Download Characterization of the protein recognized by the monoclonal

Characterization of the protein recognized by the
monoclonal antibody D6 specific for Borrelia
garinii isolates
1O.
Péter*, 1 J-C. Wyss, 1A.G.Bretz, 1L. Toutoungi, 2A. Scherl, 1X. Zhang, 2J.-C. Sanchez, 3R. Sahli
1
Institut Central des Hôpitaux Valaisans, Microbiologie (ICHV), Service of Infectious Diseases, 1950 Sion, Switzerland
2 Biomedical Proteomics Research Group, Department of Bioinformatics and Structural Biology, Geneva University, 1200 Geneva
3 Centre Hospitalier Universitaire Vaudois (CHUV), Institute of Microbiologie, 1011 Lausanne
Background :
Methods :
In Europe, Borrelia burgdorferi sensu lato isolates belong to
4 major species: B. burgdorferi sensu stricto, B. afzelii, B.
garinii and B. valaisiana.
The objective of this study was to characterize low
molecular weight proteins of B. burgdorferi sensu lato. Our
main focus was a protein around 12 kDa, that is reactive
with D6, a monoclonal antibody specific for B. garinii
isolates.
Proteins of a B. garinii isolate (VS 102) were prepared as described
schematically below.
The Bb477, Bb061, Bb390 open reading frames of 28 isolates (5 B.
burgdorferi sensu stricto, 5 B. afzelii, 13 B. garinii and 5 B. valaisiana)
was analysed by PCR and DNA sequencing using the BigDye
chemistry. Sequence alignments were done with clustalw included in
the Vector NTI advance package (Invitrogen). Genes were cloned and
expressed in E. coli PQR9. Putative epitope sequence was
synthezised and mab D6 was saturated and to observe absence of
reactivity.
Amplification by
PCR of 13-28
Borrelia isolates
Tandem mass
spectrometry
analysis
Mascot Search
Search in Genbank
Design of
primers for PCR
Sequencing of
amplicons with
ABI 310 instrument
rpsJ
B31
B
VS123
A
ACA1
VS215
A26s
Expression of rpIL and TrxA, in E.
coli PQR9
VS461
935T
387
G
NT29
VS102
Recombinants rpIL and TrxA, dilutions (1)1/2, (2)1/20, (3)1/200, (4)1/2000
Immuno-absorption of
mab D6 with synthetic
peptide (TrxA 4-20)
UK
V
Results :
Partial sequences of 3 proteins of Borrelia
burgdorferi were obtained from the tandem
mass spectrometry analysis: Bb477 (30S
ribosomal protein S10, rpsJ), BB061
(thioredoxine A, TrxA), Bb390 (50s ribosomal
protein L7/L12, rpIL). All 3 complete genes
were amplified by PCR from 13 to 28 isolates.
DNA sequences were translated and aligned.
VS116
AG1
C+
C+
TrxA
B
B31
VS123
VS215
A
VS461
ACA1
A26s
935T
C+ 1 2
3 4 1 2
……..rpIL…………….. TrxA
3
4
387
20047
A19s
A77c
G
FAR01
G25
M63
VS BM
VS BP
HP3
NT19
VS102
V
UK
VS116
AG1
rpIL
B31
B
VS123
VS215
Geho
IP1
VS461
ACA1
A
A26s
A38s
Bo23
935T
387
20047
A19s
G
A77c
FAR01
G25
M63
VS BM
VS BP
HP3
NT29
VS102
VS116
V
UK
AG1
F10.08.94
FRANK
Conclusion 1:
Conclusion 2:
Conclusion 3:
After purification of low molecular mass
proteins of B. garinii, partial sequences of 3
proteins of Borrelia burgdorferi were
obtained from the mass spectrometry
analysis: Bb390 (50s ribosomal protein
L7/L12, rpIL), Bb477 (30S ribosomal
protein S10, rpsJ), BB061 (thioredoxine A,
TrxA). All 3 complete genes were amplified
by PCR from 13 to 28 Borrelia isolates
belonging to B. burgdorferi, B. afzelii, B.
garinii and B. valaisiana.
TrxA and rpIL were expressed in E. coli
PQR9 and mab D6 showed weak reactivity
to recombinant TrxA protein and no
reactivity to recombinant rpIL. Based on
sequence alignements, epitope was
suspected to be in position 7-12 of TrxA
A synthetic peptide was ordered and was
used for immuno-absorption. It confirmed
that epitope of mab D6 corresponds to
KEDFVA sequence of Thioredoxyne A
protein (amino acids 7-12).