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. 2018 Sep 13;9:2003.
doi: 10.3389/fimmu.2018.02003. eCollection 2018.

Molecular Characterization, Phylogenetic, Expression, and Protective Immunity Analysis of OmpF, a Promising Candidate Immunogen Against Yersinia ruckeri Infection in Channel Catfish

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Molecular Characterization, Phylogenetic, Expression, and Protective Immunity Analysis of OmpF, a Promising Candidate Immunogen Against Yersinia ruckeri Infection in Channel Catfish

Erlong Wang et al. Front Immunol. .
Free PMC article

Abstract

Outer membrane porins, as the major components of Gram-negative bacterial membrane proteins, have been proven to be involved in interactions with the host immune system and potent protective antigen candidates against bacterial infection in fish. Outer membrane porin F (OmpF) is one of the major porins of Yersinia ruckeri (Y. ruckeri), the causative agent of enteric red mouth disease of salmonid and non-salmonid fish. In the present study, the molecular characterization and phylogenetic analysis of OmpF gene was studied, heterogenous expression, immunogenicity and protective immunity of OmpF were systemically evaluated as a subunit vaccine for channel catfish against Y. ruckeri infection. The results showed that OmpF gene was highly conserved among 15 known Yersinia species based on the analysis of conserved motifs, sequences alignment and phylogenetic tree, and was subjected to negative/purifying selection with global dN/dS ratios value of 0.649 throughout the evolution. Besides, OmpF was also identified to have immunogenicity by western blotting and was verified to be located on the surface of Y. ruckeri using cell surface staining and indirect immunofluorescence assays. Moreover, recombinant OmpF (rtOmpF) as a subunit vaccine was injected with commercial adjuvant ISA763, significantly enhanced the immune response by increasing serum antibody levels, lysozyme activity, complement C3 activity, total protein content, SOD activity, immune-related genes expression in the head kidney and spleen, and survival percent of channel catfish against Y. ruckeri infection. Thus, our present results not only enriched the information of molecular characterization and phylogenetics of OmpF, but also demonstrated that OmpF holds promise to be used as a potential antigen against Y. ruckeri infection in fish.

Keywords: Yersinia ruckeri; immune effect; immunogenicity; molecular characterization; ompF; phylogenetic analysis.

Figures

Figure 1
Figure 1
PCR, cloning, alignment and conserved domains analyses of OmpF. (A) PCR amplification of OmpF gene. M1: DNA marker (DL2000); lane 0: negative control; lane 1: PCR product of OmpF gene with 1095 bp. (B) Identification of cloning plasmid T-OmpF. M1: DNA marker (DL2000); lane 1: PCR identification of T-OmpF; lane 2: digestion of T-OmpF with NcoI and SacI; lane 3: digestion of T-OmpF with SacI [A and B are Figures 1A,B from Wang et al. (61) reproduced with permission from South China Fisheries Science]. (C) Amino acid sequence alignment of OmpF in this study with reference Y. ruckeri OmpF deposited in NCBI (ADK27779.1), black box presented the signal peptide sequences (1–22 a.a.), red shade regions presented the OM_channels superfamily conserved domain, red “*” indicated the trimer interface polypeptide binding sites, blue “*” indicated the eyelet of channel, “↓” indicated both trimer interface polypeptide binding sites and eyelet of channel. (D) Conserved domains of OmpF.
Figure 2
Figure 2
Conserved motifs analysis of OmpF with other 25 reference OmpFs. (A) The logos of top five conserved motifs with length ranging from five to fifty in OmpF amino acid sequences. (B) The distribution of these five motifs in the two-dimensional topology structures of OmpF (used OmpF in this study as an example). (C) The location of these five motifs in bacteria OmpFs. Red shades presented Y. ruckeri, blue shades presented other Yersiniaceae species, dotted box presented other Enterobacteriaceae species.
Figure 3
Figure 3
Multiple sequences alignment and phylogenetic tree of OmpF with other 25 reference OmpFs. (A) Multiple sequences alignment of OmpF with other 25 reference OmpFs. Red shades indicated OmpF in this study was consistent with Y. ruckeri OmpF with 99.2% identities, blue shades indicated other Yersiniaceae species OmpFs, dotted box presented other Enterobacteriaceae species. (B) Phylogenetic tree of OmpF with other 25 reference OmpFs. The numbers at each branch represent the bootstrap values obtained with 1000 replicates. “KP159420” represent the OmpF in this study.
Figure 4
Figure 4
Molecular cloning, expression, purification and western blotting analysis of rtOmpF. (A) PCR amplification of tOmpF gene. M1: DNA Marker (DL2000); lane 0: negative control; lane 1: PCR product of tOmpF gene with 1032 bp. (B) Identification of recombinant cloning plasmid T-tOmpF. M1: DNA Marker (DL2000); lane 1: PCR product of T-tOmpF; lane 2: digestion of T-tOmpF with NcoI and SacI; lane 3: digestion of T-tOmpF with SacI; M2: DNA Marker (DL10000). (C) Identification of recombinant expression plasmid P-tOmpF. M1: DNA Marker (DL2000); lane 1: PCR product of P-tOmpF; lane 2: digestion of P-tOmpF with NcoI and SacI; lane 3: digestion of P-tOmpF with SacI; M2: DNA Marker (DL10000) [C is Figure 1C from Wang et al. (61) reproduced with permission from South China Fisheries Science]. (D) SDS-PAGE analysis of recombinant protein rtOmpF. M: protein marker; lane 1~6: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-tOmpF) supernatant, uninduced BL21 (P-tOmpF) sediment, induced BL21 (P-tOmpF) supernatant, induced BL21 (P-tOmpF) sediment, lane 7: purified rtOmpF. (E) Western blotting analysis of rtOmpF with rabbit anti-6 × His antisera. M: protein marker; lane 1: specific binding between rtOmpF and rabbit anti-6 × His antisera. (F) Western blotting analysis of rtOmpF with rabbit anti-Y. ruckeri antisera. M: protein marker; lane 1: specific binding between rtOmpF and rabbit anti-Y. ruckeri antisera [D, E and F are Figure 10 from Wang et al. (61) reproduced with permission from South China Fisheries Science]. (G) The cross-protection of OmpF in Yersiniaceae species was analyzed by Western blotting with rabbit anti-rtOmpF sera. M: protein marker; lane 1~3: Y. ruckeri YRWEL01, Y. enterocolitica and Y. pestis, respectively.
Figure 5
Figure 5
Detection of OmpF localization using bacteria cell surface staining and indirect immunofluorescence. (A) Cell surface staining of Y. ruckeri. Y. ruckeri incubated with rabbit negative antisera (negative control) showed no color. (B) Indirect immunofluorescence assay. Y. ruckeri incubated with rabbit negative antisera (negative control) showed no fluorescence signal.
Figure 6
Figure 6
The detection of specific serum antibody in vaccinated fish using ELISA. Channel catfish were vaccinated twice at 2-week intervals, with PBS, PBS+ ISA763, rtOmpF and rtOmpF+ISA763 respectively. Sera were collected from 1st to 8th week psv. Data are presented as means ± SE (n = 5). Different letters above a bar denoted significant difference (P < 0.05).
Figure 7
Figure 7
Serum lysozyme activity (A) and complement C3 (B) of vaccinated fish. Channel catfish were vaccinated twice at 2-week intervals, with PBS, PBS+ ISA763, rtOmpF and rtOmpF+ISA763 respectively. Sera were collected from 1st to 8th week psv. Data are presented as means ± SE (n = 5). Different letters above a bar denoted significant difference (P < 0.05).
Figure 8
Figure 8
Serum total protein content (A) and SOD activity (B) of vaccinated fish. Channel catfish were vaccinated twice at 2-week intervals, with PBS, PBS+ ISA763, rtOmpF and rtOmpF+ISA763 respectively. Sera were collected from 1st to 8th week psv. Data are presented as means ± SE (n = 5). Different letters above a bar denoted significant difference (P < 0.05).
Figure 9
Figure 9
The melt curves and relative expression levels of immune-related genes in the head kidney and spleen of vaccinated fish. (A) The melt curves analysis of two reference genes and seven immune-related genes. (B) Heatmap analysis of the fold changes of immune-related genes determined by qRT-PCR in the head kidney and spleen. For each gene, the mRNA level of the PBS-vaccinated fish was set as 1. Data were presented as means (n = 5). Different letters in the same tissues denoted significant difference (P < 0.05) of the same gene in different groups. The color scale was shown at right of the figure, with blue color indicating low fold changes and red color indicating high fold changes.
Figure 10
Figure 10
Percent survival analysis of vaccinated fish using Kaplan-Meier method. The differences among groups were analyzed by log-rank test. Different letters denoted significant difference (P < 0.05).

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