Pavel Novotny

P.70 pertactin, an outer-membrane protein from Bordetella parapertussis: cloning, nucleotide sequence and surface expression in Escherichia coli

The gene prn encoding the outer‐membrane protein P.70 (pertactin) from Bordetella parapertussis has been cloned in Escherichia coli and its DNA sequence determined. Analysis of the DNA sequence reveals that the gene has an open reading frame comprising 922 amino acids capable of encoding a protein with a molecular weight of 95177 (P.95). In vivo processing of this precursor yields a protein with an estimated Mr of 70kDa (P.70) which is located on the surface of B. parapertussis. Homology between the prn gene from B. parapertussis and that from Bordetella pertussis is 91.3%. The homology is 93% when the protein sequence of P.95 is aligned with that of P.93 from B. pertussis. The major differences between the P.70 pertactin from B. parapertussis and the P.69 pertactin from B. pertussis occur in the number of reiterated units within the repeat motifs found in both proteins; the sequence Gly–Gly–Xaa–Xaa–Pro is repeated four times in the P.70 pertactin, and five times in the P.69 pertactin, while the sequence Pro–Gln–Pro occurs nine times in P.70 pertactin and five times in P.69 pertactin. Cloning of the gene for P.95 in an E. coli expression vector results in the synthesis of a protein that mimics native gene expression in B. parapertussis, i.e. the P.95 protein is synthesized and subsequently processed to yield the P.70 form of the protein on the surface of the cell.

Cloning, nucleotide sequence and heterologous expression of the protective outer-membrane protein P.68 pertactin from Bordetella bronchiseptica.

The prn gene encoding the 68 kDa protective outer-membrane protein of Bordetella bronchiseptica (P.68 pertactin) was cloned, sequenced and expressed in Escherichia coli. The gene was isolated by DNA:DNA hybridization experiments using a radioactively-labelled fragment of the homologous prn gene from Bordetella parapertussis. DNA sequence analysis reveals that the gene is capable of encoding a protein with a molecular mass of 93996 Da (P.94); this precursor molecule is processed to form the P.68 antigen on the surface of B. bronchiseptica. Heterologous expression of the full-length gene encoding P.94 in Escherichia coli results in similar processing, with the P.68 antigen targeted to the bacterial outer membrane. Comparison of P.94 with the P.93 and P.95 precursors, encoding homologous proteins from Bordetella pertussis and B. parapertussis, shows a high degree (greater than 90%) of homology. The major differences between all three proteins occur in the number of repeats of the two families (Gly-Gly-Xaa-Xaa-Pro)n and (Pro-Gln-Pro)n of reiterated sequence motifs.

Circulating cellular immune response to oral immunization of humans with cholera toxin B-subunit

Peripheral blood mononuclear cells were taken from subjects before and after oral immunization with cholera toxin B-subunit. Cells obtained from naive volunteers before immunization did not proliferate in vitro to B-subunit. Oral immunization induced a proliferative response in all volunteers with a peak stimulation index of 20, and was detected up to 1 year later. The proliferative response kinetics suggest the appearance in the blood of primed T cells from the gut coinciding with the disappearance of primed plasmablasts from the circulation, supporting the concept of a common mucosal immune system in man for T and B cells.