Identification of a Haemophilus influenzae 5'-nucleotidase protein: cloning of the nucA gene and immunogenicity and characterization of the NucA protein
| Autor: | Bruce A. Green, Peggy Ooi, Kevin F. Jones, Robert P. Smith, Michael J. Fiske, Robert John Zagursky |
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| Rok vydání: | 2000 |
| Předmět: |
DNA
Bacterial Nucleotidase activity Protein subunit Immunoelectron microscopy Recombinant Fusion Proteins Immunology Molecular Sequence Data Heterologous Gene Expression Biology medicine.disease_cause Microbiology Haemophilus influenzae law.invention Rats Sprague-Dawley Mice law medicine otorhinolaryngologic diseases Animals Amino Acid Sequence Antigens Cloning Molecular Microscopy Immunoelectron Escherichia coli 5'-Nucleotidase Conserved Sequence Antiserum Base Sequence Molecular biology Rats Infectious Diseases Genes Bacterial Microbial Immunity and Vaccines Recombinant DNA Parasitology Rabbits |
| Zdroj: | Infection and immunity. 68(5) |
| ISSN: | 0019-9567 |
| Popis: | We report on the identification of a surface-exposed, highly conserved, immunogenic nontypeable Haemophilus influenzae (NTHi) protein, which elicits cross-reactive bactericidal antibodies against NTHi. The protein was extracted from NTHi strain P860295 with KSCN and purified; it migrated as a single band on a sodium dodecyl sulfate-polyacrylamide gel with an apparent molecular mass of 63 kDa. Mouse antiserum generated against the purified protein was reactive on whole-cell enzyme-linked immunosorbent assay (ELISA) with seven NTHi strains and type b Eagan and Whittier strains and exhibited bactericidal activity to homologous and heterologous NTHi strains. However, the protein is made in small amounts in NTHi as corroborated by immunoelectron microscopy. To further study this protein, we cloned, sequenced, and expressed it recombinantly in Escherichia coli . The recombinant protein is localized in the periplasm of E. coli and has been purified to homogeneity. Both the recombinant and native proteins possess 5′-nucleotidase activity; hence, the protein has been called NucA. Mouse antiserum directed against the recombinant NucA protein was reactive on Western immunoblots and whole-cell ELISA with all H. influenzae strains tested including Eagan and was bactericidal for two heterologous strains tested. The antiserum also resulted in a log reduction in bacteremia, in an infant-rat protection study with H. influenzae type b as the challenge strain. These features suggest that NucA is a potential subunit vaccine candidate against NTHi disease. |
| Databáze: | OpenAIRE |
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