【作者】 孙铂光；

【导师】 唐学玺； 张晓华；

【作者基本信息】 中国海洋大学 ， 生态学， 2009， 博士

【摘要】 随着水产养殖业近年来的迅速发展，海水养殖动物的细菌性病害造成了巨大的经济损失。由弧菌属细菌(Vibrio spp．)引起的弧菌病(Vibriosis)是在世界各地鱼、虾、贝类等海水养殖动物中普遍流行、危害最大的细菌性疾病之一。弧菌是海洋环境中常见细菌类群之一，广泛分布在近岸及河口海水、海洋生物的体表与肠道中，是海水和许多海洋生物的正常菌群。在已知的弧菌中，约20种是海水养殖动物的病原菌。哈维氏弧菌作为一种重要的海水养殖动物病原弧菌，其危害日益显现。哈维氏弧菌的致病机理还不清楚。在哈维氏弧菌可能的毒力因子中，VHH溶血素——一种副溶血弧菌TLH类的溶血素，被认为与哈维氏弧菌对鱼的致病性密切相关。本研究以气相色谱技术检测哈维氏弧菌VHH溶血素磷脂酶水解磷脂酰胆碱的产物，确定了其磷脂酶活性的类型：VHH既有磷脂酶A1活性，又有磷脂酶A2活性，是一种磷脂酶B。对重组的VHH溶血素进行化学修饰，发现组氨酸和二硫键修饰试剂抑制了VHH的磷脂酶活性，说明其磷脂酶催化中心可能含有组氨酸残基，并且其构象的稳定性依赖于半胱氨酸形成的二硫键。以生物信息学手段查明了VHH磷脂酶活性中心的位点，并对其中的关键氨基酸残基——丝氨酸153进行了点突变，将其替换成无催化作用的甘氨酸残基，在大肠杆菌表达重组突变蛋白S153G，并以镍-琼脂糖树脂亲和层析纯化了该蛋白；突变的VHH溶血素S153G失去了磷脂酶活性、溶血活性和对大菱鲆的致病性，说明VHH溶血素的溶血活性和对鱼类的致病性由其磷脂酶活性赋予。对VHH溶血素N端可能的类lipocalin结构域的三个关键氨基酸残基分别进行了突变以瓦解其三级结构，突变的VHH溶血素均失去了磷脂酶活性，说明VHH溶血素N端的可能结构域对于VHH溶血素行使其磷脂酶功能是必需的。对VHH溶血素的C端磷脂酶结构域进行了同源建模，并以所得模型与VHH溶血素的底物磷脂酰胆碱进行分子拼接，查明了VHH溶血素在催化状态下与底物结合的可能位点，并对这些氨基酸残基分别进行了定点突变。其中，色氨酸174是VHH溶血素与底物的胆碱基团结合的位点，对这一氨基酸残基的突变，导致突变蛋白W174A失去了磷脂酶活性，说明色氨酸174可能是VHH溶血素在催化状态下与底物结合的关键位点。以Southern杂交技术检测了vhh溶血素基因在VIB645菌株基因组的分布，结果表明在其染色体和质粒上各有一个vhh溶血素基因。本研究促进了对哈维氏弧菌致病机制的深入理解，填补了对弧菌TLH／VHH类溶血素作用机理认识上的空白，并对促进包括基因工程疫苗在内的环境友好型的哈维氏弧菌病害防治技术有重要的指导意义。更多还原

【Abstract】 With the rapid development of aquaculture, bacterial pathogens for marineanimals have caused severe economic loss. Vibriosis is a common and widespreadepidemic disease caused by Vibrios which are normal inhabitants in aquaticenvirments. Among these bacteria, Vibrio harveyi has become recognized as a seriouspathogen for marine animals. We know little about the mechanism for thepathogenesis of Vibrio harveyi.Among the potential virulent determinants, Vibrio harveyi hemolysin, i.e. VHH,a TLH (thermolabile hemolysin from Vibrio. Parahaemolyticus) -like protein wascertainly determined to involve in fish pathogenesis. In this study, gaschromatographic (GC) analysis was used to identify hydrolytic type of VHH. And theresult indicated that VHH has either phospholipase A1 activity or A2 activity. Hence itcould be considered as a phospholipase B. The result of chemical modificationshowed recombinant VHH was sensitive to the reagents for histidine and disulfidebond modification, revealing that there might be a histidine residue within the activesite of the phopholipase domain of VHH and one or more disulfide bonds werenecessary for the stability of VHH conformation. Ser153 was putatively identified asa very important residue of active site for VHH by bioinformatic method.Site-directed mutagenesis was performed to substitute this residue for anon-functional glycine. The mutant protein was overexpressed in Escherichia coli andpurified by metal chelating affinity chromatography. Enzymatic activity assays andpathogenicity test indicated that the mutant protein lost phospholipase and hemolyticactivities and pathogenicity to fish, which indicated that both the hemolytic activityand pathogenicity to fish of VHH were due to its phospholiapse activity. 3 keyresidues of a possible lipocalin-like domain localized at N terminal of VHH weresubstituted with alanine. And all the resulting mutant proteins lost phospholiapseactivties, suggesting the potential N terminal domain was necessary element for thephospholiapse activity of VHH. A 3-D model obtained from homology modelling of VHH phospholipase domain was employed to facilitate an in silico docking analysiswith phosphatidylcholine. And the result showed the potential binding sites of VHHto the substrate in catalysis process. Among these residues, tryptophan 174, theresidue binding to the choline part of phosphatidylcholine, might play a pivotal role insubstrate-enzyme binding because its substitution with alanine resulted in VHH lostthe phospholipase activity at all. At last, we examined the distribution of vhh genesamong the genome of V. harveyi strain VIB645. And the result indicated that one inchromosome and another in plasmid. This study facilitated the understanding ofpathogenesis of Vibrio harveyi and the mode of action of VHH/TLH-like hemolysin,and would be helpful to the development of vaccine against Vibrio harveyi.更多还原