Cloning And Expression Of The Virulence FatA Gene In Vibrio Anguillarum

Vibrio anguillarum, a severe pathogen of marine fish, is the causative agent ofvibriosis which is a highly fatal hemorrhagic septicemic disease. In this paper, fatA gene,the important virulence gene of Vibrio anguillarum, was cloned by the method ofmolecular cloning and recombinant technics. The recombinant expression vectorpET-28a(+)/fatA was constructed and E.coli BL21(DE3) was transformed to express theFatA protein, which is the most important component of the iron transport system inVibrio anguillarum. The expressed protein was purified and used as antigen to immuneanimals to gain the antiserum. The titer of the antiserum was measured by the methodsof double agar diffusion. This provided important data for the designing and applicationof the living vector vaccine for Vibrio anguillarum.The pEIB1 plasmid was extracted from Vibrio anguillarum MVM425 by themethod of SDS cracking and the gene of fatA was amplified by the method of PCR fromthis plasmid. Then the recombinant expression vector pET-28a(+)/fatA was constructedafter double enzyme digestion and the host strain Dh5a was transformed. The selectedclones were verified by double enzyme digestion and DNA sequencing. DNAsequencing results showed that the sequence of DNA fragments inserted into the vectorwas identical to the one that had been reported before. The predict protein coded by therecombinant vector is a fused protein, which has a His-tag at the carboxylic end.The recombinant expression vector pET-28a(+)/fatA was then transformed intoE.coli BL21(DE3) and the FatA protein was expressed under the induction of IPTG.SDS-PAGE results showed that a protein with a molocular weight of 86 kDa wasexpressed within the host strain. Further studies showed the expression quantity of FatAprotein reached the highest level after 3-hour-IPTG induction at 37℃. Inclusion bodieswould form under this kind of condition and most of the FatA protein was containedwithin them.The expressed FatA protein was purified by the method of affinity chromatographyaccording to the manual of the producer after treatment with 2M urea. Then the purifiedFatA protein was dialyzed with PBS and quantified with comasse blue. Theconcentration of FatA was adjusted for later use as an antigen. Antiserum was gainedafter animal immunization and double agar diffusion experiments and Western blottingwere conducted afterwards. And the titer of the antiserum was measured as 1:128according to the results of double agar diffusion experiments.In brief, the fatA gene from Vibrio anguillarum was successfully expressed inE.coli BL21(DE3) contained the recombined plasmid pET-28a(+)/fatA. The molecularweight of the expressed protein was identical to the anticipant one. Then the expressedFatA protein was purified after affinity chromatography and prepared as antigen foranimal immunization, and the titer of the antiserum gained was 1:128.This research could provide an essential base for the recombined lactobacillusvaccine designed for fish vibriosis, which was constructed by the expression system ofP170. And it also provided a route for fish vibriosis protecting and curing.