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Anti-E1

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Title: Slide 1 Author: yinling lin Last modified by: dplavin Created Date: 2/28/2008 5:15:30 AM Document presentation format: On-screen Show Company – PowerPoint PPT presentation

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Title: Anti-E1


1
Supplemental Fig. 1
Anti-E1
Anti-E2
A
r E1E2p7 protein)
VEE/SIN-E1E2P7
VEE/SIN-E1E2P7
VEE/SIN-E1E2P7
rE1E2P7 protein
VEE/SIN-E1E2P7
r E1E2P7 protein
VEE/SIN-E1E2
VEE/SIN-E1E2
VEE/SIN-NS345
rE1E2p7 protein
VEE/SIN-E1E2
VEE/SIN-E1E2
VEE/SIN-NS345
35
75

33-35


68-74
25
50
35
15
25
EndoH
EndoH
B
rSOD-NS5 (NS5A/B)
rSOD-NS5 (NS5A/B)
rSOD-C33C (NS3)
rSOD-C100 (NS4)
VEE/SIN-NS345
VEE/SIN-NS345
VEE/SIN-NS345
VEE/SIN-NS345
rE1E2p7 protein
rE1E2p7 protein
rE1E2p7 protein
rE1E2p7 protein




70
NS4A/B 35

69


NS4B 27

57


NS4A 8
Anti-NS3
Anti-NS4
Anti-NS5A
Anti-NS5B
2
Legend of supplemental Fig. 1 Detection of HCV
protein expression from VEE/SIN replicon
particles by Western blotting. A. Detection of E1
and E2 expression from the BHK cell lysates with
VEE/SIN-E1E2 and VEE/SIN-E1E2p7 infection.
VEE/SIN-NS345 is a negative control. rE1E2p7
protein is a positive control. The size was
indicated corresponding to E1 (33-35kDa) and E2
(68-74kDa). EndoH protein was treated with EndoH
(Biolab) at 37ºC for 1 hour. B. Detection of NS3,
NS4, NS5A and NS5B expression from the cell
lysates with VEE/SIN-NS345 infection. rE1E2p7 is
a negative control. SOD-C33C (a.a. 1192-1457),
SOD-C100 (a.a. 1569-1931), and SOD-NS5 (a.a.
2054-2995) are recombinant proteins used as
positive controls for the detection of NS3, NS4,
NS5A and NS5B respectively. The size was
indicated corresponding to NS3, NS4A, NS4B,
NS4A/B, NS5A and NS5B. Indicated the specific
detection of the proteins.
3
Supplemental Fig. 2
A
B
PBS (1,2,3)
E1E2/MF59 (1,2,3)
PBS (1,2,3)
E1E2/MF59 (1,2,3)
VEE/SIN-E1E2 (1,2,3)
E1E2/MF59/CpG (1,2,3)
VEE/SIN-E1E2 (1,2,3)
E1E2/MF59/CpG (1,2,3)
E1E2/MF59 (1,2) VEE/SIN-E1E2 (3)
E1E2/MF59/CpG (1,2) VEE/SIN-E1E2 (3)
E1E2/MF59 (1,2) VEE/SIN-E1E2 (3)
E1E2/MF59/CpG (1,2) VEE/SIN-E1E2 (3)
IFN-g
IFN-g
CD8
CD4
4
Legend of supplemental Fig. 2 Detection of CD4
and CD8 T cells responses by ICS/FACS. BALB/c
mice (n 10) received three injections i.m. at
3-week intervals by the immunogens as indicated.
The spleen cells were harvested 2 weeks after the
last immunization and stimulated with 10 mg/ml of
HCV specific peptides for ICS and FACS analysis.
The data are presented as dot plots of ICS for
CD4 and IFN-g after E1 peptide pool stimulation
(A) and for CD8 and IFN-g after CD8 E2 peptide
stimulation (B). Significant responses are
indicated by circles in the upper-right quadrant.
5
Supplemental Fig. 3

PBS (1,2,3)
E1E2/MF59 (1,2) VEE/SIN-E1E2 (3)
E1E2/MF59 (1,2,3)
VEE/SIN-E1E2 (1,2,3)
VEE/SIN-E1E2 (1,2) E1E2/MF59 (3)
VEE/SIN-E1E2 (1,2) E1E2/MF59/CpG (3)
E1E2/MF59/CpG (1,2) VEE/SIN-E1E2 (3)
E1E2/MF59/CpG (1,2,3)
6
Legend of supplemental Fig. 3 VEE/SIN-E1E2
immunization and Prime/boost regimen with
E1E2/MF59 and VEE/SIN-E1E2 could stimulate good
CD8 T cells responses. BALB/c mice (n 10) were
received three injections i.m. at 3-week
intervals by the immunogens as indicated. The
spleen cells were harvested at 2 weeks after the
last immunization and stimulated with 10 mg/ml of
HCV specific peptides for ICS and FACS analysis.
20-mer over-lapping peptide pools for HCV-1a E1
region (E1 pool) and E2 region (E2 pool), and
single peptide to E2 for CD4 T cells (CD4 E2
pep) and CD8 T cells (CD8 E2 pep) were used for
stimulation. The data are presented as mean total
percentages of CD8IFN-g cells from two pools
(five mice per pool). Plt0.05 as compared with
PBS control group.
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