підручники. біохімія / Uchebnik_po_Biokhimii_E_S_Severin
.pdfHpa I
5' ~ ~3'
3' ~ ~5'
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PacLllenlleH111e |
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Eco RI |
5' |
3' |
3' |
~5' |
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PacLllenlleH111e |
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Hind Ill |
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5' |
3' |
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3' |
~5' |
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PacwenlleH111e |
8
0
0
PMC. 4-62. nocnE!AOBaTenbHOCTb HyKneoTltAOB B AHK, yaHaeaeMaSI TpeMSI HaM6onee '18CTOMCnonbayeMblMM pecTpMKT838MM.
Pecrp111K4111py10u.i111e HyKnea3bl nonyYa10r 1113 pa3nlllYHblX 6aKrep111~: HPA I - Haemophilus parainfluenzae; Eco RI - 1113 Escherichia coli; Hind Ill - 1113 Haemophilus inf/uenzae. Pecrp111KTa3bl rnna 1 pacu.iennHJOT ,QHK c 06pa300aH111eM «cnenblX» KOH400, a Apyrne (r111na 2) c o6pa30B8H1110M no M0CTY pa3pb1Ba OAH04enO'i0'iHblX«mmKlllX» KOHL.108.
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IIOCJIE)J,OBATE.JlbHOCTEH
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214
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( CEKBEHHPOBAHHE ,II.HK)
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.IlYT KOHKypttpoBaTh c ttopMaJihHhIMH llHT<D 3a BKJIIO'-leHHeB paCTylll,YJO IIOJTHttyKJTeOTH)lHYJO uerrh. Ilp11 BcTµaJ11BaHJ11H MHT<D BMecrn coorneTCTBYJO- w:ero HyKJieOTl1fl,a CHHTe3 JJ,HK npeKpaw:aeTCH.
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215
5'-npaillMep
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HyK11eOT11Abl: 5'-npaillMep-10-11-12-13-14-15-16-17-3' |
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AHK-norn1Mepa3a+AAT<t>, ArT<l>, ALF<!> |
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Ecrm c1-1Hre3 |
- |
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1 |
ATT<l> "1 OA"1H "13 4 AAHT<l> |
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11 |
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3aKaHY"1BaJlCf!: .QAAT<t> AQrT<t> |
AQ L.J,T<t> AQIT<!> |
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10 |
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12 |
- |
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- |
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Pa3Mep npoAyKTa |
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- |
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-14 |
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13 |
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(no 1.11-1cny HyK11eornAoB) |
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15 |
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- |
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16 |
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3neKTpocjJopea |
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17 |
"- |
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- |
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a nom1aKp1-1naM"1AHOM rene- |
'\o |
\ |
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/ |
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A |
G |
C T |
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18 |
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17 |
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PaaMep AHK-npOAYKTa |
rn |
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(no '"1"1CJlyHyK1leOT"1AOB) |
14 |
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nocneAOBareJlbHOCTb BHOBb |
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13 |
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C"1HT93"1posaHHOill L1en1-1 |
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12 |
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5'-npail!Mep-ACGTGATA-3' |
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11 |
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10
9
8
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MaTpttua. CeKBem1pyeMhii1 cf>parMeHT JJ,H K MenIT ITO cf>occf>aTHOMY OCTaTKY H ,LJ,06aBJUIIOT B 51qei1KM MaTPHUbI. <l>parMeHT JJ,HK rn6pw:unyeTC51 TOJibKO c TeMH OKTaHyKJieOTH)J,aMH, ITOCJie,LJ,OBaTeJihHOCTH KOTOPhIX KOMTIJieMeHTapHbI era yqacTKaM. TaKHM o6pa3oM, onpeL1,eJI5leTCH tta6op Bcex B03MO)!(HhIX OKTaHYKJieOTH,LJ,OB, rrpHCYTCTBYJOIUHX B HCCJie,LJ,yeMOM ¢parMeHTe JJ,HK. JJ,an:ee nptt ITOMOIUH cneIJJiall:bHOM KOMITbIOTepHOM o6pa60TKM ynop5l)J,oqHBaeTCJI paCITOJ10)KeHHe OKTaMepoB B HCCJie,LJ,yeMOM cf>parMeHTe JJ,H K.
f. fioJiyqEHME PEKOMI>MHAHTHhIX ,11,HK
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1. II0Ay1tenue peKoMounanmHblX J(HK
J],JIH rronyqeHJ15l TaKHX MOJieKyJl rrepBOHa'!aJibHO BbI,LI,eJIHIOT JJ,HK H3 2 pa3HbIX H:CTO'IHHKOB(pttc.
M MX AMIIJIM<l>MKAQMJI
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Jia )IJI5l HCCJie,LJ,OBaHH5l. 3TO HenpocTaH Ja,LJ,a'fa, oco6eHHO CCJIH HCTOl.fHHKOM JJ,HK CJI~aT TKaHH
4-65).
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216
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'tipa f\l\:HTOB ~1HK\ 1-1 Ll HK, |
cmna1-11-1 blX \H:~)KJtY |
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".HJnKll\IH» KOllLiaMlL |
Kona,1e1-111-1oe cw11- |
ua Ii HC |
rj:lpdl\ICHTOR OCYll lCCTIFIH I{)T c no~10Wbl0 |
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JJl K-,1,ffaJbl B r1pHcyTcnivrn |
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PHC. 4-65. nony'teHHepeKOM61/iHaHTHblX JlHK. L:(sa o6pasqa /]HK:
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Myrau1111, B03HttKa10m11e B y'laCTKaxY3HaBaHHH onpeneJieHHhIX pecTpttKTa3, neJiaIOT 3TH yqacTKH L(H K He'l)'BCTBttTeJihHhIMMK neiicTn1110 ¢epMeHTOB. 3TO MO)l(eT 6bITb JierKO o6Hap)')KeHOno 113MeHeH11IO MHHbl pecTpHKUHOHHbIX ¢parMeHTOB )],HK. Il)],P<l>-aHaJIH3 BKJilO'laeTcJienyrom11e 3Tanhi: BhI.[leJieH11e reHOMHOH )],HK, ee peCTpHKUHIO cneumptt'-IecKoti3H.IIOHyKJiea3oil, 3JieKTpo¢opern- 'leCKOe pa3.[leJ1eH11e o6pa3YIOilIHXC51 ¢parMeHTOB )],HK H H.[leHrn¢11KaUHIO 3TI1X ¢parMeHTOB nyreM 6JIOT-m6p11JJ,113aUHH no Cay3epHy. Ha 3JieKTpo¢o- perpaMMax np11 OTCYfCTBHH peCTPHKUHH B HCCJieJJ,yeMOH )],HK BhrnBJIHIOT OJlHH KpynHhIH ¢parMeHT, COOTBeTCTBYIOlUHM no MHHe fIOCJie,llOBaTeJibHOCTH )],HK Me)!(,Ily JlBYM51 coce,II,HHMH yqacTKaMH peCTPHKUHH M5i TOH )Ke 3H,llOHyKJiea3bl. IlpH HMM - 'lMH pecTpHKUHH B nOJIHMOPWHOM y'-IaCTKe Ha 3JieKTpo¢operpaMMe 6yneT npttcyrcrnonaTh MeHbwttti no pa3MepaM ¢parMeHT, paBHhlM paCCTOHHHIO Me)!(,Ily noJIHMOPcPHblM YlJaCTKOM pecTpHKUHH H O,!I,HHM 113 6JIH)l(aHWHX noCT051HHbJX y'-IaCTKOB peCTpMKUl1l1 (p11C. 4-68).
Il,l]P<l>-aHaJIH3 MO)l(eT 6hITb 3Ha'-IHTeJihHOynpo- lUCH B TOM cnyqae, eCJil1 B03MO)l(Ha cneutt<l>H'-IeC- KaH aMTIJIHcPl1KaQH51 y'laCTKa)],HK, CO,II,ep:xrnmero noJI11Mop¢HbIH catiT pecTpttKUHH. TecTttposaHtte COCT051Hl151 3TOfO JIOKyca B03MO)((H0 nyreM nposeneHl151 nUP 11 peCTPHKUMH aMJJJIMWMUMpOBaHHOro ¢parMeHTa. Ilp11 0Tcyrcrn11H catlrn Y3HaBaHttH B HCCJienyeMOH o6JiaCTH )],HK pa3Mepbl aMnJIHcPMUHPOBaHHOfO ¢parMeHTa He H3MeH51TC51 nocJie ero o6pa60TKM pecTPHKTa3oil. EcJIM yqacTOK y3HaBaHH51 He M3MeHeH, o6pa60TKa ¢epMeHTOM np11neneT K o6pa30BaHl1IO 2 MaJieHbKHX ¢parMeHTOB c TOM )Ke CYMMapHOM MHHOM, 'ITOH HCXO,!I,HbJM cf:>parMeHT.
IlpM o6cJie,II,OBaHHH nautteHTOB l1 'lJieHOB HX ceMeti Ha HOCI1TeJibCTBO naTOJIOfH'-IeCKHX reHOB W11pOKO HCTIOJib3YJOT 3TOT MeTO,ll, C nOMOillhlO KOTOporo:
•11,II,eHTHWHUHPYIOT ,II,eJieUHl1 B reHe ,!I,HCTpo¢ttHa, Ha ,!I,OJIIO KOTOpb!X npHXOJl,HTC51 OKOJIO 60%
ncex Myrau11tl:, Bbl3bIBalOUIH.X MHO)lHCTPOcPHIO )],10weHHa;
•n11arnocT11pyroT reMo¢HJittIO A, HeK0Topb1e TaJiacceMHH, peTHH06JiaCTOMY 11 rpaHyJieMaT03;
•KOHTPOJIHPYIOT 3!.lOPOBbe ,II,eTeH B ceMMIX, B KOTOpblX pO,!I,MTeJIH HBJIHIOTCH reTepo3HroTaMl1 no reHy cepnOBH)lHO-KJieTO'-IHOH aHeMH11 11
JlPYrHM ne<t>eKTHbIM reHaM.
2. OnpeiJe!leuue Mymau.uii c nOMOUl,blO ll.!l.!le!lbcneu,urjJu1tecKux npo6
MHor11e Myrau1111, BhI3hma10m11e B03Hl1KHOBeHtte reHHblX 6oJie3Hetl:, He nona,II,aIOT B Y'JaCTKl1, orneTcrneHHhie 3a Y3HaBaH11e ¢epMeHTOB pecTp11KUMH. B 3TOM cJiy'lae, ecJI11 nocJienonaTeJibHOCTh OCHOBaHHH B 06JiaCTH MYTaU1111 113BeCTHa, TO ee o6Hap)')KHBaIOTc noMOillhlO aJIJieJih-Cneu11¢tt'-IeC- Kl1X OJIHroHyKJJeOTH,!I,OB. L(nH 3TOro CHHTe3HPYIOT KOpOTKHe OJIHfOHyKJieOTH,!I,HbJe 30H,!I,bl' conep)l(a- ll{He 06bI'lHOOKOJ10 19 HYKJieOTH,!I,OB, KOMnJieMeHTapHbie yqacTKY HOPMaJihHoro tt MyraHTHoro aJIJieJIH B )],HK. 06JiaCTb reHOMa, CO,II,ep)Ka11.1,y10 HCCJie,II,yeMhlH reH, aMTIJIHWHUMPYIOT c nOMOUiblO OUP, 11 o6pa3UhI IlOJI)"feHHOM )],HK nepeHOCHT Ha HHTpoueJIJ1IOJI03Hbie cPHJibTpbl (JlOTHJIH CJIOT6JIOTnrnr). Ilpo6h1 Bh1Jlep:11rnna10T c 32 P-30H,II,aM11 MH BhrnBJlemrn HOpMaJibHOH HJIH MyraHTHOH nocJie)lOBaTeJlbHOCTH. y fOM03HroT no HCCJ1e,II,yeMOH MyraUHH )],HK 6y,II,eT m6pH,UH30BaTbC51 TOJibKO c 30H,IIOM, KOMrIJieMeHTapHblM MYTaHTHOH nocJienoBaTeJihHOCTl1. )],HK HOpMaJihHOfO fOM03HfOTHOro HH,!I,11BH,llyyl\1a CBH)l(eTCH c 30H,!I,OM, COOTBeTCTBYIOll{HM Hel13MeHeHHotl: Hy1<.i1eoTttnHoti noCJie,II,osaTeJihHOCTH, Torna KaK c )],HK reTep03HfOT 6y,II,yr m6pH,II,H30BaThCH o6a 30H,lla. Ha pttcyHKe 4-69 npe,IICTaBJTeHbl pe3yJibTaThl reHHOro 30H,!I,11pOBaHH51
7 nau11eHTOB Ha HOCHTeJibCTBO HaH60Jiee 'laCTO BCTpe'-Iaiowetlc51,II,eJieumi: 3 HyKJieorn,II,os (LiF508)
B re He, OTReTCTBeHHOM 3a pa3Bl1Ttte MYJ<OBHCU11 - no3a.
0JIHroHyKJieOTH,II,bI, aJIJieJih-cneu11¢11'-IHh1e no onpeneJieHHhIM MYTaUHHM, MO)l(HO HCnOJih30BaTh B Ka'leCTBenpai1Mepon B IlUP npH KJil1Hl1'-IeCKOM TeCTHpOBaHMH HaceJieHH51 Ha HaJiwme naTOJIOfH- '-IeCKOfO retta. EcJIH )],HK, noJTyqettHa51 OT nau11eHTa, aMJJJIH<l>ttu11pyeT c MyTaHTHbJM OJTHroHyKJieOTH,!I,OM, TO CJie,II,OBaTeJihHO nau11eHT HBJIHeTCH HOCHTeJieM Myrau1111. ECJTH HYKJieOTH,llHa51 nocJie- ,!I,OBaTeJThHOCTb B HCCJie)l)'eMOMrette He l13MeHeHa, TO OJIHroHyKJieOTl1Jl, CO,II,ep)l(all{HH MyraUHIO, He CB51)KeTCH C L(HK-MaTp11ueti, l1 IlUP He nOMJJ:eT.
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pecTp1.1K1..11.11.1 |
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reH 13-rno61.1Ha |
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.....GAG ...... |
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<l>parMeHT pecTp1.1K1..11.11.1 1,1 K1.1no6a3bl |
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My,-aHTHb1i;I reH |
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j3-rno61.1Ha |
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.....GTG...... |
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<l>parMeHT pecTpi.1Kl..ll-11.1 |
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1,3 K1.1no6a3bl |
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AA SS |
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1,3 K1.1no6a3bl |
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1, 1 Kl.1no6a3bl |
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PaA1.1oaerorpaMMa nocne rn6p1.1,Q1.13a1..11.1111 c 30HAOM K l3-rno61.1Hy
PMc. 4-68. PecTpMKl.IMOHHblM aHanMs JlHK reuorno6MHa <1enoeeKa, 6onbHOro cepnOBMAHO-KneTO'IHOMaHeMMeM (HbS). M111c- ceHc-My,-a4111S1, OTBeTCTBeHHa51 aa B03HlllKHOBeH111e cepnOBlllAHO-KfleTOLJHOM aHeMllllll, CB513aHa c 3aMeHOM B reHe ~-4en111 rno6111Ha TplllnneTa GAG Ha GTG. np111 3TOM y,-paLJ111eaeTC5I yLJaCTOK peCTp111K4111111 ¢epMeHTa Mstll, yaHat0ll.1ero nocne,:ioeaTenbHOCTb CCTNAGG, ri:ie N
MO>KeT 6b1Tb nt06biM OCHOBaH111eM. np111 Hanl/ILJllllll My,-a4111111 reHHblM 30HA r1116p111,:i111ayeTC5I c 6onee KpynHblM C!JparMeHTOM !lHK pa3MepoM
1,3 Klllno6a3bl, 111Mel0ll.1111M np111 sneKTpo¢opeae MeHbWYIO nOABlll>KHOCTb, LjeM npo,:iyKT pecTplllK4111111 HOpManbHOro reHa, j:lnlllHa KOTOporo paeHa 1,1 K111no6aab1.
HopManbHas:i npo6a |
Myra1..11.1s:i i1F508 |
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oeeoe |
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PMC. 4-69. reHHOe 30HAMPOB8HM8 HS HOCMT8nbCTBO MyKOBMCl.IMAOSa. c nOMOlJ.1blO nL-lP aMnn111¢1114111pyt0T reHOMHYIO
!lHK, Ill npo,:iyKT nepeHOC51T Ha 2 Hei4nOHOBblX qllllnbTpa, OAlllH 1113 KOTOpbiX rn6p111,:i111ayt0T c 32 P-30HAOM Ha HOpManbHblM annenb, a BTOpoi4 c 32 P-30HAOM Ha MYTaHTHblM annenb t.F508. 06paaoBaH111e rn6p111AOB 06Hapy>K111Ba10T paj:11110aBTOrpaC!J111LJeCKlll. npo6bi 1-3 cny>K111n111 KOHTponS1M111: nepea51 co,:iep>Kana npo,:iyKTbl nL-lP !lHK OT rOM03111roT no HOpManbHOMY reHy; BTOpa51 - OT reTepoa111roTHoro HOC111Ten51 MyTa4111111 t.F508; TpeTb51 - OT 6onbHoro MYKOBlllC4111A030M, roM03111rDTHOro no MyTa4111111 t.F508. npo6b1 4-10 nonyLJeHbl np111 06cne,:ioeaH111111 7 na4111eHTOB Ha Hoc111TenbCTBo t.F508: 4, 5, 6 111 9 OKaaan111cb roMoa111roTaM111 no HOpManbHOMY reHy, a 7, 8 Ill 10 - reTep03111rOTHblMlll HOClllTen51Mlll MyTaHTHOro reHa.
E. llcnoJih30BAHME ,IJ;HK-TEXHOJior11fi ,U.Jlll
JTOJIYllEHMH JIEKAPCTBEHHbIX JTPEIIAPATOB
II JIEqEHMH PA3JiffqHblX liOJIE3HEH
Baiill,Hllb1 - O'IBI..UeHHb1e6eJI1<H, aHTIITeHHhie .uerepMllliaHTbI PMa B036y.Il,l1reJieii BttpycHhIX tt 6aicrepttaJibHhIX HHQ:>emttll:. B rrOCJie.IJ,Hee BpeMH MX nonyllaIOT, IIOJih3Y5!Cb TeXHHKOH peKoM6MHaHTHbIX .llHK. flepooii BaKUI.fHOll, CIDITe3HpOBaHHOH 3THM cnoco6oM, 6hma BaKUMHa npoTHB Bttpyca renaTIITa B.
EeJIKH, HMe1mn:ue TepaneBTH'ICCKoe3Ha'leuue, rronyqaIOT c HCIIOJib30Bamt:eM 3TOH TeXHOJIOfHH BO MHOfHX CTpaHax MHpa. TaK, O)J,HHM ll3 rrepBblX CHHTe3ttpOBaH HHCymrn qenoBeKa (pttc. 4- 70).
B KJieTKax E. coli, rpattcQ:>opMHpOBaHHbIX TVIa3MH-
.uaMtt, co.uep)l(aI.IJ:HMH ,llHK, KOTOpa5! KO.UHPOBana A- tt B-uerrtt HHCYJIHHa, ttapa6aTbIBaIOT 6e11KOBb1e rrpo.uyKTbl A- 11 B-uerretl:. flocne OllttCTKH HX no.u- BepraIOT <Pon.umrry tt oKHcnemno, KOTopoe o6ecnellHBaeT o6pa30BaHtte COOTBeTCTBYIOI.IJ:HX )J,HCyJibQ:>IDJ.- HbIX MOCTHKOB.
222
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1. X111MepHb1e nna3MV1Abl, |
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co,lleplt<aw.111e |
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co.a.ep>Kalll,111x x111MepHb1e |
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b8KTepvianbH85'1 |
nna3M111Abl |
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xpOMOCOMa |
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A·L\erlb |
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A-L\eno-Nf-1;, |
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AKH-1 BHblVt |
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B-L\enb-Nf-1;, _j---1\-- COOH |
"1HCYf1"1H |
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A1t1cynbcp111.nHaSo1 |
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CBH3b |
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Pi.tc. 4·70. nonr1eHi.te lt1HCyni.tHa 1.1enoseKa B 1<11eTK8X E.coli. 1 - |
Tp8HCcj:JOpM8L\lllH KJ18TOK E. coli nna3MlllA8Mlll, KOTOpb18 COA8P)l(8T |
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reHbl, KOAlllPYIOLL\1118 CTPYKTYPY A- 111 B-L1enelii 111Hcyn111Ha; |
2 - ClllHTe3 A- 111 B-4enelii 111Hcyn111Ha |
B npo4ecce Bb1palL(111BaH111H KynbTYPbl |
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TpaHccpopM111posaHHb1x Kfl8TOK E. coli; 3 - BblAeneH111e 111 OYlllCTKa A- 111 B-4enelii 111Hcyn111Ha; 4 - |
npocTpaHcrneHHasi yKn8AKa A- 111 B- |
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4enelii lllHcyn111Ha Ill OKlllCneHllle OCTaTKOB 4111CT8111Ha. |
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AHaJ10fl11c!HbIM cnoco601v1 no11y1c1eH ropMoH poCTa, 11cnOJlb3yeMblM )lJUI Jie4eHl1}1 )leTeJ.i c He)lOCTaT01c!HOCTbJO 3TOro ropMotta. 6011ee CJIO)l(HbJe 6eJIKI1 noJiy4eHbl B KyllbType KJleTOK MJieKOnl1TaIOllU1X. TaK, nect>eKTbI u rette ¢aKTOpa VIII, Kon11py10w,ero OJlMH 113 6eJIKOB - Y'IacnmKoBcnepThl-
Ba10mei1 CMCTeMbl KPOlll1, OTBeTCTBeHHbl 3a B03Hl1KHOBett11e re1v10¢11J11111. Ll,o rnro KaK ¢aKTOp VII I
6bUT nOllY'IeHMeTO)laMM reHHOH JUDKettep1111, 60JlbUJOe KOJll14ecrno 6011hHbJX norn6ano OT CDl1Ll,a l1Jll1 remtTMTa, KOTOpb!Ml1 OHM 3apa>Kafll1Ch B peJyJlbTaTe BBe)leHM}I Bbl)leJieHHOro 113 KPOBI1 ¢aK-
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