Journal of Fisheries of China (水产学报) 2013/37:12 PP.1839-1847
According to OmpW gene sequence published in GenBank,primers were designed and the DNA fragment of about 645 bp(encoding 214 amino acids)was amplified by PCR from genomic DNA of Vibrio Harveyi ZJ0607.The full length product was then cloned into the prokaryotic expression vector pET-32a(+)for protein expression in Escherichia coli strain BL21(DE3).The molecular weight of expression fusion protein OmpW was about 43.8 ku.The recombinant protein was highly expressed under induction conditions of exposure at 37 ℃,in 0.1 mmol/L of IPTG for 5 h.The recombinant protein was purified by the best expression condition,purified and used as antigen to immunize the Kunming-mice,and the antibody titer reached 1∶30 000.The result of the Western-blot revealed that specific antigen-antibody reaction occurred between the antiserum and its corresponding recombinant fusion protein.In order to study the immunogenic and protective effects of DNA vaccine,plasmid DNA encoding outer membrane protein OmpW gene was used as a DNA vaccine to immunize red snapper(Lutjanus sanguineus).PCR results indicated that pcDNA-OmpW was distributed in muscle,head kidney,liver,spleen 7-28 days after vaccination.RT-PCR results indicated that the OmpW gene was expressed in all above tissues of vaccinated fish 7-28 days after vaccination.Red snapper immunized with DNA vaccine showed higher serum antibody and corresponding recombinant fusion protein by ELISA and Western-blot.In addition,fish immunized with DNA vaccine developed a protective response to live Vibrio harveyi challenged 35 days post-inoculation,as demonstrated by increased survival of vaccinated fish over the control fish.This study indicates that pcDNA-OmpW is an effective vaccine candidate against Vibrio harveyi infection.
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