepeKpHBaioT flBa οοηοββηηη. SanoJiHHioT 3-KOHiļbi, T.e., KOHCTpynpyiOT Tynbie kohiih, h nocjie 3to-ro cHOBa jinrnpyioT τβκ, φγο cooTBeTCTByioinaH nocjieflOBaTejibHOCTb yfljiHHHeTCH Ha οοηοβημθ napu. OflHOBpeMeHHO yflajiHioT Nde Ι-μθοτο, PaccTOHHne ot M.H.-nocJieflOBaTejibHocTH flo CTapTOBoro KOflOHa yn-jmHHeTCH ot 8 no 10 οοηοβηηχ nap. B najībHefiiDeM, 3KcnepnMeHTajibHoe npoBefleHne ποηοηηθτοη b npHMepe: а) KoHCTpyKLjHfl nJia3MHflbi pbf 163. flJia3MKfly pbf 161 pa3pe3aioT c πομοιιιβκ) Nde i, 3aTeM bh-CTynaiomHe kohhn 3anojiHHfOT Φρθγμθητομ KjieHOBa nojiHMepa3bi I IIHK (2). nocjie 3τογο JinrnpyiOT ο6η«ηημ o6pa30M T4-jinra3ofi h 33ΤΘΜ τρβΗΟφορΜΗριηοτ b.E. coli K 12 y M 103. nocjie KyjibTHBHpoBaHHH Ha aMnuuHJiJiHHOBOH cpene BbidMpaioT kjiohh, KOTopbie coflepffiaT ruia3-MHfly pBF 163.OHa OTJītmaeTCH ot pbf 161 0TcyTCTBH6M MecTa Nde H flOnOJIHHTeJIbHUM 00Η0Β3ΗΜβΜ -ΤΑ- B OČJiaCTH ΠΡΘΚΗβΓΟ Nde-Ī -I-MecTa. б) BKCnpeCCMTHHblR τβοτ. E. coli GRT-I TpaHC$OPMHpyiOT C nOMOlUbK) nJia3MHflH pBF 163, μ 33τθμ φθρΜβΗτκρνΒτ, Kax onncaHo b ripuMepe I g) η κοπϋΤΗΒβιοτ pe3yjibTaT 3KcnpeccnH. Βμχολη rscu-PA -npoTeHHa npenBapuTejibHofi CTaflHH, onpefleJiflioT nocjie očpaTHofi peaKunii h 3κτηβηροβ3ηηη b BHfte rtcu-PA -aKTHBHOCTH Ha KJieTOUHOii cycneHLiHH c onTtmecKoii πλοτηοοτβκ) I, yepe3 1-7 qacoB nocjie hh^kphh 62 mji HHfl0JiaKpn-jioboh khcjiotom/ji. Ohh cpaBHHMH co 3HaneHHHMH BbixoflOB, nojiyueH-HblMH ΠΡΜΜΘΗΘΗΗΘΜ rUia3MHflbI pBF 160 B TOM »Θ ΙΙΙΤΒΜΜΘ E.COli. , 19. LV 10120
ΦΟΡΜΥΛΑ H30BPETEHHA 1) Cnocod nojiyweHHH ηοβηχ iuiasMHfl λλη πρΗΜθΗβΗΗϋ npH no-JiyweHHH njia3MHH0reHH0r0 aKTHBaTOpa, OTJIHiiafOHHftCH τθμ, uto njia3MMiiy pBR 322, H3 κοτοροΑ yflajieHa nic/bom o&iacTb M/MJIM ΠΟ MeHbDiefi Μβρβ MaCTb ΓΘΗΘ ΡΘ3Η0ΤΘΗΤΗ00ΤΗ K TeTpaiJHKJIMHy, Me»fly MecTaMH paspeea eco ri h Hind m "MyjibTHKJiOHHpyioinero" caitoa BBOflflT c HyKJieoTHflHoii nocjiefloBaTejibHocTbio, KOTopan Mew MecTaMH pa3pe3a xba i h Nde i coflepiHT nocjieflOBaTe^bHocTb tae-lļajībrapHo h Ha paccTOHHHM 6-12, npe,ņno™TejibHO 8-10 HyK-JieOTHflOB, - CTapTOBH^ KOflOH ATG, H c ero nOMOIUblO BCTpaHBaiOT H3-BeCTHblM 06pa30M B OflHH Μ1Λ flBB TpaHCKpnniļHOHHbIX TepMHHaTOpa, CMHT6TnuecKyK) tiacTHWK) ποολθ,ι;οβ8τθ.πβηοοτβ scu-pa -cīpyKTypHO-ro reHa, npe^notiTHTejibHo Ha^HHafomjnocH ot 3'-K0Hua CTpyKTypHoro reHa, a τθκ«θ peryjinpyeMbift προΜοτορ, h TaKHM 00pa30M nojiytiatoT nJia3MHflu, KOTopbie ofijiajiaioT cjie,qyK)innMn OTJiHtiHTejibHbiMH πρμ3Η3Κ3μη: а) OnepoH πμθμηαν coflep*HT peryjmpyeMbifi προΜΟτορ, b Ka-uecTBe Μβοτβ οοθαμηθημη ph(5ocom - nocjieflOBaTejibHocTb UlHHe-flajib-rapHo, cīapTOBbin koaoh, cnHTeTiwecnft CTpyKTypHHH reH jijih ozi-ηοημτθβογο aKTHBaTOpa yponJia3MHHoreHa M scu-pa ", coflepaamero 411 aMHHOKHCJIOTHUX OCT3TKOB ,H, BHH3 OT CTpyKTypHOrO reHa, OflHH -flBa TepMHHaTOpa; б) flJia3MHflbl npHrOflHbl flJlfl 3KCnpeCCHH rscu-PA -ΠρΟΤΘΗΗΒ npeflBapHTejibHofi CTa,qnn co οτθπθηβιο 3KcnpeccMn no μθηβπιθΑ Mepe 10 Bec. % o(5pa30BaBDierocfl odinero npoTenHa b 3HTepo6aKTepwnx, npe,ņnOUTHTeJIbHO B ΙΙΙΤΒΜΜΒΧ Escherichia coli. 2) Cnocod no n.I, OTJiimaiomHfiCH τβΜ, ^το b KauecTBe pery-jinpyeMoro npoMOTopa BCTpanBaioT trp hjih tac -προΜοτορ c noMoinbio MyjibTHKJioHHpyiomero caiiTa. 3) . Cnocod no π.π. I n 2, oTJiimaiotnHftCH τθμ, uto b KatiecTBe pery;inpyeMoro npoMOTopa BCTpanBatoT cHHTeTnuecKHft trp -προΜοτορ b HyKJieoTHiiHoK nocjieflOBaTeJibHocTH. 4) . Cnocotf no π.π. 1 m 2, oTJiimaioinHftcH τθμ, μτο b KawecT-Be peryjinpyeMoro npoMOTopa BCTpanBaioT tac -προΜοτορ H3 πμθμμ-ΛΜ ptac SDT ( DSM 5018).
5 ). Cnoco6 no π. 1, oTjmaioinHftcfl τθμ, χιτο b κθχιθοτβθ CJieflyioinnx jņpyr 3a flpyroM TpaHCKpnniļH0HHbix τθρμηηθτοροβ βοτρθη-ΒβίΟΤ trp A-TepMHHaTOP H/HJIH tet A/orf L -ΤβρΜΗΗβΤΟρ H3 T n 20. LV 10120 10 B OnepOH ΗΟΒΗΧ nJia3MH,E(. 6). CnocoG no n.I, OTJinuaiomniicfl τθμ, χιτο c noMOiubto mjtjib-THKJioHnpyiomero cafiTa b uacTHtiHofi CTaflHH BCTpanBaeTCH οτργκτγρ-ηηη γθη c HyKJieoTHflHoii nocjieflOBaTejibHocTbH). ?). Cnoco6 ΠΟΛΥΣΗΜΗ ηοβηχ njia3MHfl no π. I, oTJimiaiomHi?- CH ΤΘΜ, tlTO Eco RI x Hind III $Ρ8ΓΜΘΗΤ H3 n0JiyiieHH0ft B C00T- βθτοτβηη c πγΗκτβΜΗ 1-6 njia3MHnbi, Hcnojib3yioT b Ka^ecTBe 3kc-npeccnoHHofi KacceTbi b npyrofi, b flpyrow, 3HTepo6aKTepHajībHHfi, npenno^THTeJibHO, b cnoco6Hyio pa3MH0*aTbCH θβτοηομηο n;ia3MHfly E. coli. 8) Cnocod nojiyweHHH ηοβηχ njia3Mn.ii no n.I, OTJiHuaiomHficfl ΤΘΜ, 4T0 B nOJiyweHHOft B COOTBeTCTBHH C ΠΥΗΚΤΒΜΜ 1-7 B nJia3MHfle yHJiHHHioT paccTOHHHe Me»fly nocJienoBaTejibHocTbio lŪHHe-HaJībrapHo H CTapTOBHM KOflOHOM ΠγΤΘΜ pa3pe3a C nOMOUIbK) Nde I, 3anOJIHeHHH no;iyqeHHbix μθοτ pa3pe3a h nocjieflyiomero jiHrHpoBaHHH noJiyweHHbix Tynux KOHUtOB. 9) ΠρπΜθΗβΗΗβ nojiyweHHofi no πυηκτβμ 1-8 njia3MHflbi npH no-jiyyeHHH njia3MHHoreHHoro aKTMBaTOpa, OTJiHwaiomHiicH τθμ, wto θτοΑ nJia3MHfloft TpaHC$opMHpyioT BHTepofiaKTepnaJībHHii ηιτβΜΜ, npennouTM-TeJIbHO UJT3MM E. coli Κ3ΒΘ0ΤΗΗΜ θ6ρβ30Μ, HHflyiiHpyiOT ΘΚΟΠρβΟΟΗΚ) scu-PA -οτργκτγρΗΟΓΟ reHa, o6pa30BaBUiHficH προτΘΗΗ-rscu-PA, npoMe?«yTowHoK cTaflHH, oTflejinioT ot cpeflu h JiH3HpyioT ČaKTepHajībHue KJieTKH, προτβΗΗ npeflBapHTejibHofi οτβληη CTaČHJiH3HpyiOT h 3aTeM nonBepraioT odpaTHOfi peaKUHH no rscu-PA b pe3yjibTaTe B03nefiCTBHH penoKc-CHCTeMbi. 10) ΠρΗΜΘΗΘΗΗΗ nOJiyweHHOft no ΠΥΗΚΤΘΜ 1-8 nJia3MHflbI npn nojiyqeHHH njia3MHHoreHHoro aKTHBaTopa b οοοτβθτοτβηη c πυηκτομ 9, OTJiHtiaroineecfl τθμ, wto θτο8 njia3MHnoft τρβηοΦορμηρυιοτ uiTaMM BHna E.coii , npenno^THTejibHo, uiTaMM K12 E.coli , h 3aTeM ocymecTBJiHK)T cnoco6 no n. 9. LV 10120 ΡΕΦΕΡΑΤ ΜθοΰρβτβΗΗβ KacaeTca πολώθηκα ηοβηχ πλθθμηα c οιίητθτμ-tjecKofi nocaeflOBaTeabHocTbio ,ΠΗΚ, κοτορΗθ πρμγοληη 9iccnpnMnpoBaTb προτβΗΗ npeflBapMTejibHofi CTafinu rscu-PA (τ.β. HerjiHKoaminpoBaH-Hyro tiacTb προτβπΗθ oflHoueriHofi npoypoKHHa3bi) b βητθρο63κτθρμηχ , B «aCTHOCTH E.coli B ΚΟΛΗΜβΟΤΒβ, KOTOpbie flaiOT BbIXOfl, 3HaWHTeJlb- ho npeBbimaromutf βηχολη, flocTuraeMbie npn H3BecTH0M πρΜΜβΗβΗΜΊ naa3MMfl. noaytiaromnecH b οοοτβθτοτβμμ c Μ3θ6ρβτβΗΗβΜ njia3MHflbi,xapax-Tepn3yioTCH τβΜ, uto hx onepoH odJiaflaeT peryjinpyeMbiM, b οοοτβθτ-CTBywm6M cjiyqae θΜΗΤβτκ«βοκΗΜ προΜοτοροΜ, UlMHe-flaabrapHo-nocjie-AOBaTeJIbHOCTblO, CTapTOBbIM KOflOHOM, ΟΜΗΤΘΤΗΙίΘΟΚΗΜ CTpyKT^pHblM γθηομ h, pacnojioffieHHbiM BHH3 ot CTpyKTypHoro reHa, ΠΟ MeHblIiefi Mepe, oflHHM TepMHHaTopoM. nojiyueHHe ηοβμχ njia3MMfl, a TaK»e ΟΜΗΤβΤΗΜβΟΚΜΧ Π00·Πββ0Β3ΤΘ.ΠΒΗ00ΤβΚ βΗΚ, OCymeCTBJIAK)T UCXOflfI H3 ΜΜβϊΟΐρΧΟΗ B npOflaffie nJia3MHfl. ΘΚΟΠρΗΜΙφΟΒβΗΗΗΛ ΠΡΗ ΗΧ ΠΡΜΜΘΗΘΗΜΗ b BucoKOfi οτβπβΗΜ προτβΗΗ npeflBapHTejibHoft CTannn κββθοτηημ 06-pasoM noflBepraraT odpaTHoii peaKUHH ao TepaneBTH^ecKH ncnojib3ye-Moro b KaqecTBe njia3MoreHHoro aKTHBaTopa rscu-PA , κοτορηίί 3a-ΤΘΜ, npM JKSJiaHHM, Τ3Κ!Κβ ΠρΗ OCymeCTBJIHeMOM Η3ΒβΟΤΗΗΜ odpa30M pacmenJieHMH, ΜΟΜβΤ dbITb HCn0JIb30BaH flJIH nOJiy^eHHA B T6XHHt|6C-kom MacuiTade flByxuennoro HerjMK03HJīnpoBaHHoro ypoKHHa3onpoTe-HHa (rtCU-PA ) .
Claims (9)
- LV10120 Izgudrojuma formula Jaunu plazmīdu iegūšanas paņēmiens·un to izmantošana plazminogēna aktivatora iegūšanā 1. Jaunu plazmīdu iegūšanas paņēmiens plazminogenā aktivatora iegūšanai, atšķiras ar to, ka plazmīdu pBR 322, no.kuras aizvākts nic/bom apgabals un/vai vismaz dala rezistences gēna pret tetraciklīnu, starp Eco RI un Hind III griezuma vietām "multiklonējošā" saitā, ·ievada ar norādīto nukleotīdu secību, kura starp Xba I un NdeI griezuma vietām satur Šinē-Daļgarno virkni un 6-12, vislabāk 8-10 nukleotīdu attālumā - starta kodonu ATG, un ar tā palīdzību ar zi nāmu paņēmienu iebūvē vienā vai divos transkripcijas terminatoros sintētisko daļējo scu-PA-struktūrgēna virkni., kas vislabāk sākas no struktūrgēna 3'-gala, kā arī regulējamo promotoru un tādā veidā iegūst plazmīdas, kurām ir sekojošas atšķirīgas pazīmes: a) plazmīdas operons satur regulējamu promotoru, ribosomu savienojuma vietā - Šinē-Daļgarno virkni, starta kodonu, vienpa-vediena uro-plazminogena "scu-PA" aktivatora sintētisko struk-tūrgēnu, kurš satur 411 aminoskābju atlikumus un lejup no struktūrgēna vienu-divus terminatorus? b) plazmīdas derīgas iepriekšējās stadijas rscu-PA proteīna ekspresijai ar ekspresijas pakāpi vismaz 10 svara % no ente-robaktērijās, vislabāk Escherichia coli celmā, veidojamā kopējā proteīna.
- 2. Paņēmiens saskaņā ar l.p., atšķiras ar to, ka kā regulējamo promotoru ar multiklonējošā saita palīdzību iebūvē trp- vai Tac-promotoru.
- 3. Paņēmiens saskaņā ar l.p. vai 2.p., atšķiras ar to, ka kā regulējamo promotoru iebūvē sintētisko trp-pro-motoru nukleotīdu virknē.
- 4. Paņēmiens saskaņā ar l.p. vai 2.p., atšķiras ar to, ka kā regulējamo promotoru iebūvē tac-promotoru no 2 pt-ac SDT (DSM 5018) plazmīdas.
- 5. Paņēmiens saskaņā ar l.p., atšķiras ar to, ka kā vienu otram sekojošos transkripcijas terminatorus jauno plazmīdu operonā iebūvē trp A-terminatoru un/vai tet A/orf L-terminatoru no Tn 10. Paņēmiens saskaņā ar l.p., atšķiras ar to, ka daļējā stadijā ar multiklonējošā saita palīdzību iebūvē struktūrgēnu ar nukleotīdu virkni.
- 7. Jaunu plazmīdu iegūšanas paņēmiens saskaņā ar l,p,, atšķiras ar to, ka plazmīdas fragmentu, kura iegūta saskaņā ar l.-6.p.p., Eco RI x Hind III izmanto kā ekspresijas kaseti citā, enterobaktēriju, vislabāk autonomi vairoties spējīgā E.coli plazmīdā.
- 8. Jaunu plazmīdu iegūšanas paņēmiens saskaņā ar l.p., atšķiras ar to, ka plazmīdā, kura iegūta saskaņā ar l.-7.p.p., pagarina attālumu starp Šinē-Daļgarno virkni un starta kodonu, sašķeļot ar Nde I, griezuma vietas·aizpildot un sekojoši trulos galus ligējot.
- 9. Saskaņā ar l.-8.p. iegūtās plazmīdas izmantošana plazminogenā aktīvatora iegūšanā, atšķiras ar to, ka ar šo plazmīdu transformē enterobaktēriju celmu, vislabāk E.coli celmu ar zināmu paņēmienu, inducē scu-PA struktūrgēna ekspresiju, atdala, starpstādijā izveidojošos rscu-PA proteīnu no vides un iepriekšējās stadijas lizētās baktēriju šūnas, proteīnu stabilizē un pēc tara redoks-sistēmas iedarbības rezultātā pakļauj atgriezeniskai reakcijai līdz rscu-PA.
- 10. Saskaņā ar l.-8.p. iegūtās plazmīdas izmantošana plazminogenā aktivatora iegūšanā saskaņā ar 9.p., atšķiras ar to, ka ar šo plazmīdu transformē E.coli celmu, vislabāk E.coli K 12 celmu, un pēc tam veic paņēmienu saskaņā ar 9.p.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3923866 | 1989-07-19 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| LV10120A LV10120A (lv) | 1994-05-10 |
| LV10120B true LV10120B (en) | 1994-10-20 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| LVP-92-204A LV10120B (en) | 1989-07-19 | 1992-11-13 | Method of producing of new plasmides and using thereof in producing of the plasminogen activator |
Country Status (30)
| Country | Link |
|---|---|
| US (1) | US5637503A (lv) |
| EP (1) | EP0408945B1 (lv) |
| JP (1) | JP3236284B2 (lv) |
| KR (1) | KR0167761B1 (lv) |
| AT (1) | ATE131534T1 (lv) |
| AU (1) | AU631662B2 (lv) |
| CA (1) | CA2020656C (lv) |
| DD (1) | DD298426A5 (lv) |
| DE (2) | DE4020438A1 (lv) |
| DK (1) | DK0408945T3 (lv) |
| ES (1) | ES2083399T3 (lv) |
| FI (1) | FI102386B1 (lv) |
| GR (1) | GR3019294T3 (lv) |
| HK (1) | HK1005193A1 (lv) |
| HR (1) | HRP920596B1 (lv) |
| HU (1) | HU214243B (lv) |
| IE (1) | IE901849A1 (lv) |
| IL (1) | IL94529A (lv) |
| LT (1) | LT3611B (lv) |
| LV (1) | LV10120B (lv) |
| MT (1) | MTP1063B (lv) |
| NO (1) | NO304952B1 (lv) |
| NZ (1) | NZ234542A (lv) |
| PL (1) | PL166199B1 (lv) |
| PT (1) | PT94776B (lv) |
| RU (1) | RU2073720C1 (lv) |
| SI (1) | SI9011347B (lv) |
| SK (1) | SK280028B6 (lv) |
| YU (1) | YU48371B (lv) |
| ZA (1) | ZA904006B (lv) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2067226A1 (lv) * | 1969-11-27 | 1971-08-20 | Cefilac | |
| DE4101736A1 (de) * | 1991-01-22 | 1992-07-23 | Gruenenthal Gmbh | Neue als plasminogenaktivatoren einsetzbare polypeptide, dafuer codierende plasmide und verfahren zu deren herstellung und deren verwendung |
| DE4440892A1 (de) * | 1994-11-17 | 1996-05-23 | Gruenenthal Gmbh | Proteine mit fibrinolytischen und gerinnungshemmenden Eigenschaften |
| DE4442665A1 (de) * | 1994-11-30 | 1996-06-05 | Gruenenthal Gmbh | Chimäre Proteine mit fibrinolytischen und thrombinhemmenden Eigenschaften |
| KR100467034B1 (ko) * | 1996-12-27 | 2006-04-07 | 주식회사 엘지생활건강 | 압축성형타입오일케익화장료및그의제조방법 |
| KR100372233B1 (ko) * | 1997-03-12 | 2003-06-11 | 주식회사 코리아나화장품 | 미백 파우더, 그의 제조방법 및 그를 함유하는 메이크업 미백 화장료 |
| KR100341638B1 (ko) * | 2000-03-17 | 2002-06-22 | 유상옥,송운한 | 기능성 표면처리 분체의 제조 방법 및 이를 함유하는메이크업 화장료 조성물 |
| JP4124639B2 (ja) * | 2002-12-17 | 2008-07-23 | 株式会社日本触媒 | 大腸菌を用いたｓ−ヒドロキシニトリルリアーゼの製造方法 |
| US7906276B2 (en) * | 2004-06-30 | 2011-03-15 | Kimberly-Clark Worldwide, Inc. | Enzymatic detection techniques |
| EP2147105B1 (en) | 2007-05-02 | 2013-04-24 | Merial Limited | Dna plasmids having improved expression and stability |
| RU2553533C2 (ru) * | 2013-10-23 | 2015-06-20 | Общество с ограниченной ответственностью "Международный биотехнологический центр "Генериум" ("МБЦ "Генериум") | Способ выделения и очистки рекомбинантной человеческой проурокиназы м5 |
| CN104555732A (zh) * | 2015-01-18 | 2015-04-29 | 河南省中原起重机械总厂 | 一种多功能门式起重机 |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4370417A (en) * | 1980-04-03 | 1983-01-25 | Abbott Laboratories | Recombinant deoxyribonucleic acid which codes for plasminogen activator |
| US4558010A (en) * | 1980-04-03 | 1985-12-10 | Abbott Laboratories | Recombinant deoxyribonucleic acid which codes for plasminogen activator and method of making plasminogen activator protein therefrom |
| FI88932C (fi) * | 1982-04-15 | 1993-07-26 | Genentech Inc | Framstaellning av funktionellt maenskligt urokinasprotein |
| US4710464A (en) * | 1984-09-27 | 1987-12-01 | Eli Lilly And Company | Transcription terminators |
| EP0210279B1 (en) * | 1985-01-25 | 1991-08-14 | Sagami Chemical Research Center | Stabilized human prourokinase |
| FR2594845B1 (fr) * | 1986-02-21 | 1989-12-01 | Genetica | Preparation par voie microbiologique de l'activateur tissulaire humain du plasminogene (t-pa) et conversion de l'enzyme ainsi obtenue en sa forme active |
| FI100106B (fi) * | 1986-12-05 | 1997-09-30 | Novartis Ag | Menetelmä plasminogeenin yksisäikeisen yhdistelmäaktivaattorin valmist amiseksi |
| EP0303028A1 (en) * | 1987-08-10 | 1989-02-15 | GRUPPO LEPETIT S.p.A. | Method for preparing single chain urokinase |
| JP2970811B2 (ja) * | 1987-10-30 | 1999-11-02 | オンコゲン ア リミテッド パートナーシップ | 原核生物細胞中にポリペプチドを調製するための発現システム |
-
1990
- 1990-05-22 IE IE184990A patent/IE901849A1/en not_active Application Discontinuation
- 1990-05-23 ZA ZA904006A patent/ZA904006B/xx unknown
- 1990-05-28 IL IL94529A patent/IL94529A/xx not_active IP Right Cessation
- 1990-06-19 NO NO902720A patent/NO304952B1/no not_active IP Right Cessation
- 1990-06-20 SK SK3067-90A patent/SK280028B6/sk unknown
- 1990-06-25 HU HU903970A patent/HU214243B/hu not_active IP Right Cessation
- 1990-06-27 DE DE4020438A patent/DE4020438A1/de not_active Withdrawn
- 1990-06-28 MT MT1063A patent/MTP1063B/xx unknown
- 1990-06-29 EP EP90112400A patent/EP0408945B1/de not_active Expired - Lifetime
- 1990-06-29 AT AT90112400T patent/ATE131534T1/de not_active IP Right Cessation
- 1990-06-29 DE DE59009961T patent/DE59009961D1/de not_active Expired - Fee Related
- 1990-06-29 DK DK90112400.8T patent/DK0408945T3/da active
- 1990-06-29 ES ES90112400T patent/ES2083399T3/es not_active Expired - Lifetime
- 1990-07-06 CA CA002020656A patent/CA2020656C/en not_active Expired - Fee Related
- 1990-07-09 AU AU58832/90A patent/AU631662B2/en not_active Ceased
- 1990-07-11 SI SI9011347A patent/SI9011347B/sl unknown
- 1990-07-11 YU YU134790A patent/YU48371B/sh unknown
- 1990-07-12 US US07/551,907 patent/US5637503A/en not_active Expired - Fee Related
- 1990-07-17 NZ NZ234542A patent/NZ234542A/xx unknown
- 1990-07-17 PL PL90286087A patent/PL166199B1/pl unknown
- 1990-07-18 DD DD90342894A patent/DD298426A5/de not_active IP Right Cessation
- 1990-07-18 RU SU4831114/13A patent/RU2073720C1/ru not_active IP Right Cessation
- 1990-07-18 JP JP18816990A patent/JP3236284B2/ja not_active Expired - Fee Related
- 1990-07-18 KR KR1019900010860A patent/KR0167761B1/ko not_active IP Right Cessation
- 1990-07-18 FI FI903640A patent/FI102386B1/fi not_active IP Right Cessation
- 1990-07-19 PT PT94776A patent/PT94776B/pt not_active IP Right Cessation
-
1992
- 1992-09-29 HR HRP-1347/90A patent/HRP920596B1/xx not_active IP Right Cessation
- 1992-11-13 LV LVP-92-204A patent/LV10120B/lv unknown
-
1993
- 1993-07-12 LT LTIP776A patent/LT3611B/lt not_active IP Right Cessation
-
1996
- 1996-03-13 GR GR960400698T patent/GR3019294T3/el unknown
-
1998
- 1998-05-19 HK HK98104295A patent/HK1005193A1/xx not_active IP Right Cessation
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