海产品中副溶血弧菌O抗原合成关键基因筛选鉴定与分子分型

Vibrio parahaemolyticus(Vp) is one of the foodborne pathogens which is widely isolated from seafoods. The O-antigen is a component of the outer member of Vp and plays an important role in its pathogenicity, immunity and survival, there are 13 kinds of O-antigen in Vp. The O-antigen synthesis genes are always complexed on chromosome as cluster, and it is called O-antigen gene cluster. Up to now, there were less studies on O-antigen, key genes associated with O-antigen synthesis were not clear, and there was no report on molecular typing method based on O-antigen cluster of Vp. A abundant of Vp isolates obtained by our previous work will be used in this study, these isolates were isolated from seafoods and were clustered into 9 kinds of O-antigen groups. First 9 kinds of O-antigen gene clusters will be cloned by long-PCR and sequenced by shotgun method, and then genetic diversity of O-antigen gene clusters will be analysised via statistical and bioinformatics softwares. Secondly, according to the result of genetic diversity, the candidate key genes in O-antigen synthesis pathway will be predicted by BLAST. To confirm the function of candidate gene, mutant strains will be constructed by gene deleting, and then chitin based transformation and complementation tests will be implemented. At last, on the basis of the above work, the targets that can distinguish different O-antigen gene clusters will be explored and the new molecular typing method of Vp will be established. This study will not only provide the solid foundation for expounding the relation between the pathogenicity and serotype of Vp, synthesis pathway and molecular evolution of O-antigen but also provide reliable theory support for supervision and epidemiological survey of Vp in seafoods.

副溶血弧菌是一种广泛存在于海产品中的食源性致病菌，O抗原是其表面的主要抗原，与其致病性、免疫性及生存等密切相关，已报道副溶血弧菌有13种O抗原。负责O抗原合成的相关基因多成簇出现，形成O抗原基因簇。目前，国内外有关副溶血弧菌O抗原的研究较少， O抗原合成的关键基因尚不明确，更缺乏相对应的分子分型方法。本项目以课题组前期从海产品中分离的副溶血弧菌(共9种O抗原类型)为研究对象，利用分子生物学和生物信息学方法，克隆并破译这9种O抗原基因簇，综合分析不同O抗原基因簇的序列，筛选并鉴定O抗原合成的关键基因，阐明副溶血弧菌O抗原多样性的遗传基础，发掘出能区分9种O抗原副溶血弧菌的分子标识，建立新的分子分型方法，为开展海产品中副溶血弧菌的监测和流行病学调查提供技术支撑，为探索副溶血弧菌的O抗原的合成途径、分子进化机制以及血清型同致病性之间关系奠定基础。

副溶血弧菌是引起我国沿海地区食物中毒的主要致病菌。致病性副溶血弧主要菌集中于几种特定的血清型中，O 抗原是副溶血弧菌表面的主要抗原，与其致病性和对环境的耐受力有非常重要的作用，也是建立分子分型的很好材料。副溶血弧菌O 抗原的多样性是由O 抗原合成相关基因决定的。.本研究以实验室前期保存的副溶血弧菌为材料，建立了一种针对副溶血弧菌O抗原合成基因簇进行指纹图谱分型的方法，并构建了O抗原合成关键基因的缺失株及回补株，通过对庚糖基转移酶基因Ⅱ（waaF基因）的功能分析，阐明了waaF基因是副溶血弧菌O抗原合成的关键基因。主要结果如下：.1）建立了一种针对副溶血弧菌O抗原进行分子分型的方法。采用长聚合酶链式反应扩增副溶血弧菌O抗原合成基因簇，使用3种限制性内切酶（EcoR V, BsmⅠ和XmnⅠ）对扩增产物进行限制性酶切，经琼脂糖凝胶电泳，NTSYS软件进行聚类分析。结果显示，该方法可得到清晰的电泳图和准确的分型。 .2）以不同O抗原基因簇的核酸序列为基础，筛选鉴定O抗原合成的关键基因。利用甲壳素介导的转化技术对实验室保存的O3:K6临床分离株的waaF基因进行了敲除。功能分析结果表明：缺失株同O和K抗血清均不发生凝集反应，说明waaF基因不仅同O抗原的合成相关，其同K血清型的表型也密切相关；向24d龄昆明小鼠灌胃0.5ml野生株和缺失株菌液，发现试验组小鼠于灌胃后2小时内出现精神萎靡，食欲下降，运动减少，贴壁，打堆，竖毛，全身震颤的现象，其中接种野生株的小鼠致死时间较缺失株短；组织切片观察，致死小鼠的肝脏和小肠组织都有不同程度的充血现象，其中野生株致死小鼠的小肠组织出现严重的裂解现象，显示O抗原同副溶血弧菌的致病能力存在关系。.3）分别了克隆野生株、O5和O10菌株的waaF基因，利用大肠杆菌S17λpir菌株与副溶血弧菌接合转移的方法，成功构建了waaF基因的回补株。结果表明：野生株及O5血清型waaF基因回补株同O和K抗血清均发生凝集反应；但O10菌株waaF基因未能恢复血清型，表明不同O抗原的waaF基因功能存在差异。溶血实验显示野生株、缺失株和回补株均呈现大小相似的β-溶血环，说明副溶血弧菌的O抗原同其TDH基因的表达没有直接的关联。.本研究为后续开展海产品中副溶血弧菌的监测和流行病学调查提供技术支撑。此外，副溶血弧菌O抗原合成关键基因的功能分析也为探索副溶血弧菌