用免疫蛋白质组学方法筛选哈氏弧菌免疫反应蛋白

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

用免疫蛋白质组学的研究方法鉴定了哈氏弧菌的免疫反应蛋白。将哈氏弧菌接种于TSB培养基28℃培养18h,利用裂解液裂解细菌,提取全菌可溶性蛋白,一向电泳采用7cmpH4～7的胶条进行等电聚焦,二向用12.5%的SDS-PAGE胶分离蛋白质,考马斯亮蓝染色,获得双向电泳图谱,再结合免疫转印技术检测免疫反应的蛋白质,利用ImageMaster 2D Platinum进行配对分析得到15个非特异性的免疫反应性蛋白点,30个特异性的免疫反应性蛋白点。经过鉴定得到13种非特异性的免疫反应性蛋白质,这其中有2对蛋白在胶上的位置不同,但鉴定为同一种蛋白;同时得到28种特异性的免疫反应性蛋白质,有一个蛋白点没有在数据库中查找到相对应的蛋白质,还有1对蛋白在胶上的位置不同,但鉴定为同一种蛋白。将特异性免疫反应蛋白鉴定结果与非特异性比较发现有1对蛋白鉴定为同一种蛋白;另外,No.17和No.19是F0F1 ATP synthase的两个亚基。特异性免疫反应蛋白鉴定结果中有6个蛋白质是已知的其他细菌的具有免疫反应的蛋白,分别为OmpN,OmpW,OmpU,alanine dehydrogenase,Elongation factor Ts(EF-Ts),cysteine synthase成功建立了哈氏弧菌的免疫蛋白质组学研究方法,筛选出28种具有特异性免疫反应的蛋白质,为哈氏弧菌的免疫蛋白研究提供理论依据和操作框架。

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.