【作者】 孙理云；

【导师】 陆承平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2005， 博士

【摘要】 猪链球菌是集约化养猪场的重要的病原之一。迄今为止,发现有35个血清型。猪链球菌2型（Streptococcus suis 2,SS2）被认为是毒力最强的。马链球菌兽疫亚种（Streptococcus equi subsp.zooepidemicus,SEZ）可感染猪、马、犬、羊、鸡、猫及奶牛,在兽医方面具有重要的意义。上述两种菌均是人兽共患病病原,也是我国猪链球菌病的主要病原。 细菌表面的疏水性是细菌重要的理化特性,与细菌表面组成物质有很大关系,一定程度上与细菌的致病性有关。 纤连蛋白（fibronectin,Fn）是由α和β链组成的一种二聚体糖蛋白,相对分子质量（Mr）约450kD,由多种类型的细胞表达产生,在动物体内两种存在方式,一种游离存在于血浆中,一种以纤维状存在于细胞外基质（ECM）;α与β链中均含有Ⅰ型、Ⅱ型和Ⅲ型三种重复序列;整合素、肝素、胶原/明胶和血纤蛋白都与Fn结合,位置各不相同。细菌表面的Fn结合蛋白在Fn上有不同的结合位置。 业已证明化脓性链球菌黏附宿主细胞的机制之一是结合Fn,进而定植和侵袭宿主。细菌表面的Fn结合蛋白与ECM的Fn的结合促进上皮细胞内化链球菌。细菌与体液中的游离Fn结合在细菌逃避宿主的免疫防御方面也有重要意义。 SEZ和SS2均有Fn结合蛋白基因,但二种细菌与游离（free）及固定（immobilized）Fn体外结合情况如何,未见报道。采用盐析法测定SEZ ATCC35246株（简称ATCC35246）和SS2江苏分离株HA9801（简称HA9801）的表面疏水性,ATCC35246在浓度为1.0M的硫酸铵溶液中2 min内析出,HA9801在0.6M的硫酸铵溶液中析出。用不同浓度的人Fn包被ELISA板,采用ELISA检测对数生长后期的ATCC35246和HA9801体外结合固定Fn的情况,ATCC35246的OD₄₁₅随细菌浓度的升高而增大,10⁸CFU/ml达到饱和浓度,而HA9801的OD₄₁₅与结合BSA对照值相比,经t检验差异不显著（p>0.05）。同时采用间接免疫荧光方法（IFA）,检测体外结合游离的人Fn的情况,在荧光显微镜下可发现两种细菌周围有绿色荧光。结果表明二菌均为表面疏水菌:ATCC35246既可结合固定的Fn,又结合游离的Fn;而HA9801仅与游离的Fn结合。 用不同pH值的PBS稀释对数生长后期的ATCC35246成不同浓度,将其加入用不同浓度的人纤连蛋白（Fn）包被好的ELISA板,加入兔ATCC35246血清与结合在Fn上的细菌作用后,加入1:2,000稀释的碱性磷酸酶标记的山羊抗兔IgG,作用1h后更多还原

【Abstract】 Streptococcus suis is an important pathogen which has been associated with a wide variety of infections in swine such as meningitis, septicemia, arthritis, pneumonia and endorcarditis. Of the 35 official serotypes described to date, serotype 2 is the most virulent and the most commonly isolated from diseased pigs. This miroorganism is also recognized as an agent of zoonosis. Streptococcus equi （Lancefield group C） comprises the two subspecies, S. equi subsp. equi and S. equi subsp. zooepidemicus. Subspecies zooepidemicus is considered an opportunistic commensal; however, after stress or a virus infection, it can cause a secondary infection, which results in strangles-like symptoms, subspecies zooepidemicus also infects a wide range of other animals, such as pigs, dogs, sheep, goats, birds, rabbits, guinea pigs, foxes, cats and cows. Human cases with infection due to subspecies zooepidemicus have also been reported .The attachment of bacteria to surface of the host is considered to be a prerequisite for subsequent colonization or invasion of host. This attachment is mediated by specific interactions between adhesins on the bacterial surface and host cell receptors. For group A streptococcui, fibronectin（Fn） is one of the receptors that is well characterized. Fn is a large glycoprotein composed of two polypeptide chains with a combined molecular mass of 450 kDa. It is found in a soluble form in bodily fluids and in an immobilized form in extracellular matrices and on the surfaces of host cells. Fn has multiple functional domains that interact with diverse substrates, such as fibrin, heparin, collagen, other components of the extracellular matrix, and a wide variety of eukaryotic and prokaryotic cells.Cell surface hydrophobicity of Streptococcus equi subsp . zooepidemicus ATCC35246 and Streptococcus suis type 2 jiangsu isolate HA9801 was determined by "salting out". The interaction of soluble and immobilized fibronectin with two species was investigated by indirect immunofluorescent assay and enzyme-linked immunosorbent assay, respectively. The results showed that ATCC35246 and HA9801 was cell surface hydrophobic and that the former could bind two forms of fibronectin, the late only solube.Aliquots of the Streptococcus equi subsp . zooepidemicus ATCC35246 cells suspended in phosphate buffer solution（PBS） of various pHs were transferred to the ELISA platesprecoated with human fibronectin of various concentrations, and incubated at 37℃ for 1 h. After the buffers and unbound cells were washed out with PBS-Tw,bound cells were detected using strain-specific rabbit antibodies, followed by alkaline phosphatase-labeled second antibody, and colour development with P -nitrophenyl phosphate（lmg/ml） in carbonate buffer（pH9.6 ,with 1 mM MgCl₂）. The results showed that adhesion was bacterial cell concentratin-dependent , affected less by fibronectin procoated within concentration range 0. 0625μg/ml¹0μg/ml and was optimal at pH7. 4.fops gene（encoding fibronectin- and fibrinogen-binding protein, FBPS ） was amplified from genomic DNA of Streptococcus suis type 2 Jiangsu isolate HA9801 by polymerase chain reaction（PCR）technique. Recombinant plasmid pMD-T-FBPS was constucted by cloning PCR product into the clone vector pMDT-18. The 1938bp recombinant fragment of pMD-T-FBPS was verified by restriction endonuclease analysis and sequencing. The fops gene nucleotide sequence of Jiangsu isolate was more than 99.99% identical to that of SS2 Netherland strain reported by Greeff and to that of human Germany strain. FBPS displayed 76.0%, 73.0%, 72.4%, 70.1% and 68.8% identity with FbpA of S.gordonii（CAA4628.2）, putative fibronectin/fibrinogen-binding protein of S. mutan （AAN59108.1）, PavA of S. pneumoniae,（AAK75087.1）, adherence and virulence protein A of S. agalactiae 2603V/R（AAN00072.1） and FBP54 of S. pyogenes（AAA57236）,respectively. Based on its structural feature , FBPS was speculated to be an anchorless adhesin.Based on the sequence of fops of Streptococcus suis type 2 HA9801, primers were designed for amplification fops gene and its frag更多还原