To identify the receptors for the outer membrane protein H (OmpH) of avian P.multocida,the membrane proteins of chicken embryo fibroblast (CEF) cells were separated by SDS-PAGE and analyzed by Ligand blot.The OmpH-binding protein was identified by MALDI-TOF mass spectrometry,and its distribution in the membrane proteins of different host esophageal mucosal cells was detected by Ligand blot,ELISA and immunofluorescence microscopy,respectively.Ligand blot analysis showed that a 49-kDa membrane protein of CEF cells bound to recombinant OmpH,and MALDI-TOF spectral results demonstrated that the OmpH-binding protein was ATP synthase β subunit.In addition,the OmpH receptor was present in the chicken and rabbit mucosal cell membranes,but was not detected in the bovine and swine mucosal cell membranes.The above results indicate that the OmpH receptor may be CEF cell-derived ATP synthase beta subunit.

The cp36 gene encoding an adhesive protein was amplified by PCR from genomic DNA of rabbit P. multocida C51-3 strain, and cloned into the pMD18-T vector and then sequenced. The mature adhesive protein without a signal peptide of cpm36 gene was amplified by PCR from the recombinant plasmid pMD18-cp36, then cloned into the prokaryotic expression vector pQE30 to provide a recombinant plasmid pQE30-cpm36. The recombinant protein of CPM36 was produced in Escherichia coli M15 harboring the recombinant plasmid pQE30-cpm36 by IPTG induction, and the recombinant protein purified by the affinity chromatography with Ni(2+)-NTA resin. The sequence analyses showed that the ORF of cp36 gene was 1032 bp in length, and DNA homology of the cp36 genes between the C51-3 strain and the previously reported different serotype strains of P. multocida in GenBank was 76.9 to 100%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 37 kD, and the Western blotting analysis demonstrated that the recombinant protein CPM36 and native 36 kD protein of C51-3 were recognized specifically by an antiserum against the recombinant protein, suggesting that the recombinant protein is an antigenic protein.
Adhesins, Bacterial ; biosynthesis ; genetics ; immunology ; Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Pasteurella multocida ; chemistry ; Rabbits ; microbiology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology