characterization of the protein recognized by the monoclonal antibody d6 specific for borrelia...
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Characterization of the protein recognized by the monoclonal antibody D6 specific for Borrelia
garinii isolates1O. 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, Switzerland2 Biomedical Proteomics Research Group, Department of Bioinformatics and Structural Biology, Geneva University, 1200 Geneva3 Centre Hospitalier Universitaire Vaudois (CHUV), Institute of Microbiologie, 1011 Lausanne
Background : 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.
Methods :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.
Tandem mass spectrometry analysis
Design of primers for PCR
Mascot Search
Search in Genbank
Amplification by PCR of 13-28 Borrelia isolates
Sequencing of amplicons with ABI 310 instrument
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.
B31
VS123
VS215
A26s
ACA1
VS461
935T
387
NT29
VS102
UK
VS116
AG1
B
A
G
V
Recombinants rpIL and TrxA, dilutions (1)1/2, (2)1/20, (3)1/200, (4)1/2000
C+ 1 2 3 4 1 2 3 4 ……..rpIL…………….. TrxA
Expression of rpIL and TrxA, in E. coli PQR9
Immuno-absorption of mab D6 with synthetic peptide (TrxA 4-20)
Conclusion 1: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.
Conclusion 2: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
Conclusion 3: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).
rpsJ
TrxA
rpIL
B
A
G
V
B31VS123
VS215
VS461
ACA1
A26s
935T
387
20047
A19s
A77c
FAR01
G25
M63
VS BM
VS BP
HP3
NT19
VS102
UK
VS116
AG1
C+ C+
B
A
G
V
B31
VS123
VS215
Geho
IP1
VS461
ACA1
A26s
A38s
Bo23
935T
387
20047
A19s
A77c
FAR01
G25
M63
VS BM
VS BP
HP3
NT29
VS102VS116
UK
AG1
F10.08.94
FRANK