Cloning and bioinformatics of outer membrane protein a gene of Aeromonas hydrophila GYK1 strain

Objective: To clone the outer membrane protein A (OmpA) gene of Aeromonas hydrophila GYK1 strain and analyze its bioinformatics. Methods: According to the sequence of published ompA gene, a pair of degenerate primers were designed to amplify the core sequence of ompA gene of GYK1 strain by PCR, based on which thermal asymmetric interlaced PCR (TAIL-PCR) was performed to amplify further flanking regions of the gene. The amplified ompA gene fragments were spliced by using Vector NTI 9.0 software, predicted for basic parameters and signal peptide of OmpA by Vector NTI 9.0 and SignalP 3.0 software, and analyzed for homologies to the ompA gene of other bacterial strains by BlastN software, based on which a phylogenetic tree was constructed, and the three-dimensional structure of OmpA was analyzed. Results: The cloned ompA gene of GYK1 strain, at a full-length of 2 004 bp, consisting of an ORF at a length of 1 059 bp encoding 352 amino acids, a 5′-terminal flanking sequence at a length of 271 bp and a and 3′-terminal flanking sequence at a length of 574 bp. The relative molecular mass and isoelectric point of OmpA were about 37 900 and 4.94, respectively, while the signal peptide consisted of amino acid residues 1-20 at N-terminus. The phylogenetic tree constructed based on the OmpA sequences of 22 bacterial strains revealed highest homology (95.5%) of GYK1 strain to SSU strain of A. hydrophila. Three-dimensional structure model showed that the N-terminal domain of OmpA was β-barrel consisting 8 anti-parallel β-sheets, while the average flexibility of four loop regions in segment Asn25 ∼ Ala205 were significantly higher than those of other regions. Conclusion: The ompA gene of A. hydrophila GYK1 strain was successfully cloned, of which the bioinformatics was analyzed. It laid a foundation of development of recombinant vaccine based on A. hydrophila-delivered exogenous proteins.