ASU TVDC Technical Report
6/22/10
Kathryn F. Sykes and Stephen A. Johnston
and Phillip Stafford
Completed Milestones: 25, 26, 28* and 32, 33, 34, 35,
36
Active Milestones: UNM 29*/ASU28*, ASU 37/38
Currently Inactive Milestones: 30
Slide 1
MILESTONE 28
Build SCHU S4
proteome
Gray: (sub)milestone title
Yellow: Re-opened
Build ORF expression
library corresponding
to proteome
Generate complete
protein-fragment library
Array protein-fragments
for T cell stimulation
assays
Re-opened
Re-opened
Re-opened
Slide 2
Generation of polypeptide
antigens for UNM T cell assays
SOP for detecting T cell
stimulation with synthetic
(IVTT) antigens
Production of
antigens (ASU)
Production
Assay
development
(UNM)
T cell proliferation
IFNg ELISpot assay
Screening
(UNM)
Identification of
stimulatory proteins &
peptides
Confirmation
Assay optimization
using ivt proteins
Slide 3
Evaluating alternative IVTT
protein capture beads
• In our initial test of the small vs. large diameter capture beads,
immune cells from LVS-immunized NHP were used for the IFNgamma ELISPOT assays.
• We determined that background was low using either bead.
• A second test was designed to test specificity of stimulated
response, against IglC1-immune rat splenocytes.
• Large scale synthesis of IglC 1 (putative positive)and ASFV053
(negative control) was repeated on both small MyOne (1micron) and
large M-280 (2.8 micron)beads.
Slide 4
New Preps of IglC1 and ASFV053 on the two bead formats
0.25x
0.75x
0.25x
BSA, ug
2 1 0.5
ASFV053
IVT-rx
250150100755037kDa
37-
33kDa
2520-
MyOne
MyOne
10-
M-280
MyOne
15M-280
Precision Plus Standard (Bio-Rad)
kDa
0.75x
4
0.75x
iglC1 IVT-rx
Slide 5
Production of LEE Templates
for New IVTT FTT Library
• New vs. old prep of genomic FTT was
compared in PCRs.
• Titrating optimal new template amount
for PCRs.
Slide 6
Evaluation of new Schu-S4 genomic DNA preparation (May 2010) as a template for ORF amplification
L
1
2
3
4
5
1
2
FTT1008A
3
4
5
L
1
2
FTT1800A
3
4
5
1
2
FTT1001A
3
4
5
FTT1016A
bp
1000 –
500 –
250 –
bp
1000 –
750 –
PCR
by-products
500 –
250 –
PCR
by-products
L
1
2
3
4
FTT0549A
5
1
2
3
4
5
L
FTT0633A
1
2
3
4
FTT0511A
5
1
2
3
4
5
FTT1215B
bp
1000 –
bp
1000 –
750 –
500 –
250 –
500 –
250 –
PCR
by-products
PCR
by-products
L - 1kb DNA Step Ladder (Promega)
1 - 4 ng “new” gDNA prep template (May 2010)
2 - 9 ng “new” gDNA prep template (May 2010)
3 - 22 ng “new“ gDNA prep template (May 2010)
4 - 100 ng “new” gDNA prep template (May 2010)
5 - 60 ng “old” gDNA prep template (March 2006)
Slide 7
Interpretations
• ORF PCR products are considerably cleaner;
there are few to no byproducts.
• Significantly less DNA is required. As little as
4ng of starting genomic DNA was sufficient to
amplify most ORFs, at high yields and
acceptable specificity.
• However,100ng of starting template appears to
be least likely to generate the lower migrating by
product
Slide 8
Testing parameters for optimal LEE
construction
• Assembly of LEEs from ORFs generated
with old vs. new gDNA.
• Methods of purifying ORFs before
assembly
• Amount of PCA template for final
production step
Slide 9
Overlapping PCR optimization for LEE template assembling with potentially problematic ORFs
AMP
12 11 12 11 12 13 14
AMP
11 12 11 12
AMP
AMP
AMP
AMP
GP
GP
1 step PCA
Test 3
AMP
AMP
AMP
AMP
EP
UP
GP
AMP
Test 2
GP
AMP
EP
UP
UP
GP
AMP
EP
GP
AMP
EP
UP
Test 1
1kb DNA
Step Ladder
(Promega)
GP
GP
AMP
AMP
1kb DNA
Step Ladder
(Promega)
bp
10005001 2 3 4
7
8
14 13 14
14
14
2
AMP
AMP
AMP
AMP
AMP
AMP
AMP
AMP
1 2
AMP
GP
GP
AMP
AMP
AMP
AMP
GP
GP
AMP
AMP
2 step PCA
EP
UP
GP
AMP
EP
6
4
UP
GP
AMP
3
UP
2
GP
AMP
EP
GP
AMP
EP
UP
1
5
10005009
5
6
7
10
1
2
4
5
7
2
1 2
1
2
3 4
3
4 3
4
3
4
3 4
8
8
- “Old” (March 2006) gDNA template
for ORF amplification
- GP – gel purified products for PCA;
AMP – AMPure purified products for PCA ;
EP – ethanol precipitated PCR products for PCA;
UP – un-purified PCR products for PCA
- 1step PCA: 15x cycles (Ta=58C) for LEE (P+O+T)
- 1/5 PCA template for PCR
1
- “Old” (March 2006) gDNA template
for ORF amplification
- AMPure purified products for PCA only
- 1 step PCA: 15x cycles (Ta=58C) for LEE (P+O+T)
- 2 step PCA: 10x cycles (Ta=53C) for LEE (P+O)
+ 10x cycles (Ta=58C) for LEE (PO+T)
- 1/25 PCA template for PCR
- “New” (May 2010) gDNA template
for ORF amplification
- GP – gel purified products for PCA;
AMP – AMPure purified products for PCA
- black - 160 fmole, blue – 320 fmole,
purple – 480 fmole of each component (P,O,T)
in PCA
- red - 160 fmole ORF with byproduct after
AMPure purification
- 1 step PCA: 10x cycles (Ta=58C) for LEE(P+O+T)
- 1/50 PCA template for PCR
Genes: 1- FTT1001A; 2- FTT1068A; 3- FTT0511A; 4- FTT1215B; 5- FTT0639A; 6- FTT1329A; 7- FTT0877A; 8-FTT1712A; 9- FTT0162A; 10- FTT1589A;
11- FTT1008A; 12- FTT1800A; 13- FTT0549A; 14- FTT0633A
Slide 10
Continued:
Evaluation of quality of different re-array and dilution primer sets
from 2007-2008 to produce potentially problematic ORFs
- 5 and 10 uM primer and primer-mix subsets significantly
degraded during storage and can not be use for ORF
amplification.
- 50 uM original primer stocks and 0.8 uM primer-mix
subsets had demonstrated decent quality during test-ORF
amplification. However both subsets are partial and can
cover amplification of 70-80% of the targeted (problematic)
ORFs.
Slide 11
Evaluation of primer quality from 50 uM stock plate subset
1
2
3
4
5
6
7
8
bp
1000500-
PCR
by-products
Genes: 1- FTT1166A; 2- FTT0478B; 3- FTT1401A; 4- FTT0451A; 5- FTT0611A; 6-FTT1036B;
7- FTT0420A; 8-FTT1715A
Slide 12
Overlapping PCR optimization for LEE template assembling with potentially
problematic ORFs
3
AMP
AMP
7
AMP
UP
AMP
AMP
UP
AMP
UP
AMP
UP
AMP
UP
AMP
UP
AMP
UP
Test 4
bp
15001000-
500-
1
2
3
4
5
PCA in 50 ul
6
4
5
PCA in 30 ul
- “New” (May 2010) gDNA template for ORF amplification
- UP – un-purified PCR products (P,O,T )for PCA
AMP – AMPure purified products (P,O,T) for PCA
- 160 fmole of each component (P,O,T) in PCA
- 1 step PCA: 10x cycles (Ta=58C) for LEE(P+O+T)
- 1/50 or 1/30 PCA template for PCR (20x cycles)
Genes: 1- FTT1166A; 2- FTT0478B; 3- FTT1401A; 4- FTT0451A; 5- FTT0611A; 6-FTT1036B; 7- FTT0420A
Slide 13
Additional Points
• Description of deliverables completed for each active
milestone:
– MS 35: Microarray results with qPCR verification
– MS 36: Set of 9 proteins for testing as vaccine candidates
• List of relevant publications from the past month
•
ASU reports no TVDC publications in May 2010
• MSCR status
– MS33: NIAID provided review on 6/2/10; ASU edited 10 SOPs; NIAID MSCR
comments under review at UNM on 6/17/10.
– MS34: Is overdue; At ASU site visit, Mitch planned to submit to UNM by 3/31/10
Wed.
– MS35: Mitch- completed work; ASU will write MSCR (5/31/10- overdue)
– MS36: Mitch- completed work; ASU will write MSCR (5/31/10) – note, MSCR36
doesn’t include additional analysis ; overdue
Slide 14
MILESTONE 37/38
Generate and purify mg
quantities of 12
selected
FTT proteins
Gray: (sub)milestone title
Green: open
Transcriptome
Ag discovery
Proteome
Ag discovery
Live vector-based
Ag discovery
Active
Active
Active
Slide 15
Construction of bacterial expression construct for IglC1 in pET32b
pJet2.1
TOPO
bp
3000-
1000640-
iglC1
500-
BamHI and HindIII digest of iglC1 sequence-perfect
pJet2.1/blunt and pCR-BluntII-TOPO clones
Slide 16
MILESTONE 36
Final integration of expression data
and informatics analysis
Gray: (sub )milestone title
Red: completed
Green: in progress
Felgner and Expression and Western Analysis
Slide 17
Just received annotations for protein array data, compiling data for
data mining to come. Early visualizations are presented here.
TOPO
pJet2.1
Slide 18
First Look: Felgner Array Data
NHP
TOPO
Early analysis:
pJet2.1
FTT1282, FTT1776c are
two commonly high
(multi-species) genes
highly expressed in
expression in mouse.
Slide 19
First Look: Felgner Array Data
TOPO
Human
pJet2.1
Slide 20
First Look: Felgner Array Data
TOPO
Mouse
pJet2.1
Slide 21
First Look: Felgner Array Data
TOPO
Rat
pJet2.1
Vaccine and Naïve and doses are intermingled
Slide 22