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Molecular analysis of a haemagglutinin of Haemophilus paragallinarum^a

Department of Microbiology and Parasitology, School of Molecular and Microbial Sciences, University of Queensland, St Lucia, QLD 4072, Australia¹
Agency for Food and Fibre Sciences, Animal Research Institute, Department of Primary Industries, Yeerongpilly, Australia²
National Veterinary Assay Laboratory, Dobutsu Iyakuhin Kensajo, 1-15-1 Tokura, Kokubunji-shi, Tokyo, Japan³

Author for correspondence: Michael P. Jennings. Tel: +61 7 3365 4879. Fax: +61 7 3365 4620. e-mail: jennings{at}mailbox.uq.edu.au

The gene encoding a haemagglutinin of H. paragallinarum, hagA, has been identified and the full-length nucleotide sequence determined. A 39 kDa protein, recognized by an anti-haemagglutinin monoclonal antibody, mAb4D, was purified from H. paragallinarum strain 0083 and the N-terminal sequence obtained. The full-length nucleotide sequence was obtained by inverse PCR and the deduced amino acid sequence of the protein encoded was shown to be similar to other outer-membrane proteins of closely related organisms in the HAP group (Haemophilus, Actinobacillus, Pasteurella), especially the P5 protein of Haemophilus influenzae. The hagA gene was cloned into a His-tag expression vector and overexpressed in Escherichia coli strain M15(pREP4). The identity of the purified recombinant protein as a H. paragallinarum haemagglutinin was confirmed by haemagglutination of chicken red blood cells and reactivity, in a Western blot, with the monoclonal antibody specific for the serovar A haemagglutinin.

Keywords: serotyping antigen, infectious coryza, outer-membrane protein

Abbreviations: AP, alkaline phosphatase; HA, haemagglutination activity; HI, haemagglutination inhibition

^a The GenBank accession numbers for the sequences determined in this work are AF491817–AF491827.