【作者】 胡巧云；

【导师】 陈焕春； 金梅林；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2009， 博士

【摘要】 猪链球菌是一种重要的人畜共患病,能引起猪的脑膜炎、败血症、关节炎和急性死亡,每年给养猪业造成重大的经济损失。根据链球菌的荚膜可分为35个血清型,32和34血清型后来被证明实际上是属于Streptococcus orisratti链球菌。其中,2型链球菌的毒力最强,危害最大。近年来,也不乏猪链球菌感染人的报道,主要因为接触到感染的猪或组织。人感染链球菌后,会引起脑膜炎、心内膜炎、败血症和永久性失聪,严重时能引起人的死亡。目前对猪链球菌的控制仍然止步于对其致病机制知之甚少,缺少有效地疫苗和诊断方法。尽管猪已经鉴定出猪2型链球菌的一些毒力因子,但其入侵和破坏宿主的致病机理还很不明了。为进一步了解链球菌与宿主相互作用的关系,本研究利用体内诱导抗原技术鉴定了链球菌感染宿主过程中表达的蛋白,并对部分基因的功能做了进一步的研究。1.猪2型链球菌体内抗原的筛选和鉴定本研究使用体内诱导抗原技术去挖掘猪链球菌感染过程中活动的蛋白。使用pET28a, b, c载体构建了基因组猪2型链球菌的基因组表达文库,库容分别为8000,12000,11000,达到了基因覆盖率为99.9%的要求。收集了实验感染与临床采集的康复血清,采用膜吸附法,依次吸附了康复血清里的菌体表面抗体、菌体裂解物的抗体以及与大肠杆菌交叉反应的抗体,获得了体内表达抗体的探针。使用探针筛选基因组表达文库大于12000个克隆,经第二轮第三轮筛选鉴定,最终获得了包括已知毒力因子溶血素在内20种体内诱导。功能包括代谢、细胞分离的酶类,转运相关基因,细胞表面蛋白及其他未知功能的蛋白。选取不同亚细胞定位的5个基因,一个细胞壁蛋白(sspA),3个细胞质蛋白(secA, mod and moeA)和一个未知结构的蛋白(orf28)使用实时荧光定量PCR技术,通过比较感染小鼠体内与体外培养细菌的基因表达差异,发现验证的5个基因均有不同程度的上调表达。也从另一方面验证了体内诱导抗原技术的可靠性。这些鉴定出的蛋白值得更进一步的研究其对致病的作用。2.猪2型链球菌枯草菌素样丝氨酸蛋白酶(Subtilisin-like serine protease, SspA)的鉴定和致病功能研究SspA是通过体内诱导抗原技术筛选出来的,其与康复血清的反应性最强。表达纯化了SspA基因内部不同的片段ssp328-1624, sspA1610-2800,sspA3655-4470,使用点杂交的方法验证了SspA确实只在康复血清中存在抗体,而免疫血清中抗体检测为阴性。进一步使用荧光定量PCR方法验证了sspA基因在感染猪体内的上调表达。使用软件InterProScan预测SspA蛋白属于枯草杆菌素家族,含有motifⅠ(Asp 251), motifⅡ(His 311), and motifⅢ(Ser 649)。并使用间接免疫荧光的方法证实了含有LPXTG结构的SspA蛋白位于细胞的表面,为细胞壁相关蛋白。分析了SspA的参考菌株和临床菌株中存在情况,发现在参考菌株中,有29株存在sspA基因,同时使用间接免疫荧光方法验证该基因在参考菌株表面的表达。在扩增的112株2型分离株和53株其他血清型菌株中(包括1/2,1,7,9,4,11,13,12,19,21,23,25,28,6,10,14,15和20,结果都为PCR阳性。该基因在链球菌中相对保守。构建了sspA的基因突变株与恢复突变株。发现突变株的生长曲线、溶血性以及亚细胞结构并没有明显的改变。将突变株、野生菌株与基因回复突变株以同样的剂量感染本动物猪后,突变株的毒力相对于野生菌株大大减弱,而回复突变株的毒力与野生菌株相当。表明是SspA单基因缺失导致的毒力变化,提示了该基因在猪2型链球菌致病中发挥一定的作用。以突变株与野生菌株混合,感染本动物猪,比较它们在组织定植的能力。结果发现,突变株仍然可以在猪的扁桃体中定植,但扩散至深层组织形成感染的能力降低。突变株不具备与野生菌株竞争在扁桃体定植的能力。这些数据表明细菌表面蛋白SspA是一种保守的毒力因子,参与猪链球菌的致病过程。更多还原

【Abstract】 Streptococcus suis is an important cause of meningitis, septicaemia, arthritis and sudden death in pigs. So far,35 serotypes were described based on variation of the capsular polysaccharide (CPS). S. suis infections especially the serotype 2, caused great economic loss in pig industry worldwide every year. Moreover, S. suis can infect humans, mainly associated with exposure to infected pigs or tissues. A recent outbreak in China caused by a serotype 2 strain resulted in 38 deaths among 215 infected humans, an 18% mortality rate. The bacterium has caused sporadic human illness in other countries as well, including the United Kingdom, and has been identified as a leading cause of bacterial meningitis in Hong Kong Special Administrative Region and Vietnam. S. suis infections raised considerable international concerns among the public health professionals.Currently, attempts to control the diseases caused by S. suis are still hampered by insufficient knowledge of pathogenesis mechanism, and a lack of effective vaccines and sensitive diagnostic methods. Althogh a set of virulence traits was identified. Knoledge about he molecular mechanism of invasion and damage of the host tissue by the bacteria is still limited. In order to put an insight to the interaction of host and pathogen, we use in vivo induced antigen technology to identify of Streptococcus suis serotype 2 antigens which were uniquely expressed during infection.1. Identification of Streptococcus suis antigenic determinants specifically expressed during infectionStreptococcus suis is an important swine and human pathogen with adhesive and invasive properties. Pathogenesis mechanism of S. suis infection has not been completely defined due to limited information on its virulence factors. In this study, in vivo antigen technology (IVIAT) was used to identify S. suis antigens expressed during pig infection. In total,20 in vivo induced (ivi) genes were identified, including suilysin, a known putative virulence factor; enzymes function in metabolism/housekeeping, cell division/replication and cell wall degradation, transporting related genes, cell wall-associated proteins, and hypothetical proteins of unknown function. Five genes were selected to test the in vivo expression by relative real-time PCR in mice. SecA, putative modification enzyme (mod), moeA and orf28 were apparently upregulated in vivo. Of the twenty ivi genes, three parts of a subtilisin-like serine protease (ssp) gene were identified. The three segments (sspN, sspM and sspC) were expressed and purified. The anti-SSP antibodies in various phages after infection were evaluated. It was found that the antibody developed as early as at the first week post infection. Such proteins might be explored as markers of S. suis infection. S. suis antigens identified by IVIAT warrant further evaluation on their contributions to pathogenesis, and may probably be developed for diagnostc and preventive applications.2. Identification of a cell wall-associated subtilisin-like serine protease involved in the pathogenesis of Streptococcus suis serotype 2Streptococcus suis is an important swine and human pathogen, and also an emerging zoonotic agent. A surface-associated subtilisin-like serine protease (SspA) of S. suis was identified by screening a genomic expression library as fragments of this protein reacted most strongly with convalescent-phase pig sera. The sspA gene is present in 29 of 33 S. suis serotypes reference strains and is expressed on the surface of S. suis. Relative real-time quantitive PCR assay demonstrated that sspA mRNA expression in vivo was several thousand fold of that in vitro. A sspA- mutant was generated from a S. suis serotype 2 strain SC19 by allelic exchange. The mutant was not different from the wild type strain in subcellular structures and in hemolytic phenotype. However, the virulence of the sspA mutant was markedly lower than the wild type in pigs as demonstrated in experimental infections. These data indicated that the surface associated protein SspA is a conserved virulence factor of S. suis and is involved in the pathogenesis of S. suis.更多还原