The plasmid-located gene cafl encoding the capsular antigen fraction I (Fl) of Yersiniu pestis was cloned and sequenced. The gene codes for a 170 amino acid peptide with a deduced M, of 17.6 kDa. The signal peptide sequence was highly homologous to the E. co/i consensus signal sequence. The Fl was assumed to have p-sheet structure for the most part. The region located between amino acids 100 and 150 was suggested to contain putative antigenic determinants and to stimulate T cells.
1, INTRODUCTIONThe ultimate goal of infectious disease research is their prevention. Vaccination is one of the most effective ways in which that goal can be attained. It is necessary to know the gene structure and putative immunogenic surface structures of antigens to create recombinant vaccines.More than 10 antigens have been isolated from Yersinia pestis. The capsular antigen fraction 1 (Fl) was shown to be a highly protective antigen among such thermolabile antigens as D, Fl, T, V and W [I). Some properties of the Fl structure have been studied recently [2], but the nucleotide and amino acid sequences have been unknown so far. Here, we report the cloning and sequencing of the Y. pestis cafl gene coding for the Fl , and the predicted secondary structure with potential antigenic determinants.
MATERIALS AND METHODS
Bacterial strains, plasmids and DNA manipulafionsThe E. co/i strains LE392 and HBIOI were used as transistent hosts for cosmid pHC79 [3], and pUC18 or f9 [4], respectively. Y. pestisF1 positive vaccine strain EV was obtained from the Culture Collection, All-Union Antiplague Institute 'Microb', USSR. Cultures were grown overnight while shaking at 37°C in liquid LB or on solid medium supplemented with the relevant antibiotics for plasmid selection.
Construction of a gene library, screening, and subcloningThe Y. pestis plasmid pFra DNA (about 110 kb in size) was partially digested with EcoRI, ligated with EeoRIdigested cosmid pHC79 and packaged in vitro. The library was amplified in E. coli LE392 and ApRTcR colonies selected were further screened for Fl production by enzyme immunoassay. The isolated cosmid ~153 containing a 40-kb fragment of pFra DNA was then digested with EcoRl and an 8.6kb fragment was cloned into pHC79. The resulting cosmid pFS2 was digested with SalI and Hind111 and a 4.5kb fragment was cloned into pUCI9. The plasmid pFS2-13 generated was used for gene sequencing. The 1 .O-kb Alul fragment of pFS2-I 3 was cloned into the SmaIdigested pUCl8 (plasmid pF18L) and sequenced.
Protein sequencingThe Fl protein was isolated from culture medium and purified by polyacrylamide gel electrophoresis as described [2]. The N-terminus of the mature protein was identified by a PTH-amino acid analyzer (Model 120A, Applied Biosystems).
Secondary structure and anrigenic determinant analysis
The effective synthesis of the envelope antigen Fl of Y. pestis in E. coli HBlOl is mediated by the expression of the cuflM gene. This gene was sequenced, and the protein encoded was found to have a significant homology with the chaperone protein PapD of uropathopnic E. coli. The data presented allow one to suppose CaflM and PapD proteins perform similar functions in the biogenesis of the Y. pestis capsule and E. coli P-pili, respectively.
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