Jian Jichang

Cloning and prokaryotic expression of flaB gene from Vibrio alginolyticus strain HY9901, the causative agent of vibriosis in Lutjanus sanguineus.

To investigate the possibility of flaB as a candidate antigen for vaccine production,primers were designed based on flaB gene sequences published in GenBank. The flaB gene of Vibrio alginolyticus strain HY9901,the causative agent of vibriosis in Lutjanus sanguineus,was amplified by PCR and cloned into pMD19-T vector. Sequence analysis revealed that flaB gene is 1 134 bp and encodes a putative protein of 377 amino acids. The amino acid sequence of FlaB of V. algonilyticus showed highest identity to V.parahaemolytus (92%). The flaB gene was linked into prokaryotic vector pET-32a(+),and the His-FlaB fusion protein with 60.5 ku molecular mass was successfully expressed in E. coli BL21. The soluble recombinant protein was highly expressed under induction conditions of exposure to IPTG (0.4 mmol/L) at 28 ℃ for 10 h and successfully purified on Ni2 +-IDA column. The purified fusion protein was injected into SPF mice to produce anti-FlaB serum. Western blot analysis revealed that the prepared antiserum not only specifically reacts to the FlaB fusion protein,but also specifically reacts to natural total protein extracted from V.alginolyticus. This result indicates that the FlaB may be one of the important protective antigens of V.alginolyticus,which could provide a basis for further study on the immunogenecity of FlaB and vaccine preparation.

Identification for immuno-reactive proteins of Vibrio harveyi by two-dimensional electrophoresis method.

Vibrio harveyi is one of the most serious marine pathogen that can infect a number of aquaculture species.It has caused severe losses to aquaculture industries worldwide.Attempts to control the infection are hampered by lack of effective vaccines and rapid diagnostic kits,the formulation of which could be facilitated by the identification of immunogenic proteins.In this study,an immunoproteome-based approach was developed to identify candidate antigens of Vibrio harveyi for vaccine development.A 2-DE map has been constructed for Vibrio harveyi,in the pI range of 4.0 to 7.0.Strain HY99 was grown in TSA medium for 18 hours at 28 ℃.Total soluble proteins were extracted using lysis buffer and purified with a 2-D clean-up kit.Protein concentrations were determined by 2-D Quant Kit,and the proteins were separated by 2-DE under immobilized pH gradients(IPG).The 2-DE map was obtained from 3 gels run with 7 cm immobilized pH gradient strips and 12.5% SDS-PAGE gels.The electrophoregrams were obtained by coomassie brilliant blue staining.2-DE gels were scanned with Image Master 2D Platinum analyzed by 300 dpi 2-DE image analysis revealed(429 ± 18) protein spots.Then Vibrio harveyi HY99 anti-sera was analyzed for reactivity by Western-blotting against Vibrio harveyi total soluble proteins separated by 2-DE.The 3 maps analyzed revealed 45 pair protein spots by ImageMaster 2D Platinum.15 spots are non-specific-immunoreactive proteins of Vibrio harveyi,and 30 spots are specific-immunoreactive proteins of Vibrio harveyi.These 30 spots were chosen for mass spectrometry identification,and 29 spots were successfully matched with the proteins of NCBInr database(http://www.matrixscience.com).Two isoforms of formate acetyltransferase were proposed.The 30 spots from the 2-DE map corresponded to 28 proteins.None of these identified proteins have previously been reported as immunogenic in Vibrio harveyi.6 proteins are known from other bacterial immunoproteomic analyses.They may be considered to be cross-reactive antigens from other bacterial infections.OmpN were identified a number of times during the immunoproteome analysis of other bacteria,such as Shigella flexneri,Pasteurella multocida,Escherichia coli.OmpW is one of the major outer membrane proteins of Vibrio alginolyticus,and it is an immunoprotein in the report.OmpU is an important virulence factor involved in the adherence of Vibrio vulnificus to the host cells.alanine dehydrogenase,Elongation factor Ts(EF-Ts),cysteine synthase were recognized by anti-sera of Staphylococcus epidermidis.To the best of our knowledge,there are no reports about the immunogenicity of the other remaining 18 identified proteins.Their role in immunoreaction is not fully understood.It is suggested that this study may be valuable for the immuno-proteomics research on Vibrio harveyi.These immunoreactive proteins could be novel candidates for vaccine development.Future studies will evaluate the protection of the 28 proteins by a nasal immunization and challenge.