During canine viscero-cutaneous leishmaniasis the anti-Hsp70 antibodies are specifically elicited by the parasite protein.
A Leishmania infantum cDNA library was screened with sera from dogs with viscero-cutaneous leishmaniasis. Sequence analysis of a positive clone isolated from the library revealed that it coded for the carboxyl-terminal region of a member of the 70-kDa heat-shock protein family. The full-length sequence of the L. infantum hsp70 gene was determined after isolation of genomic clones. This protein shows a high degree of sequence conservation with the homologous protein from other organisms. To test its antigenicity a recombinant Hsp70 protein fused to the maltose-binding protein was produced in Escherichia coli using the expression vector pMAL-cRI. By FAST-ELISA assays it was observed that while the complete recombinant protein was recognized by 100% of the sera, the 20 carboxyl-terminal amino acids of the protein were only recognized by 30% of those sera. Thus, although a B-cell epitope must be present within the carboxyl terminal end of the protein other antigenic determinant(s) must reside out of this region. The analysis of the cross-reactivity with mouse Hsp70 by Western blotting strongly suggests that the anti-Hsp70 antibodies generated by infection with L. infantum are directed at specific determinants of the L. infantum Hsp70. Thus, our results indicate that anti-Hsp70 autoantibodies are not induced during Leishmania infection.
Quijada
L
L
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Spain.
Requena
J M
JM
Soto
M
M
Alonso
C
C
D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000096:biosynthesis*; D000917:Antibody Formation; D000918:Antibody Specificity; D001323:Autoantibodies / Q000096:biosynthesis*; D015153:Blotting, Western; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary; D004283:Dog Diseases; D004285:Dogs; D015723:Gene Library; D017125:Genes, Protozoan; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D018314:Leishmania infantum / Q000235:genetics / Q000276:immunology; D016773:Leishmaniasis, Cutaneous / Q000276:immunology / Q000662:veterinary*; D007898:Leishmaniasis, Visceral / Q000276:immunology / Q000662:veterinary*; D051379:Mice; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D017386:Sequence Homology, Amino Acid
D000913:Antibodies, Protozoan; D001323:Autoantibodies; D018076:DNA, Complementary; D018840:HSP70 Heat-Shock Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins