Molecular characterization of a novel 32-kDa merozoite antigen ofBabesia gibsoniwith a better diagnostic performance by enzyme-linked immunosorbent assay

Parasitology ◽  
2007 ◽  
Vol 134 (9) ◽  
pp. 1185-1194 ◽  
Author(s):  
G. O. ABOGE ◽  
H. JIA ◽  
K. KURIKI ◽  
J. ZHOU ◽  
Y. NISHIKAWA ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Diagnostic Performance ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protozoan Parasites ◽  
Babesia Gibsoni ◽  
Gene Encoding ◽  
Reading Frame ◽  
Immunodominant Antigen ◽  
Gst Fusion Protein

SUMMARYWe cloned and expressed a novel gene encoding a 32-kDa merozoite protein ofBabesia gibsoni(BgP32). The length of nucleotide sequence of the cDNA was 1464 bp with an open reading frame of 969 bp. The truncated recombinant BgP32 (rBgP32) without a signal peptide and C-terminal hydrophobic sequence was expressed inEscherichia colias a soluble glutathione-S-transferase (GST) fusion protein. Western blotting demonstrated that the native protein was 32-kDa, consistent with molecular weight of the predicted mature polypeptide. Enzyme-linked immunosorbent assay (ELISA) using rBgP32 detected specific antibodies from 8 days to 541 days post-infection in the sequential sera from a dog experimentally infected withB. gibsoni. Moreover, the antigen did not cross-react withB. canissubspecies and closely related protozoan parasites, indicating that rBgP32 is a specific diagnostic antigen. Analysis of 47 sera taken from dogs with anaemic signs revealed that rBgP32 detected a higher proportion ofB. gibsoniseropositive samples (77%) than its previously identified rBgP50 (68%) homologue. These results indicate that the BgP32 is a novel immunodominant antigen ofB. gibsoni, and rBgP32 might be useful for diagnosis ofB. gibsoniinfection.

scholarly journals High-Level Expression of Truncated Surface Antigen P50 of Babesia gibsoni in Insect Cells by Baculovirus and Evaluation of Its Immunogenicity and Antigenicity

2003 ◽  
Vol 10 (4) ◽  
pp. 596-601 ◽  
Author(s):  
Shinya Fukumoto ◽  
Xuenan Xuan ◽  
Kimie Kadota ◽  
Ikuo Igarashi ◽  
Chihiro Sugimoto ◽  
...  
Keyword(s):  
Insect Cells ◽  
Recombinant Baculovirus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Babesia Gibsoni ◽  
Gene Encoding ◽  
Hydrophobic Region ◽  
Immunodominant Antigen ◽  
Diagnostic Potential ◽  
Highly Hydrophobic ◽  
High Level

ABSTRACT Previously, we identified an immunodominant antigen, P50 of Babesia gibsoni. In the present study, the gene encoding the truncated P50 (rP50t) without a C-terminal hydrophobic region (29 amino acids [aa]) was expressed in insect cells by a recombinant baculovirus. The highly hydrophobic C-terminal 20-aa regions seems to be a transmembrane region, which was evidenced by the fact that rP50t was effectively secreted into the supernatant of insect cells infected with the recombinant baculovirus. N-terminal amino acid sequence analysis of rP50t indicated that N-terminal 19 aa function as a signal peptide. The expression level of rP50t reached up to 2 mg per 108 cells infected with the recombinant baculovirus. The immunogenic property of rP50t was evaluated by an immunization test in mice. Mice immunized with rP50t induced a high-level antibody titer against the B. gibsoni merozoite. Monoclonal antibodies (MAbs) to rP50t were produced in mice to determine the immunogenic regions of P50. The epitope(s) recognized by all five MAbs were located between aa 190 and 273, suggesting that the central part of P50 is a highly immunogenic region. The diagnostic potential of rP50t was evaluated using an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate clearly (P < 0.0001) between B. gibsoni-infected dog serum and B. canis-infected dog serum or noninfected dog serum. Our results indicated that the rP50t may provide a useful potential immunogenic reagent for use in diagnosis and as a subunit vaccine to control B. gibsoni infection in dogs.

scholarly journals Cloning, Nucleotide Sequence, and Expression of the Gene Encoding the Bacteriocin Boticin B from Clostridium botulinum Strain 213B

2000 ◽  
Vol 66 (12) ◽  
pp. 5480-5483 ◽  
Author(s):  
Sean S. Dineen ◽  
Marite Bradshaw ◽  
Eric A. Johnson
Keyword(s):  
Amino Acids ◽  
Nucleotide Sequence ◽  
Clostridium Botulinum ◽  
Shuttle Vector ◽  
Open Reading Frame ◽  
Gene Encoding ◽  
Reading Frame ◽  
Heat Stable ◽  
Group I

ABSTRACT Boticin B is a heat-stable bacteriocin produced byClostridium botulinum strain 213B that has inhibitory activity against various strains of C. botulinum and related clostridia. The gene encoding the bacteriocin was localized to a 3.0-kb HindIII fragment of an 18.8-kb plasmid, cloned, and sequenced. DNA sequencing revealed the boticin B structural gene,btcB, to be an open reading frame encoding 50 amino acids. A C. botulinum strain 62A transconjugant containing theHindIII fragment inserted into a clostridial shuttle vector expressed boticin B, although at much lower levels than those observed in C. botulinum 213B. To our knowledge, this is the first demonstration and characterization of a bacteriocin from toxigenic group I C. botulinum.

Babesia gibsoni: Serodiagnosis of infection in dogs by an enzyme-linked immunosorbent assay with recombinant BgTRAP

2008 ◽  
Vol 118 (4) ◽  
pp. 555-560 ◽  
Author(s):  
Youn-Kyoung Goo ◽  
Honglin Jia ◽  
G. Oluga Aboge ◽  
M. Alaa Terkawi ◽  
Ken Kuriki ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Babesia Gibsoni

Production and Characterization of a Monoclonal Antibody to Campylobacter jejuni

2009 ◽  
Vol 72 (4) ◽  
pp. 870-875 ◽  
Author(s):  
S. A. HEO ◽  
R. NANNAPANENI ◽  
M. G. JOHNSON ◽  
J. S. PARK ◽  
K. H. SEO
Keyword(s):  
Monoclonal Antibody ◽  
Campylobacter Jejuni ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Campylobacter Coli ◽  
Carbonate Buffer ◽  
Horse Blood ◽  
Microaerobic Conditions ◽  
Immunoglobulin Subclass

Campylobacter species are a group of spiral-shaped bacteria that can cause disease in humans and animals. We developed a high-affinity monoclonal antibody (MAb) probe that recognizes Campylobacter jejuni cells. Cell suspensions grown under microaerobic conditions at 42°C for 20 h on Bolton agar plates with lysed horse blood were used as live and heat-killed preparations, centrifuged at 8,000 × g for 20 min, and resuspended in carbonate buffer (pH 9.6) for coating on the enzyme-linked immunosorbent assay plates. BALB/c mice were immunized with C. jejuni sonicated cells at 107 CFU/ml to generate MAb-producing hybridoma clones. Of about 500 initial hybridoma clones, MAb 33D2, which reacted with C. jejuni and Campylobacter coli, was selected for further evaluation. MAb 33D2 is in the immunoglobulin subclass G2a and had relatively weaker reactivity with the C. coli strains tested. MAb 33D2 did not show any cross-reactions with the nine non-Campylobacter bacteria tested in the enzyme-linked immunosorbent assay and had a stronger affinity for C. jejuni as live versus heat-killed cells. In Western blot assays, MAb 33D2 recognized two major antigens of 62 and 43 kDa in extracts from C. jejuni cells but only one antigen of 62 kDa in extracts from C. coli cells.

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