Characterization of a recombinant transferrin-binding protein A (TbpA) fragment from Haemophilus parasuis serovar 5.
Haemophilus parasuis, the etiological agent of Glässer's disease in pigs, possesses iron acquisition pathways mediated by a surface receptor that specifically bind porcine transferrin. This receptor is composed of transferrin-binding protein A (TbpA) and TbpB. As it has been reported for other gram-negative organisms, H. parasuis TbpA could be useful as a candidate target for H. parasuis vaccination. In this study, a 600-bp tbpA fragment of the gene encoding TbpA from H. parasuis serovar 5, the Nagasaki strain, was amplified by PCR and cloned into a pBAD/Thio-TOPO expression vector, generating the pBAD-Thio-TbpA-V5-His (TbpA-His) construction. Escherichia coli LMG194-competent cells were transformed with this construction, followed by the induction of protein expression with arabinose. A band (38.5 kDa) corresponding to a 200-amino acid recombinant TbpA (rTbpA) fragment was seen on the sodium dodecyl sulfate polyacrylamide gel electrophoresis and confirmed by immunoblotting. Polyclonal antibodies raised against this fragment were specific for H. parasuis and Actinobacillus pleuropneumoniae, reacted at the cell surface with H. parasuis, and a significant bactericidal activity was also detected. Therefore, this rTbpA fragment induces an immunological response and might be useful as an antigen for vaccination against Glässer's disease.
Martínez
Sonia
S
Departamento de Sanidad Animal, Facultad de Veterinaria, Unidad de Microbiología e Inmunología, Universidad de León, León, Spain.
Frandoloso
Rafael
R
Rodríguez-Ferri
Elías F
EF
González-Zorn
Bruno
B
Gutiérrez-Martín
César B
CB
D016977:Actinobacillus pleuropneumoniae / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D044137:Haemophilus parasuis / Q000235:genetics*; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D033881:Transferrin-Binding Protein A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D000942:Antigens, Bacterial; D011994:Recombinant Proteins; D033881:Transferrin-Binding Protein A