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-Tạo Và Khảo Sát Hoạt Tính Sinh Học in Vitro Của Thụ Thể Interleukin 33 Dạng Tự Do Được Biểu Hiện Từ Tế Bào Người Hek293
Transcript of -Tạo Và Khảo Sát Hoạt Tính Sinh Học in Vitro Của Thụ Thể Interleukin 33 Dạng Tự...
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I HC QUC GIA THNH PH H CH MINH
TRNG I HC KHOA HC T NHIN
TRNG TH HONG DIU
TO V KHO ST HOT TNH SINH HC IN VITRO
CA TH TH INTERLEUKIN-33 DNG T DO
C BIU HIN T T BO NGI HEK293
Chuyn ngnh: Di truyn hc
M s: 60 42 70
LUN VN THC S SINH HC
NGI HNG DN KHOA HC:
TS. NGUYN NG QUN
THNH PH H CH MINH 2012
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LI CM N
u tin, em xin gi li CM N THY! chn thnh n
TS. Nguyn ng Qun, cm n Thy vi nhng kin thc chuyn ngnh
mi, nhng phng php nghin cu hay v tinh thn am m trong cng
vic Khng ch gii hn trong lun vn ny m n s c ngha rt ln i
vi em trong nhng bc i tip theo.
Cm n nhng ngi bn trong nhm ti anh L Gia Bo v bn
Nguyn Th Thanh Tho, mi ngi mt tnh cch, mi hng gii quyt vn
, nhng s hp tc gip chng ti khc phc kh khn v ln lt hon
thnh tng th nghim. Rt vui khi c lm vic cng mi ngi.
Xin gi li cm n n tt c cc anh ch phng CNSH Y Dc v
CNSH Thy Sn Trung tm Cng ngh Sinh hc Tp.HCM gip em
trong thi gian qua.
Lun vn ny s khng th thc hin nu khng c ngun kinh ph
nghin cu khoa hc ca Trung tm Cng ngh Sinh hc Tp.HCM. Ti xin
chn thnh cm n qu Trung tm cng nh Ban Gim c Trung tm to
iu kin cho ti thc hin ti ny.
Con vn lun c gng sng tt khng ph lng mong mi ca Ba
M. Cm n Ba M ca con tht nhiu!
Tp. H Ch Minh, thng 05 nm 2012
Trng Th Hong Diu
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MC LC
Li cm n ............................................................................................................. i
Mc lc ................................................................................................................. ii
Danh mc cc k hiu, cc ch vit tt ................................................................. vi
Danh mc cc bng ............................................................................................ viii
Danh mc cc hnh ............................................................................................... ix
LI M U ...................................................................................................... 1
CHNG 1 TNG QUAN TI LIU ........................................................... 2
1.1. Vai tr ca IL-33 trong sinh l v bnh l .................................................. 2
1.1.1. Vai tr ca IL-33 trong sinh l ............................................................ 2
1.1.2. Vai tr ca IL-33 trong bnh l ........................................................... 3
1.2. c im sinh hc ca IL-33 ...................................................................... 6
1.2.1. Cu trc ca IL-33 ............................................................................ 6
1.2.2. T bo sinh tng hp v t bo ch ca IL-33 .................................. 7
1.2.3. IL-33 ngoi bo v ni bo ................................................................ 8
1.2.4. Con ng truyn tn hiu ca IL-33 thng qua IL-33R .................... 9
1.3. c im sinh hc ca IL-33R ................................................................. 10
1.3.1. Cu trc ca IL-33R ........................................................................ 10
1.3.2. Tng tc ca IL-33 vi IL-33R ..................................................... 11
1.3.3. IL-33R dng t do ........................................................................... 12
1.4. c ch hot ng ca IL-33 l mt liu php tm nng iu tr
cc bnh vim d ng ............................................................................. 12
1.5. S lc v cc dc phm c ch hot ng cytokine da trn nguyn tc
tng tc cnh tranh .................................................................................. 14
1.6. H thng t bo ng vt biu hin protein ti t hp ............................... 16
CHNG 2 VT LIU V PHNG PHP ............................................. 18
2.1. Vt liu ..................................................................................................... 18
2.1.1. Plasmid ............................................................................................. 18
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2.1.2. Ha cht to dng plasmid biu hin msIL-33R:Fc hIgG1 ................ 19
2.1.3. Ha cht dng trong nui cy vi khun E. coli................................... 19
2.1.4. Ha cht nui cy t bo HEK293 v EL-4 ....................................... 19
2.1.5. Ha cht dng bin np plasmid vo E.coli v HEK293 ............... 20
2.1.6. Ha cht li gii t bo, nh lng protein v thy phn
nhm ng trn glycoprotein ........................................................... 20
2.1.7. Ha cht dng cho SDS-PAGE, Western Blot v
ng kt ta min dch ...................................................................... 20
2.1.8. Ha cht ELISA ................................................................................ 21
2.2. Phng php ............................................................................................ 22
2.2.1. To plasmid biu hin protein ti t hp msIL-33R:Fc hIgG1 ......... 22
2.2.1.1. Khuch i on gen m ha cho sIL-33R ca chut ............... 22
2.2.1.2. Phn tch DNA bng in di trn gel agarose ........................... 23
2.2.1.3. Tinh sch DNA ........................................................................ 23
2.2.1.4. Ct hn ch DNA bng NotI v HindIII ................................... 24
2.2.1.5. Phn ng ni DNA bng ligase ................................................ 25
2.2.1.6. Bin np plasmid vo E. coli DH5 kh np ............................ 25
2.2.1.7. Tch chit plasmid t E. coli .................................................... 26
2.2.2. Phng php nui cy vi khun ...................................................... 27
2.2.2.1. Nui cy E. coli DH5 ............................................................. 27
2.2.2.2. Bo qun E. coli DH5 mang plasmid ..................................... 27
2.2.2.3. To t bo kh np E. coli DH5 bng phng php
ha bin np ............................................................................. 27
2.2.3. Phng php nui cy t bo HEK293 ........................................... 28
2.2.4. Phng php nui cy t bo EL-4 ................................................. 29
2.2.5. Phng php bin np plasmid vo t bo HEK293 ........................ 29
2.2.6. Phng php phn gii t bo ......................................................... 30
2.2.7. Phng php Bradford nh lng protein ...................................... 30
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2.2.8. Phng php thu nhn msIL-33R:Fc hIgG1t dch nui cy
t bo HEK293............................................................................... 31
2.2.9. Phng php thy phn nhm ng trn glycoprotein
msIL-33R:Fc hIgG1 bng PNGase F ............................................... 32
2.2.10. Phng php to IL-33 chut nh du biotin ............................... 32
2.2.11. Phng php SDS-PAGE, Western Blotting ................................. 33
2.2.11.1. in di SDS-PAGE ................................................................ 33
2.2.11.2. Western Blotting .................................................................... 35
2.2.12. Phng php ng kt ta min dch (co-immunoprecipitation) .... 36
2.2.12.1. ng kt ta min dch bio-mIL33 v msIL-33R:Fc hIgG1 ... 36
2.2.12.2. ng kt ta min dch bio-mIL33 v FLAG-IL33R ............. 36
2.2.13. Phng php nh gi kh nng c ch IL-33 ca
msIL-33R:Fc hIgG1 trn m hnh t bo nui cy ......................... 37
2.2.14. Phng php ELISA nh lng mIL-2 ........................................ 38
CHNG 3 KT QU V THO LUN ................................................... 39
3.1. To dng plasmid pmsIL33R-Fc mang gen m ha protein
msIL-33R:Fc hIgG1 ................................................................................ 39
3.1.1. Khuch i, thu nhn on gen m ha th th IL-33 dng t do
ca chut v chun b vector Signal pIgplus cho to dng ................. 39
3.1.2. To plasmid Signal pIgplus ti t hp mang gen m ha protein
msIL-33R:Fc hIgG1 .......................................................................... 40
3.2. Biu hin v thu nhn protein ti t hp msIL-33R:Fc hIgG1 t t bo
ngi HEK293......................................................................................... 42
3.3. Phn tch mt s c im ca protein ti t hp msIL-33R:Fc hIgG1 ..... 44
3.3.1. S ng ha ca msIL-33R:Fc hIgG1 ti t hp biu hin
t HEK293 ........................................................................................ 44
3.3.2. Protein msIL-33R:Fc hIgG1 c biu hin v tit ra mi trng
ngoi bo dng dimer ..................................................................... 46
3.4. Biu hin IL-33 chut c nh du biotin t vi khun E.coli .................... 48
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3.5. S tng tc ca msIL-33R:Fc hIgG1 vi bio-mIL33 trong iu kin
in vitro .............................................................................................................. 50
3.6. S cnh tranh ca msIL-33R:Fc hIgG1 vi IL-33R biu hin b mt
t bo trong vic tng tc vi bio-mIL33 ............................................... 52
3.7. Kh nng c ch hot ng IL-33 ca msIL-33R:Fc hIgG1 trn m hnh
t bo EL-4 nui cy in vitro .................................................................... 55
CHNG 4 KT LUN V NGH ....................................................... 59
4.1. Kt lun .................................................................................................... 59
4.2. ngh ..................................................................................................... 59
TI LIU THAM KHO ................................................................................. 60
PH LC ........................................................................................................... 71
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DANH MC CC K HIU, CH VIT TT
APC Antigen Presenting Cells
BMMC Bone marrowderived mast cell
bp base pair
BSA Bovine serum albumin
CD2 Cluster of differentiation 2
cs cng s
CTLA-4 Cytotoxic T-Lymphocyte Antigen 4
DMEM Dulbecco's Modified Eagle Medium
DNA Deoxyribonucleic acid
dNTP Deoxynucleotide triphosphate
ELISA Enzyme-linked immunosorbent assay
Fc Fragment crystallizable region
FCS Fetal calf serum
HEK293 Human Embryonic Kidney 293
hIgG1 Human immunoglobulin G1
HRP Horseradish peroxidase
IFN Interferon
Ig Immunoglobulin
IL Interleukin
IL-1RAcP Interleukin-1 receptor accessory protein
IL-33R Interleukin-33 receptor
IRAK IL-1 Receptor Associated Kinase
IB Inhibitor of NF-B
JNK c-Jun N-terminal kinase
KDa Kilodalton
LB Luria Bertani
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LFA-3 Lymphocyte function-associated antigen 3
M Marker
MAP kinase Mitogen-activated protein kinase
mIL-2 Mouse interleukin-2
mIL-33 Mouse interleukin-33
mRNA Messenger ribonucleic acid
msIL-33R Mouse soluble interleukin-33 receptor
MyD88 Myeloid differentiation primary response gene 88
NCBI National Center for Biotechnology Information
NF-HEV Nuclear factor high endothelial venule
NFB Nuclear factor kappa B
OD Optical density
PBS Phosphate buffered saline
PCR Polymerase chain reaction
RPMI Roswell Park Memorial Institute
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel
electrophoresis
TAB TAK1 binding protein
TAK1 TGF--activated kinase 1
Th T helper cell
TIR Toll-like Interleukin-1 Receptor
TNF Tumor Necrosis Factor
TPO Thrombopoietin
TRAF-6 TNF Receptor Associated Factor 6
WB Western Blot
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DANH MC CC BNG
Bng 1.1. Cc bit dc c bn cht l protein lai mang vng Fc IgG1 .............. 15
Bng 2.1. Thnh phn gel polyacrylamide dng trong SDS-PAGE ..................... 20
Bng 2.2. Cc khng th c s dng trong th nghim Western Blot ............... 21
Bng 3.1. p ng tit mIL-2 do tc ng ca mIL-33 t t bo EL-4 khi c x
l vi msIL-33R:Fc hIgG1 nhiu nng khc nhau ....................... 57
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DANH MC CC HNH
Hnh 1.1. Cu trc khng gian IL-33 ca ngi .................................................... 6
Hnh 1.2. Con ng truyn tn hiu ca IL-33 thng qua IL-33R ...................... 10
Hnh 1.3. Cu trc khng gian vng ngoi bo ca IL-33R v tng tc gia
IL-33R vi IL-33 ................................................................................ 11
Hnh 1.4. sIL-33R (ST2) c cu trc tng ng vi vng ngoi bo ca IL-33R
(ST2L) v ngn cn con ng truyn tn hiu ca IL-33 .................. 12
Hnh 1.5. M hnh c ch IL-33 ca msIL-33R:Fc hIgG1 ................................... 14
Hnh 2.1. S ca cc plasmid c s dng trong ti ............................... 18
Hnh 3.1. Thu nhn on gen m ha msIL-33R v vector Signal pIgplus .......... 40
Hnh 3.2. Kt qu sng lc cc dng plasmid ti t hp ..................................... 41
Hnh 3.3. Biu hin v tinh sch s b protein msIL-33R:Fc hIgG1 t h thng
t bo ngi HEK293 ......................................................................... 44
Hnh 3.4. S glycosyl ha ca msIL-33R:Fc hIgG1 c biu hin t t bo
ngi HEK293 .................................................................................... 46
Hnh 3.5. Protein msIL-33R:Fc hIgG1 c biu hin dng dimer ................. 48
Hnh 3.6. Biu hin IL33 chut nh du biotin ................................................. 50
Hnh 3.7. Protein msIL-33R:Fc hIgG1 biu hin t HEK293 tng tc vi
bio-mIL33 iu kin in vitro ............................................................ 52
Hnh 3.8. msIL33R:Fc hIgG1cnh tranh vi FLAG-IL33R biu hin trn b mt
t bo trong vic tng tc vi bio-mIL33 ......................................... 54
Hnh 3.9. msIL-33R:Fc hIgG1 c ch hot ng ca IL-33 trn m hnh t bo
EL-4 nui cy ............................................................................................. 57
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LI M U
Interleukin(IL)-33 l thnh vin mi nht ca h cytokine tin vim IL-1 c
vai tr quan trng trong hot ng ca h thng min dch bo v c th. Tuy nhin,
hot ng bt thng ca IL-33 c chng minh l c lin quan n cc bnh d
ng v vim nh hen suyn, vim da d ng, vim thp khp... Do , c ch hot
ng ca IL-33 c th l mt gii php mi gp phn iu tr cc bnh nu trn.
Mc tiu ca nghin cu: to ra protein lai msIL-33R:Fc hIgG1 (th th IL-33
dng t do ca chut gn vi vng Fc IgG1 ca ngi) biu hin t t bo HEK293
(Human Embryo Kidney) c hot tnh sinh hc c ch hot ng ca IL-33. msIL-
33R:Fc hIgG1 c ch IL-33 bng cch bt ly IL-33 t do qua hn ch s tc
ng ca cytokine ny ln t bo ch thng qua th th ca n trn b mt t bo.
Ni dung nghin cu bao gm:
1. To dng plasmid pmsIL33R-Fc mang gen m ha protein msIL-
33R:Fc hIgG1
2. Biu hin v thu nhn protein ti t hp msIL-33R:Fc hIgG1 t t bo
ngi HEK293
3. Phn tch mt s c im ca protein ti t hp msIL-33R:Fc hIgG1
4. Biu hin IL-33 chut c nh du biotin (bio-mIL33) t vi khun
E.coli
5. Kho st s tng tc ca msIL-33R:Fc hIgG1 vi bio-mIL33 trong
iu kin in vitro
6. Kho st s cnh tranh ca msIL-33R:Fc hIgG1 vi IL-33R biu hin
b mt t bo trong vic tng tc vi bio-mIL33
7. nh gi kh nng c ch hot ng IL-33 ca msIL-33R:Fc hIgG1
trn m hnh t bo EL-4 nui cy in vitro
Phn tng quan ti liu s tm tt cc hiu bit hin nay v h IL-33/IL-33R
v mt s vn c lin quan n ti nghin cu.
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CHNG 1: TNG QUAN TI LIU
1.1. Vai tr ca IL-33 trong sinh l v bnh l
1.1.1. Vai tr ca IL-33 trong sinh l
Interleukin-1 (IL-1) l mt h cytokine tin vim gm cc thnh vin IL-1,
IL-1, IL-18 v IL-1Ra (IL-1 receptor antagonist) ng vai tr quan trng trong h
thng phng v ca c th, trong iu ha min dch v phn ng vim. Nm 2005,
Schmitz v cs pht hin mt thnh vin mi ca h IL-1 l IL-33 [64].
IL-33 u tin c tm thy trong t bo thnh mch ni m, c chc nng
iu ha biu hin gen v c t tn l NF-HEV (nuclear factor high
endothelial venule) [7]. Protein ny c xc nh c trnh t amino acid tng
ng cao vi IL-18 v cu trc phin ging vi cc thnh vin trong h IL-1 [64].
IL-33 c vai tr quan trng trong h thng phng v ca c th, n c th
hot ha cc t bo trong h thng min dch bm sinh v min dch thch ng. IL-
33 c th hot ha trc tip t bo Th2 v qua kch thch cc t bo B gii phng
cc cytokine tin vim IL-4, IL-5, IL-13 v khng th IgM (immunoglobulin M)
[30], [64]. Pht hin gn y trn m hnh chut cho thy t bo tua (dendritic cell)
khi b kch thch bi IL-33 c th bit ha t bo T thnh t bo Th2 [60]. IL-33 tc
ng ln t bo bch cu ht a acid (eosinophil) lm cho cc t bo ny sn xut
IL-8, gii phng ht v gip cho s tn ti ca t bo [15], [64]. i vi tng bo
(mast cell) v bch cu ht a kim (basophil), IL-33 cm ng s trng thnh, s
gii phng ht, s tn ti v sn xut mt s cytokine tin vim nh IL-8, IL-13, IL-
4, IL-6 v histamin [5], [26], [65]. IL-33 lm gim tnh trng nhim trng do tng
cng bch cu ht trung tnh (neutrophil) ti v tr m b xm nhim do kch thch
tnh ha hng ng ca loi t bo ny [6]. IL-33 cn tng cng s bit ha i
thc bo (macrophage) theo hng p ng min dch t bo Th2 v tng s sn
xut TNF (Tumor Necrosis Factor ) t cc i thc bo c cm ng bi LPS
(lipopolysaccharide) [19], [35].
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Ngoi ra, hot ng ca IL-33 cn ng vai tr bo v h thng tim mch
chng li cc bnh nh cao huyt p [63] v x va ng mch [44]. Gn y, c
cng b cho rng IL-33 lin quan n qu trnh to m xng khi n c kh nng
bit ha bch cu n nhn (monocyte) thnh t bo xng (osteoblast) v kch
thch cc t bo khc tng cng s biu hin cc tc nhn hot ha t bo xng
[49]. Tuy nhin cng c mt s bo co ph nhn kt lun ny [62], [66].
1.1.2. Vai tr ca IL-33 trong bnh l
Nh thy trn, IL-33 cng vi cc cytokine trong h IL-1 c vai tr
quan trng trong hot ng ca h min dch. Thng thng cc cytokine ny c
kim sot cht ch trong vim cp tnh trnh s hy hoi m do tng cng cc
hot ng d ha.
Tuy nhin, s biu hin qu mc ca IL-33 c th gy ra nhiu bnh l vim
v d ng. Hen suyn l mt bnh vim mn tnh c xc nh do cc p ng qu
mc phi, vim d ng, tng nng IgE (immunoglobulin E) trong huyt thanh
v tng cng sn xut cc cytokine lin quan n Th2. Do vy, IL-33 l nhn t
quan trng trong bnh hen suyn qua con ng p ng min dch Th2 [39]. IL-33
c biu hin tng cao t bo biu m ph qun ca bnh nhn hen suyn
(asthma) v c bit cao cc bnh nhn hen nng [58]. Mt s nghin cu trn m
hnh ng vt cho thy vai tr quan trng ca con ng IL-33/ST2L trong hen
suyn v vim kh qun d ng. Tim IL-33 vo chut b gy vim kh qun lm
tng nng tnh trng ca bnh [34]. IL-33 kch thch cc p ng qu mc, kch
thch s tng sinh t bo hnh tr, cm ng bch cu ht a acid v i thc bo,
tch t cc cytokine IL-4, IL-5 v IL-13 trong phi [31], [35], [71].
Tc ng ca IL-33 nh mt yu t tin vim cng c xc nh trong bnh
vim khp do s hot ha qu mc nguyn bo si v tng bo trong cc khp.
IL-33 c tm thy trong t bo ni m v nguyn bo si mng hot dch cc
bnh nhn vim thp khp, vim khp xng v vim khp vy nn [74]. IL-33
cng biu hin trong nguyn bo si mng hot dch t bnh nhn b vim khp c
nui cy trong iu kin in vitro [80]. IL-33 cng c mt trong huyt thanh nhng
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bnh nhn vim thp khp v nhiu hn so vi trng hp b vim khp xng [48],
[74]. Tc ng ca IL-33 trong vim khp cng c chng minh m hnh bnh l
trn chut. S biu hin mRNA ca IL-33 tng ln trong m khp ca chut vim
khp c cm ng bi collagen v tng mnh trong thi k u ca bnh [80]. Khi
gy vim khp trn chut khng biu hin ST2 (th th ca IL-33), kt qu cho thy c
s gim sn xut cc cytokine tin vim (IL-17, TNF v IFN- Interferon ) v khng
th so vi khi gy vim khp chut hoang di. Ngc li, tim IL-33 vo chut b
gy vim khp s lm gia tng hin tng vim [53], [80]. Hot ng ca IL-33 gy
vim khp c chng minh c lin quan n tng bo (mast cell) v khi x l IL-33
trn chut thiu tng bo th khng thy tng tnh trng bnh vim khp [79].
IL-33 cng c thy l c lin quan n bnh vim da. IL-33 c biu
hin nhiu hn m da ca nhng bnh nhn vim da so vi ngi bnh thng
[59]. Nng IL-33 cng tng ln trong huyt thanh ca cc bnh nhn b x ha
da [83]. Hn na, x l IL-33 lm tng mc x ha da m hnh bnh l vim
da trn chut [61]. Ngc li, khi gy cm ng vim da chut khng biu hin
protein ST2 cho thy mc bnh l nh hn so vi khi gy vim da chut hoang
di c biu hin ST2 [25].
Hot ng p ng min dch qu mc trong h thng ng rut cng gy
ra cc triu chng vim rut nh vim lot i trng v bnh Crohn. Mt s bo co
cho thy c s lin quan gia IL-33 v cc dng bnh vim rut. IL-33 biu hin
nhiu nguyn bo si v t bo biu m rut ca cc bnh nhn b vim lot i
trng v bnh Crohn, ng thi IL-33 v sST2 (th th IL-33 dng t do) cng c
tm thy trong huyt thanh [9], [11], [54]. Chut c x l IL-33 c biu hin tng
sinh biu m rut, gia tng s lng bch cu ht a acid v bch cu ht trung tnh
di chuyn t mu vo nim mc rut [64]. IL-33 lm tng tnh trng bnh trong m
hnh chut b vim rut lin quan n Th1/Th2 v cm ng sn xut IL-17 t cc t
bo hch bch huyt (lymph node cell) [54].
Hin nay, vai tr ca IL-33 bc u cng c nghin cu trn mt s bnh
nh thn kinh, tim mch, bo ph v ung th. C s gim biu hin IL-33 trong no
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ca bnh nhn Alzheimer, iu ny cho thy IL-33 c th c vai tr trong hot ng
bo v h thng t bo thn kinh [13]. IL-33 lm gim tnh trng phnh i v x
ha tim, gim chng nhi mu c tim v ci thin chc nng tm tht [67]. IL-33
lm gim s tin trin ca chng x va ng mch do hot ha con ng sn
xut cc cytokine Th2, c ch con ng min dch Th1 nn gim hm lng IFN
trong huyt thanh v cc t bo hch bch huyt [44]. IL-33 hin din trong cc t
bo m v c chng minh l lm gim tnh trng tch ly m ca cc t bo ny.
Trong iu kin nui cy in vitro, khi b kch thch bi IL-33 t bo m m tng
biu hin cc cytokine Th2 (IL-5, IL-13, IL-10) v gim biu hin cc protein ca
con ng sinh tng hp v tch ly lipid. Hn na, mc bo ph ca m hnh
chut s gim xung khi chng c tim IL-33. Ngc li, vi ch n nhiu
cht bo, chut khng biu hin ST2 c mc bo ph cao hn so vi chut hoang
di. Nh vy, IL-33 c th c hot tnh chng li s bo ph [43]. IL-33 cn c
cho l c vai tr trong bnh ung th. Kt qu thc nghim cho thy p ng min
dch t bo chng khi u tng ln m hnh chut ung th khng biu hin gen ST2
so vi chut ung th c biu hin ST2 [27]. iu ny c th c gii thch l do
con ng tn hiu IL-33/ST2 hot ha p ng min dch th dch Th2 v c ch
p ng min dch t bo thng qua Th1, nn khi con ng IL-33/ST2 b c ch
th p ng Th1 s tng mnh ln dn n kt qu l kh nng khng khi u cao
hn. Bn cnh , IL-33 ni bo c thy biu hin nhiu trong cc t bo ni m
trng thi ngh nhng protein ny khng c biu hin t bo ung th [33].
iu ny cho thy IL-33 ni bo c th c chc nng kim sot s phn chia ca t
bo v ngn chn s bin i t bo bnh thng thnh t bo ung th. Tuy nhin,
nhiu nghin cu hn cn c thc hin lm r gi thit ny.
Cc kt qu nghin cu nu trn cho thy IL-33 c lin quan n c ch sinh
bnh ca nhiu bnh khc nhau c bit l cc bnh vim t min v d ng. Do vy,
IL-33 c th l phn t ch c hng ti trong cc liu php iu tr mi cho
nhng bnh ny.
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1.2. c im sinh hc ca IL-33
1.2.1. Cu trc ca IL-33
Tin IL-33 (preIL-33) ngi gm 270 amino acid (aa) c kch thc 30 kD,
u N (aa 1-111) ca protein ny c tn hiu nh v nhn v u C (aa 112-270) c
cu trc tng ng vi h IL-1. IL-33 ca chut gm 266 aa, c trnh t aa tng
ng cao vi IL-33 ngi (55%). Trong iu kin in vitro, IL-33 ngi c th b
caspase-1 phn ct v tr aa 111 to thnh IL-33 trng thnh (matIL-33) kch
thc 18kD, IL-33 chut b caspase-1 phn ct v tr aa 108 to matIL-33 t aa 109-
266. preIL-33 v matIL-33 u c hot tnh cytokine kch thch t bo ch thng qua
tng tc vi th th ca n l ST2L hay cn gi l IL-33R [3], [5], [8], [11], [64].
Tuy nhin, mt s nghin cu gn y chng minh rng preIL-33 c th b phn ct
bi caspase-3 v caspase-7 v tr aa 178 trn ngi v v tr aa 175 trn chut to
nn sn phm ct khng c hot tnh sinh hc [3], [41], [73].
Cu hnh khng gian ca IL-33 gm 12 chui xon sp xp thnh cu trc -
trefoil v hai chui xon nm trc 8 v 12. Vng li -trefoil c bo tn cao
so vi cc cu trc trung gian ni gia cc chui , c bit l on ni gia 4- 5
gm 10 aa rt linh ng v khng c cu trc n nh. Ngoi ra, mt s vng trong
cu trc protein c kh nng lm tng ng lc ca phn t trong qu trnh tng
tc nh u N t gc aa 1 n 9, on ni 3- 4 t gc aa 40 n 46, on ni 11-
12 t gc aa 143 n 150 v u C t gc aa 159 n 160 [37].
Hnh 1.1. Cu trc khng gian IL-33 ca ngi [37]
-
7
1.2.2. T bo sinh tng hp v t bo ch ca IL-33
u tin, IL-33 c gi l yu t nhn ca t bo thnh mch ni m
(Nuclear Factor High Endothelial Venule: NF-HEV) v n c tm thy trong
nhn ca cc t bo ny [7], [11]. T bo thnh mch ni m l nhng t bo c
bit ha t cc m bch huyt th cp, chng biu hin cc phn t bm dnh cho
php cc t bo bch cu di chuyn t mu vo m bch huyt. Gn y, IL-33
cng c tm thy trong nhn ca cc t bo biu m, c bit l da, thnh rut
v phi [47].
IL-33 sau khi c sn xut cc t bo ni m v biu m, c tit ra ngoi
t bo v bin i thnh dng c hot tnh sinh hc nh mt cytokine hoc c th
vo nhn thc hin chc nng nh mt yu t iu ha phin m [11], [64]. Tuy
nhin, c ch tit ca IL-33 hin nay vn cha r. V trong cu trc ca IL-33
khng cha trnh t tit nn IL-33 khng th tit theo c ch thng thng l qua h
thng ni cht nhm v th Golgi. V vy, c nhiu kh nng l IL-33 c tit theo
c ch hoi t ca t bo di dng tin phn t, sau c bin i v tc ng
ln cc t bo ch qua phc hp th th ca n [41].
IL-33 kch thch t bo ch thng qua th th c hiu IL-33R v ng th th
l IL-1RAcP (IL-1 Receptor Accessory Protein) hin din trn mng mt s t bo
ch ca n nh bch cu ht a kim, bch cu ht a acid, tng bo, t bo git t
nhin v lympho bo Th2 [2], [12], [64]. Trong , lympho bo Th2 v tng bo l
nhng t bo biu hin th th IL-33 nhiu nht. IL-33 cm ng lympho bo Th2 tit
IL-5 v IL-13, cm ng tng bo tit mt s cytokine v chemokine nh IL-1, IL-
6, IL-13, TNF v MCP-1 (Monocyte Chemotactic Protein-1) [24], [46], [64]. Bch
cu ht a kim biu hin IL-33R t hn so vi lympho bo Th2 v tng bo, ngoi
ra s biu hin th th IL-33 y c kch thch bi IL-3. Do IL-33 cng c th
tc ng trc tip ln bch cu ht a kim kch thch t bo ny to cc cytokine
Th2, nhng vai tr quan trng hn l n phi hp vi IL-3 v IgE trong tng cng
p ng min dch ca bch cu ht a kim [55], [72]. IL-33 tc ng trc tip bch
cu ht a acid kch thch sn xut IL-8 v cc cht oxid ha, ng thi phi hp vi
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8
GM-CSF (Granulocyte-Macrophage Colony Stimulating Factor) lm tng cng hot
ng tit IL-3, IL-5 v IL-8 ca bch cu ht a acid [15], [55]. IL-33 hot ha t bo
git t nhin lm tng biu hin v tit IFN- [10]. Ngoi ra, IL-33 cn tc ng ln
t bo tua lm tng cng tit IL-6, kch thch s biu hin IL-8 t bo biu m v
ni m thng qua phc hp th th ca n trn b mt t bo [60], [82].
Nh vy, IL-33 c to ra ch yu t cc t bo ni m, biu m v kch
thch nhiu loi t bo bch huyt to ra cc cytokine ng vai tr quan trng trong
hot ng ca h thng min dch bm sinh v thch ng.
1.2.3. IL-33 ngoi bo v ni bo
Da vo chc nng m IL-33 c chia thnh hai dng l IL-33 ngoi bo v
IL-33 ni bo.
IL-33 ngoi bo l IL-33 c phng thch ra ngoi t bo v thc hin chc
nng nh mt cytokine tin vim bng cch tc ng ln cc t bo ch thng qua
phc hp th th IL-33R c hiu ca n hin din trn b mt t bo. u C t v
tr aa 112-270 cha cu trc -trefoil l vng quyt nh c tnh cytokine ngoi bo
ca IL-33. Do vy, c IL-33 trng thnh do s phn ct ca caspase-1 v tin IL-
33 u c hot tnh cytokine [41], [64]. Vai tr nh mt cytokine tin vim ngoi
bo ca IL-33 c lm r trong nhiu nghin cu cho thy IL-33 c vai tr
quan trng trong sinh l c th, cng nh trong nhiu dng bnh l lin quan n
vim v d ng do kch hot h thng cytokine ca t bo Th2.
Tuy nhin, chc nng ni bo ca IL-33 l vn mi c tm hiu trong
thi gian gn y. IL-33 ni bo c cho l yu t trong nhn c vai tr iu ha
phin m v n c mang cu trc helix-turn-helix gip IL-33 gn vo DNA trong
nhn t bo. Helix-turn-helix nm u N ca tin IL-33 t v tr aa 1-65 v c tnh
bo tn cao gia cc loi. Do vy, c u N v ton b phn t IL-33 u c kh
nng lin kt vi DNA c ch phin m [11]. Tuy nhin, c ch c ch phin m
ca IL-33 ni bo gn y c chng minh l do protein ny tng tc vi nhn t
phin m NFB v ngn khng cho NFB tng tc vi trnh t c hiu trn vng
promotor ca cc gene do NFB iu ha [4].
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9
1.2.4. Con ng truyn tn hiu ca IL-33 thng qua IL-33R
IL-33 tng tc vi th th c hiu IL-33R v ng th th l IL-1RAcP biu
hin trn b mt t bo ch, to thnh phc hp khi u s truyn tn hiu thng
qua con ng NFB v MAP kinase [2], [64]. Vng TIR (Tool Interleukin 1
Receptor) ca IL-33R v IL-1RAcP thu ht protein MyD88 v qua huy ng
IRAK-4 v IRAK-1 (IL-1 Receptor Associated Kinase) vo phc hp. Trong phc
hp trn, IRAK-4 t phosphoryl ha tr nn hot ng v hot ha IRAK-1. IRAK-
1 c hot ha s t phosphoryl ha chnh n v tch ra khi phc hp th th IL-
33. Trc khi tch khi phc hp protein, IRAK-1 tng tc vi TRAF-6 (TNF
Receptor Associated Factor 6) v phosphoryl ha hot ha TRAF-6. Ti lt
mnh, TRAF6 phosphoryl ha v hot ha TAK1 (TGF--activated kinase 1) trong
phc hp vi hai protein TAB2 v TAB3 (TAK1 binding protein). TAK1 tip tc
hot ha cc MAP kinase JNK v p38, t dn n s hot ng ca mt s yu
t phin m. ng thi, TRAF-6 cng tng tc vi IB gii phng NFB khi
trng thi c ch trong t bo cht. Cc nhn t phin m c hot ha nh MAP
kinase JNK, p38 v NFB di chuyn vo nhn khi ng s phin m ca cc
gen biu hin mt s cytokine tin vim [50].
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10
Hnh 1.2. Con ng truyn tn hiu ca IL-33 thng qua IL-33R [50]
1.3. c im sinh hc ca IL-33R
1.3.1. Cu trc ca IL-33R
IL-33R l mt glycoprotein xuyn mng thuc h IL-1R, do gen ST2 m ha.
IL-33 c t l tng ng vi IL-1R1 l 28% v nu xt ring vng ni bo th t l
ny l 38%. IL-33R ca chut c kch thc 567 aa gm ba vng cu trc: vng
ngoi bo c 328 aa (1-328), vng xuyn mng v cn mng c 38 aa (329-366) v
vng ni bo c 201 aa (367-567). Vng ngoi bo hnh thnh ba cu ni disulfide
ti 6 gc cysteine l cc v tr xy ra tng tc vi IL-33 v quyt nh tnh cht
immunoglobulin ca phn t. ng thi, vng ngoi bo cng cha 9 v tr
asparagine c kh nng b glycosyl ha [77], [84]. Cu trc khng gian vng ngoi
bo ca IL-33R gm ba vng immunoglobulin D1, D2 v D3. D1 v D2 lin kt
cht bng cu ni disulfide v tch bit vi D3 bng mt lin kt m rng vi 8 gc
aa. Nh c im ny m D3 c th xoay gip c nh IL-33 vo vng lm ca
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11
phc hp D1-D2 [37]. Vng ni bo ca IL-33R mang vng TIR vi cu trc 5
chui xon bao quanh 5 phin , vng TIR ny l cu trc quan trng khi u
con ng truyn tn hiu thng qua tng tc vi vng TIR trn ng th th IL-
1RAcP v trn adaptor MyD88 [2], [81].
1.3.2. Tng tc ca IL-33 vi IL-33R
IL-33 tng tc vi IL-33R vng ngoi bo ca IL-33R lin quan n s
thay i ha hc v in tch ca cc gc aa. Trn IL-33 hnh thnh hai vng tng
tc do s thay i ha hc mnh ca cc nhm aa. Vng th nht mang in tch
m, c s tham gia ca cu trc kp tc 2-3, cc on ni 10-11 v 3-4.
Vng th hai mang in dng, c to thnh bi cc gc u N ca 1 v 5,
on ni 8-9 v u C ca 12. Trn IL-33R, vng D1-D2 to thnh b mt lm
mang in tch dng v vng D3 c mt phn k nc mang in tch m. Do vy,
IL-33 v IL-33R hnh thnh hai vng tng tc c hiu tng ng, vng th nht
ca IL-33 vi vng D1-D2 ca IL-33R v vng th hai ca IL-33 vi vng D3 ca
IL-33R [37].
Hnh 1.3. Cu trc khng gian vng ngoi bo ca IL-33R v tng tc gia
IL-33R vi IL-33 [37]
A. Cu trc khng gian ca IL-33
B. Cu trc khng gian ca vng ngoi bo IL-33R
C. M hnh tng tc gia IL-33 vi vng ngoi bo IL-33R
C A B
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12
1.3.3. IL-33R dng t do
IL-33R dng t do (soluble IL-33R: sIL-33R) c cu trc ging ht vng ngoi
bo ca IL-33R. sIL-33R v IL-33R cng c m ha t gen ST2 v hnh thnh do
qu trnh bin i sau phin m. sIL-33R c kch thc 337 aa, c c 3 vng
immunoglobulin v 9 v tr asparagine c kh nng b glycosyl ha nh vng ngoi
bo ca IL-33R, thiu vng xuyn mng v ni bo [77], [84]. sIL-33R tn ti t do
trong mi trng ngoi bo v thc hin vai tr c ch con ng truyn tn hiu ca
IL-33 bng cch cnh tranh vi IL-33R trong tng tc vi IL-33 [22].
Hnh 1.4. sIL-33R (ST2) c cu trc tng ng vi vng ngoi bo ca IL-33R
(ST2L) v ngn cn con ng truyn tn hiu ca IL-33 [36], [84]
1.4. c ch hot ng ca IL-33 l mt liu php tm nng iu tr cc bnh
vim d ng
IL-33 tc ng ln t bo ch bng cch gn vo th th ca n l IL-33R trn
b mt t bo, sau huy ng ng th th l IL-1RAcP to thnh phc hp th
th truyn tn hiu hot ha t bo ch [2]. Hin ti, phng thc c ch hot ng
ca IL-33 c tip cn ph bin l ngn chn s tng tc gia IL-33 vi IL-
33R. thc hin iu ny, khng th khng IL-33R v khng th khng IL-33
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13
thng c p dng ngn cn s hnh thnh phc hp IL-33 v IL-33R, qua
ngn cn con ng truyn tn hiu ca IL-33 trn cc t bo ch.
X l vi khng th khng vng ngoi bo IL-33R cho thy c s c ch hot
ng trn p ng min dch nim mc phi nh gim s xm nhim ca bch
cu ht a acid, gim lng IL-4, IL-5 v IgE trn m hnh chut hen suyn [17],
[23], [28], gim triu chng vim khp v thoi ha khp trn m hnh chut vim
khp c cm ng collagen [53].
Hng th hai l dng khng th khng IL-33 cng mang li nhiu kt qu kh
quan. Khng th khng IL-33 lm gim cc triu chng hen suyn d ng trn m
hnh chut v lm gim mt s tc nhn nh IgE trong huyt thanh, bch cu ht a
acid, lympho bo v cc cytokine IL-4, IL-5 v IL-13 trong dch nhy ph qun [38].
Nh trnh by mc 1.3.3, ngoi dng biu hin trn b mt t bo th IL-
33R cn tn ti dng t do (sIL-33R). sIL-33R c th c vai tr t nhin l iu ha
m hot ng ca IL-33 bng cch cnh tranh vi th th trn b mt t bo trong
vic tng tc vi IL-33 [22], [52]. Da trn pht hin ny, sIL-33R nhn to c
tng hp v th nghim hot tnh c ch IL-33. Kt qu cho thy tim sIL-33R vo
chut b cm ng hen suyn lm c ch con ng hot ha Th2 ca IL-33, lm gim
s to thnh cc cytokine nh IL-4, IL-5, IL-13 trong mu v gim triu chng bnh
hen suyn d ng chut [22]. Ngoi ra, khi gy hen suyn d ng trn chut chuyn
gen biu hin sIL-33R, lng IL-4, IL-5 v bch cu ht a acid c to ra thp hn
so vi m hnh hen suyn trn chut hoang di [51]. Hn na, x l sIL-33R:Fc cn
lm gim triu chng ca bnh vim khp trn m hnh chut [53].
Cc kt qu bc u nu trn cho thy c ch hot ng ca IL-33 l mt
liu php mi nhiu tim nng trong iu tr cc bnh hen suyn, vim thp khp.
Cho ti nay, cc nghin cu pht trin cc phn t c ch hot ng ca IL-33 ang
c thc hin giai on u v cn nhiu vn cn phi c lm r trc khi
c mt dc phm khng IL-33 c cng nhn v a vo s dng lm sng.
Nhm tip cn vi hng nghin cu trn, mc tiu ca ti l bc u to
ra v xc nh hot tnh protein ti t hp msIL-33R:Fc hIgG1 s dng h thng
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14
biu hin t bo HEK293 (Human Embryo Kidney). Bn cht ca protein ti t hp
ny l vng ngoi bo ca th th IL-33 chut c mang vng Fc ca IgG1 ngi
u C. Nu c biu hin thnh cng protein ny s tn ti dng dimer gm 2
phn t lin kt vi nhau thng qua cu ni disulfide vng Fc IgG1. Cu trc dimer
ny gip msIL-33R:Fc hIgG1 tng tc tt v bt cc phn t IL-33 t do dch
ngoi bo, t hn ch s tc ng ca cytokine ny ln t bo ch thng qua th
th ca n trn b mt t bo. Kt qu l gim s sn xut cc cytokine gy vim t
cc t bo ch ca IL-33 nh bch cu ht a kim, bch cu ht a acid, tng bo,
t bo git t nhin v lympho bo Th2. Bng cch ny, msIL-33R:Fc hIgG1 c th
lm gim tc ng c hi ca IL-33 gip ci thin tnh trng cc bnh vim t min
v d ng. M hnh hot ng ca msIL-33R:Fc hIgG1 c tm tt Hnh 1.5.
Hnh 1.5. M hnh c ch IL-33 ca msIL-33R:Fc hIgG1
1.5. S lc v cc dc phm c ch hot ng cytokine da trn nguyn tc
tng tc cnh tranh
Ti nay, c 5 loi bit dc c bn cht l mt vng protein c hot tnh
gn vi vng Fc IgG1 c s dng iu tr bnh trn ngi. Cc bit dc ny
c trnh by Bng 1.1.
msIL-33R: Fc hIgG1
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15
Bng 1.1. Cc bit dc c bn cht l protein lai mang vng Fc IgG1
TT Tn bit dc Hng sn xut Bn cht iu tr bnh C ch hot ng
1 Amevive (alefacept) Biogen Idec
LFA-3: Fc IgG1 Vy nn mn tnh
LFA-3:Fc IgG1 tng tc vi CD2 trn t bo T, ngn khng cho t bo T b hot ha do tip xc vi t bo trnh din khng nguyn (APC) biu hin LFA-3 trn b mt [32]
2 Arcalyst (rilonacept) Regen-eron
sIL1RAcP-sIL1R: Fc IgG1
Hi chng t vim do nhit thp c tnh gia nh, hi chng Muckle-Wells v bnh vim a h thng khi pht tr s sinh
sIL1RAcP-sIL1R:Fc IgG1 bt ly IL-1 t do, vn c sn xut qu mc cc bnh c cp, qua ngn khng cho cytokine ny tc ng ln t bo ch nn hn ch c tc ng gy vim ca IL-1 [70]
3
Enbrel (Etanercept) Amgen v Wyeth
sTNFR: Fc IgG1
Vim thp khp, vy nn v x cng ct sng
sTNFR:Fc IgG1 bt ly TNF-/ t do, vn c sn xut qu mc cc bnh c cp, qua ngn khng cho cytokine ny tc ng ln t bo ch nn hn ch c tc ng gy vim ca TNF-/ [78]
4 Nplate (romiplostim) Amgen
TPO: Fc IgG1
Xut huyt do thiu tiu cu
TPO:Fc IgG1 to phc hp vi th th TPO (thrombopoietin) trn mng t bo megakaryocyte ty xng lm gia tng lng tiu cu lu thng trong mu gip ci thin tnh trng xut huyt [56]
5
Orencia (abatacept) Bristol-Myers Squibb
sCTLA-4: Fc IgG1
Vim thp khp t p ng vi thuc khng TNF-
sCTLA-4:Fc IgG1 gn ln protein B7 trn t bo APC ngn khng cho B7 tng tc vi CD28 trn t bo T t hn ch s hot ha t bo T bi APC [14]
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16
Cch tip cn c s dng pht trin protein th th IL-33 dng t do gn
vng Fc IgG1 (sIL33R:Fc hIgG1) nhm c ch IL-33 ca nghin cu ny tng t
nh c ch hot ng c ch IL-1 v TNF- ca bit dc Arcalyst v Enbrel.
1.6. H thng t bo ng vt biu hin protein ti t hp
Nh trnh by, vng ngoi bo ca IL-33R c 3 cu ni disulfide v 9 v tr
glycosyl ha cng vi vng Fc IgG1 s to thnh mt protein c cu trc phc tp v
cn c bin i sau dch m. Hin nay c nhiu h thng biu hin protein c s
dng ph bin nh vi khun, nm men, nm si, t bo cn trng v t bo ng vt
hu nh. Mi h thng c u nhc im khc nhau v cht lng sn phm protein
ti t hp, kh nng tng qui m trong sn xut cng nghip v gi thnh [18]. Trong
, h thng t bo ng vt c nng sut biu hin protein ti t hp thp nht, vi
iu kin nui cy phc tp v tn km nht so vi cc h thng biu hin protein ti
t hp khc. Nhng h thng ny c u im l to ra protein ti t hp c cht lng
cao nht thch hp s dng lm dc phm ngi. Do c im ny nn h thng t
bo ng vt vi b my bin i sau dch m y , l thch hp nht biu hin
sIL33R- Fc IgG1 ti t hp c hot tnh sinh hc. Trong lun vn ny dng t bo
HEK293, t bo c dng ph bin to protein ti t hp trong nghin cu cng
ngh sinh hc c s dng lm h thng biu hin msIL-33R:Fc hIgG.
Dng t bo ngi HEK293 (Human Embryonic Kidney 293) c to ra t s
bin np t bo thn phi ngi vi DNA ca adenovirus loi 5 [21]. DNA 4,5 kb ca
adenovirus loi 5 to lin kt vi b gen t bo ngi v tr gen PSG 4 (pregnancy-
specific -1-glycoprotein) trn nhim sc th s 19 (19q13.2) [40].
HEK293 c th b xm nhim mnh bi adenovirus so vi dng t bo HEK s
cp. HEK293 c kh nng cm ng to khi u sau khi tim vo chut 3-6 tun vi t
l 90-100% [21]. C nhiu bng chng cho thy t bo HEK293 biu hin tnh cht
ca t bo biu m nh c s biu hin ca cc nhn t c hiu cho qu trnh bit
ha t bo biu m giai on sm: vimentin, keratin 8 v keratin 18. Hn na, s
biu hin ca nhiu receptor tyrosine kinase trn b mt t bo cho php t bo p
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17
ng vi mt s kch thch nh yu t tng trng nguyn bo si, yu t tng trng
biu m, insulin, yu t tng trng ging insulin, ephrin [69]. Ngoi ra, t bo
HEK293 th hin c tnh ca t bo thn kinh rt r khi biu hin ba trong bn nhn
t c hiu cho t bo thn kinh l NF-M (Neuroflament- Medium subunit), NF-L
(Neuroflament- Light subunit), -internexin [69] hay biu hin tnh cht ca t bo
to mu [57]. Trong t bo HEK293, nhn t tin vim IL-8 c sn xut theo con
ng NF-B nh s hot ha TLR4 (Toll-like receptor 4) trn b mt t bo [16],
[42] hay nh s cm ng ca IL-33 thng qua th th IL-33R v ng th th IL-
1RAcP c biu hin nhn to trn b mt t bo [1].
H thng t bo HEK293 c ng dng thnh cng trong phng th nghim v
c quy m sn xut ln vi rt nhiu sn phm nh cc protein tit, protein ti t hp,
protein mng, cc vector virus nh adenovirus, retrovirus v lentivirus, cc khng th
n dng v vaccine ng dng trn ngi nh interferon , influenza; trong nghin cu
c tnh sinh l v c ch iu ha ca nhiu phn t bm dnh, cc cytokine v receptor.
Dng t bo HEK293 l h thng biu hin protein ti t hp c s dng rng
ri, nh b my sinh ha c kh nng tin hnh cc qu trnh dch m v bin i sau
dch m mt cch chnh xc to protein trng thnh c chc nng sinh hc. Ngoi ra,
t bo HEK293 cn mang nhng thuc tnh quan trng khc nh pht trin nhanh, bo
qun d dng, c th bin np gen bng nhiu phng php, hiu sut bin np cao [76].
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18
CHNG 2: VT LIU V PHNG PHP
2.1. Vt liu
2.1.1. Plasmid
Plasmid Protein c m ha Trnh t nh du Vector Ngun gc
pFL-mIL-33R IL-33R chut
Pro18 Phe567
3X FLAG u N
Signal pIgplus
(Novagen)
GS. Michael Martin H Giessen c
pmsIL1RAcP-Fc
IL-1RAcP chut dng t do
Ser21 Val347
Fc IgG1 ngi u C
Signal pIgplus
(Novagen)
GS. Michael Martin H Giessen c
pmIL33-Bio
Mat IL33 chut & BirA
Ser109-Ile266 _
pET-Duet (Novagen)
GS. Michael Martin H Giessen c
Phng php
Hnh 2.1. S ca cc plasmid c s dng trong ti
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19
2.1.2. Ha cht to dng plasmid biu hin msIL-33R:Fc hIgG1
B kt tch chit plasmid t vi khun (#27106, Qiagen)
B kt tinh sch DNA t sn phm PCR v gel agarose (#28106, Qiagen)
Cc enzyme ct gii hn: NotI, AvrII, HindIII v buffer phn ng (New
England Biolabs).
Phn ng ni: Buffer DNA T4 ligase 10x, DNA T4 ligase (#M0202S, New
England Biolabs).
Phn ng PCR: Pfu polymerase, Pfu buffer + MgSO4 10x, dNTP (Fermentas)
in di DNA: Agarose (Bio-Rad), Tris base (Promega), Acid acetic (Merck),
Bromophenol blue (Merck), Ethidium Bromide (Merck), thang DNA
(GeneRuler 1kb plus, Fermentas).
2.1.3. Ha cht dng trong nui cy vi khun E.coli
Chng E.coli DH5
Mi trng LB: 1% trypton (Bio-Rad), 0,5% yeast extract (Bio-Rad), 1%
NaCl, nc ct; pH7, hp v trng.
Mi trng LB agar: 1,5% bacto- agar (Bio-Rad) trong mi trng LB.
Ampicillin (Mekophar) 1000x: 50mg/ml ampicillin trong nc, lc v trng.
2.1.4. Ha cht nui cy t bo HEK293 v EL-4
T bo HEK293 do i hc Korea Seoul Hn Quc cung cp
T bo EL-4 c cung cp bi ATCC M
Mi trng nui cy t bo HEK293: DMEM (#G0001-3010- PAA) b sung
10% FCS (#A15-151, PAA), 3,7g/l NaHCO3 v 1x penicillin/streptomycin
(#P11-010, PAA).
Mi trng nui cy t bo EL-4: RPMI1640 (#G0029-3010, PAA) b sung 5%
FCS, 2g/l NaHCO3 v 1x penicillin/streptomycin
Trypsin EDTA (#L11-003, PAA)
PBS: 8g/l NaCl; 0,2g/l KCl; 1,44g/l Na2HPO4; 0,24g/l KH2PO4; pH 7,4; hp
v trng
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20
2.1.5. Ha cht dng bin np plasmid vo E.coli v HEK293
E.coli: glycerol 10%, CaCl2 1M trong nc ct, hp v trng.
HEK293: Polyethylenimine (PEI) (#40,872-7, Aldrich) 1mg/ml trong nc,
pH 7, lc v trng.
2.1.6. Ha cht li gii t bo, nh lng protein v thy phn nhm ng trn glycoprotein
Dung dch li gii t bo: 50mM HEPES; 0,25M NaCl, 20mM -Glycerophosphat,
5mM Na2-4-Nitrophenylphosphat.6H2O, 1mM Na2EDTA.2H2O, 10% glycerol,
0.5% IGEPAL (Sigma).
Dung dch Bradford: Bio-Rad protein assay (#500-0006, Bio-Rad)
BSA (#500-0007, Bio-Rad)
Ha cht thy phn nhm ng: buffer bin tnh glycoprotein 10X, buffer
phn ng G7 10X, NP-40 10% v PNGase F (#P0704, New England Biolabs),
2.1.7. Ha cht dng cho SDS-PAGE, Western Blot v ng kt ta min dch
Protein A-sepharose (GE Healthcare) bt c hiu vng Fc ca IgG1 ngi.
Anti FLAG bead (Sigma) bt c hiu vng FLAG trn FL-mIL33R
Gel SDS-PAGE mini (7x9cm): Tris HCl (Merck), SDS (Promega),
polyacrylamide v bis polyacrylamide (Merck), TEMED (Merck), APS (Merck)
Bng 2.1. Thnh phn gel polyacrylamide dng trong SDS-PAGE
Thnh phn Gel phn tch (5,5ml/gel) Gel gp
(2ml/gel) 7,5% 10% 12,5% 15% - H2O - Tris-HCl 1,5M - pH 8,8 - Tris-HCl 1M - pH 6,8 - SDS 1% - AA/Bis-AA 30%/0,8% - TEMED - APS 40%
2,2ml 1,38ml - 0,55ml 1,37ml 8l 8l
1,74ml 1,38ml - 0,55ml 1,83ml 8l 8l
1,28ml 1,38ml - 0,55ml 2,29ml 8l 8l
0,82ml 1,38ml - 0,55ml 2,75ml 8l 8l
1,22ml - 0,25ml 0,2ml 0,33ml 5l 5l
Laemmli buffer 3X: 5,2ml Tris 1M pH 6,8; 6g SDS; 11,9ml glycerol, 15ml -
Mercaptoethanol, 24mg bromphenol blue, nc ct 100ml.
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21
Dung dch in di SDS-PAGE: 192 mM Glycin (Sigma), 25 mM Tris Base
(Sigma) - pH ~8,5, 0,1% SDS (Merck).
Dung dch chuyn mng: 192mM Glycin, 25 mM Tris Base - pH ~8,5, 10%
Methanol (Merck)
TBS/T: 20mM Tris, 137mM NaCl - pH 7,6 , 0,1% Tween-20 (Merck)
Thang protein: prestained protein ladder (#SM0671 Fermentas), unstained
protein ladder (#SM0661 Fermentas)
Mng nitrocellulose (#162-0115, Bio-Rad)
Cht hin mu DAB (#D5905, Sigma)
Streptavidin HRP (RPN.1231)
Bng 2.2. Cc khng th c s dng trong th nghim Western Blot
STT Epitope nh du Loi Ngun gc pha long
1 Fc IgG1 ngi HRP D Sigma #A0170 1:2000
2 ST2 (IL-33R) chut Khng Th Abcam #ab25877 1:1000
3 IgG th (khng th th cp) HRP La Abcam
#ab16284 1:5000
2.1.8. Ha cht ELISA
Kit ELISA mIL-2 ca BD biosciences (#555148)
Dung dch m ph ging sodium carbonate 0,1M: 8,4g NaHCO3, 3,56g
Na2CO3/lt H2O, pH 9,5
Dung dch ra ging: 0,05% tween 20 trong PBS
Dung dch kha ging: 10% fetal bovine serum (FBS) trong PBS
Dung dch m c cht ELISA: 0,1M sodium acetat, 0,1M citric acid - pH 4,9
Dung dch c cht ELISA: 9ml dung dch m c cht + 1ml TMB (1mg/ml
trong DMSO) + 15l H2O2 3%
3,3,5,5-Tetramethyl-Benzidine (TMB) (#T2885, Sigma)
Dung dch ngng phn ng: H2SO4 1M
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22
2.2. Phng php
2.2.1. To plasmid biu hin protein ti t hp msIL-33R:Fc hIgG1
2.2.1.1. Khuch i on gen m ha cho sIL-33R ca chut
on gen m ha sIL33R (Pro18 Arg332) ca chut c khuch i
bng phn ng PCR da trn khun mu l plasmid pFL-mIL-33R vi mi
xui mIL33R-P18s-HindIII (5 GCGGAAGCTTCCCATGTATTTGACAG
TTACGGAG-3) v mi ngc mIL33R-R332as-NotI (5-AAAGCGGCCG
CTCGGTGATCAATTGGTTGTTTC-3).
Thnh phn phn ng PCR:
Thnh phn Nng cui Th tch cho 1 phn ng (V=50 l) Plasmid 1ng 1 l
Pfu buffer + MgSO4 10X 1X 5 l
dNTPs 25mM 0,2 mM 0,4 l
Mi xui 10 M 0,5 M 2,5 l
Mi ngc 10 M 0,5 M 2,5 l
Pfu polymerase 5U/l 0,1 U/l 1 l
H2O 37,6 l
i chng m cho phn ng PCR c thnh phn tng t nh trn
nhng plasmid c thay th bng nc.
Chng trnh nhit PCR:
95 2 pht
95 45 giy
54 30 giy 35 chu k
72 2 pht
72o 10 pht
Sn phm PCR c kim tra bng phng php in di trn gel agarose 1%.
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23
2.2.1.2. Phn tch DNA bng in di trn gel agarose
10l sn phm PCR c ha vi 1l dung dch np mu 10X (5mM
Tris-HCl - pH 8, 0,0025% Bromphenolblue, 0,0025% Xylen Cyanol, 5%
Glycerol) v in di kim tra trn gel agarose 1% trong buffer TAE 0,5X
(20mM Tris, 0,5mM EDTA, 10mM acetic acid) vi hiu in th 100V trong
30 pht. Gel c nhum vi ethidium bromide (0,05%) trong 15 pht v c
kt qu bng thit b Geldoc (Bio-Rad).
2.2.1.3. Tinh sch DNA
Trc khi thc hin bc tip theo, DNA c tinh sch t phn ng
PCR hoc t gel agarose bng b kit ca hng Qiagen .
Quy trnh tinh sch DNA t sn phm PCR:
Cng 5 ln th tch PBI vo 1 th tch mu PCR v trn u
Cho hn hp trn vo ct tinh sch
Ly tm ct vi tc 13000 vng/pht trong 1 pht, loi b dch sau ly tm
Ra ct bng 750 l PE v ly tm vi tc 13000 vng/pht trong 1 pht,
loi b dch sau ly tm.
Ly tm tc 13000 vng/pht trong 1 pht lm kh ct v chuyn ct
sang eppendorf 1,5ml mi
Cho 30-50 l EB hoc nc v trng vo gia mng trong ct v gi yn
trong 1 pht
Ly tm 13000 vng/pht trong 1 pht v thu nhn dch sau ly tm trong
eppendorf
Quy trnh tinh sch DNA t gel agarose:
Ct phn gel agarose c cha on DNA mc tiu
Cho gel vo eppendorf v cn. Thm 3 ln th tch dung dch QG vo mu
gel (~300 l QG cho 100mg gel)
hn hp 50oC trong 10 pht gel tan hon ton
Thm 1 ln th tch isopropanol vo hn hp v trn u
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24
Cho hn hp trn vo ct tinh sch
Ly tm ct vi tc 13000 vng/pht trong 1 pht, loi b dch sau ly tm
Ra ct bng 750 l PE, ly tm vi tc 13000 vng/pht trong 1 pht,
loi b dch sau ly tm
Ly tm tc 13000 vng/pht trong 1 pht lm kh ct v chuyn ct
sang eppendorf 1,5ml mi
Cho 30-50 l EB hoc nc v trng vo gia mng trong ct v gi yn
trong 1 pht
Ly tm 13000 vng/pht trong 1 pht v thu nhn dch sau ly tm trong
eppendorf
2.2.1.4. Ct hn ch DNA bng NotI v HindIII:
Sn phm PCR khuch i on gen m ha msIL-33R (Pro18 Arg332)
sau khi tinh sch v plasmid pmsIL1RAcP-Fc c ct bng hai enzyme ct
hn ch NotI v HindIII to nguyn liu cho phn ng ni tip theo.
Thnh phn phn ng ct sn phm PCR msIL-33R v plasmid
pmsIL1RAcP-Fc:
msIL-33R pmsIL1RAcP-Fc
Sn phm PCR msIL-33R 10,6 l (5 g) - Plasmid GI0273 - 6,19 l (5 g) NEBuffer 2 10X 5 l 5 l BSA 100X 0,5 l 0,5 l HindIII 20U/l 0,25 l 0,25 l NotI 10U/l 1 l 1 l H2O 32,65 l 37, 06 l
Phn ng c 37oC trong 2 gi. Sn phm ct c kim tra li
bng in di trn gel agarose v tinh sch qua ct, xc nh nng DNA
bng my quang ph.
Thnh phn
Phn ng ct hn ch (V=50 l)
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25
2.2.1.5. Phn ng ni DNA bng ligase
Sn phm ct ca on gen msIL-33R sau khi tinh sch c chn vo
Sn phm ct ca on gen msIL-33R sau khi tinh sch c chn vo
plasmid pmsIL1RAcP-Fc ( c ct b vng sIL1RAcP) v tr ngay trc
on gen Fc IgG1 to plasmid ti t hp m ha protein lai msIL-33R:Fc
hIgG1.
Thnh phn phn ng ni gia sn phm PCR msIL-33R v plasmid
pmsIL1RAcP-Fc ct bng hai enzyme ct hn ch HindIII v NotI:
Thnh phn Nng phn ng Th tch cho 1 phn ng (V=10 l) pmsIL1RAcP-Fc 200 ng 3,8 l msIL-33R 91 ng 0,7 l T4 DNA ligase buffer 10X 1X 1 l T4 DNA ligase 2 weiss unit/l 2 weiss unit 1 l
H2O 3,5 l
Phn ng ni c tin hnh 22oC trong 2 gi. Sn phm ni c
ha bin np vo E.coli DH5.
2.2.1.6. Bin np plasmid vo E.coli DH5 kh np
Mt trong nhng phng php n gin v hiu qu chuyn
plasmid vo t bo E.coli l sc nhit. Qu trnh to t bo E. coli kh np
lm cho cu trc mng ca t bo b yu i, nn khi b sc nhit s hnh thnh
nhng l nh trn mng t bo to iu kin cho cc phn t DNA kt
dnh trn mng i vo bn trong t bo cht mt cch d dng.
Cc bc c tin hnh chuyn plasmid vo t bo E.coli DH5:
Trn u, nh nhng 5 l sn phm phn ng ni (Mc 2.2.1.5) vo 100 l
t bo E. coli kh np. Gi trong 10 pht.
Chuyn eppendorf cha dch t bo trn vo b n nhit 42oC trong 45 giy.
Chuyn nhanh eppendorf cha dch t bo trn vo li b trong 1 2 pht.
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26
Thm vo eppendorf 900 l mi trng LB. Lc vi khun 37oC trong 1 gi.
Ly tm 2500 vng/pht trong 5 pht. Ht b 700 l dch ni, huyn ph
phn cn li v ht 100 l tri trn mi trng a LB c cha ampicillin
nng 50g/ml
37oC qua m, cc t bo E.coli mang plasmid s mc c trn
mi trng chn lc
2.2.1.7. Tch chit plasmid t E.coli
Plasmid c tch chit t E. coli bng b kit Miniprep ca hng
Qiagen theo quy trnh ca nh sn xut, tm tt nh sau:
Tng sinh mt khun lc n E.coli trong 5ml mi trng LB c b sung
ampicillin vi nng 50g/ml iu kin lc 250 vng/pht, 37oC trong 16 gi.
Thu sinh khi bng cch ly tm 8000 vng/pht trong 3 pht nhit phng
Huyn ph sinh khi trong 250 l buffer P1.
Thm 250 l buffer P2 v o ngc khong 4-6 ln.
Thm 350 l buffer N3 v o ngc khong 4-6 ln.
Ly tm vi tc 13000 vng/pht trong 10 pht, chuyn ton b dch ni
vo ct.
Ly tm vi tc 13000 vng/pht trong 1 pht, b dch sau ly tm
Ra ct bng 750 l buffer PE, ly tm vi tc 13000 vng/pht trong 1
pht, b dch sau ly tm.
Ly tm vi tc 13000 vng/pht trong 1 pht lm kh ct v chuyn
ct sang eppendorf 1,5ml mi.
Cho 30-50 l EB hoc nc v trng vo gia mng trong ct v gi yn
trong 1 pht.
Ly tm 13000 vng/pht trong 1 pht v thu nhn dch sau ly tm trong
eppendorf.
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27
2.2.2. Phng php nui cy vi khun
2.2.2.1. Nui cy E.coli DH5
E. coli DH5 mang plasmid c nui cy trong mi trng LB hoc
LB agar c b sung ampicillin duy tr plasmid. Mi trng lng c
37oC v lc vi tc 250 vng/pht qua m. a mi trng agar c
37oC qua m.
2.2.2.2. Bo qun E.coli DH5 mang plasmid
750l canh trng vi khun nui cy qua m c trn vi 750l
glycerol 86% v trng v bo qun lnh -80oC.
2.2.2.3. To t bo kh np E. coli DH5 bng phng php ha bin np
T bo E. coli c kh nng hp th DNA ngoi bo giai on pha log.
B mt t bo mang in tch m do c s hin din ca cc phn t
phospholipids v lipopolysaccharides. iu kin thng, cc phn t DNA
mang in tch m rt kh lin kt vi b mt t bo, nhng khi t bo c
x l vi Ca2+ th cc phn t DNA ngoi bo s d dng dnh vo b mt t
bo qua trung gian Ca2+. T , cc phn t DNA c th c hp th vo
trong t bo cht nh c ch sc nhit hay thc bo.
Cc bc chun b t bo kh np:
Cy 1 khun lc E. coli DH5 vo 5ml mi trng LB lng. Nui cy qua
m iu kin lc 250 vng/pht, 37oC.
Cy chuyn 1ml dch nui cy vo 100ml mi trng LB lng. Nui cy lc
250 vng/pht, 37oC cho n khi gi tr OD590 ca dch nui cy t
khong 0,3-0,4.
Chuyn dch nui cy vo cc falcon 50ml lnh v trong 5 10 pht.
Cc bc tip theo phi thc hin trong lnh.
Ly tm thu sinh khi t bo 2500 vng/pht, 7 pht, 4oC. B dch ni.
Huyn ph sinh khi t bo trong 10ml dung dch CaCl2 0,1 M lnh. Ly tm
2500 vng/pht, 5 pht, 4oC. B dch ni.
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28
Huyn ph sinh khi t bo trong 10ml dung dch CaCl2 0,1 M lnh.
trong 30 pht. Ly tm 2500 vng/pht, 5 pht, 4oC. B dch ni.
Huyn ph sinh khi trong 2ml dung dch CaCl2 0,1 M. B sung 2ml
glycerol lnh 50% v trn u.
Chia lng nh 100 l vo eppendorf 1,5ml v tr -80oC
2.2.3. Phng php nui cy t bo HEK293 T bo HEK293 c nui cy trong mi trng DMEM b sung 10%
huyt thanh bo thai b (FCS: fetal calf serum), penicillin/streptomycin v trong
t m 37oC vi 10% CO2. T bo c cy vo bnh nui t bo 75 cm2 cha
20ml mi trng mt 2x106 /bnh v c cy truyn sau 3-4 ngy nui cy.
cy truyn, t bo c ra 2 ln vi PBS v trng v x l vi 2ml
dung dch trypsin/EDTA 1x trong 2-5 pht 37oC. 8ml mi trng nui cy c
huyt thanh c b sung vo bnh ngng ht ng ca trypsin. T bo c
huyn ph bng pipet. 20l dch huyn ph c nhum bng 20l trypan blue
0,4%. T bo sng bt mu xanh s c m bng bung m hng cu. 2x106
t bo c cy vo bnh nui cy mi.
bo qun t bo, dch huyn ph t bo c ly tm 500xg trong 5
pht loi b mi trng. Cn t bo c huyn ph mt 2x106/ml trong
FCS b sung 10% DMSO. 1ml (2x106 t bo) dch bo qun t bo c cho vo
tup bo qun v c ng lnh chm -80oC qua m v sau c chuyn
vo bo qun bnh nit lng.
gii ng t bo, tup t bo t nit lng c gii ng lp tc
37oC. Dch bo qun t bo c ha vo 9ml mi trng nui cy c FCS v ly
tm 500xg trong 5 pht loi b dch ni. Cn t bo c huyn ph vo
20ml mi trng v nui cy nh m t trn.
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29
2.2.4. Phng php nui cy t bo EL-4 T bo EL-4 c nui cy trong mi trng RPMI1640 b sung 5%
FCS, penicillin/streptomycin v trong t m 37oC vi 5% CO2. T bo c
cy vo bnh nui t bo 25 cm2 cha 10ml mi trng mt 5x104 /ml v
c cy truyn sau 3-4 ngy nui cy.
cy truyn, dng pipet trn u dch t bo, sau ly 20l m
nng nh thc hin t bo HEK293. Chuyn lng dch t bo tng
ng 5x104 t bo/ml vo bnh nui cy mi.
Thao tc bo qun v gii ng t bo EL-4 c thc hin tng t nh
thc hin t bo HEK293.
2.2.5. Phng php bin np plasmid vo t bo HEK293 Phng php bin np plasmid vo t bo dng polyethylenimine (PEI)
c p dng trong nghin cu ny [2].
PEI ng gi phn t DNA to thnh cc ht mang in tch dng, cc
ht ny lin kt vi cc gc mang in tch m trn b mt t bo v c a
vo t bo thng qua c ch nhp ni bo. Cc bng mng cha phc hp PEI-
DNA b ph v do tc ng ca p sut thm thu hoc lysosome gii phng
DNA t do vo t bo cht v sau DNA c khuch tn vo nhn.
Quy trnh bin np plasmid vo t bo:
Chun b a petri (100mm) nui cy 3,6x106 t bo trong 10ml mi
trng trc 1 ngy.
Chun b 360 l hn hp gm 6g plasmid m ha cho protein cn biu
hin, 12l PEI 1mg/ml v mi trng DMEM. Ht b 7ml mi trng trong a t
bo trn v chuyn 360l hn hp vo a.
a trong t CO2 37oC, 10% CO2 trong 4 gi. Sau , b sung 7ml
mi trng nui cy t bo vo a v li trong t CO2
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30
2.2.6. Phng php phn gii t bo T bo HEK293 nhn plasmid biu hin protein FLAG-mIL33R c
phn gii nh sau:
T bo trong a 100mm c ra 2 ln bng PBS lnh.
1ml dung dch li gii t bo (Mc 2.1.6) c cho vo a v t bo c
thu nhn bng tc ng c hc v cho vo 1 eppendorf.
Dch t bo c 1 gi trn phn gii hon ton.
Ly tm dch phn gii t bo tc 13000 vng/pht, 4oC trong 10
pht thu dch ni.
2.2.7. Phng php Bradford nh lng protein Bradford l phng php nh lng protein da trn s thay i bc sng
hp thu cc i ca thuc nhum coomassie brilliant blue khi to phc hp vi
protein. Trong dung dch c tnh acid, khi cha lin kt vi protein th thuc nhum
c bc sng hp thu cc i l 465 nm, khi kt hp vi protein th thuc nhum c
bc sng hp thu cc i l 595 nm. Do , hp th bc sng 595 nm c lin
h trc tip ti nng protein.
Phng php Bradford c thc hin trong a 96 ging vi cc bc
nh sau:
Dng ng chun vi BSA (bovine serum albumin) dy nng : 0;
3,125; 6,25; 12,5; 25; 50; 100; 200g/ml. Tng th tch mu trong mi ging l
100 l, lp li hai dy ng chun.
Cho vo mi ging 100 l cc mu cn o nng protein, mi mu lp
li hai ln. C th pha long mu bng nc nng nm trong ng chun.
Pha thuc nhum Bradford vi t l th tch H2O:Bradford reagent l 3:2
Cho 100 l thuc nhum trn vo mi ging c chun b mu sn
Kt qu c c bng my Elisa bc sng 595nm.
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31
2.2.8. Phng php thu nhn msIL-33R:Fc hIgG1 t dch nui cy t bo HEK293
msIL-33R:Fc hIgG1 do t bo HEK293 biu hin v tit ra mi trng
ngoi bo c thu nhn v tinh sch bng ht Protein A-sepharose.
Sepharose l cc ht gel agarose c cu to t cc phn t
polysaccharide, c kh nng hnh thnh lin kt cng ha tr vi protein A nh gc
OH trn phn t ng. Protein A lin kt c hiu vi vng Fc ca IgG1 thng
qua cc tng tc k nc v tnh in trn chui nng. Do , khi cc ht
sepharose lng xung s ko theo protein mc tiu v t c th thu nhn protein
ny d dng.
Qu trnh thu nhn msIL-33R:Fc hIgG1 t dch nui cy t bo HEK293
c tin hnh nh sau:
Ra 100 l ht Protein A-sepharose 3 ln bng PBS lnh, ly tm 1000
vng/pht trong 1 pht.
Cho ton b ht Protein A-sepharose vo 50 ml dch nui cy t bo
HEK293 biu hin msIL-33R:Fc hIgG1.
Lc o lin tc hn hp 4oC trong 16 - 18 gi.
Ly tm 1000 vng/pht trong 1 pht, b dch ni v thu nhn ht Protein
A-sepharose.
Ra ht Protein A-sepharose 5 ln bng PBS lnh.
Thi gii msIL-33R:Fc hIgG1 ra khi ht Protein A-sepharose bng
dung dch glycin 100mM, pH2. Trn u 100l dung dch thi gii vo ht Protein
A-sepharose, gi trn trong 15 pht, ly tm 1000 vng/pht trong 1 pht v thu
nhn dch ni cha msIL-33R:Fc hIgG1, thm 10l dung dch Tris base 1,5 M,
pH 8,8 trung ha dch protein. Lp li bc thi gii vi glycin pH2 thu trit
protein v gi mu protein -80oC.
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32
2.2.9. Phng php thy phn nhm ng trn glycoprotein msIL-33R:Fc hIgG1 bng PNGase F
Bn cht ca c protein msIL-33R v vng Fc IgG1 ngi l nhng
glycoprotein. Hn na, glycosyl ha l qu trnh bin i sau dch m cn thit cho
s gp cun v biu hin hot tnh ca cc phn t protein c bit l cc protein
th th. lm r c tnh glycosyl ha, protein msIL-33R:Fc hIgG1 c x l
loi b cc nhm ng bng enzyme PNGase F. y l mt amidase c hot
tnh ct t lin kt gia GlcNAc (N-Acetylglucosamine) v amino acid
asparagine nn loi c cc gc ng khi cc phn t glycoprotein.
Phn ng thy phn nhm ng bng enzyme PNGase F c tin hnh
nh sau:
To hn hp 20l gm 10g msIL-33R:Fc hIgG1 v 2l buffer bin
tnh glycoprotein 10X. Gi hn hp trn 100oC trong 10 pht
Mu ngui li ti nhit phng th b sung thm 10l hn hp gm
3l buffer phn ng G7 10X, 3l NP40 10%, 2l PNGase F v 2l H2O. Trn
u hn hp v 37oC trong 2 gi.
2.2.10. Phng php to IL-33 chut nh du biotin IL-33 trng thnh ca chut nh du biotin c to ra bng cch ng
biu hin IL-33 vi enzyme BirA trong vi khun E.coli. IL-33 c thit k c
mang trnh t acid amin GLNDIFEAQKIEWH u N. V tr lysine (K) trong
trnh t trn s c gn nhm biotin nh xc tc ca enzyme BirA.
C th qu trnh biu hin IL-33 nh du biotin c thc hin nh sau:
Bin np plasmid pmIL33-Bio biu hin ng thi protein IL-33 dng
trng thnh ca chut v enzyme BirA vo vi khun E. coli BL21(DE3) kh np.
Mt khun lc n E.coli BL21(DE3) mc trn mi trng LB Agar
penicillin (c mang plasmid pmIL33-Bio) c tng sinh qua m trong 5ml mi
trng LB cha ampicillin vi nng 50g/ml iu kin lc 250 vng/pht, 37oC.
Chuyn 500l dch tng sinh vi khun vo 50ml mi trng LB -
ampicillin v nui cy lc 160 vng/pht, 37oC, qua m.
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33
Chuyn ton b 50ml dch tng sinh trn vo 1 lt mi trng LB -
ampicillin v nui cy lc 160 vng/pht 30oC trong 30 pht.
Cm ng biu hin protein bng IPTG vi nng 10M.
B sung 50l biotin nng 2mM vo dch nui cy sau mi 20 pht
trong vng 4 gi iu kin lc 160 vng/pht, 30oC.
Ly tm thu sinh khi vi khun vi tc 8000 vng/pht trong 10 pht, 4oC.
Sinh khi t bo thu nhn trn c ra 5 ln bng PBS lnh, ly tm
8000 vng/ pht trong 10 pht, 4oC, loi dch ni.
Ra sinh khi ln cui bng buffer ly gii pH 7,9 cha Tris HCl 50mM
v EDTA 1mM, ly tm 8000 vng/pht trong 10 pht, 4oC.
Ha sinh khi t bo trong 5ml buffer ly gii trn, tin hnh ph v
mng t bo bng sng siu m (sonicator) trong thi gian 2 pht 30 giy vi
hiu in th 30V.
Ly tm vi tc 10000 vng/pht trong 10 pht 4oC, thu dch ni
cha protein tng s, gi -80oC.
2.2.11. Phng php SDS-PAGE, Western Blotting
2.2.11.1. in di SDS-PAGE
in di trn gel polyacrylamide vi s c mt ca SDS l k thut phn
tch cc phn t protein b bin tnh theo kch thc ca chng. Gel
polyacrylamide c hnh thnh do s polymer ha cc phn t acrylamide v
s lin kt cho ca methyleneacrylamide do ammonium persulfate (APS)
di s xc tc ca TEMED. Gel polyacrylamide to cc l trng cho php
cc phn t protein i qua, c chn lm mi trng in di v n c tnh tr
v mt ha hc v c to hnh nhanh chng.
SDS l tc nhn chnh gy bin tnh protein. SDS c in tch m rt
ln, c kh nng bc xung quanh khung polypeptide lm bin tnh cc phn t
protein cu trc bc 2 v bc 3 (khng c cu ni disulfide). Phc hp SDS-
protein mang in tch m t l vi khi lng v kch thc ca phn t
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34
protein, khi t trong in trng th protein s chy t cc m sang dng
vi tc khc nhau ty thuc vo kch thc: protein c kch thc cng nh
th chy cng nhanh. Ngoi ra, hn hp in di c un nng n gn nhit
si vi s c mt ca mt s cht kh khc nh 2-mercaptoethanol (BME)
hay dithiothreitol (DTT) lm bin tnh protein bng cch kh lin kt disulfide
dn n tho xon cu trc bc 3 v lm gy cu trc bc 4 ca protein.
Quy trnh in di SDS-PAGE:
Lp khun to gel theo hng dn ca nh sn xut
Pha gel acrylamide phn tch nng mong mun theo thnh phn nu
mc 2.1.7
nhanh hn hp trn vo khe hp gia hai ming knh trn khun v ph
nhanh ln b mt gel mt lp mng isopropanol.
Ch khong 15-30 pht gel ng. v ra sch lp isopropanol trn.
Pha gel gp theo thnh phn nu mc 2.1.7 v nhanh gel gp ln trn
gel phn tch v t lc vo gel to ging.
Khi gel kh th rt lc ra nh nhng v lp khun gel vo b in di.
Cho buffer chy in di vo bn trong khun gel v bn in di ti ng
vch.
Chun b mu protein bin tnh in di: trn 20 l mu protein vi 10 l
laemmli buffer 3X (Mc 2.1.7), gia nhit 95oC trong 10 pht.
Np mu protein bin tnh vo tng ging trn gel, y np bn in di v
chy in di vi hiu in th 100V trong khong 1-2 gi.
Sau khi chy in di xong, tch gel ra nh nhng khi tm knh, c th xem kt
qu bng cch nhum coomassie brilliant blue hoc dng gel chuyn mng
lm Western Blotting.
Quy trnh SDS-PAGE khng bin tnh (non-reducing SDS-PAGE) c
thc hin nh trn nhng s dng laemmli buffer khng c -Mercaptoethanol.
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35
2.2.11.2. Western Blotting
Western Blotting l phng php pht hin protein mc tiu trong phc
hp protein sau khi chuyn ln mng lai nh lin kt c hiu khng nguyn-
khng th. y l phng php c nhy cao, cho php xc nh s c mt,
trng lng phn t v nh lng protein trong cc mu khc nhau. Sau khi chy
in di, protein trong gel c chuyn ln mng lai, ngm mng lai vi khng th
c hiu c mang enzyme hoc c nh du phng x. Phc hp khng
nguyn-khng th c hnh thnh ti v tr protein mc tiu v c pht hin
bng c cht to mu hoc p phim X quang.
Cc bc c tin hnh trong phng php Western Blotting:
in di mu protein trn gel SDS-PAGE
Ngm gel, mng nitrocellulose v giy thm trong dung dch chuyn mng
(Mc 2.1.7) trong 15 pht.
Chuyn protein qua mng nitrocellulose bng my chuyn mng semi dry vi
cng dng in 1mA/cm2 trong 2 gi.
Ngm mng trong TBS/T (Mc 2.1.7) 5% sa gy hoc 5% BSA trong 2 gi
trn my lc ngang nhit phng.
Ra mng 3 ln bng TBS/T, 5 pht/ln trn my lc.
mng lai vi khng th s cp c hiu cho protein mc tiu trong TBS/T vi
t l pha long thch hp (Bng 2.2) trong 2 gi trn my lc nhit phng.
Trong trng hp Western Blotting pht hin bio-mIL33 hay msIL-33R:Fc
hIgG1 th khng th s cp c thay th bng streptavidin-HRP hay anti-Fc
hIgG1-HRP.
Ra mng lai 3 ln bng TBS/T, 10 pht/ln trn my lc.
Hin mu mng lai bng hn hp 3ml TBS/T + vin DAB 10mg + 2,4l H2O2
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36
2.2.12. Phng php ng kt ta min dch (co-immunoprecipitation) Phng php ng kt ta min dch c s dng nh gi s tng
tc gia msIL-33R:Fc hIgG1 hoc FLAG-IL33R vi bio-mIL33. Nguyn tc ca
phng php l cc protein cn kho st c cho vo cng mt hn hp, sau
msIL-33R:Fc hIgG1 hoc FLAG-IL33R c phn tch khi hn hp bi tng
tc c hiu vi protein A-sepharose (tng tc Fc IgG) hay anti-FLAG bead
(tng tc FLAG epitope). Nu bio-mIL33 tng tc vi msIL-33R:Fc hIgG1
hoc FLAG-IL33R, n s c thu nhn khi hn hp cng vi protein ny gi
l s ng kt ta min dch (co-immunoprecipitation). Phng php c tin
hnh c th nh sau:
2.2.12.1. ng kt ta min dch bio-mIL33 v msIL-33R:Fc hIgG1
Cho 10g msIL-33R:Fc hIgG1 v 30g bio-mIL33 vo 1ml PBS c BSA 2%
v 37oC trong 30 pht.
Cho vo hn hp trn 40l Protein A-sepharose, lc o lin tc trong 4 gi 4oC.
Ly tm vi tc 1000 vng/pht trong 1 pht 4oC thu nhn sepharose v
b dch ni.
Ra sepharose 5 ln bng PBS lnh, loi b dch ni bng ly tm 1000
vng/pht trong 1 pht, 4oC.
Cho vo sepharose 50 l laemmli buffer 3X, un nng 95oC trong 10 pht.
Ly tm 1000 vng/pht trong 1 pht, thu dch ni v phn tch mu protein
bng SDS-PAGE v Western Blotting vi khng th c hiu vng Fc IgG1
ca ngi v streptavidin HRP pht hin bio-mIL33.
2.2.12.2. ng kt ta min dch bio-mIL33 v FLAG-IL33R
T bo HEK293, biu hin protein FLAG-IL33R trn mng t bo, c phn
gii theo phng php m t mc 2.2.6 thu nhn dch protein trong c
FLAG-IL33R.
FLAG-IL33R c thu nhn bng cch lc lin tc qua m 4oC dch phn
gii t bo vi anti-FLAG bead vi t l 10l bead/1ml dch phn gii.
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37
20l anti-FLAG bead c gn FLAG-IL33R trn c cho vo hn hp
cha 2g bio-mIL33 khng c hoc c msIL-33R:Fc hIgG1 vi lng tng
dn (6,6g v 66g) trong 1ml PBS 2% BSA. Hn hp c lc o lin
tc trong 4 gi 4oC.
Ly tm eppendorf vi tc 1000 vng/pht trong 1 pht 4oC thu nhn
anti-FLAG bead v b dch ni.
Ra anti-FLAG bead c 5 ln bng PBS lnh, loi b dch ni bng li tm
1000 vng/ pht, 1 pht, 4oC.
Cho vo bead 50 l laemmli buffer 3X, un nng 95oC trong 10 pht. Ly tm
1000 vng/pht trong 1 pht, thu dch ni v phn tch mu protein bng SDS-
PAGE v Western Blotting vi khng th c hiu IL-33R ca chut v
streptavidin HRP pht hin bio-mIL33.
2.2.13. Phng php nh gi kh nng c ch IL-33 ca msIL-33R:Fc hIgG1 trn m hnh t bo nui cy
Di tc ng ca IL-33, t bo EL-4 biu hin v gii phng mIL-2 ra
ngoi mi trng. Khi msIL-33R:Fc hIgG1 c b sung vo mi trng nui cy,
nu c hot tnh sinh hc n s bt ly IL-33 t do lm gim lng IL-33 c th tc
ng ln th th trn t bo do lm gim lng mIL-2 tit ngoi bo.
Quy trnh th nghim:
Cy 105 t bo EL-4 vo mi ging ca a 96 ging trong 100l mi
trng RPMI1640 5% FCS v 37oC 5% CO2.
B sung 50l mi trng RPMI1640 5% FCS cha 2M Ca ionophore
A23187 vo mi ging.
Kch thch t bo bng mIL-33 thng mi nng 10ng/ml khi khng
c mt hoc c mt msIL-33R:Fc hIgG1 vi t l msIL-33R:Fc hIgG1: IL-33 tng
dn 1, 10, 100, 500, 1000 ln. Lu : mIL-33 v msIL-33R:Fc hIgG1 c
chung vi nhau 37oC trong 15 pht trc khi cho tip xc vi t bo EL-4.
T bo c 37oC, 5% CO2 trong 8 gi. Thu nhn dch ni trong
mi ging v lu gi mu -80oC.
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38
2.2.14. Phng php ELISA nh lng mIL-2 Nguyn l ca phng php ELISA l da trn tnh c hiu khng
nguyn-khng th. Khng nguyn c xc nh da vo s to mu ca c cht
di tc dng ca enzyme lin kt vi khng th. ELISA c nhy cao nh
hot tnh khuch i ca enzyme.
Quy trnh thc hin theo hng dn s dng b kit ELISA mIL-2 ca BD
biosciences:
Ph a ELISA bng 100l khng th s cp (capture antibody) c
pha long trong m ph ging vi t l 1:500, a 4oC, qua m.
Ra ging 3 ln bng 300 l dung dch ra ging (Mc 2.1.8) cho mi
ging trong mi ln ra, loi b dung dch ra ging.
Cho 200 l/ging dung dch kha ging (Mc 2.1.8), nhit phng
trong 1 gi. Ra a nh trn.
Chun b ng chun mIL-2: ng chun c pha long bc 2 trong
dung dch kha ging vi dy nng
Cho vo mi ging 100l mu mIL-2 chun cc nng 200; 100; 50;
25; 12.5; 6.3; 3.1 pg/ml, mu dch nui cy t bo th nghim (pha long 10 ln
trong dung dch kha ging) v i chng m. a nhit phng trong 2 gi.
Ra a 5 ln bng 300 l dung dch ra ging / ging/ mt ln ra., Loi
b ht dung dch ra ging.
Cho 100 l khng th th cp ( pha long 1:500 trong dung dch kha
ging ) v streptavidin-HRP ( pha long 1:500 trong dung dch kha ging) vo
mi ging, nhit phng trong 1 gi.
Ra a 7 ln nh trn v loi b ht dung dch ra ging.
Thm 100 l dung dch c cht ELISA (Mc 2.1.8) vo mi ging, a
trong ti, nhit phng khong 30 pht.
Thm 50 l dung dch H2SO4 1M vo mi ging dng phn ng
o gi tr mt quang bc sng 450nm trn my c ELISA.
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39
CHNG 3: KT QU V THO LUN
3.1. To dng plasmid pmsIL33R-Fc mang gen m ha protein
msIL-33R:Fc hIgG1
3.1.1. Khuch i, thu nhn on gen m ha th th IL-33 dng t do
ca chut v chun b vector Signal pIgplus cho to dng
Mc tiu ca ti l to protein msIL-33R:Fc hIgG1 bao gm th th IL-
33 dng t do ca chut (mouse soluble IL-33 receptor: msIL-33R) gn vi vng
Fc IgG1 ca ngi. msIL-33R ca chut di 337 aa, trong trnh t t Met1 ti
Pro328 ging ht nh trnh t vng ngoi bo ca mIL-33R biu hin trn mng
t bo. Do vy, on gen m ha cho msIL-33R c th c thu nhn bng phn
ng PCR vi khun mu l gen m ha cho mIL-33R. Phn ng PCR c thc
hin vi mi xui mIL-33R-P18s-HindIII v mi ngc mIL-33R-R332as-NotI
(Mc 2.2.1.1) trn khun mu l plasmid pFL-mIL-33R mang trnh t nucleotide
ca ton b gen m ha protein mIL-33R (Mc 2.1.1). Sn phm PCR thu c
c kch thc d on l 962 bp m ha mIL-33R vng Pro18-Arg332 tng
ng vi vng ngoi bo ca protein ny v cng l th th IL-33 dng t do.
Vng Met1-Leu17 ca mIL-33R l trnh t tn hiu tit ngoi bo t nhin ca
protein IL-33R. Trnh t ny b loi b khi sn phm khuch i PCR v n
khng cn thit cho protein msIL-33R ti t hp do trong vector biu hin Signal
pIgplus mang sn trnh t tn hiu tit hCD33.
Kt qu in di phn tch trn gel agarose 1% cho thy sn phm PCR c
kch thc tng ng vi kch thc l thuyt ca on gen m ha mIL-33R
Pro18-Arg332 (Hnh 3.1A). Sn phm PCR ny c tinh sch qua ct thc
hin tip cc bc to dng plasmid pmsIL33R-Fc.
chun b vector cho to dng, plasmid pmsIL-1RAcP-Fc vector Signal
pIgplus c mang on gen m ha cho protein msIL-1RAcP c ct bng hai
enzyme ct hn ch l HindIII v NotI (Mc 2.2.1.4). Sn phm ct c kim tra
bng in di trn gel agarose 1% (Hnh 3.1B), kt qu cho thy on gen m ha
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40
msIL-1RAcP (1054bp) c ct khi vector Signal pIgplus (6292bp). Vector
Signal pIgplus c tinh sch khi gel agarose bng b kit tinh sch DNA
(#28106, Qiagen) v c kim tra li bng in di (Hnh 3.1C). Tng t, on
gen m ha msIL-33R thu c trn cng c ct bng hai enzyme HindIII v
NotI v c tinh sch sn sng cho phn ng ni vo vector Signal pIgplus.
3.1.2. To plasmid Signal pIgplus ti t hp mang gen m ha protein
msIL-33R:Fc hIgG1
on gen m ha cho msIL-33R c chn vo vector Signal pIgplus bng
phn ng ni c xc tc bi enzyme T4 DNA ligase (Mc 2.2.1.5) to plasmid
ti t hp biu hin protein msIL-33R:Fc hIgG1. Sn phm ca phn ng ni c
bin np vo E. coli DH5 bng phng php ha bin np (Mc 2.2.1.6). V
plasmid ti t hp mang gen khng ampicillin nn nhng khun lc E. coli DH5
M Signal pIgplus
7000 bp 5000 bp
C
Hnh 3.1. Thu nhn on gen m ha msIL-33R v vector Signal pIgplus
A. Sn phm PCR khuch i on gen m ha mIL-33R Pro18-Agr332 bng cp mi c hiu trn khun mu l plasmid pFL-mIL33R, mu nc c dng lm i chng m.
B. Plasmid pmsIL-1RAcP-Fc c ct bng HindIII v NotI loi b on gen m ha msIL-1RAcP v thu nhn vector Signal pIgplus
C. Vector Signal pIgplus dng thng c tinh sch khi gel agarose v kim tra li bng in di
pmsIL-1RAcP-Fc
M _ HindIII/NotI
B
7000 bp 5000 bp
1500 bp 1000 bp
Vector Signal pIgplus (6292 bp)
msIL-1RAcP (1054 bp)
msIL-33R (962 bp) 700 bp
1000 bp
M H2O pFL-mIL33R A
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41
c bin np thnh cng c kh nng pht trin trn mi trng thch LB vi nng
ampicillin 50g/ml. By khun lc c chn tng sinh trong mi trng LB
lng c ampicillin v plasmid c tch chit t cc dng vi khun ny. kim tra
s c mt ca on gen m ha msIL-33R trong cc dng vi khun c chn, cc
mu plasmid c ct bng 2 enzyme ct hn ch HindIII v NotI v c kim tra
bng in di trn gel agarose 1%. Kt qu th hin Hnh 3.2 cho thy 5/7 dng
plasmid b ct bng HindIII v NotI cho sn phm ct l mt bng c kch thc
tng ng vi kch thc on gen m ha msIL-33R (962bp). Nh vy, 5/7 dng
E.coli DH5 c chn c mang plasmid ti t hp pmsIL33R-Fc. Mt trong s cc
dng plasmid ti t hp ny c kim tra bng gii trnh t ti cng ty Macrogen
(Hn Quc). Kt qu gii trnh t cho thy dng plasmid pmsIL33R-Fc c chn
mang on gen m ha ng trnh t vng ngoi bo IL-33R ca chut t v tr
Pro18 n Arg332 (E08652.1 trn Genbank ca NCBI) v ng khung dch m.
Nh vy, plasmid pmsIL-33R-Fc biu hin protein lai msIL-33R:Fc hIgG1 c
to dng thnh cng v s c s dng cho cc th nghim tip theo.
Hnh 3.2. Kt qu sng lc cc dng plasmid ti t hp By dng plasmid thu nhn t phn ng ni c ct bng enzyme HindIII v
NotI kim tra s hin din ca on gen msIL-33R c chn vo vector pIgplus. Cc mu i chng ln lt l vector Signal pIgplus dng thng, sn phm PCR khuch i on gen smIL-33R v vector ti t pmsIL33R-Fc (dng s 1) khng b ct bi enzyme ct gii hn. Kt qu cho thy cc dng plasmid 1, 2, 5, 6, 7 c mang on chn msIL-33R trong khi dng 3 v 4 khng mang on chn.
700 bp 1000 bp
Trnh t gen m ha
msIL-33R (962bp)
M 1 2 3 4 5 6 7
Cc dng plasmid c ct bng HindIII-NotI pIgplus thng
pmsIL33R-Fc msIL-33R
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42
3.2. Biu hin v thu nhn protein ti t hp msIL-33R:Fc hIgG1 t t bo
ngi HEK293
Sau khi c to dng thnh cng, plasmid pmsIL33R-Fc m ha protein
msIL-33R:Fc hIgG1 c bin np vo t bo HEK293 bng phng php PEI nh
m t Mc 2.2.5. T bo HEK293 mang plasmid c nui cy iu kin 37oC,
10% CO2 trong 72 gi. Trong thi gian nui cy, t bo HEK293 biu hin v tit
protein msIL-33R:Fc hIgG1 ra ngoi mi trng do plasmid pmsIL33R-Fc c
thit k biu hin tn hiu tit hCD33 u N ca protein mc tiu.
Dch nui cy t bo c c c bng cch lc qua ct lc amicon kch
thc 50KDa. Dch nui cy t bo sau c c c xc nh hm lng protein
tng s bng phng php Bradford (Mc 2.2.7). Protein msIL-33R:Fc hIgG1
cng c thu nhn t dch nui cy t bo bng phng php kt ta min dch
thng qua tng tc gia vng Fc hIgG1 v protein A trn ht protein A-sepharose
(Mc 2.2.8). Bn cnh , mu i chng m l dch nui cy t bo HEK293
c chuyn vector rng (khng mang on gen m ha cho protein ti t hp)
cng c thu nhn, c c v thc hin kt ta min dch vi protein A-
sepharose. Cc mu protein th nghim v i chng m nu trn c phn tch
bng phng php in di SDS-PAGE v nhum coomassie blue. Kt qu c
trnh by Hnh 3.3A cho thy khng c s khc bit r rt trong thnh phn
protein ca mu dch nui cy t bo HEK293 biu hin msIL-33R:Fc hIgG1 v
mu dch nui t bo i chng (Sup). Nh vy, vi 100g protein tng s, phn
tch SDS-PAGE khng cho thy c s c mt ca protein ti t hp msIL-
33R:Fc hIgG1 trong dch nui cy t bo HEK293. Tuy nhin, mu tinh sch s
b bng kt ta min dch vi protein A-sepharose (IP: immunoprecipitation),
thnh phn protein ca mu th nghim v i chng c s khc bit. mu IP
dch nui cy t bo mang vector rng, kt qu in di cho 2 bng protein kch
thc khong >25KDa v >55KDa. Vi mu IP dch nui cy t bo mang
plasmid pmsIL33R-Fc, ngoi 2 bng protein nu trn kt qu in di cho thy
thm 1 bng protein c kch thc khong 100KDa. Bng protein kch thc
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43
100KDa ny c th l protein ti t hp msIL-33R:Fc hIgG1 do HEK293 tng hp
v tit ra ngoi mi trng. Kt qu ny cng cho thy qu trnh IP vi protein A-
sepharose thu nhn c protein mc tiu v loi b c phn ln cc protein
c mt trong mi trng nui cy t bo - ch yu l t huyt thanh FCS - ra khi
mu protein sau tinh sch s b.
Do bng protein nhn thy mu IP c kch thc 100KDa ln hn so vi
kch thc l thuyt ca msIL-33R:Fc hIgG1 l 65KDa, kho st SDS-
PAGE/Western Blot c thc hin vi khng th c hiu cho vng Fc hIgG1
khng nh y chnh l protein mc tiu. Ging nh d on, kt qu trnh by
Hnh 3.3B cho php khng nh bng protein kch thc ~100KDa l msIL-33R:Fc
hIgG1. Kt qu tng t cng c thy khi lm Western Blot cc mu IP vi
khng th c hiu cho IL-33R chut (kt qu khng trnh by trong lun vn).
Khng c tn hiu no c nhn thy mu i chng t bo mang vector rng
(EV) khi nhum mng vi khng th khng Fc hIgG1v khng IL-33R. Nh vy, 2
bng protein kch thc >55KDa v >25KDa c trong mu IP dch th nghim v
dch i chng khng lin quan n protein ti t hp mc tiu m l nhng protein
c sn trong mi trng nui cy t bo v c ta vi protein A-sepharose. Da
vo kch thc ca chng v kh nng tng tc vi protein A c th d on 2
bng protein ny l chui nng (~50KDa) v chui nh (~25KDa) ca khng th c
trong huyt thanh bo thai b vn l mt thnh phn thit yu trong mi trng nui
cy t bo HEK293.
Nh vy, protein ti t hp th th IL-33 dng t do ca chut gn vi vng
Fc IgG1 ca ngi (msIL-33R:Fc hIgG1) c biu hin v thu nhn thnh cng
t h thng t bo ngi HEK293.
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44
Hnh 3.3. Biu hin v tinh sch s b protein msIL-33R:Fc hIgG1
t h thng t bo ngi HEK293 T bo HEK293 c bin np bi plasmid pmsIL33R-Fc m ha protein msIL-33R:Fc
hIgG1 hoc vector rng (EV: empty vector). Sau 72 gi, 10ml dch nui cy t bo c thu nhn v c c bng lc amicon kch thc l 50KDa (Sup), hoc c trc tip vi 20l ht protein A-sepharose (IP) thu nhn protein ch msIL33R:Fc hIgG1. 100g protein tng s ca dch nui cy t bo c c (Sup) v 20l ht protein A-sepharose sau IP c x l trong laemmli buffer v phn tch bng SDS-PAGE v Western Blot. (A) Cc mu protein c phn tch bng SDS-PAGE v nhum coomassie blue cho thy bng protein msIL33R:Fc hIgG1 (mi tn en) v chui nng - chui nh ca khng th b (mi tn xm). (B) Protein msIL33R:Fc hIgG1 trong mu IP c pht hin bng Western Blot vi khng th c hiu vng Fc hIgG1. Th nghim c lp li 3 ln vi kt qu tng t.
3.3. Phn tch mt s c im ca protein ti t hp msIL-33R:Fc hIgG1
3.3.1. S ng ha ca msIL-33R:Fc hIgG1 ti t hp biu hin t HEK293
Theo kt qu trnh by Mc 3.2, protein msIL-33R:Fc hIgG1 c biu
hin t HEK293 c kch thc khong 100KDa, kch thc ny ln hn kh nhiu
so vi kch thc l thuyt ca protein c tnh bi cng c Compute pI/Mw
(www.expasy.org/proteomics) l 65KDa. S gia tng kch thc thc t ca
protein ti t hp msIL-33R:Fc hIgG1 biu hin t t bo HEK293 so vi kch
thc tnh ton c th l do protein ti t hp tri qua qu trnh glycosyl ha.
B
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45
Glycosyl ha l qu trnh bin i sau dch m cn thit cho s gp cun v biu
hin hot tnh ca cc phn t protein c bit l cc protein th th.
lm r c tnh glycosyl ha ca protein ti t hp msIL-33R:Fc hIgG1,
protein c x l loi b cc nhm ng bng enzyme PNGase F (New
England Biolabs) nh phng php nu Mc 2.2.9. y l mt amidase c hot
tnh ct t lin kt gia GlcNAc (N-Acetylglucosamine) v amino acid
asparagine nn loi c cc gc ng khi cc phn t glycoprotein. Mu
protein msIL-33R:Fc hIgG1 c x l v khng x l vi enzyme PNGase F
c phn tch bng SDS-PAGE/Western Blot vi khng th c hiu vng Fc
IgG1 ngi. Ging nh mong i, phn tch Western Blot cho thy mu msIL-
33R:Fc hIgG1 c x l vi PNGase F c trng lng thp hn mu protein
khng x l vi enzyme (Hnh 3.4). iu ny cho thy protein msIL-33R:Fc
hIgG1 biu hin t bo HEK293 c glycosyl ha. ng ch l mu
msIL-33R:Fc hIgG1 loi b cc gc ng c kch thc ng vi kch thc
tnh ton l 65KDa.
Kt qu ny hon ton hp l v nh bit msIL-33R l mt
glycoprotein vi 9 v tr c th c ng ha v vng Fc hIgG1 cng l mt
glycoprotein. Hin tng thay i kch thc do ng ha cng c thy
trong nghin cu ca Senn v cs nm 2000 biu hin T1-Fc c kch thc 97KDa
t t bo gan chut [68]. Nhiu nghin cu cho thy vng ngoi bo msIL-33R
t kch thc 38,5 KDa c th b glycosyl ha ti 65 KDa trong t bo HEK293
v fibroblast, vng Fc hIgG1 t 26 KDa cng b glycosyl ha ti 32-35 KDa
trong t bo HEK293 [45], [22], [85].
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46
Hnh 3.4. S glycosyl ha ca msIL-33R:Fc hIgG1 c biu hin t t bo ngi HEK293
10g protein msIL-33R:Fc hIgG1 c x l loi b cc nhm ng bng 1000 unit enzyme PNGase F iu kin 37oC trong 2 gi. Mu i chng c x l tng t nhng PNGase F c thay th bng 1 th tch tng ng PBS. Cc mu protein c phn tch SDS-PAGE/Western Blot vi khng th c hiu vng Fc hIgG1. Th nghim c lp li 2 ln vi kt qu tng t.
3.3.2. Protein msIL-33R:Fc hIgG1 c biu hin v tit ra mi trng ngoi
bo dng dimer
Trong nghin cu v sn xut protein ti t hp th vic biu hin protein c
hot tnh sinh hc v n nh v mt cu trc c xem l yu t quan trng nht.
Khng th c tnh n nh cao khi tn ti dng dimer c chng minh qua
mt s nghin cu, nh Goto v Hamaguchi kho st trn vng CL IgG ca
ngi vo nm 1979 [20] hay Thies v cs kho st trn vng CH3 IgG ca
chut vo nm 2002 [75]. c mt s th th t do dng phn t lai c mang
vng Fc hIgG1 c pht trin thnh cng v c ng dng trong tr liu nh
Amevive, Arcalyst (Mc 1.5). Vng Fc hIgG1 c gn vo mt protein c
hot tnh sinh hc ngoi mc ch thun tin trong tinh sch v thu nhn protein
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ti t hp th n cn c vai tr lm tng thi gian tn lu ca protein trong c th
v lm tng i lc ca protein vi phn t ch. Vng Fc hIgG1 trn 2 phn t
protein lai s lin kt vi nhau thng qua ba cu ni disulfide c hnh thnh
gia cc gc cystein (hai cu ni pha trc vng CH2 v mt cu ni trong vng
CH3), nh cc protein lai ny c biu hin dng dimer. Do vy, cu trc
dimer l c im quan trng m protein msIL-33R:Fc hIgG1 c to ra trong
nghin cu ny cn c c.
kim tra protein msIL-33R:Fc hIgG1 c c t bo HEK293 biu hin
dng dimer hay khng, protein ti t hp c phn tch bng SDS/PAGE
trong iu kin khng bin tnh (s dng laemmli buffer khng c -
mercaptoethanol) v bin tnh (s dng laemmli buffer c -mercaptoethanol). -
mercaptoethanol trong laemmli buffer l nhn t lm t gy cc cu ni
disulfide gia 2 n phn smIL-33R:Fc hIgG1 nn lm bin i cu trc dimer
thnh dng monomer. Do vy, nu protein msIL-33R:Fc hIgG1 tn ti dng
dimer th phn tch SDS-PAGE khng bin tnh s cho bng protein c kch
thc cao hn nhiu so vi khi phn tch bng SDS-PAGE bin tnh. Kt qu ghi
nhn Hnh 3.5, bng protein thy mu in di khng bin tnh c kch thc
ln hn hn so vi bng protein thy mu in di bin tnh. Nh vy protein
msIL-33R:Fc hIgG1 biu hin t t bo HEK293 tn ti dng dimer.
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Hnh 3.5. Protein msIL-33R:Fc hIgG1 c biu hin dng dimer
20ml dch nui cy t bo HEK293 mang plasmid pmsIL33R-Fc c thu nhn v chia lm 2 phn bng nhau. Protein msIL-33R:Fc hIgG1 trong mi 10ml dch ni c thu nhn bng IP vi 20l protein A-sepharose. Sau khi ra nhiu ln, cc ht protein A-sepharose c gia nhit 95oC - 10 pht trong laemmli buffer khng bin tnh (khng c -mercaptoethanol: 1) hay bin tnh (c -mercaptoethanol: 2) v c phn tch bng SDS/PAGE. Kt qu th hin trn l i din ca 3 ln th nghim lp li c kt qu tng t.
3.4. Biu hin IL-33 chut c nh du biotin t vi khun E.coli
IL-33 nh du biotin (bio-mIL33) l nguyn liu cn thit cho bc th
nghim hot tnh ca protein msIL-33R:Fc hIgG1 biu hin t t bo HEK293
trn. tng hp bio-mIL33, plasmid pmIL33-bio (do GS. Michael Martin i
hc Giessen c tng) m ha ng thi cho protein mIL-33 dng trng thnh
(mature IL-33) v enzyme biotin ligase BirA c bin np vo dng E. coli
BL21(DE3). Dng E.coli BL21(DE3) mang plasmid c chn lc v tng sinh
trn mi trng LB c ampicillin v cm ng biu hin protein mIL-33 v BirA
bng IPTG c b sung biotin vo mi trng. Trong t bo E.coli enzyme BirA xc
tc qu trnh gn biotin vo v tr amino acid lysine trong 1 trnh t peptide c bit
1. SDS/PAGE khng bin tnh 2. SDS/PAGE bin tnh
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c thit k u N ca mIL-33 (Mc 2.2.10). Sau thi gian nui cy biu hin
protein, E.coli c thu nhn bng ly tm, ra bng PBS lnh v c ly gii bng
sng siu m trong dung dch Tris HCl pH 7,9. Nng protein tng s trong dch ly
gii E.coli l 32mg/ml c xc nh bng phng php Bradford. Mu protein i
chng m cng c chun b tng t t E. coli BL21(DE3) khng mang plasmid
pmIL33-bio. Hai mu protein ny c kim tra bng phng php in di SDS-
PAGE vi cng hm lng protein l 50g. Kt qu nhum coomassie blue cho thy
mu protein biu hin bio-mIL33 c bng protein v tr ~25KDa m hn so vi
bng protein tng ng mu i chng m (Hnh 3.6A). y c th l vch protein
bio-mIL33 ti t hp biu hin E.coli. kim chng s hin din ca bio-mIL33
trong mu dch ly g
