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Biotechnology and Applied Biochemistry (2000) 32, (1–8) (Printed in Great Britain)
B-cell epitope mapping of the Neisseria meningitidis P64k protein using overlapping peptides
Sonia González*1, Lisel Viña*2, Consuelo Nazábal*, Glay Chinea†, Evelin Caballero* and Alexis Musacchio*
*Division of Vaccines, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 10600, Cuba, and †Division of Physical-Chemistry, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 10600, Cuba
Key words: meningococci, P64k protein, synthetic peptide. Abbreviations used: mAb, monoclonal antibody; 3-D, three-dimensional; ABTS, 2,2´-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid); HRP, horseradish peroxidase; H P64k, holoenzyme form of P64k; A P64k, apoenzyme form of P64k.1 To whom correspondence should be addressed (e-mail sonia.gonzalez@cigb.edu.cu). 2 Current address: Center of Molecular Immunology, P.O. Box 16040, Havana 11600, Cuba 
A common meningococcal antigen designated P64k has been identified, cloned and expressed in Escherichia coli. The recombinant antigen is highly immunogenic in several animal species and its immunogenicity in healthy human volunteers is under investigation. Recently, P64k has been used as an immunological carrier for weak immunogens. To characterize the B-cell epitopes on P64k, recognized by immune sera obtained from mice, rabbits and monkeys, multiple overlapping peptides were synthesized and screened for antibody binding. Peptides covering the complete sequence of the P64k protein, 59 in all, of 20 amino acids each (overlapped by 10 residues), were synthesized. A number of continuous epitopes were detected with all sera, when immune and pre-immune bleeds were compared. For mouse and monkey sera, a few major antigenic peptides were identified, while the recognition of the rabbit serum was much more heterogeneous. Despite variation in the exact location of continuous epitopes defined by different anti-P64k sera, we found an immunogenic core region within the molecule, composed of amino acids Asp524–Gly533. Consistently, in this protein segment there was an amino acid stretch located in a b-hairpin loop, which is exposed to the solvent in the previously determined three-dimensional structure of the protein. This region is protruding and accessible to a sphere with a radius of 9 Å.
Received 15 December 1999; accepted 2 March 2000
Portland Press Ltd © 2000 |
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