biu hin protein ti t hp NS3, E.

coli chng XL1- blue c in bin np

plasmid pQE30NS3 theo hng dn s dng ca Qiagen. Dng t bo bin np cha
pQE30NS3 c nui cy qua m 37
o
C, mi trng LB cha 50g/mL ampicillin.
Sau khi nui cy qua m, tin hnh pha long 100 ln trong mi trng LB v cc
khng sinh sau , 37
o
C n khi gi tr OD600= 0.5. sau khi biu hin DENV2-NS3,
cm ng bng cch b sung 1mM IPTG (Sigma-Aldrich, USA), trong 4h, 37
o
C. phn
tch biu hin ca protein NS3, cc mu c ly tm 10000 g, 10 pht 4
o
C. cn t bo
c phn tch bng in di.
Thc hin tng t i vi XL1-blue c chuyn plasmid pQE-30
Tinh sch
Cc t bo thu c em ra li trong m A (50 mM TrisHCl, pH 8, 5 mMb
mercaptoethanol (bMeOH), 500 mM NaCl, and 1 mM PMSF). T bo b ph v ngay lp
tc nh qu trnh ng bng, tan bng trong N2 lng v sau ly tm 15000g, 20 pht,
4
o
C tch ra phn protein ha tan v cn cha protein khng tan. Phn khng tan cha
cc th vi c ra li trong m B (8 M urea, 50 mM TrisHCl, pH 8, 5 mMbMeOH,
200 mM NaCl, v 1 mM PMSF), qua m 4
o
C. dch thy phn sau c ly tm
15000g, 1h, 4
o
C. phn protein ha tan c load vo ct i lc HisTrap
TM
(GE Health
care Life Sciences, USA) s dng h thng sc k i lc HPLC (Amersham Biosci-ences,
USA). Cc protein gn trn ct c ra bng m C (m B, pH 6.3). tin hnh ra gii
protein vi gradient s dng m D (8 M urea, 50 mM TrisHCl, pH 8,5mM MeOH,
200 mM NaCl, 1 mM PMSF, and 500 mM imidazole). Cc phn on peak (3ml) c
gp li v pha long n khi hp th 280nm t 0.8 1. Protein NS3 b bin tnh
c ti gp bng phng php thm tch trong m E (50 mM TrisHCl, pH 8, 500 mM
NaCl, 10 % glycerol, 5mM MeOH, v 0.05 % CHAPS) cha urea cc nng khc
nhau, urea gim dn mi gi t 6,4,2,1 v 0.01 M, v khng c urea, qua m, 4
o
C.
sn phm thm tch thu hi bng siu lc s dng mng YM-30 (Amicon, USA), sau
cho qua ct gel HiPrep 26/60 Sephacryl S-100 HR gel ltration column (GE Healthcare
Life Sciences) vi tc 1.0mL/pht. Cc phn on peak c gp li v c c ti
nng 5mg/mL. ton b cc mu protein c phn tch bng in di.
Phn tch protein
Cc mu protein c in di SDS-PAGE 12%. Cc band protein c quan st nh
nhum vi Coomassie brilliant blue R-250 (Sigma). Nng protein c xc nh bng
phng php Bradford (Bio-Rad Laboratories, USA).
Cc phng php min dch xc nh protein ti t hp NS3
WB
Cc mu protein c chuyn t gel polyacrylamide ln mng nitrocellulose. Mng c
qua m vi 4% sa tch bo trong PBS-T (0.8 % NaCl, 0.02 % KCl, 0.014 %
KH2PO4, 0.009 % Na2HPO4,pH 7.4, and 0.05 % Tween-20) at 4oC, ra 3 ln vi PBS-
T, sau 2h nhit phng (RT) vi khng th s cp anti-DENV2 HMAF t l
1:100 vi 4% skimmed milk trong PBS-T. sau khi ra sch, mng c phn ng vi
IgG peroxidase conjugate (GE Healthcare Life Sciences) vi t l 1:2000 trong PBS-
Tween trong 1 h nhit phng. Sau , ra li mng cc band c pht hin bng
cch vi c cht mu diaminobenzidine (DAB) n khi thay i mu sc c cht.
ELISA
xc nh phn ng ca cc protein ti t hp DENV2-NS3 chng li HMAFs tng
ng vi 4 type huyt thanh, phng php ELISA c p dng. a ELISA gm 96 ging
(Greiner bio-one,Germany) ph 5g/mLprotein ti t hp NS3 trong 2h, 37 v c i
chng dng; 100 g/mL khng nguyn DENV2 pha long trong m (0.16 %
Na2CO3,0.29 % NaHCO3, and pH 9.5). i chng m, ph 5 g/mL E.coli v 100L
Mock. Sau , cc a c ph BSA,1 gi, 37 trong cng dung dch m. ra 3 ln
vi PBS-T sau mi bc ELISA. ..
diluted and incubated for 1 h at 37 C. Goat anti-mouse
IgG peroxidase conjugate at 1:2,500 dilution was added
and plates were again incubated for 1 h at 37 C. After
washing, the reaction was developed with 100 lL of
500 lg/mL ortho-phenylenediamine dihydrochloride
(OPD) as chromogen and 0.015 % hydrogen peroxide as
substrate in 0.1 M citrate buffer (2 % Na2HPO4, 1 % citric
acid, pH 5.0). Plates were kept for 30 min at 25 C and the
reaction was stopped with 12.5 % H2SO4. The absorbance
was measured at 490 nm using an ELISA plate reader
(Bio-Rad Laboratories). Mean values and standard errors
of the mean for each group were calculated. The positive
cut-off value was taken as twice the absorbance value of
the negative control
Nui cy t bo v kch thc virus
T bo lch ca chut c tim chng DENV2 thu trong iu kin v trng v
cc hng cu c ly gii bng cch cho thm 0.83% dung dch NH4Cl. Cc t
bo ng vt c ra 2 ln vi PBS v 2% HFBS (PAA Lab-oratories, Ontario,
Canada) v dch ni cha 2x10^6 t bo trong mi trng RPMI-1640 b sung
100 U/mL penicillin, 100 g/mL streptomycin (Gibco,USA), 2 mM glutamine
(Gibco), 5 9 105 M MeOH (Sigma), and 5 % HFBS. Kt thc, mi ging c
2x10^5 t bo c nui cy trong 96 ging cng vi khng nguyn (DENV2
hoc Mock v 10g/mL protein NS3 tinh ch. ConA(sigma) c dung lm i
chng dng. Sau 4 ng, vi 5% CO2, 37 , thu dch ni v bo qun -20 .
Tt c cc phng php lp li 3 ln.

























Kt qu
To dng, biu hin v tinh sch protein ti t hp DENV2-NS3