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

Swine respiratory diseases induce severe economic losses in the swine industry worldwide. Several methods have been developed and applied to control these diseases. However, there are still problems of disease control in the swine industry. Recently, egg yolk antibodies have been found to offer several advantages for disease control in animals and humans. In a previous study (24), antibodies to several causative pathogens of swine respiratory diseases were developed. However, several problems remained, especially in terms of reduced laying rates. Therefore, experimental vaccines were reformulated with various bacterial antigens of the swine respiratory diseases. After immunizing hens with the antigens, antibody profiles and other effects including laying rates were investigated and compared to those of the previous study. Profiles of antibody titers were very similar with those of the previous study. However, side effects, such as depression, weakness, reduction of laying rates and mortality, were dramatically lowered and laying rates were increased in hens injected with certain experimental vaccines. In particular, laying rates of hens injected with vaccines against atrophic rhinitis were increased up to 84% by injecting a vaccine composed of only the DNTs of B. bronchiseptica and P. multocida D:4. Efficacies of the vaccines against swine pneumonic pasteurellosis and pleuropneumonia were very similar with those of the previous study. These results suggest that new vaccines could be effective in the production of egg yolk antibodies against the causative agents of swine respiratory diseases.
Actinobacillus pleuropneumoniae/classification/genetics/*immunology ; Animals ; Antibodies, Bacterial ; Antibody Formation ; Bacterial Outer Membrane Proteins/genetics/isolation & purification ; Bordetella bronchiseptica/classification/genetics/*immunology ; Egg Yolk/microbiology ; Female ; Immunoglobulins/*genetics ; Oviposition ; Pasteurella multocida/classification/genetics/*immunology ; Serotyping ; Swine

Outer membrane proteins of Pasteurella (P.) multocida have been known to be protective immunogens. Pasteurella lipoprotein E (PlpE) has been reported to be an important cross reactive outer membrane protein in P. multocida. The gene encoding the PlpE of P. multocida serotypes A: 3, B: 2 and D: 1 was amplified from the genomic DNA. The amplified products were cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clones revealed a single open reading frame of 1,011 bp, 1,008 bp and 1,017 bp encoding a protein with a calculated molecular mass of 37.829 kDa, 37.389 kDa and 37.965 kDa for serotypes A: 3, B: 2 and D: 1 respectively. The comparison of the plpE sequence in different capsular types revealed a high degree (>90%) of homology. Furthermore, the plpE gene of Haemorhhagic septicaemia causing serotype (B: 2) was expressed in E. coli and recombinant PlpE was strongly immunostained by antiserum against whole cell antigen, indicating that the protein is expressed in vivo.
Animals ; Bacterial Outer Membrane Proteins/*genetics/immunology/metabolism ; Base Sequence ; Blotting, Western ; Cattle ; Cattle Diseases/*microbiology ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; *Genetic Variation ; Hemorrhagic Septicemia/microbiology/*veterinary ; India ; Lipoproteins/*genetics/immunology/metabolism ; Molecular Sequence Data ; Open Reading Frames/genetics ; Pasteurella multocida/*genetics/immunology ; Sequence Analysis, DNA ; Sequence Homology ; Serotyping ; Species Specificity