【作者】 张茜；

【导师】 王大鹏； 田桢干；

【作者基本信息】 上海交通大学 ， 食品工程， 2015， 硕士

【摘要】 致病性副溶血弧菌(Vibrio parahaemolyticus,Vp)是重要的食源性致病菌之一。但是,关于其主要致病因子-直接耐热溶血毒素(Thermostable direct hemolysin,TDH)调控机制的研究报道较少。本研究以6因素5水平正交实验筛选提高TDH诱导表达的培养条件,并通过单因素及正交实验证实各因素对TDH表达的作用。本研究获得2种可提高TDH表达的Vp培养基。其中,培养温度、pH值、牛磺胆酸(Taurocholicacid,TCA)和NaCl浓度均可影响TDH的表达。本研究运用双亲本接合法,将Mini-Tn5-Km2/Cm片段随机插入致病性Vp(ATCC 33846,tdh+)基因组,构建随机插入突变的Vp突变体库;以含有相应抗生素(Km/Cm)和氨苄青霉素(Amp)的硫代硫酸盐柠檬酸盐胆盐蔗糖琼脂培养基(Thiosulfate citrate bile salts sucrose agar,TCBS)进行突变株(KmR/CmRAmpS)筛选;并以PCR方法对突变菌株进行Km/Cm基因筛查,成功构建包含490株Vp突变株的突变体库。以我萋氏血平板筛选溶血表型变化菌株,共获得5株溶血表型变化稳定的菌株(2株为溶血能力上调,3株为下调)。对5株突变菌株进行生长曲线、菌膜形成能力和运动能力测定发现:2株溶血能力上调菌株及1株溶血能力下调菌株在运动能力、生长速率和菌膜形成能力方面较亲本株显著降低(P<0.05);另2株溶血能力下调菌株菌膜形成能力较亲本株显著提高(P<0.05)。通过质粒拯救法,仅获得1个促进TDH表达的基因(pstS);该基因对Vp溶血能力具有促进作用,而对菌膜形成能力具有抑制作用。本研究获得的影响TDH表达的环境因素、溶血表型突变株以及促进TDH表达的基因(pst S)将为进一步探讨Vp tdh的调控机制奠定基础。更多还原

【Abstract】 Pathogenic Vibrio parahaemolyticus(Vp) is one of the most important foodborne pathogenic bacteria. The thermostable direct hemolysin(TDH) is the main pathogenic factor, which coded by tdh gene. However, little information on regulation is known about the hemolysin. In this study, factor, which regulated TDH expression, were evaluated by a 6 factors 5 levels orthogonal experiment. The factors were confirmed by the expression of TDH using the single factor and orthogonal experiment. The results demonstrated that various factors affect the expression of TDH, including different medium, temperature, pH value, the concentration of taurocholic acid and NaCl.In this study, the Mini-Tn5-Km2/Cm was inserted randomly into the genome of pathogenic Vp(ATCC 33846, tdh+) by bioparental mating. The conjugants(KmR/CmRAmpS) were investigated using thiosulfate citrate bile salts sucrose agar medium with appropriate antibiotics(Km/Cm) and ampicillin(Amp) respectively. After detection of the Km/Cm gene by polymerase chain reaction, library of mutation was established, which conjugants with Tn5 in different gene locus. After evaluating hemolytic activity with human blood agar, the phenotype of 5 strains was changed, including 2 up-regulated and 3 down-regulated strains. There was significant difference between the 5 conjugants and parent strain on growth rate, biofilm formation and motility ability. Among 3 down-regulated strains, the biofilm formation ability of 2 conjugants was increased(P<0.05). The residual 3 strains demonstrated the significant difference(P<0.05) on growth rate, biofilm formation ability and motility, comparing with their parent strain. Using the plasmid rescue method, one gene(pst S) that regulated the TDH expression was obtained. The results indicated that pst S gene enhances the hemolytic activity of pathogenic Vp, and reduces the ability of biofilm formation.The conjugants, various factors and up-regulated gene on TDH expression will contribute on investigation of regulation system and molecular mechanism of tdh in further study.更多还原