Cloning and expression of genes encoding transferrin-binding protein A and B from Actinobacillus pleuropneumoniae serotype 5.
Actinobacillus pleuropneumoniae is an important primary pathogen in pigs, which causes a highly contagious pleuropneumonia. As an adaptation to the iron-restricted environment of the host, A. pleuropneumoniae possesses iron acquisition pathways mediated by surface receptors that specifically bind transferrin from the host. The receptor is composed of two receptor proteins, transferrin-binding protein A and B (TbpA and B), which are both capable of binding to transferrin. An impairment of iron uptake mechanisms is likely to reduce virulence. For this reason, these two proteins can be useful as a candidate target for A. pleuropneumoniae vaccination. To do this, genes encoding the TbpA and B from a serotype 5 isolate of A. pleuropneumoniae were amplified from genomic DNA template by PCR and cloned into a pRSET prokaryotic expression vector, generating the pRSET-A.pp-TbpA and B. Escherichia coli BL21(DE3)pLysS competent cells were transformed with each construct followed by the induction of protein expression by the addition of IPTG. Bands corresponding to the predicted sizes (110 and 60 kDa) were seen on the SDS-PAGE. Polyclonal antibodies raised against recombinant TbpA and B from mice were reacted with bacterial proteins. This result indicates that the recombinant proteins can induce immunological responses and might be useful as candidate targets for A. pleuropneumoniae vaccination.
Kim
Tae jung
Tj
College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea.
Lee
Jae il
Ji
D016977:Actinobacillus pleuropneumoniae / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D008970:Molecular Weight; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013045:Species Specificity; D033881:Transferrin-Binding Protein A / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D033882:Transferrin-Binding Protein B / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D011994:Recombinant Proteins; D033881:Transferrin-Binding Protein A; D033882:Transferrin-Binding Protein B