ABSTRACT
Vibrio mimicus is the causative agent of ascites disease in fish. The heat-labile hemolytic toxin designated VMH is an immunoprotective antigen of V. mimicus. However, its epitopes have not been well characterized. Here, a commercially available phage displayed 12-mer peptide library was used to screen epitopes of VMH protein using polyclonal rabbit anti-rVMH protein antibodies, and then five positive phage clones were identified by sandwich and competitive ELISA. Sequences analysis showed that the motif of DPTLL displayed on phage clone 15 and the consensus motif of SLDDDST displayed on the clone 4/11 corresponded to the residues 134-138 and 238-244 of VMH protein, respectively, and the synthetic motif peptides could also be recognized by anti-rVMH-HD antibody in peptide-ELISA. Thus, both motifs DPTLL and SLDDDST were identified as minimal linear B-cell epitopes of VMH protein. Although no similarity was found between VMH protein and the consensus motif of ADGLVPR displayed on the clone 2/6, the synthetic peptide ADGLVPR could absorb anti-rVMH-HD antibody and inhibit the antibody binding to rVMH protein in enhanced chemoluminescence Western blotting, whereas irrelevant control peptide did not affect the antibody binding with rVMH. These results revealed that the peptide ADGLVPR was a mimotope of VMH protein. Taken together, three novel B-cell epitopes of VMH protein were identified, which provide a foundation for developing epitope-based vaccine against V. mimicus infection in fish.
Subject(s)
Bacterial Proteins/immunology , Epitopes, B-Lymphocyte/immunology , Hemolysin Proteins/immunology , Vibrio mimicus/immunology , Amino Acid Sequence , Animals , Antibodies, Bacterial , Bacterial Proteins/genetics , Bacterial Vaccines/genetics , Bacterial Vaccines/immunology , Enzyme-Linked Immunosorbent Assay , Epitopes, B-Lymphocyte/genetics , Fish Diseases/immunology , Fish Diseases/microbiology , Fishes , Hemolysin Proteins/genetics , Peptide Library , Rabbits , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Vibrio Infections/immunology , Vibrio Infections/microbiology , Vibrio Infections/veterinary , Vibrio mimicus/genetics , Vibrio mimicus/pathogenicityABSTRACT
Sinibotia pulchra (Cypriniformes: Cobitidae) is a small cyprinid fish. In this study, the complete mitochondrial genome sequence of S. pulchra is sequenced. The S. pulchra complete mitochondrial genome (GenBank accession no. KT362179) was a circular molecule of 16 568 bp in length, with 13 protein-coding genes (PCGs), 2 ribosome RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, an L-strand replication origin (OL) and a control region (D-loop). The nucleotide acid composition of the entire mitogenome was 31.69% for A, 25.63% for T, 26.94% for C and 15.74% for G, with an AT content of 57.32%. The AT content of 12S rRNA, 16S rRNA and D-loop was 50.21%, 56.86% and 66.74%, respectively.
