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-
.
(MSc.)
2007
-
.
(MSc.)
.
.
.
.
.
.
.
ii
-
... , , , , , , , . , , ,
! !
,
. , . , . . , , . !
, . . , . . . , . , . , , , .
. . , , . , , . , - , , .
. , , ! . !
(ESF), ( ) , .
, , , , !
iii
-
....................................................................................................iv
.....................................................x
.......................................................................................... 1
1......................................................................................................... 1
1. ................. 1
1.1 -..................................................................................... 1
1.2 .......................... 1
1.3 -............................................................ 2
1.3.1 ............................................... 3
1.3.2 ............................................ 5
1.3.2.1 ................... 6
1.3.2.2 .............. 7
1.4 ...................................................... 8
1.4.1 ................................................................... 8
1.4.2 ....................................... 11
1.4.3 ............................................................... 13
1.5 ........................................... 17
1.5.1 .............................. 17
1.5.2 .................................................................................... 18
1.5.3 .
................................................................. 20
1.5.4 .......... 21
1.5.4.1 ........... 22
1.5.4.2 ..... 24
1.6
........................................................................................ 26
1.7 ...................................... 30
2....................................................................................................... 32
O
....................................................................... 32
iv
-
2.1 ............................................................................................................... 32
2.2 AChR ................................ 32
2.2.1 (myasthenia gravis) ...................................................... 32
2.2.1.1 ..................................................................................... 32
2.2.1.2 ........... 34
2.2.1.3 ............................................................. 36
2.2.1.4 ..................................................................... 37
2.2.1.5 : -
............................................................................... 39
2.2.1.6 ........................................................................ 44
2.2.1.7 ............................... 45
2.2.2 ..................................................................... 49
2.3 AChR ............................. 50
2.3.1 Alzheimer.................................................................................. 50
2.3.2 Parkinson ................................................................................. 52
2.3.3 ........................................................................................................ 53
2.3.4 .................................................................................................. 54
2.3.5 ................................................................................... 54
3....................................................................................................... 56
- .................................. 56
3.1 ............................................................................ 56
3.1.1. ...................................................................... 56
3.2 ............................................................................................... 57
3.2.1 ...................................................................................... 57
3.2.2 ...................................................................... 58
3.2.3 ................................................................................... 59
3.3 .......................... 61
.............................................................................................................. 63
..................................................................................... 65
................................................................................................................... 65
1. ............................................................................. 65
2. ........................................................................................................ 66
3. - ............................................................................ 67
3.1 .................................................................................... 67
v
-
3.2 (kits) ........................................... 68
3.3. (primers)- .......................................... 68
3.4 ..................................................................................................... 69
3.5. ....................................................................................................... 70
4.
........................................................................................ 73
5.
............................ 76
6. .................................................................................................. 76
............................................................................................................ 77
1. (PCR) .................................................. 77
2. DNA ..................... 78
2.1. DNA
(boiling miniprep of plasmids)................................................................. 78
2.2. DNA
(maxi preparation of plasmids-alkaline lysis) ......................... 79
3. DNA ............................................................. 81
4. DNA
............................................................................................... 82
5. DNA ........................ 83
6. DNA ............................................................................................. 83
6.1. ............................ 83
6.2. ............................................................ 85
7. DNA.................................................................................. 85
8. DNA () ...................... 86
9. DNA ........................... 87
10. ................................................................................. 89
10.1. Escherichia coli89
10.2. Pichia pastoris ....... 89
11. . 90
12. Pichia pastoris
DNA ........... 91
13. .
(clone screening)....................................................................... 93
vi
-
14. .
...................................................................... 94
15.
Pichia pastoris
..................................................... 94
16.
SDS (SDS-PAGE) ................................................................................................. 95
17. (dot blot) ................. 96
18. Ni-
NTA- ...................................................................................................... 97
19. ............... 98
20. 125--Bgtx ......... 99
21.
................................. 99
22.
................................................................................................................. 100
23. (RIA)............................................................... 102
24. 671 ............................................. 103
25.
671 . ............................................................... 103
26. ,
................................. 105
........................................................................................... 107
1. cDNA hk6
pBluescript KSII ................................................................................................. 107
2. cDNA 1
(PCR)............................................................................................ 110
3. cDNA 1
pBluescript KSII, hk6 .............. 112
4. -
pBluescript KSII, hk6
cDNA 1 AChR ............................................................... 115
vii
-
5. hk6-linker-1 pPIC9,
- Pichia
pastoris ................................................................................................................. 116
5. hk6-linker-1 pPIC9,
- Pichia
pastoris ................................................................................................................. 117
6. Pichia pastoris
pPIC9 hk6-linker-1......................................................................... 121
7. .
.................................................................................................. 122
8.
....... 125
9.
................................................................................................................. 128
10.
....................................................... 129
11.
AChR
..................................................................... 131
12. AChRs
,
.......................................................................................... 134
13. AChRs
,
1 ............................................... 137
14.
.......................................................................................... 138
14.1 ........................................................................................................... 138
14.2
MG2. in vivo
1
.............................................................................................................. 139
viii
-
14.3
MG3. in vivo
.............................................................................................................. 141
....................................................................................................... 142
....................................................................................................... 143
........................................................... 153
........................................................................................................ 183
SUMMARY ......................................................................................................... 185
ix
-
ACh:
AChR:
nAChR:
mAChR:
..:
..:
Ca2+:
-Bungarotoxin (-Bgtx): -
Main Immunogenic Region (MIR):
ACh-binding protein (AChBP):
muscle-specific kinase (MuSK):
Ig:
Antigenic Modulation:
mAb:
complementarity-determing region,
(CDR):
Fetal Bovine Serum (F.B.S):
Radioimmunoassay (RIA):
Counts per minute (cpm):
Human kallikrein-6 (k6): 6
x
-
1
1.
1.1 -
.
, .
,
.
,
.
-,
.
, .
,
(Kalamida et al., 2007).
1.2
- (Ligand-gated ion channels
LGICs).
.
5- (5-) (Maricq et al., 1991),
(Gly) (Grenningloh et al., 1987), - (GABA)
1
-
(Schofield et al., 1987), (ACh) (Stroud et al.,
1990; Betz 1990a).
, ,
.
-
,
(Betz 1990b; Ortells and Lunt 1995).
,
(nAChRs)
(Changeux et al., 1987; Karlin, and Akabas,
1995).
1.3 -
(AChRs) ( 1)
ACh, ,
. AChRs,
,
,
:
) (nAChRs),
(Changeux, and Edelstein, 2001; Lindstrom,
1997)
) (mAChRs),
(Wess, 1996; Ishii, and Kurachi,
2006).
G-.
nAChRs ,
,
,
,
2
-
(..) (..),
(Lindstrom, 1997).
-
- AChRs
AChRs
ACh
1: . , , , 2 4 ( Lindstrom et al. 2000)
1.3.1
AChR
- .
:
) ,
,
Torpedo ( , )
Electrophorus.
3
-
) - ( )
.
(, , ) 2 2 (Wess, 1996; Ishii, and
Kurachi, 2006) ( 2). 2
Torpedo Electrophorus
, 2
. ,
, (Mishina et
al., 1986; Schuetze, 1986).
(Horton
et al., 1993).
2: ( Wess, 1996)
1970
AChR
(Patrick, and Lindstrom, 1973; Lindstrom et al., 1976a).
4
-
,
,
,
.
1.3.2
AChRSs
, ,
.
(2-10)
(2 - 4), ,
(McGehee, and Role, 1995).
- ,
,
.
,
.
(Role, 1992).
,
.
: )
(
nM), -,
2-6
2-4 )
( M) -
5
-
,
7-9 (Lindstrom, 2000).
,
,
,
, ,
, ,
(Gotti et al., 1997).
, Alzheimer, Parkinson, ,
,
(Lindstrom, 1997).
1.3.2.1
AChR, ,
.
,
.
,
,
,
. 4, 5, 6, 2 4,
(mesostriatal
dopaminergic neurons), , ,
,
(Champtiaux et al., 2003; Salminen et al., 2004).
7 -
, ,
GABA-
(Breese et al., 1997; Chen, and Patrick, 1997; Kawai et al.,
2002). 8
6
-
7 , 9
(Elgoyhen et al., 1994). , 10
9
(Elgoyhen et al., 2001; Plazas et al., 2005).
, 7
(Wang et al., 2003).
1.3.2.2
AChR
-, -, - (Lena et al., 1993; Wonnacott,
1997). - - -
,
, , , , 5-
- ...
,
..,
AChR (Sher et al., 2004; Jensen et al., 2005).
7 (Sher et al., 2004).
...,
.
ACh
,
7, 42 34
(Levy, and Aoki, 2002; Sher et al., 2004).
AChRs
,
Ch ,
7
-
(Smith et al., 1979; Arenella et al., 1993; Corriveau, and Berg, 1993;
Devay, and Role, 1994; Howard et al., 1995).
,
c-fos
PC12 (Greenberg et al., 1986).
Ch
,
. ,
-
AChRs .
(7, 42,
34) (Alkondon, and Albuquerque, 2004).
,
, , , ,
, , ,
(Whaley et al., 1981; Sharma, and
Vijayaraghavan, 2002; Gahring, and Rogers, 2005; Skok et al., 2006).
AChRs
(Borovikova et al., 2000)
,
(Heeschen et al., 2001, 2002).
1.4
1.4.1
DNA
DNA (cDNA)
8
-
AChR. cDNA
, , , Torpedo (Noda et al.,
1982, 1983a, b; Claudio et al., 1983). cDNA
cDNA DNA
, (Claudio, 1989).
.
Torpedo californica (, , )
56 - ,
35-50%,
(Raftery et al., 1980; Noda et al., 1983).
(Nef et al., 1986).
. 1
80% Torped (Hucho et al., 1996).
,
(Sargent,
1993). , 7 ,
93%
97% .
.
(
)
, 128 142
Torpedo.
15 (Stroud
et al., 1990).
.
9
-
(Ortells, and Lunt, 1995).
Cys- (Karlin et al., 1995).
, .
192 193
Torpedo (Kao et al., 1986).
. 1, , 7
, .
,
.
1 1 , , , , 3 2
2 , 2, 5, 7 3 4
(Mattson, and Heilbronn, 1975).
,
(Nomoto et al., 1986; Poulter et al., 1989; Shoji et al., 1992; Strecker et al.,
1994).
AChR
.
Torpedo
(Strader et al., 1979; Noda et
al., 1983b; Devillers-Thiery et al., 1983; Finer-Moore, and Stroud, 1984).
,
-.
1-4 (
15-20 )
. 210 ,
- .
, 3 4
10
-
.
(Finer-Moore, and Stroud,
1984; Yee, and Huganir, 1987).
( - 4-28 )
(DiPaola et al., 1989).
(Schofield et al., 1987; Karlin et al., 1995).
1.4.2
AChR
,
Fourier (FTIR), (CD),
Raman (Hucho et al., 1996).
Torpedo .
,
, ,
. ,
,
.
AChR -
- . FTIR
- 32-33%, -
36-43%, - ( 14-
24%) (Naumann et al., 1993).
,
- (Noda et al.,
1983b; Devillers-Thiery et al., 1983; Finer-Moore, and Stroud, 1984).
9
5 -
, - ,
(Unwin, 1993). 5 -
,
11
-
2 (Hucho et al., 1996;
Imoto et al., 1988; Leonard et al., 1988).
2 -
1, 3
4 - .
- -
(Grne-
Tschelnokow et al., 1994).
,
-
100% 25%
9 (Claudio et al., 1983; Noda
et al., 1983b; Devillers-Thiery et al., 1983; Unwin, 1993).
,
4,
4 -
. - 5
. - 2
. - (15 ) 1, 3, 4
,
(Miyazawa et al., 2003).
,
.
-
-. -
,
ACh (Unwin, 1993).
ACh -
(Miyazawa et al., 1999).
12
-
1.4.3
. AChR,
,
. ,
(
)
Torpedo (Mitra et al., 1989).
Torpedo
(Reynolds, and Karlin, 1978; Lindstrom et al., 1979; Raftery et al., 1980).
( 17),
,
.
85 (Toyoshima, and Unwin, 1990).
,
,
( 3, 4).
9,
Torpedo,
,
. 65,
.
120-125 60 ,
20 .
80 20-
25 (Unwin, 2003).
13
-
ACh
ACh
3: Torpedo. , , . . ( Unwin, 1993) 9
, ,
. ,
,
.
-
, 20-25
,
7-8.
4. ,
,
14
-
1, 3 4
. -
2 , ,
(Miyazawa et al., 2003).
ACh 4: . . , , , . (1-4), ( Changeux, 1993)
,
Torpedo, ,
1:1. 43kDa (rapsyn
receptor associated protein of the synapse),
, ( 3)
(Sealock, 1982; LaRochelle, and Froehner, 1986; Frail et al., 1988; Froehner et al.,
1990; Phillips et al., 1991, 1993; Apel et al., 1995).
.
, ,
,
15
-
(Schiebler, and Hucho, 1980; Wise et al., 1981).
(Wise et al., 1981). ,
.
Torpedo
,
(Hamilton et al., 1985; Kubalek et al., 1987; Chatrenet et al.,
1990).
(Wise et al., 1981; Holtzman et al., 1982; Karlin et al., 1983).
,
ACh ,
(Kurosaki et al.,
1987; Blound, and Merlie, 1989; Pedersen, 1990; Sine, and Claudio, 1991).
-
L , ,
,
. L: low,
ACh H: high,
(Machold et al., 1995).
,
.
,
.
16
-
1.5
1.5.1
.
: ,
,
, .
.
,
( 3,5 nm),
.
,
.
.
.
20 40nm.
.
,
.
-
.
(Kandel et al., 1995).
( )
.
(
) ,
.
17
-
(Kandel
et al., 1995).
1.5.2
, ,
.
,
Schwann (Peper et al., 1974).
2m
.
,
.
, ,
( 5).
ACh.
ACh
.
, 104
(Kuffler, and Yoshikami, 1975).
(Ca2+) .
Ca2+ ACh (Heuser et al., 1979).
. ACh
,
( )
.
18
-
5: . , ( ). . , ( ). . ( Despopoulos, 1989)
1msec
5104 . ,
,
.
. ,
- ,
,
.
(Vincent, and Wray,
1992). ACh
, .
19
-
.
- , ACh (Zubay, 1993).
1.5.3 .
AChR
,
( 6) (Katz et al., 1957; Sakmann et al., 1980;
Neubig et al., 1982; Heidmann et al., 1983; Jackson, 1989; Auerbach, 1993; Hess,
1993).
i) : ,
.
.
ii) : (. ACh)
msec.
, .
iii) :
.
ACh, .
20sec ACh
,
20msec
(Colquhoum, and Sakman, 1985; Matsubara, 1992).
,
-
,
,
ACh (Unwin, 1995).
20
-
( )
( )
( )
( )
( )
( )
6: AChR. () () (). , - ( Changeux, et al., 1993)
, .
-, ACh
, -
, .
,
(. Ca2+), ,
,
, ,
ACh.
1.5.4
- AChR
,
:
21
-
i) :
ACh ,
.
ii) : ,
ACh,
,
.
iii) : ,
ACh
.
(Cytisine),
AChR (Papke, and Heinemann,
1994).
1.5.4.1
ACh .
,
. ACh
,
AChR.
ACh
.
,
(Kurosaki et al., 1987; Karlin et
al., 1995).
(Cys192 - Cys193)
(Kao et al., 1984).
,
(Tyr93, Trp149, Tyr190, Tyr198)
(Dennis et al., 1988; Abramson et
22
-
al., 1989; Galzi et al., 1990; Cohen et al., 1991; Middleton and Cohen, 1991).
(Cys192-
Cys193)
ACh 5-100 (Mishina et al., 1985; Galzi et al.,
1991; Tomaselli et al., 1991; OLeary, and White, 1992; Aylwin, and White, 1994).
,
,
(Mishina et al., 1985).
Asp174
Asp180 Glu189 ,
ACh,
(Czajkoski et al., 1993;
Martin et al., 1996).
- (A,
B, C)
, ,
- (D, E, F),
. ACh
.
AChRs.
,
ACh (Corringer et al., 1998).
,
ACh,
(AChE).
23
-
AChR.
Epipedobates tricolor
- - (Badio, and
Daly, 1994).
1.5.4.2
- (-Bungarotoxin, -Bgtx)
- Bungarus
multisinctus. 74 8KDa,
(Kd 5X10-10 - 2X10-11)
AChR. ACh
(Karlin et al., 1986; Arias, 1997).
AChR.
( Xenopus )
AChR,
7, 8, 9
(~10-9),
/ 7/8.
-
Torpedo,
.
ACh
-
.
(Tzartos, and Changeux, 1983, 1984; Wilson et al., 1985;
Neumann et al., 1986),
(Barkas et al., 1986, 1987, 1988; Aronheim et al., 1988),
(Wilson, and Lentz, 1988; Gotti et al., 1988; Lentz, 1995).
,
24
-
Cys192 Cys193. 189-195
Torpedo
(Tzartos, and Remoundos, 1990).
Tyr189, Tyr190, Cys192, Cys193
Asp185 -,
Cys192-Cys193 (Tzartos, and Remoundos, 1990; Chaturvedi et al., 1993; Vincent et
al., 1998).
- (-Bungarotoxin, -Bgtx)
- 6,5KDa,
Bungarus multisinctus,
- (Chiappinelli, 1983). -
- / AChR
(Chiappineli, 1993; Gotti et al., 1997). -
ACh ,
(ciliary ganglia)
( , -,
kiliaris ) (Chiappinelli,
1983). 3.1 (Ravdin, and
Berg, 1979) F (Loring et al., 1984).
D- ( )
,
.
ACh,
.
(MLA)
7
(Palma et al., 1996).
25
-
1.6
AChR .
,
. 1g
(Lindstrom et al.,
1976b). ,
,
,
(. ) ,
(Sargent et al., 1984; Froehner, 1986).
,
, Torpedo
Electrophorus, ,
, (Main
Immunogenic RegionMIR) (Tzartos, and Lindstrom, 1980; Tzartos et al., 1981, 1983).
o ,
MIR,
(Tzartos et al., 1986).
,
MIR - (Whiting
et al., 1986; Heidenreich et al., 1988).
, , ,
MIR
(Ratnam et al., 1986; Barkas et al., 1987; Tzartos et al., 1988).
MIR,
,
26
-
MIR .
(Conti-Tronconi, et al., 1981; Swanson et al., 1983; Tzartos et al., 1987;
Kubalek et al., 1987; Beroukhin, and Unwin, 1995).
MIR
,
,
MIR. MIR
, ,
(Tzartos, and Lindstrom, 1980;
Tzartos et al., 1981). -
- ,
, MIR
67-76 Torpedo
(Barkas et al., 1987; Tzartos et al., 1988; Barkas et al., 1986, 1988; Tzartos, and
Remoundos, 1990). 68-71
MIR (Bellone et al., 1989;
Papadouli et al., 1990, 1993).
Torpedo (Beroukhim, and Unwin, 1995),
67-76
. MIR
,
(
).
MIR
, .
, (Tzartos et al.,
1998).
MIR , MIR
27
-
(Kontou et al., 1996).
MIR , (
) 67-76 Torpedo
,
. 67-76 Torpedo
, MIR
400 (Tzartos, and Lindstrom, 1980; Tzartos et
al., 1981).
Lymnaea stagnalis
ACh
, d-tubocurarine -Bgtx.
ACh (ACh-binding protein, AChBP),
ACh, ( 7). AChBP
- . 210
AChR,
. AChBP
MIR , 1 .
, 2,7 ,
MIR,
MIR AChR (Brejk et al.,
2001; Smit et al., 2001).
MIR
,
. MIR
,
1-3 .
50% AChRs (Tzartos, and Lindstrom,
1980; Tzartos et al., 1987; Loutrari et al., 1992; Asher et al., 1993).
28
-
) ) 7: ) ) Lymnaea stagnalis ( Brejk, et al., 2001) ,
MIR .
(Tzartos et al., 1987; Graus et al., 1995).
MIR
(Conti-
Tronconi et al., 1981; Tzartos et al., 1985; Tzartos, and Starzinski-Powitz, 1986;
Sophianos, and Tzartos, 1989; Mamalaki et al., 1993).
MIR ,
2-3 ,
. ,
. , 5
29
-
(Tzartos et al., 1985; Sophianos, and Tzartos,
1989; Tzartos, and Starzinski-Powitz, 1986).
in vivo , F(ab)2 (
) MIR
, .
F(ab)2 ,
.
,
MIR ,
(Loutrari et al., 1992).
,
MIR ,
.
MIR
(. Fab) (
,
)
(Tzartos et al., 1985; Sophianos, and Tzartos, 1989; Mamalaki et
al., 1993).
1.7
AChR
.
,
- .
,
,
.
Golgi,
.
30
-
2 , (Kao,
and Karlin, 1986; Blount, and Merlie, 1990; Anderson, and Blobel, 1981; Anderson et
al., 1982; Merlie et al., 1982; Merlie, and Lindstrom, 1983; Olson et al., 1984; Smith et
al., 1987; Green et al., 1991).
- (125I--Bgtx).
.
, ,
,
. ,
2 2.
,
2 2 (Blount et al., 1990; Blunt, and Merlie, 1991; Gu et al., 1991;
Verall, and Hall, 1992).
,
Torpedo
.
, .
,
.
ACh.
,
ACh (Green, and Claudio, 1993; Green and
Wanamaker, 1998).
AChR ,
.
1 1\2 .
10-12
(Steinbach et al., 1979).
31
-
2
2.1
ACh
,
, AChR.
,
AChRs. , ..
AChRs,
.
,
AChRs.
,
.
,
AChR,
AChRs.
2.2 AChR
2.2.1 (myasthenia gravis)
2.2.1.1
Thomas Willis ( 8),
,
.
,
.
32
-
200
Herard (Erb, 1879; Cambell, and Bramwell, 1900).
) )
8: Thomas Willis () Wilhelm Erb () ( Hughes, 2005) Friedrich Jolly,
1895
myasthenia gravis pseudo-paralytica.
.
, gravis
- .
,
14 15 .
,
,
(Jolly, 1895).
19 . 1910
, 1930
.
1935,
(Walker, 1934), -
33
-
-
(Blalock et al.,
1939). 1960 (Simpson,
1960), 1970
(Engel, and
Santa, 1971; Fambrough et al., 1973).
,
,
Electrophorus electricus (Patrick,
and Lindstrom, 1973). ,
AChR, -,
,
,
,
(Lindstrom et al., 1976; Engel, 1984; Lisak et al., 1987; Lindstrom et al., 1988;
Schonbeck et al., 1990).
,
.
,
,
.
2.2.1.2
,
( ) 125-400
(Kurtzke, and Kurland, 1992; MacDonald et al.,
2000). ( )
, .
, (Drachman, 1998; Poulas
et al., 2000, 2001).
34
-
,
,
.
,
. ,
.
(. )
(Grob et al., 1987).
-,
( 9). 15%
(ocular myasthenia gravis),
- .
,
(Grob et al., 1987).
, , .
,
,
(Grob et al., 1987). 85%
.
.
( )
(Oosterhuis, 1984).
35
-
9 : .
2.2.1.3
AChRs
.
125I -Bgtx
(Fambrough et al., 1973).
,
33%
(Pestronk et al., 1985).
.
,
.
,
(Engel et al., 1976).
. AChRs
,
(Engel et al., 1977).
.
36
-
.
,
. ,
,
,
.
, (Waud, 1971).
,
.
,
ACh
,
. ,
,
ACh
. ,
,
(Barrett, and Magleby, 1976).
2.2.1.4
,
.
37
-
,
(Vincent et al., 2000).
. (~75%)
,
(Castleman, 1966).
,
(Drachman, 1994).
AChR,
-
AChR (Sommer et al., 1990).
, .
AChRs (Kao, and Drachman, 1977).
.
-
(CD4+),
. ,
CD4+ ,
(Drachman, 1994). , ,
(p153) AChR,
10 ,
1 .
,
(Marx et al., 1990).
,
.
,
38
-
,
,
. ,
1
(Diepering, and Stepfansson, 1989; Schwimmbeck et al., 1989). 40
6
1 .
,
AChR,
(Stefansson et al., 1985; Schwimmbeck et al., 1989).
.
,
(Pirskanen, 1977).
,
.
,
.
D-
(Smith et al., 1983; Kuncl et al., 1986).
2.2.1.5 : -
(~85%)
AChR,
.
, ,
. 1
,
39
-
(Vincent, and Newsom-Davis, 1985; Fostieri et
al., 2000).
15%
AChR,
. ,
,
(Vincent, and Newsom-Davis,
1985).
IgG ( IgG4)
(muscle-specific kinase, MuSK),
.
,
MuSK
AChR (Vincent et al., 2000).
AChRs ,
:
AChRs (
)
AChRs
, in vivo -
.
, ,
(
10).
.
90%
2-3 (Kao, and Drachman, 1977; Appel, et al., 1977; Stanley, and Drachman,
40
-
1978; Drachman, et al., 1978, 1982; Loutrari et al., 1991).
AChR
.
AChRs ( )
,
(Sideris et al., 2007).
.
,
(Engel et al., 1976).
(Engel et al., 1979; Engel, and Arahata, 1987). ,
,
,
(Toyka et al., 1977).
ACh ,
( 10).
( ),
ACh,
,
.
, ,
(Bender et al., 1975; Drachman et al., 1982; Pachner, 1989).
MuSK
. ,
,
,
. MuSK,
41
-
,
(Vincent et al., 2003).
10: AChR . , ( ) .
AChR ,
.
(
) (Lindstrom et al., 1976a;
Appel et al., 1977).
.
,
,
.
,
(.
AChR) (Drachman et al., 1982).
42
-
,
,
()
.
AChR,
,
(Tzartos et al., 1982; Vincent et al., 1987; Lindstrom et al., 1988; Tzartos et al., 1991).
AChR (Lindstrom et al.,
1988).
MIR
67-76 (Tzartos, and Lindstrom, 1980; Tzartos et al., 1981, 1982, 1983,
1988, 1991).
, .
MIR, ,
(Beroukhim, and Unwin, 1995). MIR 1
,
.
. ,
. , 1
, .
,
AChR,
.
43
-
2.2.1.6
:
:
,
, .
(tensilon).
( )
(tensilon test). ( 30
) ( 5 )
(Daroff, and Seybold, 1986).
AChR:
AChR 85-90%
(Vincent, and Newsom-Davis, 1985).
(Lindstrom et al., 1976a; Appel et al., 1977),
,
.
,
125I--Bgtx (Lindstrom et al.,
1976a; Vincent, and Newsom-Davis, 1985).
:
3
/
.
.
.
>15%
(Keesey, 1989).
44
-
:
, .
.
,
(Stalberg, and Trontelj, 1979).
,
,
.
2.2.1.7
, ,
.
, ,
,
. ,
(Stalberg, and Trontelj, 1979;
Drachman, 1998).
:
ACh
, .
ACh
,
(Daroff, and Seybold, 1986).
,
(Mestinon)
45
-
.
, AChRs
.
,
35% ,
(Buckingham et al., 1976). 10-
40
(Rowland, 1987). -AChR ,
.
,
,
(Drachman et al., 1998):
.
(Kao, and Drachman, 1977; Drachman, 1994),
.
AChR.
,
.
.
o
.
.
,
.
:
46
-
. , . T-
-
(Drachman et al., 1998).
. (Prednisone) -AChR
-AChR (Tindal, 1980).
. (Sandimmune)
L-2 - (Drachman et
al., 1998).
( )
,
.
, 3-6 .
. -
.
.
4-5
, .
(down regulation)
(Drachman et al., 1998).
,
.
.
,
.
,
.
.
,
(
47
-
), AChR
( ,
).
,
-,
(Ma et al.,
1995; Barchan et al.,1999; Im et al.,1999; Lindstrom et al., 2002).
(Sophianos, and Tzartos, 1989;
Fostieri et al., 2005) (humanized
mAbs) (Papanastasiou et al., 2000),
(- ) AChR ( MIR)
,
.
Drachman ,
, -,
AChR,
(Drachman et al., 2003).
, -2 (IL-2),
. IL-2
-
.
, ,
(Lindstrom, 2002).
FTY720,
(Kohno et al., 2005).
,
,
.
(Boneva et al., 2006).
,
AChR .
, , ,
48
-
AChR ,
Pichia pastoris Cr-
. ,
(Psaridi-Linardaki et al., 2002, 2003; Kostelidou et al., 2006).
2.2.2
.
AChR, ,
ACh .
( 60) .
,
,
,
.
,
(Engel et al., 1993, 1996a, 1996b;
Ohno et al., 1995, 1996, 1997).
,
ACh,
,
. P121L (Ohno et
al.,1996), , R311W (Ohno
et al.,1997), .
(fast channel
syndromes), .
.
ACh,
(Sine et al., 1995).
49
-
.
2 ,
(Gomez, and Gammack, 1995;
Gommez et al., 1996; Croxen et al., 1997).
1
(Engel et al., 1996a; Ohno et al., 1997),
ACh 2.
,
(slow channel syndromes).
.
(Ohno et al.,1997).
,
, .
2.3 AChR
2.3.1 Alzheimer
Alois Alzheimer
1907 .
,
( 65
) (
65 ). ,
, ,
, , .
: ) (
50
-
)
) ,
(, , )
.
(40-42 ),
-,
, -.
.
Alzheimer
,
.
,
ACh (Kihara et al., 1997; Martin-Ruiz et al., 1999; Wang et
al., 2000).
( 90%) ACh (Kihara et
al., 1997; Hardy, 1997; Li, and Buccafusco, 2003).
.
42 .
,
4 7
30%.
(mRNAs) (Warpman, and
Nordberg, 1995; Martin-Ruiz et al., 1999).
7
Alzheimer.
51
-
. ,
PC12 7 ,
, (Kihara et
al., 1997; Li, and Buccafusco, 2003). ,
( ),
AChRs
(Sahakian et al., 1989; Benowitz, 1996; Hardy et al., 1997;
Newhouse et al., 2001). -
AChR,
.
( ) ,
(Arneric et al., 1994; Martin et al., 1994).
,
( )
, ,
(Collins,
and Marks, 1996; Peng et al., 1997).
2.3.2 Parkinson
Parkinson
,
(Lloyd et al., 1975).
.
AChR,
52
-
.
L-DOPA,
(Coleman, 1992).
, ,
Parkinson (Morens et al., 1995).
,
,
AChR,
.
2.3.3
,
2% .
,
(autosomal dominant nocturnal frontal lobe epilepsy),
4 AChR.
, (Scheffer
et al., 1995; Steinlein et al., 1997).
AChRs
.
,
, .
7 (15q14) AChR
(rolandic epilepsy) (Neubauer et al., 1998).
53
-
2.3.4
,
.
, ,
(Arnold, and
Trojanowski, 1996).
.
,
ACh.
.
7 ACh
(Freedman et al., 1997).
-
- 7
,
(Freedman et al., 1997).
2.3.5
- -
AChRs,
.
400.000 ,
250.000.000
(Koop, 1988; Peto et al., 1992).
AChRs ,
,
AChRs
.
,
54
-
(Dani, and Heinemann, 1996; Pich et
al., 1997).
, .
, ,
,
.
,
,
.
AChRs .
,
, ,
.
,
AChRs.
AChRs
. , 42 7
. ( AChRs),
(
),
,
( )
(Henningfield, 1984; Collins, and Marks,
1996; Peng et al., 1997).
,
. ,
(
42) (Peng et al., 1997).
55
-
3
-
3.1
3.1.1.
.
,
.
( )
, ( 11).
,
,
. ( ,
),
(),
.
,
.
, .
,
.
, -
-, .
56
-
11: T . , ( Janeway- Travers, 1999)
3.2
3.2.1
,
.
.
,
(Janeway
et al., 1999).
57
-
. 1950
,
:
,
.
DNA
,
.
-
, .
- .
, ,
-
. ,
- ,
,
.
3.2.2
T
.
, .
, ,
,
.
: IgA, IgD, IgE, IgG IgM.
IgA 15-20%
. ,
IgA1 IgA2. , IgA ,
, .
58
-
IgD 1% ,
-.
.
IgE
.
, , .
IgG (70-
75% ). IgG1, IgG2 , IgG , IgG4
(- -),
.
.
IgM 10%
970 kD.
(Janeway et al.,
1999).
3.2.3
,
. (L)
(H) 25 50kD
, L2H2.
: L H
, H S-S
( 12).
, (Amzel, and Poljak, 1979;
Chothia, and Lesk, 1987). L H
(Variable, V) (Constant, C) .
L , VL
CL , H VH
59
-
CH1, CH2 CH3. H
,
.
L 214 . VL
108 ,
CL 109-214.
,
, 40% .
, 191
.
446 . (V)
(C) (VH CH). (C) 3 ( 4)
, CH1, CH2, CH3 ( CH4),
L.
(108 )
.
, H L ,
.
(hypervariable regions),
(CDR, complementarity-determing regions)
(antigen-binding site)
(Wang et al., 1991). -
H L o
.
30, 50 95 (CDR-1 CDR-2 CDR-3)
.
.
Fv ,
.
().
, .
60
-
.
12: . Fab Fc , Fab (Fab)2 ( Janeway- Travers, 1999)
( 12). ,
Fab Fc , (Fab)2 .
Fab VH CH1
, Fc CH2 CH3 . (Fab)2
VH CH1
. Fab
-
, .
3.3
.
61
-
, Kohler
Milstein, 1975
.
. -
.
,
. -
. ,
.
.
-,
.
:
.
.
-.
, .
.
-,
.
.
.
, ,
,
(Kohler, G., and Milstein, C., 1975).
62
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Kohler+G%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Milstein+C%22%5BAuthor%5D
-
.
.
( ) (Vincent,
A., 2002).
.
100.000 (: 1/10.000
) (Poulas, et al., 2001; Vincent, A., 2002).
.
,
(Vincent, A., 2002).
AChR , -
,
(Devillers-Thiery, et al., 1993). 5 (2 2)
ACh.
-AChR ,
AChR (Tsouloufis et al., 2000).
- AChR
AChR
.
AChR (
)
(Tzartos et al., 1982; Heidenreich et al., 1988; Graus et al., 1997).
-AChR
63
-
,
.
(
) -AChR
(Vincent, A., 2002; Tzartos et al., 1998).
-
AChR -
( )
.
,
AChR.
,
,
( AChR)
.
,
in vitro,
(671 ) in vivo
.
64
-
1.
,
:
P.C.R, MJ Research PTC-200 ( )
SHEL LAB
,
MRC
/ JEN WAY 6305
3
GENE PULSER, PULSE CONTROLLER GENE TRANSFORMER,
BIORAD.
: Sorvall RC5C ( GS3 SS34)
(corex)
Juan CR422S SORVAL RT 6000B
Eppendorf mini spin, HETTICH MIKRO 120
Beckman L8-80M ( Vti-80)
Hetovac VR-I, Heto
Mini-Protean III BIORAD
Gel Air Dryer BIORAD
Amicon RGS Millipore Minitan
system
LKB 2115
SELECTA - PRECISTERM
- WALLAC 1275 MINIGAMMA, Kontron, Analytical
MDA 212
65
-
, THERMO
FORMA CO2 incubator
, LAB CAIRE HLF-12 UV
, Raypa STEAM STERILIZER
, MEMMERT
2.
GREINER
15 50 mL
FALCON CORNING
1,5 mL GREINER
0,25 mL COSTAR
(tips),
1,0, 2,0, 5,0, 10,0 25,0 mL, Cellstar
GREINER
(tips) EPPENDORF
(5 mL quick-seal ultracentrifuge tubes)
BECKMAN
0,1 cm BIORAD 0,2 cm
NVITROGEN
1cm 0,25cm,
SARSTEDT
0,22 0,45 m WHATMAN
PVDF, Hybond C Hybond C-extra
DE 81 3
WHATMAN
SPECTRUM
Kodak X-Omatic AR Polaroid Type 57 Amersham International
P.L.C
66
-
3. -
3.1
(analytical grade)
APPLICHEM, SERVA, SIGMA, B.D.H Chemicals Ltd, DIFCO,
PHARMACIA, MERCK FLUKA
SIGMA
Taq DNA Minotech Molecular Biology Products (IMMB)
GIBCO BRL
Pfu DNA PROMEGA
MINOTECH, Molecular Biology Products (IMMB)
dNTPs IPTG PROMEGA
BRL, FERMENTAS, MINOTECH,
Molecular Biology Products (IMMB), New England Biolabs PROMEGA
: Prestained Protein Molecular Weight
Marker, FERMENTAS
DNA:
pGEM/(HinfI RsaI SinI) PROMEGA
174 RF DNA/HaeIII fragment GIBCO, BRL
/HindIII Promega
GeneRuler DNA Ladder 100bp, 1kb, FERMENTAS
(CIAP, 26 U/l) 4 DNA
(1U/l) GIBCO, BRL
, RNAse A, SIGMA
Bacto-tryptone, Bacto yeast extract Bacto agar DIFCO Laboratories
Bristol Mayers Squib, Invitrogen
TEMED, APS, DAB, DTT, ABTS SIGMA
(BSA) SIGMA
(developer) (fixer) Kodak
Ni2+-NTA- QIAGEN Inc.
, CNBr-activated
Sepharose 4B (PHARMACIA-BIOTECH)
-Bgtx SIGMA
67
-
(FBS), GIBCO
3.2 (kits)
QIAquick Gel Extraction Kit QIAGEN Inc. :
QUAquick spin QG, PE, EB (
QIAquick Spin Handbook)
QIAquick PCR Purification it QIAGEN Inc, :
QUAquick spin PB, PE ( QIAquick
Spin Handbook)
QIAquick Nucleotide Removal Kit QIAGEN Inc. :
QUAquick spin PN, PE ( QIAquick
Spin Handbook)
3.3. (primers)-
:
) 1 (1-211) cDNA
pcDNA 3.1
1 Forward (30 )
5 gct ggc ctc gtc tct aga tcc gaa cat gag 3 Xba
1 Reverse
(38 )
5 atg acg ttg acg gcg gcc gct cag ggc agg cgc tgc at 3 NotI
, Forward
Xba, Reverse
NotI.
1,
pBluescript II KS.
) (DNA-sequencing)
DNA pPIC9
hk6 :
68
-
hk6-BamHI (44 )
5 gat cca gaa gac cat tca ggc caa gac tag tgg gac tcc gtt ct 3
3 1 (21 )
5 gca aat ggc att ctg aca tcc 3
) - (linker peptide)
hk6,
:
Linker Forward (44 )
5 gat cca gaa gac cat tca ggc caa gac tag tgg gac tcc gtt ct 3 BamHI SpeI
Linker Reverse (44 )
5 cta gag aac gga gtc cca cta gtc ttg gcc tga atg gtc ttc tg 3 XbaI
BamHI, SpeI, XbaI.
3.4
:
mAb198 (produced in rats) (Tzartos et al., 1982):
,
.
anti-FLAG mAb M2 (Sigma, St Louis, MO, USA):
,
FLAG (DYKDDDDK)
mAb73 (Tzartos et al., 1982):
mAb67 (Tzartos et al., 1982),
Goat-anti-human polyclonal IgGs (Lampire Biological Laboratories, Pipersville, PA,
USA):
.
69
-
Rabbit-anti-rat polyclonal IgG-HRP (peroxidase conjugated) (Lampire Biological
Laboratories, Pipersville, PA, USA):
, DAB
.
3.5.
-
(ddH2O).
(E. coli P. pastoris)
(20 min 15 lb/inc.2) 0,2
m.
LB (Luria Bertani) LB-agar
1% (w/v) NaCl, 1% (w/v) , 0,5% (w/v) , pH 7,0 LB 1,5% (w/v) agar
SOC 0,5% (w/v) , 2% (w/v) , 10 mM NaCl, 2,5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM
YPD 1% (w/v) , 2% (w/v) , 2% (w/v)
YPD-agar YPD 2% (w/v)
YPDS 1% (w/v) , 2% (w/v) , 2% (w/v) , 1
YPDS-agar YPDS 2% (w/v)
BMGY 1% (w/v) , 2% (w/v) , 100 mM , pH 7,0, 1,34% (w/v) , 4x10-5% (w/v) , 1% (v/v)
70
-
BMMY 1% (w/v) , 2% (w/v) , 100 mM , pH 7,0, 1.34% (w/v) , 4x10-5% (w/v) , 0,5% (v/v)
DMEM Dulbeccos Modified Eagles Medium, TE671
89 mM Tris-H3BO3, pH 8,0, 2 mM EDTA TAE 40 mM Tris-CH3COOH, pH 8,0, 5 mM EDTA TE (10:10) 10 mM Tris-HCl, pH 8,0, 10 mM EDTA TE (10:1) 10 mM Tris-HCl, pH 8,0, 1 mM EDTA TE (1:0,1) 1 mM Tris-HCl, pH 8,0, 0,1 mM EDTA PBS 60 mM pH 7,4, 137 mM
(0,8% ..) NaCl, 2,7 mM (0,02% ..) KCl TBS 25 mM Tris-HCl, pH 7,4, 137 mM (0,8% ..) NaCl, 2,7
mM (0,02% ..) KCl TBST 10 mM Tris-HCl, pH 7,5, 10 mM (0,058% ..) NaCl, 0.1%
(v/v) Tween-20 PB 25 mM Tris-base, 200 mM , 1% (w/v) SDS, pH 8,5 0,1 M Tris-HCl, pH 7,5, 0,1 M MgCl2 filter assay
20 mM Tris, pH 7,5, 0,5% (v/v) Triton
STET 10 mM Tris-HCl, pH 8,0, 100 mM NaCl, 1 mM EDTA, 5%
(w/v) Triton X-100 GTE 50 mM , 25 mM Tris-HCl, pH 8,0, 10 mM EDTA
45 mM Tris-HCl, pH 6,8, 10% (v/v) , 1% (w/v) SDS, 0,01% (w/v) , 50 mM DTT [ 5% (v/v) -]
10% (w/v) CH3COOH, 40% (v/v) , 0,25% (w/v) Coomassie R-250
40% (v/v) , 10% (w/v) CH3COOH
Ponceau S
0,2% (w/v) Ponceau, 0,5% (v/v) TCA
DAB
0,03% (w/v) DAB, 0,03% (w/v) NiCl2, 0,009% (v/v) H2O2 PBS
71
-
0,2 M Glycine-HCl pH 2,8
1mM HCl
0,1M NaHCO3, pH 8,3, 0,5M NaCl
0,1M Tris-HCl, pH 8,0
pH
0,1M sodium acetate, pH 4,0, 0,5M NaCl
pH
0,1M Tris-HCl pH 8,0, 0,5M NaCl
pH
0,1M sodium acetate, pH 4,5, 0,5M NaCl
pH
0,1M Tris-HCl buffer pH 8,5, 0,5M NaCl
.
65C, .
0,1% .. ,
,
,
. , 0,5 M
Tris-HCl, pH 8,0 15 min. ,
.
0,1 Tris-HCl, pH 8,0, pH
7,8. 1/10
10 mM Tris-HCl, pH 8,0, 1 mM EDTA.
4C.
72
-
4.
E. coli
E. coli XL1-Blue, 12, TOP10F
LB
.
DNA E. coli pBluescript II KS,
( 13):
13 : pBluescript II KS : (i) (APr), (ii) (multiple cloning site, M.C.S) , (iii) - (beta-galactosidase alpha peptide) (blue/white colour selection)
73
-
Pichia pastoris P. pastoris ,
(Gregg et al., 2000).
, ,
.
P. pastoris
,
Saccharomyces cerevisiae.
, ,
P. pastoris ,
, .
, P. pastoris ,
.
P. pastoris ,
,
.
.
. ,
1 2, . AOX1
.
, P. pastoris ,
, .
P. pastoris ,
GS115 KM71. TOP10F
E. coli. , pPIC9 ( 14),
AOX1, .
74
-
14: pPIC9. : (i) (Ampicillin), (ii) , (iii) (iv) o AOX1
75
-
5.
in vitro
ChR,
671.
(medulloblastoma),
RD
. AChRs
,
(Luther et al., 1989).
ATCC (American Type Culture Collection),
Dulbeccos Modified Eagles Medium (DMEM), 10-15%
(fetal bovine serum-FBS), 370C
, 5% CO . 2
6.
in vivo
AChR,
(experimental myasthenia gravis-
EAMG)
LEWIS.
7.
,
.
,
.
76
-
1. (PCR)
,
,
DNA.
, (primers)
- DNA-
DNA , ,
(Chien et al., 1976; Mullis, and Faloona, 1987).
DNA,
5
, DNA, .
PCR
DNA, .
DNA PCR, 10 ng DNA
25 pmoles . dNTPs
2,5 mM .
20 mM Tris-HCl, pH 8,8, 10 mM
KCl, 10 mM (NH4)2SO4, 2mM MgSO4, 0,1% .. Triton X-100, 5 g BSA.
5U Taq DNA .
( ), Pfu ,
. ,
- ,
DNA,
DNA.
PCR
,
,
DNA
.
MJ Research PTC-200.
, 94C, 5 min,
77
-
DNA. 30
( ):
94C, 1 min
60C, 1 min
72C, 2 min
31 , PCR (5 l
) , 1 2% ..
2. DNA
DNA ,
DNA.
DNA , (1:0,1)
4C 20C 70 C
.
2.1. DNA
(5 g) , DNA
, DNA,
,
(Triton -100) (Holmes, and
Quigley, 1981). ,
DNA,
. DNA
,
.
, ,
DNA ,
,
.
3 mL (LB 100 g/mL ),
37C
. 1,5 mL 10.000 rpm, 1 min,
78
-
. ,
600 l STET. ,
.
, 75 l 10 mg/mL
, 2 min. ,
. 10.000 rpm,
10 min, , DNA
, DNA RNA
. 400 l ,
, .
80C, 60 min.
10.000 rpm, 15 min,
70% ..
40 l (1:0,1).
,
20 l . ,
DNA, (Rse)
20 g/mL, 65C, 5 min,
.
2.2. DNA
( mg)
DNA ,
(Birnboim, and Doly, 1979). O ,
, (PEG) (Lis, 1980).
, NaOH,
SDS. K CH3COOK, DNA
,
DNA , ,
SDS +. RNA
RAse. DNA
PEG.
79
-
() DNA
5 mL (LB 100 g/mL LB
50 g/mL ),
, 37C, 16 h. 1 mL
1 L
. , 37C, 16 h.
5.000 rpm, 15 min, 4C, Sorvall,
GS3. 40 mL
GTE ,
.
, 200 mg
5 min . DNA,
80 mL 0,2 NaOH / 1% (w/v)
SDS , 10 min. ,
60 mL 3 , pH 5,5,
, 10 min.
( , , DNA RNA)
5.000 rpm, 10 min, 4C,
Sorvall, GS3.
.
0,6 , , 30 min,
10.000 rpm, 15 min, ,
Sorvall, GS3. DNA
70% .. , 15 mL
(10:10).
() DNA
(PEG)
DNA Corex
5 M LiCl, RNA.
10.000 rpm, 10 min, 4C,
Sorvall, SS34.
Corex, ,
10.000 rpm, 10 min, ,
80
-
Sorvall, SS34. DNA 70%
.. , , 1 mL (1:0,1)
(Rase) (20 g/mL) -
(Dase). 30 min, .
, 1 mL 1,6 NaCl, 13% (w/v) PEG 8.000.
10.000 rpm, 5 min, 4C.
800 l (1:0,1)
: ) ) :
, 1:1 ) .
. DNA, 1/4
10 CH3COONH4
, 10 min, 10.000 rpm, 2 min,
4C. DNA 70%
.. 500 l (1:0,1).
3. DNA
DNA
,
, DNA (Sambrook et al., 1989).
260 nm
:
DNA 1 .D.260nm 25 g DNA/mL (1)
DNA 1 .D.260nm 50 g DNA/mL (2)
100
C = A
1,5 x NA + 0,71xNC + 1,2xNG+0,84xNT (3)
, C , ,
pmol/l, , , NC, NG NT
, , , .
81
-
4. DNA
DNA
, DNA.
, DNA
. , ,
( ),
(McDonell et al., 1977; Southern, 1979).
, ,
. DNA
(260-360 nm),
( )
DNA.
, DNA,
.
.
)
.
, 0,5 g/mL
.
)
DNA,
1-2% .. .
0,5 g/mL ,
, 100 mV.
DNA,
1% ( DNA>1000 bp), 2% ( DNA
-
DNA.
Polaroid Type 57.
DNA,
,
20-30 mA.
-0,5 g/mL
, ,
DNA.
5. DNA
DNA o
260 nm 280
nm. DNA .D.260nm/O.D.280nm = 1,8.
1,5 50% : 50%
DNA.
DNA 1:0,1 ddH2O,
0,1 1,0.
,
(260 280 nm) O.D.260/O.D.280
.
6. DNA
6.1.
DNA, ,
. :
,
.
DNA
, .
, 10 g
DNA.
83
-
QIAquick Gel Extraction Kit (QIAGEN Inc.)
DNA ,
,
, ,
.
( )
DNA. (
)
(.. 100 mg 300 l
) 50C, 10 min,
. QIAquick spin column
, 13.000 rpm, 60 sec,
DNA .
0,75 mL (
), 80% .. .
13.000 rpm, 60 sec. , ,
DNA
ddH2O 10 mM Tris/HCl pH 8,5
13.000 rpm, 60 sec.
DNA .
(QIAquick spin columns), ,
10 g DNA, 70bp 10 kbp. DNA,
, 70-80% , .
QIAquick PCR purification Kit (QIAGEN Inc.)
DNA
,
, .
5 mg DNA.
PCR ,
.
PCR 5 (
)
. 10.000 rpm, 60 sec,
. ,
84
-
0,75 mL 10.000 rpm,
60 sec, . (
) DNA
50 l ddH2O 10 mM Tris-HCl, pH 8,5
10.000 rpm, 60 sec .
6.2.
,
DNA , DNA
.
.
,
DNA , ,
, - 1:1 .
(10.000 rpm, 1 min). ,
, .
7. DNA
,
, , ,
.
,
, ,
. DNA,
, :
85
-
1/10 3 , pH 5,2
.
1/4 5 CH3COONH4, pH 7,5
.
MgCl2, 10mM 5 ,
DNA, ..
( ), PCR.
, ,
80C, 30 min 20C, ,
. 10.000 rpm,
15 min, 4C
70% .. .
, ddH2O
(1:0,1).
8. DNA ()
, 1 g DNA
1-3 U , 10-20 l,
,
3-4 h. (1U)
1 g DNA 60 min.
,
.
1/10 ,
, ,
.
,
.
86
-
9. DNA
4 DNA
,
DNA , ,
DNA . ,
(complementary
ends), (blunt ends). ,
,
. ,
, :
DNA
, DNA,
DNA
,
,
, DNA ,
DNA () .
, ,
DNA
, .
, ,
,
(calf intestine alkaline phosphatase, CIAP).
5 DNA. DNA
,
.
E. coli ,
.
DNA, 90 l
10 mM Tris-HCl, pH 8,3 15 g (
QIAquick) DNA 10 l
87
-
, 1 l (0,1 U) CIAP
(Fermentas) 37C, 30 min.
EDTA, pH 8,0 5 mM
75C, 10 min. ,
/ 1:1 .
DNA
50 l (1:0,1).
4 DNA
50 mM Tris-HCl, pH 7,6, 10 mM MgCl2, 1 mM ATP, 1
mM DTT, 5% (w/v) PEG 8.000, 100 ng DNA
DNA.
DNA 10-30 l (
,
DNA). ,
DNA, .
DNA,
,
E. coli
.
,
DNA
1:1 3:1.
, ,
DNA.
,
DNA
. , DNA
10:1.
1l (1U/l) 4 DNA ,
16C. ,
,
88
-
DNA .
20C .
DNA
, DNA
(5 3 3 5).
DNA.
10.
10.1. Escherichia coli
E. coli XL1-Blue, 12, TOP10F
LB
. 37C,
. , , ,
15 50 mL,
, 500, 1000 2000 mL
, , .
1620 h.
50% .. ,
20C 20% , 80C. ,
, , 4C 1-2
4C
.
10.2. Pichia pastoris
Pichia pastoris, GS115 71.
GS115
(histidinol dehydrogenease gene (his4)) .
KM71 (arg4)
89
-
.
YPD ,
30C.
P.
pastoris , 20C 80C,
YPD, 48 h, 30C 15%
.. [ O.D.600 nm = 50-100 ( 2,5-5,0 104 /mL
)]. , ,
4C 1-2 4C,
.
11.
CaCl2, ,
,
(Sambrook et al., 1989).
,
.
.
0,5-2,0 108 /g DNA.
(CaCl2)
10 mL LB
, 37C, . 0,5 mL
100 mL LB. ,
37C, .D.600 nm = 0,5. ,
, 10 min, 50 mL
4.000 rpm, 10 min, 4C.
. 20 mL
0,1 CaCl2. 4.000
rpm, 10 min, 4C 4 mL
0,1 CaCl2.
20% .. ,
.
90
-
200 l 1,5 mL,
- , .
80C, 6 .
100 l
5 ng DNA
, , 37C.
. 50 l
1-2 l
( ) ,
15 mL , 30 min.
42C 90 sec,
1-2 min. 1 mL 37C
LB , 37C, 1 h.
, , Petri,
, 37C, .
12. Pichia pastoris
DNA
pPIC9
P. pastoris (AOX1). K
,
DNA (Dower et al., 1988).
,
,
,
. ,
,
, .
103-104 /g DNA.
91
-
5 mL YPD
P. pastoris
YPD. , 30C,
16 h. 50 l 500
mL YPD 30C, 16 h.
1.500 g, 5 min, 4 C
500 mL , dd2.
, , 250 mL
ddH2O 20 mL 1 .
1 mL
1 ,
1,5 mL. 24 h
.
P. pastoris
80 l
, 5-10 g DNA
( ),
, , .
, 10 .
0,2 cm 5 min,
4C. ,
GENE PULSER 25 F 1,5 kV
PULSE CONTROLLER 200 ohms.
, 1 mL 1 ,
15 mL , , 30C
1-2 h. , YPDS 10
g/mL . 30C, 48-72 h.
92
-
13. .
hk6 (1-244)-
-1 (1-211),
pPIC9,
.
(signal peptide- Saccharomyces cerevisiae),
.
. Pichia pastoris,
, BMGY
50 mL .
GS115 5 mL BMGY,
71 12 mL BMGY. (~280 rpm),
16-20 h, 30C.
2.500 rpm, 10 min, 20C 5 mL
BMMY 0,5% (v/v) , .
5 , (~280 rpm), 30C.
,
0,5% (v/v), .
24, 48, 72, 96 h, ,
.
(
1 ) (dot blot)
PVDF (mAb198)
1 .
- DAB
-
(Rabbit-anti-rat polyclonal IgG-HRP-peroxidase conjugated, Lampire
Biological Laboratories, Pipersville, PA, USA).
93
-
( )
, .
14. .
, Pichia pastoris,
5 mL BMGY
(~280 rpm), 16-20 h, 30C. 0,5 mL
500 mL BMGY
(~280 rpm), 16-20 h, 30C.
O.D.600nm ~2,0. ,
3.500 rpm, 10 min,
,
0,5 % (w/v)
. P. pastoris
, , 250 500 mL
1 2L ,
.
,
4.500 rpm, 20 min, 4C.
15.
Pichia pastoris
.
. , NH4(SO4)2,
~60% , 0,05% (w/v) NaN3, 1 mM PMSF
1mM NaF. NH4(SO4)2 ,
. O
5000 rpm, 4C, , 4C.
ddH2O
94
-
50 mM, pH 8,0
, 24 h.
16. SDS
(SDS-PAGE)
, ,
(SDS) (Laemmli, 1970). ,
SDS, (
SDS
)
.
,
( ),
,
( ).
,--- (-
). 0,1% (w/v) SDS Tris-HCl 0,375,
pH 8,8 Tris-HCl 0,125, pH 6,8
.
- 0,05% v/v
,,',',- (TEMED), 0,05% (w/v)
(APS) .
45mM
Tris-HCl, pH 6,8 10% (v/v) , 0,5% (v/v) -
50mM DTT, 1% (w/v) SDS, 0,01% (w/v)
2-3 .
(- DTT)
.
.
Mini Protean
II (Biorad). 25mM Tris pH
95
-
8,5, 1% SDS (w/v) 0,2 M ,
150 Volts .
(
).
: 0,25% (w/v) Coomassie 10% (v/v)
45% (v/v) .
10% (v/v) 40% (v/v) ,
.
17. (dot blot)
(western
blotting), ,
PVDF,
. ,
, , (dot blot).
,
. .
5% .. PBS ( TBS),
, 30 min, 37C. ,
, ,
, ,
. ,
5% .. PBS ( TBS), 16 h,
4C, . ,
mAb198
AChR. ,
PBS 10 min .
(HRP) -
(-rat IgG-HRP), 1:500 5% .
PBS, 1 h, , .
96
-
, PBS, 0,1% (w/v) Triton X-
100 10 min .
DAB. 3,3- (DAB)
, H2O2, .
,
, .
2-3 min
. ddH2O,
.
18. Ni2+-NTA-
,
(6xHistag), , ,
,
, , (Hochuli
et al., 1987). 6xHistag Ni2+-NTA
,
, pH ,
, o
.
Ni2+-NTA
2 mL, 50% ..
Ni2+-NTA- , 1.000 rpm, 5 min,
. Ni2+-NTA
: ddH2O 50 mM
(PB), pH 8,0, , ~10 min.
1.000 rpm, 5 min,
.
100 mL P. pastoris,
, 50 mM
PB, pH, 8,0, Ni2+-NTA-.
97
-
,
, .
, 50 mM
PB, pH 8,0, 0,5 M NaCl, 0, 3, 7 10 mM .
, 30 min,
, , 1.000 rpm, 10 min, 4C.
Ni2+-NTA-
.
50
mM PB, pH, 8,0, 0,5 M NaCl, 50, 100, 150 1000 mM ,
.
SDS-PAGE ,
,
-.
19.
,
. ,
.
280 nm (A280nm )
(Mach et al., 1992):
Molarity = (A280 x ) / (5500 NW + 4910 NY + 125 NS-S)
(NW NY
) S-S .
98
-
20. 125--Bgtx
125--Bgtx
1 125--Bgtx-1ECD
DE81. 125--Bgtx
.
50,000 cpm 125--Bgtx
10 mM PB-0.2% BSA 50 l, 4C,
.
1-2 DE81,
, 1 mL Tris-Triton.
3 1 mL
Tris-Triton
,
. ,
, .
- - ,
.
21.
~1 mg
(1: 1-210, :
1-221, : 1-218 : 1-219)
0,3 g CNBr-Sepharose 4 (Pharmacia
Biotech) 0,1 NaHCO3, pH 8,3, 0,5 M NaCl, 1
h ( 4 0C).
( )
0,1 Tris-HCl, pH 8,0, 0,5
NaCl 2 h .
0,1 , pH 4,0, 0,5 M NaCl,
0,1 M Tris-HCl, pH 8,0, 0,5 M NaCl. , 1 mg BSA
0,5 g CNBr-Sepharose 4 (PharmaciaBiotech).
99
-
.
PBS, pH 7,4
0,05% (w/v).
22.
0,5mL ( PBS, pH 7,4),
( ~0,5 mg
), 4 0C, .
,
.
,
.
,
. ,
,
.
(RadioImmunoAssay-RIA).
: )
(%)
(. -1 , -
) ) ,
, in vitro in vivo
.
1
100
-
.
pH ( ~ 1 min 0,2M
Glycine pH 2,8). 0,2M PB
pH 7,4 pH, PBS, pH 7,4.
1
,
(cross-reactive subpopulations).
-
. -1
, ,
, .
1 .
Sepharose 4B CNBr
.
, .
.
.
,
(Sargent et al., 1984; Froehner, 1986). , IR
1 . ,
.
,
125--
Bgtx.
101
-
23. (RIA)
)
( BSA),
14 fmoles (
: TE671- , CN21-
) 50,000 cpm (800,000 cpm/pmole) 125I--
gtx ( 4 0C), 50 l PBS, 0,2% BSA. 2h
4C. 10 l - - (goat
anti-human IgGs) 1,5 h 4C. 3
1 mL PBS, 0,5% Tween-20 -.
: 100 x [(cpm
Sepharose-BSA) (cpm Sepharose-)]/
(cpm Sepharose-BSA).
) () -
14 fmoles 50,000 cpm (800,000
cpm/pmole) 125I--gtx.
PBS, 0,2% BSA, 50 l. 2h
4C. 10 l - -
1,5 h 4C. 3 1 mL PBS, 0,5%
Tween-20
-.
, (14 fmoles),
(
).
(moles/L) ,
, :
102
-
[ (cpm) /
(cpm) )] /
[ (cpm)
(cpm) )] 14 fmoles / / .
24. 671
671
DMEM R+ 10% FBS
(100U/ml) (100g/ml).
, 37 C 5% CO2 .
(scraping)
(confluency).
5 7
confluency . Trypan
blue test (Cook et al., 1989) 90%.
25.
671
(671),
AChRs
, ( ,
)
.
.
5 7 confluency ,
48 (~5105 /) ( 15).
103
-
A
+ -
Bgt
x+1
25I
-Bgt
x
A +
125 I -
Bgt
x
A
+ -
Bgt
x+1
25I
-Bgt
x
A +
125 I -
Bgt
x
15: 48 . . , . O 500/ .
37 C,
. 24h
200
(
). 37 C.
(A, B, C )
-Bgtx (cold toxin)
100 l ( - ).
A, B, C , 100 DME
R+ . 15min
125--Bgtx (hot toxin) 50 mM.
1h .. 3 500 /
DMEM 125--Bgtx
. 0.5 NaOH (200 / ),
104
-
-.
.
cpm, cpm
tetraplicates.
cpm = cpm (hot toxin) cpm (hot + cold toxin)
:
. (%) = 1- [cpm / / cpm ] 100
26.
, Lewis.
, 3-4
( 16).
LEWIS rats E 16: :
) AChR.
0.2,
.
) ,
AChR (
).
105
-
) ,
AChR, .
- ,
, .
, -
.
.
,
.
, .
,
.
,
. (grasping
test) , .
(double blind test)
.
:
(0) : -
(0,5) :
(1) : ,
(2) :
(3) : , ,
(4) :
106
-
,
AChR, ,
,
in vitro in vivo .
1
AChR, hk6 (human kallikrein-6) (Xavier
Gomis-Ruth et al., 2002) .
c-DNA 1
( 1-211) hk6 ( 1-
244) pPIC9.
DNA, .
- hk6 (1-244),
-, 4 1
(1-211).
hk6
Pichia pastoris
~ 10mg/ L (Xavier Gomis-Ruth et al., 2002).
1 (0,2- 0,5 mg/ L ,
Psaridi-Linardaki et al., 2002)
,
hk6.
1. cDNA hk6 pBluescript
KSII
2-
() , .
hk6
107
-
, neurosine . ( 223
) 244 ,
16
5 (Xavier Gomis-Ruth
et al., 2002).
)
1 mkklmvvlsl iaaawaeeqn klvhggpcdk tshpyqaaly tsghllcggv lihplwvlta
61 ahckkpnlqv flgkhnlrqr essqeqssvv ravihpdyda ashdqdimll rlarpaklse
121 liqplplerd csanttschi lgwgktadgd fpdtiqcayi hlvsreeceh aypgqitqnm
181 lcagdekygk dscqgdsggp lvcgdhlrgl vswgnipcgs kekpgvytnv crytnwiqkt
241 iqak
) 1 atgaagaagc tgatggtggt gctgagtctg attgctgcag cctgggcaga ggagcagaat
61 aagttggtgc atggcggacc ctgcgacaag acatctcacc cctaccaagc tgccctctac
121 acctcgggcc acttgctctg tggtggggtc cttatccatc cactgtgggt cctcacagct
181 gcccactgca aaaaaccgaa tcttcaggtc ttcctgggga agcataacct tcggcaaagg
241 gagagttccc aggagcagag ttctgttgtc cgggctgtga tccaccctga ctatgatgcc
301 gccagccatg accaggacat catgctgttg cgcctggcac gcccagccaa actctctgaa
361 ctcatccagc cccttcccct ggagagggac tgctcagcca acaccaccag ctgccacatc
421 ctgggctggg gcaagacagc agatggtgat ttccctgaca ccatccagtg tgcatacatc
481 cacctggtgt cccgtgagga gtgtgagcat gcctaccctg gccagatcac ccagaacatg
541 ttgtgtgctg gggatgagaa gtacgggaag gattcctgcc agggtgattc tgggggtccg
601 ctggtatgtg gagaccacct ccgaggcctt gtgtcatggg gtaacatccc ctgtggatca
661 aaggagaagc caggagtcta caccaacgtc tgcagataca cgaactggat ccaaaaaacc
721 attcaggcca agtga
17:
-6. ) , ) DNA
( Pampalakis G. & Sotiropoulou, G., 2005)
108
-
hk6 ( 17) pPIC9,
Xho EcoRI.
, ,
Xho BamHI.
16-237
hk6 1-237. 15
,
( 238-244)
. c-DNA , BamHI
EcoRI ,
-. cDNA hk6
(1-237), pBluescript II KS,
pPIC9, Xho
BamHI. .
5 (CIAP,
26 U/l, GIBCO, BRL)
QIAquick Nucleotide Removal Kit (QIAGEN
Inc.). cDNA hk6
4 DNA (1U/l, GIBCO, BRL).
E. coli XL-
1. DNA
,
. 18 cDNA
hk6 pBluescript II KS
DNA
:
109
-
hK6 ( 1-237)
pBluescript-hK6 XhoI
BamHI
A)
5
3
M 1 2
hk6 (673 bp)500 bp
2000 bp
750 bp
B)
hK6 ( 1-237)
pBluescript-hK6 XhoI
BamHI
A)
5
3hK6 ( 1-237)
pBluescript-hK6 XhoI
BamHI
A)
5
3
M 1 2
hk6 (673 bp)500 bp
2000 bp
750 bp
B) M 1 2
hk6 (673 bp)500 bp
2000 bp