Cloning, Expression, and Genus‐Specificity Analysis of 28‐kDa OmpK from Vibrio alginolyticus

Abstract

Some Vibrio species are universal marine pathogens and Vibrio infections are often encountered due to consumption of raw or uncooked seafood. The outer membrane proteins, playing a key role in interaction between bacteria and hosts, are potential candidates for development of vaccine and markers of the genus Vibrio. In this study, the ompK (outer membrane protein K) genes of Vibrio alginolyticus, V. vulnificus, V. parahaemolyticus, V. fluvialis, and V. mimicu were cloned with 798 to 822 nucleotides encoding 266 to 274 amino acids. The ompK gene from V. alginolyticus was expressed in Escherichia coli using pET-22b expression vector. The recombinant fusion OmpK protein with 6xHis tag was purified with nickel chelate affinity chromatography. The polyclonal antibody (titer, 1:102400) against V. alginolyticus OmpK was developed in guinea pigs and it positively reacted with each of 5 Vibrio species but negative to other 18 Gram-negative bacterial strains. The result suggests that Vibrio OmpK protein could be a genus-specific antigen, which can be used for developing vaccines and rapid detection of multiple Vibrio species.

DETECTION OF VIRULENCE GENES IN Vibrio alginolyticus ISOLATED FROM Green Mussel, Perna viridis.

“…Among the virulence genes, all isolates acquire ompK followed by toxR and collagenase genes as confirmed by PCR and subsequence analysis. Furthermore, this result showed that ompK gene could be used as gene marker for Vibrio alginolyticus compared to collagenase [7,12]. Besides, in previous study by Zulkifli et al [24] toxR function as regulator for expression of the virulence factor genes in other species of Vibrio such as V. parahaemolyticus.…”

Comparative immunogenicity of outer membrane protein K and whole‐cell antigens of Vibrio parahaemolyticus for diagnosis

“…2010a); guinea pig serum against V. alginolyticus OmpK was cross‐reactive with all five Vibrio species (Li et al . 2010c) but the murine mAbs prepared in their following study were also specific to V. alginolyticus (one mAb also reacted with V. parahaemolyticus ). OmpK was further investigated as a candidate for a fish subunit vaccine; however, the relative percent survival (RPS) of homogenous strains in different studies was highly variable.…”

“…Moreover, OmpK antibodies for diagnostic purposes have also been reported with discrepant specificity in different reports (Li et al . 2010a,2010c). In these studies, recombinant OmpK has been expressed as inclusion bodies, while the immunogenicity of soluble OmpK remains unknown.…”

Protein transduction domain of transactivating transcriptional activator fused to outer membrane protein K of Vibrio parahaemolyticus to vaccinate marbled eels (Anguilla marmorata) confers protection against mortality caused by V. parahaemolyticus

“…The outer membrane proteins of the fish pathogen, V. parahaemolyticus , which play important roles in the interaction between bacteria and hosts, are potential candidates for the development of vaccines. It was reported that the outer membrane protein, ompK could elicit immune protection in fish species against V. parahaemolyticus infections (Ningqiu et al ., ; Li et al ., 2010; 2013). However, the inconvenience of vaccination application hindered its successful development as an effective vaccine against the deadly infection by V. parahaemolyticus in fish (Collado et al ., ; Romoren et al ., ; Clark and Cassidy‐Hanley, ; Companjen et al ., ; Plant and Lapatra, ; Fredriksen and Grip, ; Ruyra et al ., ; Vimal et al ., ).…”