Äîêóìåíò âçÿò èç êýøà ïîèñêîâîé ìàøèíû. Àäðåñ îðèãèíàëüíîãî äîêóìåíòà : http://biochem.bio.msu.ru/assets/docs/diploma/2008/Karpova.pdf Äàòà èçìåíåíèÿ: Sun Oct 23 15:45:39 2011 Äàòà èíäåêñèðîâàíèÿ: Mon Oct 1 20:16:26 2012 Êîäèðîâêà: Ïîèñêîâûå ñëîâà: vallis

. ..


(proBNP)

V .. : ... .., .. 2008 .



.......................................................................................................................4 .........................................................................................................................................6 ..........................................................................................................................8 ................................................................................8 BNP ...........................................................................................................................9 BNP. ...................................................................10 proBNP .................................................................................................13 ..............................................................................19 .........................................................20 BNP ............................................................................................22 BNP .........................................................................22 BNP - ..............................23 BNP ................................................................................................................23 BNP ....................................................................................................24 BNP ..............................................................................................24 BNP ....................................................................................................25 ...............................................................................................................................28 ...................................................................................................................29 ..................................................................................30 TEKES..................................................................30 (-) ....................................................30 proBNP - .............................................31 - ......................................................................................................................31 ......................................................................................................32 FPLC .................................................................32 () () - .........................................................33 GelCode Glycoprotein Staining Kit ...................................................................................................................................................33 Western blotting ........................................................................................................................33 , ...................................................................................................................................34 proBNP .................................................................................................35 (Enhanced Chemioluminescence, ECL) ....................................................................................34 recproBNP .................................................35 proBNP in vitro .................................................................................36 ...........................................................................................................37 proBNP 37 proBNP 293 ................................................................................................................................37 proBNP HEK 293, pCMV/hproBNP, - ..............38 recproBNP, 293.............................................................41 proBNP proBNP ............................................................................................................43 recproBNP ( 293) .................46 recproBNP ( 293) ...............................................................................................................................................48 2


recproBNP, ..................................................................................49 NT-proBNP, proBNP, 293, -1 NIH 3T3 ........51 recproBNP 293, 1 NIH 3T3 ........................................................................................................................54 NT-proBNP, .............................................................................................56 recNT-proBNP ECL........58 recproBNP ( 293) ........................59 proBNP, -1 NIH 3T3 .................................................................................................................................61 proBNP in vitro........................64 proBNP recproBNP (E. coli) 65 proBNP, recproBNP (E. coli) in vitro, - .......................................................................................67 recproBNP in vitro .........................................................................68 ...................................................................................................................................71 : ........................................................................................................................................72 ......................................................................................................................73

3



ANP -- ATP -- BNP -- cGMP -- CNP -- DMEM -- Dulbecco's modified Eagle's Medium DNP -- D HEK 293 (human embryonic kidney) -- NPR-, , -- , , NT-proANP -- N- NT-proBNP -- N- BNP preproBNP -- proBNP -- recNT-proBNP (CHO-1) -- NT-proBNP, -1 recNT-proBNP (E. coli) -- NT-proBNP, E. coli recNT-proBNP (HEK 293) -- NT-proBNP, 293 recNT-proBNP (NIH 3T3) -- NT-proBNP, NIH 3T3 recproBNP (CHO-1) -- proBNP, -1 recproBNP (E. coli) -- proBNP, E. coli recproBNP (HEK 293) -- proBNP, 293 recproBNP (NIH 3T3) -- proBNP, NIH 3T3 TEKES -- -- -- -- 4


-- -- -- - -- -- -- -- -- -- -- -- -- () -- -- -- - -- - , Tween-20 --

5



(BNP) , . , . BNP ( proBNP), 108 (). -- N- (NTproBNP, 1-76 ) BNP (77-108 ). - , , BNP NTproBNP , . (), , . , , NTproBNP proBNP . , proBNP , BNP. , proBNP, , proBNP . , BNP, , proBNP. , proBNP . , NTproBNP proBNP . (Schellenberger et al., 2006). , proBNP -1, -, 7 (T36, S37, S44, T48, S53, T58, T71). , NT-proBNP proBNP . 6


NT-proBNP proBNP . , NT-proBNP /, proBNP -- / . proBNP proBNP, , , . , proBNP, , , , , BNP.

7




(brain natriuretic peptide, BNP) , . () (atrial natriuretic peptide, ANP), (-type natriuretic peptide, CNP) [1]. , , D (dendroaspis natriuretic peptide, DNP) (. 1), [3].

. 1. [2].

, 17 (), [4]. ANP 28 , BNP 32 , NP 22 53 [1]. BNP ANP . CNP [5]. , ANP, BNP CNP [1]. . , . , , - , [ 1]. , 8


, -, (cGMP) cGMP- .

BNP
BNP 1988 . (26 ) (70%) ANP. , , . [ 7] , BNP . , BNP , ANP. , , BNP - ANP -- . 1989 . , BNP , BNP [ 8]. BNP . , (), BNP [9]. 1990 . BNP [10]. , , 32 . , , - ( 77 108 ) BNP, . [11] , -- proBNP. (), BNP [12], BNP (). , BNP , . BNP . 9 (4 ), BNP, (13 -- 15 ),


, , N- (NT-proBNP) . , - , [13].

BNP.
BNP , (. 2) [14]. BNP 3' , AUUUA. , , BNP [14].

. 2. BNP [14].

BNP . BNP BNP . , BNP [15]. , ANP [16]. [17] BNP . , BNP . , , , , , , 1-3 % BNP . , 10


BNP 5 48 . , 3-4 . , , , BNP. BNP 3 ( , ). [18] , BNP . BNP 12 . , ANP. , BNP , , BNP . , , BNP. BNP . BNP , ANP, . [19] , BNP 52 % , , , BNP 77 % . , , BNP. , 10 . 4 6 52 80 . , , , (2), (2) , . Nothern blot BNP . , , - , : > > = . 52% . BNP 3-4 , . 11


, BNP 30 , ANP 3-4 [20]. , BNP ANP. , , BNP . , , BNP . ,

. BNP . [20] , BNP . , BNP BNP , BNP 1-2 . , 1000 , , 10-20 . , BNP . , ( 20 ) BNP BNP . BNP BNP . , BNP, . , BNP . BNP . -I, -II BNP [21]. , -1 . -1 , NF-kB -1. BNP cis - GATA-4, , . [22] -1 BNP. 12


, -1 BNP , - . -1 Ras-, Rac- 38- . , BNP . BNP tatttat, BNP BNP [21].

proBNP
BNP , 134 . preproBNP , 26 , - . proBNP (108 ) N- (NT-proBNP) BNP (. 3). , : BNP, proBNP NT-proBNP [13]. , proBNP , . .

-2 6 Pre -p ro BNP -2 6 -1 1

1 08 1 08 Pro BNP

. .



1 76 77 1 08 BNP

NTp ro BNP -

. 3. BNP. .

proBNP , [23]. . 13


, proBNP, . , , proBNP. , , , proBNP. , , BNP [24]. BNP, , [25]. [26] , BNP, proBNP. , , proBNP 6-8 BNP. , proBNP . , proBNP BNP [26]. , BNP proBNP BNP, proBNP , proBNP . , proBNP / BNP. , , BNP , cGMP NPR-A. . proBNP . in vitro. , [23]. , proBNP , BNP. , proBNP Arg-X-X-Arg 73-76 , . , in vitro proBNP [23].

14


, . 1989 [ 27]. , c-fes/feps. Kex2 ( ). , - . . . , , , , , , , , . , - , , [27]. (), N- . , , [27]. [28] , BNP. . , , BNP. 14 28 . BNP , . proBNP . , , , proBNP, -, . proBNP , . , BNP/proBNP 2:1 1:1, . BNP/proBNP 3:1, . , , RXXR , [29]. RXXR proBNP. 15


proBNP. , , , , - . -- . , in vitro proANP proBNP [30]. , , , . , , proBNP BNP NT-proBNP . , proBNP . , proBNP proANP . , , proANP BNP [23]. , , . [31] proANP , , , ANP. 98, 101 102 Arg proANP , , - proANP . , . , II . . Western blotting , , . , proANP , , ANP, , . , , , , , , proANP . , , . . , ANP 16


, . , ANP, . , , , [32]. , . , 866 Arg Ala . , . , , , , , . Western blotting , . , . , , , , , proANP. , . , , . , N-. . , Asn-968 1087 . [33] . , . , , , proANP. , , . , R801A , , proANP. , : Arg801-Ile802. . 17


, , , - . , . II . , , . , . , . , . , , , , , , . 1 proBNP [23]. proBNP . [34] , BNP. , BNP 2-3 , BNP 2 . , BNP . BNP . BNP ANP, , , BNP ANP, BNP . , 2 ANP BNP . 10 , . , BNP , . 18


, BNP ANP - Ca2+.


, . : (NPR-A), (NPR-B) (NPR-C). , NPR- ANP BNP, NPR-B NP, NPR-C . , - (. 4). , , , , [ 36].

. 4. [35].

NPR-A NPR-B . , : - , , . - -

19






,





, 41 [37]. - , , . NPR-A , , , , . - , , , , . NPR-A. , , . . - . , . . - . GMP-

[37]. NPR-C : , G- . , NPR-C Gi- [35]. : NPR-C , , , . . , , - (Npr3), , [ 38]. [39] , NPR-C : ANP CNP BNP.


: , NPR-C, ; , . 20


, [2]. BNP 13-20 . , BNP , . [40] NPR-C .

- , . ANP BNP , , . ,

NPR-C , . NT-proBNP . , NT-proBNP , BNP. , NT-proBNP , , NT-proBNP , BNP [2]. [41] , , , BNP . [42] , proBNP BNP NT-proBNP, , , , proBNP . proBNP , , , ( 1 4 ) . , , , proBNP, . , , proBNP , proBNP. [43] , , BNP, . , BNP , BNP. , 21


, 2 , BNP. BNP. , , BNP
7-32

,

6 N- BNP. , BNP , BNP. BNP , . , . , , , , . , BNP, , , . , , BNP , 6 N- BNP, NPR-B. , BNP , BNP. , BNP -- .

BNP
BNP
, BNP . , . BNP , . , BNP , II [35]. BNP BNP. [44] 48 BNP 1, 2 4 / . BNP , . . 22


BNP -
BNP ,

, . , [45] BNP . , BNP ANP. BNP [35]. , , BNP [13].

BNP
proBNP, , [23]. , BNP . , BNP N- (Ser-Pro). , X-Pro , . , BNP N- . , N- BNP BNP . [46] - :

. , BNP, -- proBNP. [47] - NT-proBNP. , , NT-proBNP, NT-proBNP [48].

23


[49] , , proBNP 25 , 12 -. , , , proBNP. - proBNP. , , proBNP, NT-proBNP BNP, .

BNP
BNP , . , BNP , . BNP 0,5 30 / ( 0,15 8,7 /) . BNP 80 / 55 . NT-proBNP 68 112 / ( 8,2 13,3 /) [2]. , BNP . BNP , BNP [50]. , BNP , , , . , . , BNP . BNP .

BNP
[52] . - , BNP . BNP: - . , -, 24


- 13-15 4 , . , proBNP, BNP. [53]. -, BNP , BNP . BNP: 13 3 . BNP. [10] BNP, . , proBNP, BNP, . , , proBNP BNP.

BNP
, . . , . . BNP NT-proBNP . BNP NT-proBNP [ 55]. BNP NT-proBNP . [56, 57, 58] , BNP NT-proBNP , NYHA (New York Heart Association). [56] 105 . 67 . NT-proBNP BNP. 25


, NT-proBNP , BNP. , . . , NT-proBNP BNP [56, 59], . [ 59] BNP, NT-proBNP NTproANP . 57 . , BNP NT-proBNP. 40 % BNP NT-proBNP, 41 488 /, . 73 % 70 %, -- 77 % 73 %, -- 75 % 72 % BNP NT-proBNP, . , , BNP NT-proBNP . . , BNP , [ 56, 57]. [60], NT-proBNP . NT-proBNP , , . NT-proBNP, NT-proBNP . BNP NT-proBNP [2, 61]. BNP , , NYH, . .

26


BNP . , BNP [62]. -- BNP , , .

27



(proBNP) . proBNP BNP , proBNP, . , : 1. proBNP . 2. proBNP . 3. proBNP. 4. , .

28



NT-proBNP proBNP, , .. . , NT-proBNP BNP ; , , , TEKES; CMV/myc/cyto (Invitrogen, ), proBNP NT-proBNP , « ». DMEM (Dulbecco's modified Eagle's Medium) -- ICN Biomedicals (). -56


--

Unifect ( , ). - Superdex 75 10/300 GL; -- Low Molecular Weight Gel Filtration Calibration Kit; CL-4B -- Amersham Biosciences (); -- ECL Advance Western Blotting Detection Kit; Hybond-ECL 0,45 -- Amersham Biosciences (); 96 -- COSTAR (); 0,22 -- Bio-Rad (); - -- QA-Bio (); -- Qiagen Plasmid Midi Kit (); -- GelCode Glycoprotein Staining Kit Pierce (). SIGMA (). (SIGMA) , - , 67.

29



(HEK 293, -1 NIH 3T3) DMEM (Dulbecco's modified Eagle's Medium), 10% , 40 / , 37° 2- ( 2 5%). -56


.



CMV/myc/cyto, proBNP NT-proBNP . 90-95% , .

TEKES
. . , - , 0,9 % NaCl, - NAP 5, . 0,9 % NaCl 2 /. 0,5 Na- , 9,8, 100 0,75 . TEKES. S 1 /. TEKES 1 1 . 16 + 4 . NAP-10 , 0,01 ­ HCl, pH 7,8, 0,15 NaCl, 0,1% NaN3. .

(-)
, . ( ). TEKES . - . 96- 1 , 100 30


- (10 , 150 NaCl, 7,4), 30 . (0,01 -HCl, 0,15 NaCl, 0,025 % Tween 20, 0,05 % NaN3, 7,8). 200 , , 50 , 50 -HCl, 0,9 % NaCl, 0,01 % Tween 40, 0,5 % , 0,05 % NaN3, 7,75. 50 30 . , 200 LANFIA (1,75 NaSCN, 1M NaCl, 50 NaOH, 0,5 NaHCO3, 5% , 20% -1, 10). 3 . Victor 1420 Multilabel counter.

proBNP -
proBNP -. 501, 102-108 proBNP , -- 16F3, 13-20 proBNP . (. 5) proBNP, E.coli.
10000000

y = 12541x 1, 1156 R2 = 0,9926

1000000

Cp s

100000

10000

1000 0,1 1 C, / 10 100

. 5. proBNP - 50E1-16F3. proBNP.

-
- 293, -1 FPLC äKTApurifier 10 (P-903). Superdex 75 10/300 GL. , 31


0,1 NaH2PO4, 0,7 NaCl, 5 , 7,4. 150 . 0,7 /. , : ( 67 ), ( 43 ), ( 25 ), ( 13,7 ), ( 6,517 ).


CL-4B , proBNP . , , 15 . , 0,1 , 2, , 20 -HCl, 150 NaCl, 7,5. 10000 g 15 , , 5 : , PMSF, ( 1:1000 ). , 20 -HCl, 700 NaCl, 7,5, , 20 -HCl, 100 NaCl, 7,5. 0,01 HCl. , 0,1 , 2.

FPLC
FPLC (fast protein liquid chromatography). (). HiTrap
TM

CM FF 1

äKTApurifier 10 (P-903) Amersham Biosciences (). , 50 Na-, 4,2. 1/. , 50 Na-, 1 NaCl, 4,2 0 60%, 50 .

32


() () -
- . [63]. () (): = 10% = 3%, = 10% = 3%, = 4% = 3%. , 3,0 -HCl, 8,45, 0,3% . , , 1,0 -HCl, 8,45, 0,1% , 0,75 -HCl, 8,45, 0,075% . , 13% ( ) . , 0,1 -HCl, 0,1 , 8,25, 0,1% ; , 0,2 HCl, 8,9. 3:1 , 250 -HCl, 6,8, 40% , 8% , 10% -, . 100° 5 . 150 1 ­ 1,5 .

GelCode Glycoprotein Staining Kit
, GelCode Glycoprotein Staining Kit. , , , .

Western blotting
30 100 , , 25 -HCl, 192 , 10 % ( ), 8,3. 33


, Ponceau C. , Ponceau C.

,
10% , (10 24, 7,4, 150 NaCl, 0,1% Tween-20), 30 +4° . , , , , 5% . 6 /. . 5-15 , 0,3 / 3,3'- (), 50 -HCl, 7,6, 0,03% 22, 0,1 / NiCl2. .

(Enhanced Chemioluminescence, ECL)
, . ECL , , , . . . ECL Advance Western Blotting Detection Kit. ECL Western blotting 2 % , (10 24, 7,4, 150 NaCl, 0,1 % Tween-20), 34


, 2 + 4° . 3 , , , 2 % . 25 /. . , . . . , , , . 0,5 ­ 5 . . , .

proBNP
, proBNP, , - , -- . 50 Na- , 5,0. - 0,75 1,5 200 proBNP, . 70 . 4 37°.

proBNP
recproBNP HEK 293 (-Arg-Val-Lys-Arg-) , . 0,05 . , .

35


proBNP in vitro
proBNP , 30 proBNP . , 100 HEPES, 3 CaCl2, 0,1 % Triton X-100, 7,2. 35 . 37°. , -70°. proBNP, , .

36



proBNP
proBNP 293
proBNP (recproBNP) . HEK 293, . HEK 293 . , 293 recproBNP ( ) proBNP, . recproBNP, 293 (recproBNP ( 293)), proBNP . , , . recproBNP 293 pCMV/myc/cyto, proBNP (pCMV/hproBNP). 24 10 . proBNP -. , , 501, proBNP 102-108 (501 13-20 (16F3 (5011
02-108 102-108

).

16F3, proBNP
13-20

). , ) proBNP 37

-16F3

13-20


.







recproBNP





. 6.
2,5

proBNP , /

2,0

1,5

1,0

0,5

0,0 0 24 48 72 96 120 144 168 192 216 240 ,

. 6. proBNP 293 . recproBNP - 50E1102-108-16F31320.

. 6, recproBNP 293 . recproBNP ( 293), (120 ) . , , , , , , . , 24 5 , , , ( 72-96 ) , proBNP .

proBNP HEK 293, pCMV/hproBNP, -
, recproBNP HEK 293, , , HEK 293 pCMV/hproBNP, -. - 38


proBNP - : 50E11
02-108

-16F3

13-20

, 24C587-98-Ab2 15C4

63-71

-13G12

13-20

. -16F3
13-20

50E1 50E1

102-108



() proBNP,
102-108

, , , , N- proBNP

- proBNP ( BNP-), 16F3 24C5
13-20

( NT-proBNP- proBNP).
87-98

-Ab2 proBNP,

BNP. , 24C587-98, proBNP 87-98 , 15C4 BNP- -13G12 proBNP, Ab2 , 245 BNP.
63-71 13-20



NT-proBNP, , NT-proBNP, proBNP , . , NT-proBNP proBNP NT-proBNP proBNP, 63-76 . , 15C4
63-71

NT-proBNP , proBNP - proBNP. , - recproBNP recproBNP HEK 293. , -, . 7.

39


. 7. , (Superdex 75) HEK 293, pCMV/hproBNP. - : 24587-98-Ab2 (anti-BNP), 50E1102-108-16F313-20 (anti-proBNP), 15C463-71-13G1213-20 (anti-NTproBNP). BNP.

, 24C587-98-Ab2, . -- proBNP, 24C5
87-98-Ab

,

, -16F3

BNP.





2 50E1
102-108 13-20

. ,
87-98

, 24C5 50E1
102-108

-Ab2 ,

-16F3

13-20

, ,

proBNP. BNP, BNP. , 50E110
108 2-

-16F3

13-20

15C463-71-13G12

13-20

, , -

. , 50E1 16F3
13-20 102-108

(anti-proBNP), , 40


15C4

63-71

-13G12

13-20

, .

-, , , 15C4
63-71

-13G12

13-20

, ,

N- (NT-proBNP). , NT-proBNP, , NT-proBNP ( ) proBNP. , - HEK 293, pCMV/hproBNP, proBNP: proBNP , BNP NT-proBNP. HEK 293 , proBNP. , , .

recproBNP, 293
recproBNP ( 293), . 293, recproBNP HEK

pCMV/hproBNP, . CL-4B 50111
02-108

245

87-98

, BNP- .

, , - , recproBNP , [49] recproBNP , , . recproBNP - (. « »). recproBNP - 501102-108-16F313-20. 41


recproBNP . , proBNP - Western blotting. . 8.

. 8. ­ R -250 . 1- proBNP, 293, 2 - proBNP, E. coli. ­ proBNP, 293, . ­ 15F11, proBNP 13 -24 . 1 proBNP, 293, 2 - proBNP, E. coli.

R-250 (. 8 ) (. 8 ). . 8 ( 1) , recproBNP ( 293) recproBNP, E. coli (recproBNP (E. coli) (. 8 , 2). , proBNP -- , (E. coli) . , recproBNP ( 293) recproBNP (E. coli), , , recproBNP (HEK 293), - , . recproBNP ( 293) (. 8 , 1) recproBNP (HEK 293) . Western blotting , 15F1113-24, N- proBNP , recproBNP ( 293) (. 8 , 1). , recproBNP ( 293) (. 8 , 1), 42


recproBNP (E. coli) (. 8 , 2), , , recproBNP 293 , / . , N- recproBNP ( 293), , , 15F11
13-24

recproBNP ( 293)

. , Western blotting , , recproBNP, 293, .

proBNP proBNP
proBNP proBNP, , proBNP . , recproBNP ( 293) proBNP. recproBNP ( 293) proBNP -. 6 , proBNP ( 1). , N- proBNP ( 1-76 , 245 NT-proBNP). .
87-98

, - proBNP , ,

, , [ 49], . , 245
87-98

,

proBNP . recproBNP (E.coli).

43


1. , proBNP .
() 5B 6 1-12 1D4 13-24 11D1 31-39 16D10 46-56 15C4 63-71 2 4 1 1 67-76

recproBNP 293, pCMV/hproBNP; proBNP ­ . proBNP NT-proBNP BNP, N- proBNP ( NT-proBNP), - proBNP ( BNP). , proBNP proBNP . - recproBNP ( 293) proBNP, recproBNP (E. coli). 1D4
13-24

-24587-98,





proBNP, . proBNP , 1D4
13-24

-24587-98, % (. 9).

44


rec proBNP (HEK 293) 120

proBNP

proBNP, %

100 80 60 40 20 0

5B6 (1-12)

1D4 (13-24)

11D1 (31-39)

16D10 (46-56)

15C4 (63-71)

. 9. proBNP. , proBNP (), ; , 1D413-24-24587-98, %.

. 9 proBNP, . , recproBNP proBNP . proBNP. , 63-76 proBNP recproBNP. , recproBNP 63-76 . recproBNP , 1-12 . , , recproBNP , proBNP N- . proBNP, , , () proBNP ( ). , , , 293 recproBNP 45

24E11 (67-76)


, , , proBNP proBNP.

recproBNP ( 293)
recproBNP ( 293) . , proBNP, proBNP . , recproBNP (HEK 293) - recproBNP. , recproBNP recproBNP, recproBNP ( 293) , -. recproBNP (HEK 293) , - , -- - (. « »). recproBNP ( 293), , - , . 1D4 15F11 -245
13-24

15F11

13-24



.
13-24 87-98

, proBNP,



.

. 10.

46


120

proBNP

proBNP

proBNP, %

100

80

60

40

20

0

. 10. recproBNP ( 293) . , proBNP (), ; , - 15F11-245, %.

(. 10), recproBNP ( 293) (31-76 ) . recproBNP , recproBNP . , 48-56 67-76 60 %, 31-39 63-71 -- 16 18 %, . N- (124 ) recproBNP ( 293) . N- (1-24 ) proBNP . , , BNP.

24E11 (67-76)

11D1 (31-39)

15D7 (48-56)

15F11 (13-24)

15C4 (63-71)

5B6 (1-12)

47


recproBNP ( 293)
recproBNP ( 293) Western blotting. recproBNP ( 293). recproBNP (. « »). recproBNP ( ) Western blotting , proBNP , (. 11).
. 11. recproBNP ( 293) 29D12 5-12 (), 15F1113-24 (), 241167-76 (). 1 ­ , ; 2 ­ recproBNP ( 293).

(. 11) , , N- proBNP (29D12
25-12

15F11

13-24

)

, recproBNP ( 293). , N- recproBNP . , proBNP recproBNP, . recproBNP -,
67-76







. . 11 , 24E11

, ,

proBNP, recproBNP ( 293), recproBNP . , recproBNP - recproBNP. , - , , recproBNP ( 293)

48
133


, proBNP ( ), .

recproBNP,
, recproBNP 293 proBNP, proBNP. , reproBNP. proBNP 293 recproBNP : -1 NIH 3T3. . -1 (Chinese hamster ovary) , NIH 3T3 (National Institutes of Health 3T3) -- . recproBNP, , proBNP . . recproBNP 293, -1 NIH 3T3 pCMV/hproBNP. , proBNP 293, proBNP NT-proBNP,

. , recproBNP ( 293). , recproBNP 293, -1 NIH 3T3, pCMV/hproBNP, . 12.

49


120

HEK293

CHO-K1

NIH 3T3

proBNP, %

100

80

60

40

20

0

5B6 (1-12)

1D4 (13-24)

11D1 (31-39)

16D10 (46-56)

15C4 (63-71)

. 12. recproBNP 293, -1 NIH 3T3, pCMV/hproBNP. , proBNP (), ; , - 1D4-245, %.

(. 12), recproBNP, , proBNP 1-56 . , N recproBNP (1-24 ) , , - . recproBNP (31-56 ), , , , -, . - recproBNP (63-76 ), , . recproBNP 63-76 : recproBNP ( 293), recproBNP (NIH 3T3) recproBNP (-1). , , recproBNP, .

24E11 (67-76)

50


, .

NT-proBNP, proBNP, 293, -1 NIH 3T3
, recproBNP , HEK 293 pCMV-hproBNP, , NT-proBNP, (proBNP), recproBNP. NT-proBNP, recproBNP, 293, -1 NIH 3T3. , recproBNP NTproBNP, , BNP. -. 6 , NT-proBNP . NT-proBNP, E. coli (recNT-proBNP (E.coli)). , NT-proBNP . . , N- NT-proBNP (1-24 ), 2411
67-76,

- NT-proBNP. , - NT-proBNP (28-76 ), -- 13G12
13-20

, N-

NT-proBNP. , , , , , , , , NT-proBNP. 51


recNT-proBNP 293, -1 NIH 3T3, pCMV/hproBNP. NT-proBNP , , proBNP, - recproBNP. , BNP- proBNP (24C5
87-98

501

102-108

), -16F3 ,

proBNP . proBNP, - 501
102-108 13-20

5 % . , proBNP NT-proBNP, 293, -1 NIH 3T3. - NT-proBNP proBNP , recNT-proBNP (E. coli). 154 13G12
13-20 63-71

-

, NT-proBNP,

. NT-proBNP , (. 13).

52


120

HEK293

CHO-K1

NIH 3T3

NT-proBNP, %

100

80

60

40 20

0

1D4 (13-24)

5B6 (1-12)

11D1 (31-39)

16D10 (46-56)

15C4 (63-71)

. 13. NT -proBNP 293, -1 NIH 3T3 pCMV/hproBNP. , NT -proBNP (), ; , - 15C463-71-13G1213-20.

(. 13), NT-proBNP . , N- NT-proBNP (1-24 ) , , -, NT-proBNP. NT-proBNP (31-56 ) , , , NT-proBNP. - NT-proBNP , , -, . (. 12 . 13) recproBNP, , NT-proBNP, recproBNP. , - NT-proBNP (63-76 ) recproBNP, . , NT-proBNP, 53

24E11 (67-76)


recproBNP, 293, -1 NIH 3T3, , proBNP , (63-76 ). proBNP recproBNP ( 293) proBNP . , , , . . proBNP, . , , NT-proBNP proBNP, , proBNP, , (63-76 ) . proBNP 63-76 . , , , , proBNP.

recproBNP 293, -1 NIH 3T3
recproBNP, , , recproBNP ( 293) 63 -76 recproBNP (-1) recproBNP (NIH 3T3). , proBNP ( 61-76 ), proBNP. recproBNP [NT-proBNP] [NT-proBNP + proBNP]. proBNP, . - 5B6
1-12

-13G12

13-20

. 54


561

-12

13G12

13-20

N- proBNP NT-proBNP, ,

- proBNP, NT-proBNP. [NT-proBNP + proBNP] 293, -1 NIH 3T3 pCMV/hproBNP. NTproBNP , proBNP, , , BNP proBNP (24C5
87-98

5011

02-108

).

recproBNP (E. coli). . 14.

[NT-proBNP]/([NT-proBNP]+ [proBNP]), %

45 40 35 30 25 20 15 10 5 0 HEK 293 CHO-K1 NIH 3T3

. 14. proBNP 293, -1 NIH 3T3.

, , recproBNP . , 2 93 13 % proBNP, -1 NIH 3T3 38 36 % proBNP, . , recproBNP, , , recproBNP ( 55


293) recproBNP (1) recproBNP (NIH 3T3). proBNP , proBNP (63-76 ). , 63-76 proBNP .

NT-proBNP,
NT-proBNP, recproBNP, 293, -1 NIH 3T3, NT-proBNP (recNT-proBNP), pCMV/myc/cyto, NT-proBNP (pCMV/hNT-proBNP). recNT-proBNP -. 5 , NT-proBNP. recproBNP (E. coli). , NT-proBNP ( 13-30 ). 13G12
20 13-

, N- NT-proBNP .

NT-proBNP, , (. « NT-proBNP, proBNP, 293, -1 NIH 3T3»), , . . ( ) - , 61-76 . 293, -1 NIH 3T3, pCMV/hNT-proBNP. recNT-proBNP (E. coli). 5B6
1-12

-13G12

13-20

,

NT-proBNP, .

56


NT-proBNP , 5B6
120 100 80 60 40 20 0
5B6 (1-12) 11D1 (31-39) 16D10 (48-56) 15C4 (63-71) 24E11 (67-76)
1-12

-13G12

13-20

, % (. 15).

HEK 293

CHO-K1

NIH 3T3

. 15. 293, -1 NIH 3T3, pCMV/hNTproBNP. , proBNP (), ; , -

NT-proBNP, %

NT -proBNP, NT 561-12-13G1213-20.

(. 15), (31 -56 ) recNT-proBNP, . recNT-proBNP. , 63-76 recNTproBNP, 293, , NT-proBNP, -1 NIH 3T3, ( 40 %). , , NT-proBNP, proBNP, (. 13), NT-proBNP. , NTproBNP, , - . recNT-proBNP 57


40 %, , recNT-proBNP, , . NT-proBNP, , , . , , , - . , recNT-proBNP, , NT-proBNP proBNP. NT-proBNP, , , 63-76 NT-proBNP . , , proBNP, , NT-proBNP, , .

recNT-proBNP ECL
293 recNTproBNP,



-1,

Western blotting. NT-proBNP 24E11 NT-proBNP .
67-76

,



recNT-proBNP (E. coli). Western blotting . 16.
. 16. Western blotting. 1 ­ recNT-proBNP (-1), 2 ­ recNTproBNP ( 293), 3 ­ recNT-proBNP (E. coli).

(. 16), , 2411

67-76,

- NT-proBNP , recNT-proBNP (-1) ( 1 . 16), recNT-proBNP ( 293) ( 1 . 16) , , -, - recNT-proBNP ( 293). 58


recNT-proBNP -, recNT-proBNP (-1) 2411 ,
67-76



40 %, . recNT-proBNP, , - recNT-proBNP. , recNT-proBNP NTproBNP, recproBNP, . , , proBNP .

recproBNP ( 293)
recproBNP ( 293), , , , recproBNP 293 BNP, / . , recproBNP ( 293) . , , 293, pCMV/hproBNP, BNP, proBNP BNP NT-proBNP, . recproBNP, , , 63-76 proBNP . , BNP HEK 293, , proBNP 293 10 % 63-76 proBNP, (. 14). , 293 pCMV/hproBNP, , . ,

59


, , . recproBNP, 293, , , (. « proBNP proBNP»). , -, . 17.
proBNP, proBNP,

120

100

proBNP, %

80

60

40

20

0

1D4 (13-24)

5B6 (1-12)

11D1 (31-39)

16D10 (48-56)

15C4 (63-71)

. 17. recproBNP ( 293), . , proBNP (), ; , - 1D4-245, %.

(. 17), , recproBNP, , 63 -76 proBNP, 293 . , 2411
67-76



24E11 (67-76)



6% 1,5 %

proBNP, proBNP,





,



. recproBNP, , 60


. , , 2411
67-76

-24587-98,

6 % 293 . (10 %). , , 293. , , 293 proBNP, 63-76 . , , -, proBNP, 63-76 . , 63 -76 proBNP.

proBNP, -1 NIH 3T3
, recproBNP, , , recproBNP , . -1 NIH 3T3, pCMV/hproBNP, - 2411
67-76

-245

87-98

, ,

, , 43 % 20 %, (. 12). 63-76 recproBNP , , -1 NIH 3T3, pCMV/hproBNP, recproBNP, , (63-76 ) («» 63-76 proBNP). proBNP, ( ) 63-76 , recproBNP -1 NIH 3T3 pCMV/hproBNP, , proBNP. 2411
67-76

,

61


proBNP, . , «» 63-76 recproBNP, , recproBNP, («» 63-76 proBNP), , ( «»). , , 63 -76 recproBNP (HEK 293) , , recproBNP 63-76 . - 241167-76-245
87-98

«» , , . , ,

2411

67-76

recproBNP, -- recproBNP, 2411
67-76

-1 NIH 3T3, pCMV/hproBNP, recproBNP, , . , 2411
67-76

,
102-108

501

245

87-98

, BNP.

proBNP, «», , , - proBNP, . , recproBNP: «» «» 63-76. recproBNP - , (. « proBNP proBNP»). proBNP . 18 . 19.

62


" " 63-76 proBNP (-1)
proBNP, %

180 160 140 120 100 80 60 40 20 0
5B6 (1-12) 1D4 (13-24)

" " 63-76 proBNP (-1)

. 18. proBNP («») («») 63-76 proBNP, -1.

proBNP, %

" " 63-76 proBNP (NIH 3T3) 180 160 140 120 100 80 60 40 20 0 " " 63-76 proBNP (NIH 3T3)

5B6 (1-12)

1D4 (13-24)

11D1 (31-39)

15D7 (48-56)

15C4 (63-71)

. 19. proBNP («») («») 63 -76 proBNP, NIH 3T3.

24E11 (67-76)

24E11 (67-76)

11D1 (31-39)

15D7 (48-56)

15C4 (63-71)

63


. 18 . 19 , 63-76 «» recproBNP «» recproBNP. «» «» recproBNP . , , proBNP, - recproBNP -1 NIH 3T3, pCMV/hproBNP, recproBNP , .

proBNP in vitro
( 63-76 ) recproBNP. recproBNP (E. coli). , ( 90 % proBNP ) 30 recproBNP (E. coli). 0,25 2,5 . 2 37°. ­ 70°. - 501
102-108

-16F3

13-20

recproBNP.
102-108

501

-16F3

13-20



proBNP, BNP. proBNP (. 20).

64


100

100
% proBNP

% proBNP

80 60 40 20 0 0,0 0,5 1,0 1,5 2,0 2,5

80 60 40 20 0 0 1 2 , 3 4



,



. 20. proBNP .

(. 20 ) , 30 recproBNP ­ 1 . . , 30 proBNP 1 , 0,5 4 37°. proBNP. (. 20 ) recproBNP. , ­ 1 30 recproBNP, ­ 2,5 37°.

proBNP recproBNP (E. coli)
proBNP, recproBNP (E. coli) in vitro, Western blotting. proBNP, recproBNP (E. coli) : 245 BNP), 2411
87-98,

- proBNP ( 11-22
67-76

, BNP
87-98

( - NT-proBNP). 245 BNP. 241167

proBNP
-76

,

proBNP NT-proBNP. recproBNP (E. coli) in vitro . 21.

65


. 21. ECL Western blotting. ­ 24587-98, ­ 241167-76. 1 ­ recproBNP (E. coli), , 2- recproBNP (E. coli) .

(. 21), , 245

87-98,

BNP, recproBNP (E. coli), , proBNP ( 1 . 21 ), ( 2 . 21 ) , -, BNP, . 2411
67-76



, . , recproBNP (E. coli) ( 2 . 21 ) recproBNP (E. coli), ( 1 . 21 ). , 245
87-98

241167-76,

, proBNP, recproBNP (E. coli), , , , proBNP. , 245
87-98

recproBNP (E. coli)

( 2 . 21 ), , 241167
-76

( 2 . 21 ). ,

recproBNP (E. coli) 10 % proBNP. , , -, , 2411
67-76

recproBNP (E. coli) ,
87-98,

NT-proBNP, , , 245 BNP. recNT-proBNP , BNP (67-76 ), NT-proBNP, , recproBNP (E. coli) , proBNP . , ECL 66


Western blotting , recproBNP (E. coli) in vitro BNP NT-proBNP.

proBNP, recproBNP (E. coli) in vitro, -
, recproBNP (E. coli) , , proBNP, recproBNP (E. coli) . recproBNP (E. coli), in vitro. - - 245 proBNP, BNP, 245 24587-98.
rec proBNP (E. c oli) 6,52 BNP ( ) 13,7 67 43 25

87-98

-Ab2 (.

22).
87-98

BNP-

proBNP, Ab2 , BNP

0,40 0,35 0,30 0,25

,

0,20 0,15 0,10 0,05 0,00 10 15 20 25 30 35



. 22. - 24587-98Ab2 , - recproBNP (E. coli), in vitro. , BNP recproBNP (E. coli).

67


(. 22), , , - recproBNP (E. coli), , 245
87-98

-Ab2

. BNP. , proBNP, , recproBNP (E. coli) BNP. - BNP recproBNP (E. coli) in vitro. , - , recproBNP (E. coli) in vitro BNP NTproBNP.

recproBNP in vitro
recproBNP (-1), ( 63-76 ) recproBNP ( 293). recproBNP, 501
108 102-

-16F3

13-20

. recproBNP

proBNP. proBNP - 501
102-108

-16F3

13-20

. . 23.

68


120

ProBNP , % proBNP,

100

80

60

40

20

0 rec proBNP ( 293) " " 63-76 proBNP (- 1) " " 63-76 proBNP (- 1) proBNP (E. c oli)

. 23. recproBNP in vitro.

. 23 , «» 63-76 recproBNP (-1) , «» recproBNP (-1) recproBNP (E. coli). , 70% «» proBNP. . 23 , recproBNP ( 293) . , , 98 % proBNP . recproBNP ( 293) , 63-76 , , -, BNP. , recproBNP in vitro , recproBNP , recproBNP, .

69


proBNP proBNP.

70



recproBNP, (HEK 293, CHO-K1 NIH 3T3) proBNP. , proBNP . - , . 63 -76 proBNP NT-proBNP, . , proBNP, 63-76 . recproBNP . , recproBNP, 1 NIH 3T3, : proBNP, 63 76 . , proBNP, , in vitro, proBNP . , , proBNP 293 6 % 63 -76 proBNP, proBNP . , , , 63 -76 proBNP BNP.

71


:
1. recproBNP 293. 2. , recproBNP 293 NT-proBNP BNP. 3. recproBNP ( 293). recproBNP ( 293) proBNP. 4. recproBNP, 293, -1 NIH 3T3. 5. recproBNP NT-proBNP, . 6. 63-76 recproBNP . 7. proBNP. 8. recproBNP, -1 NIH 3T3. 9. 63-76 proBNP BNP.

72



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