Cloning,expression and DNA vaccine analysis ofOmpWofVibrio harveyiZJ 0607

Abstract

According to OmpW gene sequence published in GenBank,primers were designed and the DNA fragment of about 645 bp( encoding 214 amino acids) w as amplified by PCR from genomic DNA of Vibrio Harveyi ZJ0607. The full length product w as 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 w as about 43. 8 ku. The recombinant protein w as highly expressed under induction conditions of exposure at 37 ℃,in 0. 1 mmol / L of IPTG for 5 h. The recombinant protein w as 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 betw een 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 w as used as a DNA vaccine to immunize red snapper( Lutjanus sanguineus). PCR results indicated that pcDNA-OmpW w as distributed in muscle,head kidney,liver,spleen 7- 28 days after vaccination. RT-PCR results indicated that the OmpW gene w as expressed in all above tissues of vaccinated fish 7- 28 days after vaccination. Red snapper immunized w ith DNA vaccine show ed higher serum antibody and corresponding recombinant fusion protein by ELISA and Western-blot. In addition,fish immunized w ith 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.