Molecular cloning, bioinformatics and immunogenicity analyses of outer membrane protein ompF gene of Yersinia ruckeri

To screen potential protective antigen genes for developing genetic engineering vaccine of Yersinia ruckeri, we amplified the outer membrane protein ompF gene of Y.ruckeri isolated from channel catfish (Ictalurus punctatus) with specific primers, followed by molecular cloning, bioinformatics analyses of ompF nucleotide and amino acid sequences with bioinformatics tools and online servers. Then we conducted prokaryotic expression and immunogenicity analysis of target recombinant protein ompF. The results show that ompF gene contained a complete opening reading frame of 1 095 bp (GenBank No. KP159420) in length and encoded 364 aa. Sequence homology analysis reveals that ompF amino acid sequence was highly conserved and shared a closest genetic relationship with ompF of Y.ruckeri (GenBank No.ADK27779.1), sharing sequence identity of 99.2% and was the same branch on the phylogenetic tree. The protein had a conserved gram-neg-porins domain, a signal peptide and a transmembrane region, which suggests that it was a transmembrane protein. Moreover, the ompF protein had 12 potential antigenic determinant regions which were related to protective immunity. In addition, the results of SDS-PAGE indicate that ompF recombinant protein mainly existed in sediment in form of inclusion body, and Western bolt results show that recombinant fusion protein ompF was constructed and expressed successfully, and had good immunogenicity against Y.ruckeri infection. The results indicate that ompF can be selected as a candidate protective antigen gene of Y.ruckeri genetic engineering vaccine.