Expression of <I>OmpK</I> gene of <I>Vibrio alginolyticus</I> and development of a detection method of indirect ELISA

Abstract

Vibrio alginolyticus is the main Vibrio pathogen in aquaculture in the south of China,the number of which is the largest in marine Vibrio class.In the present study,the outer membrane protein K gene(OmpK)of V.alginolyticus strain ATCC17749 was amplified by PCR and cloned into high efficient expression vector pET28a.The results of sequencing and restriction enzyme analysis combined with agarose gel electrophoresis showed that the open reading frame sequence was correct and fully consistent with what had reported.The recombinant plasmid of pET-28a-OmpK was successfully constructed and transformed into E.coli BL21 pLys E.The fusion protein was expressed under the IPTG inducing condition,and the expression product was purified by an affinity chromatographic method.Mouse anti-OmpK poly-clonal antibodies(PAbs)were obtained via applying protein OmpK as antigen to immune mice.Western-blotting analysis proved the recombinant protein has a good reactive ability against OmpK positive serum.Then,an indirect Enzyme-Linked Immunosorbent Assay(ELISA)for the rapid diagnosis of V.alginolyticus has been developed using the PAbs.The lowest V.alginolyticus suspension was 104 CFU/mL.Cross reactions of antisera with other bacteria were detected,and all results were negative.The results indicated the suitability and simplicity of the test as a rapid,field diagnostic tool for V.alginolyticus and it can be used for the rapid detection of V.alginolyticus.Further investigation will be focused on the development of an indirect ELISA Kit for the detection of V.alginolyticus.