Wu Zaohe

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.

Construction and analysis of subtractive cDNA library of head kidney in humphead snapper,Lutjanus sanguineus

A subtracted cDNA library of humphead snapper (Lutjanus sanguineus) was constructed by suppression subtractive hybridization technology (SSH) to screen immune-related EST.The cDNA library has been constructed by the mRNA of the test group and driven by SSH.Differential ESTs from the subtracted cDNA library have been identified by both PCR technology and dot blot hybridization.Six hundred and eighty positive clones were sequenced by Sangon Biological Engineering Technology Services Co.,Ltd.The homology of the sequences was analyzed by BLASTx tool and BLASTn tool in GenBank database.Functional distribution was performed based on the features of ESTs by gene ontology annotation (GO) and 30 immune-related ESTs of L.sanguineus,such as major histocompatibility complex gene (MHC Ⅰ and MHC Ⅱ),immunoglobulin gene (IgH and IgL),heat shock protein gene (HSP10,HSP70 and HSP90) and so on,were found.In this study,the subtracted cDNA library was successfully constructed by SSH and a number of immune-related ESTs of L.sanguineus were identified,which laid a foundation for the immune-related genes research of L.sanguineus.

The effects of salinity and nutrition on molt and growth of Litopenaeus vannamei

By the methods of experimental ecology,a 5×3 factorial experiment was conducted to determine the effects of salinity and dietary protein levels on molt and growth of Litopenaeus vannamei.The experiment lasted 30 days.L.vannamei at an initial average weight of(2.01±0.02)g were divided into triplicate groups and were observed at salinities of 6,12,18,24,30 and protein levels of 30%,36%,42%.The results showed that the relative weight gain of molting decreased with increasing salinity,being the highest at salinity of 6.The relative weight gain of molting was affected significantly by different salinities and dietary protein levels,and the interactions of them(P0.05).The specific growth rate increased with increasing salinity,it was affected significantly by the different salinity and dietary protein levels(P0.05),but it was not affected significantly by the interactions of them.The molting frequency increased with increasing salinity low salinity levels(P0.05),being the highest at salinity of 18,then it decreased with further increasing salinity.At the different dietary protein levels,the higher molting frequency was obtained in experimental group with feed to the medium dietary protein levels diet(36%).The molting frequency was affected significantly by the different salinity and dietary protein levels(P0.05),it was not affected significantly by the interactions of them.The intermolt period was first prolonged,then shortened with the increase of salinity.The intermolt period was affected significantly by the difference salinties(P0.05),but not affected significantly by the dietary protein levels and the interaction of them.

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.