【作者】 吴宗福；

【导师】 陆承平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2009， 博士

【摘要】 猪链球菌(Streptococcus suis)是猪的重要致病菌,在全世界广泛分布,可以引起猪的急性败血症、脑膜炎、关节炎及急性死亡,也可导致生猪从业人员感染和死亡。根据荚膜多糖抗原的不同,猪链球菌分为35个血清型(1-34型,1/2型),其中猪链球菌2型(SS2)流行最广,从发病动物中分离率最高。此外,猪链球菌9型(SS9)也是一个重要致病血清型,与断奶仔猪的败血症、脑膜炎和肺炎的爆发有关,在澳大利亚、荷兰、比利时、德国等国家SS9流行较广。流行病学调查表明,在中国SS9也经常从病猪中分离到,并逐步呈上升趋势。目前,猪链球菌毒力因子及免疫原性蛋白的研究大都限于SS2, SS9的相关研究很少。本研究应用蛋白质组学方法筛选SS9毒力因子、SS9免疫原性蛋白、SS2与SS9共同抗原及基因芯片方法对猪链球菌斑马鱼模型的验证。1 SS9分泌蛋白比较蛋白组学研究应用比较蛋白组学方法,从两株不同毒力的SS9菌株分泌蛋白中检测致病性差异相关蛋白,以期发现SS9新的候选毒力因子。SS9菌株GZ0565与SH040917分别分离自病猪与健康猪。用斑马鱼模型评价两株细菌毒力,GZ0565对斑马鱼半数致死量为3.8×105cfu/鱼,而SH040917对斑马鱼不致病。同时,这两株细菌在猪链球菌已知毒力因子分布方面也存在差异。将病猪分离株GZ0565中有而健康猪分离株SH040917中没有的差异分泌蛋白经基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS),或MALDI-TOF-TOF-MS鉴定,分属于13种蛋白。其中5种蛋白报道与毒力相关(DNA核酸酶、O-乙酰丝氨酸裂解酶、含LysM结构的肽聚糖结合蛋白、磷酸甘油酸变位酶、5’-核苷酸酶),再用Southern杂交从基因水平验证,其中4种候选毒力因子对应基因在健康猪分离株SH040917中没有,这些蛋白的发现有助于进一步了解SS9的致病机制。2 SS9表面蛋白免疫蛋白组学研究利用免疫蛋白组学方法,以SPF小鼠抗SS9免疫血清作为一抗,与通过二维凝胶转印到PVDF膜上的SS9菌株GZ0565细胞壁蛋白杂交,从中鉴定12个免疫反应性蛋白斑点。从凝胶中切割相应的蛋白斑点,经MALDI-TOF-MS或MALDI-TOF-TOF-MS鉴定,属于8种免疫原性蛋白。它们分别是：精氨酸脱亚氨酸酶、胞外溶解物结合蛋白、延长因子TS、脑啡肽、多肽ABC运载体多肽结合蛋白、丙酮酸激酶、磷酸转乙酰酶、果糖二磷酸醛缩酶。上述免疫原性蛋白对应基因在SS9菌株中较保守,可作为SS9亚单位疫苗的候选成分。3应用免疫蛋白组学方法筛选SS2与SS9共同抗原SS2及SS9都可致猪链球菌病。研制共同疫苗是防控由SS2与SS9引起猪链球菌病的有效手段。采用免疫蛋白组学的方法,鉴定SS2与SS9共同抗原,为研制共同疫苗提供理论基础。分别以SPF小鼠抗SS2免疫血清、SS9免疫血清及免疫前对照血清作为一抗,与通过二维凝胶转印到PVDF膜上的SS2菌株HA9801细胞壁蛋白杂交,通过比较免疫转印蛋白图谱的差异,从中鉴定7个共同免疫反应性蛋白斑点。从二维凝胶中切割相应的蛋白斑点,经MALDI-TOF-MS分析鉴定,分属于4种蛋白：精氨酸脱亚氨酸酶、延长因子TS、O-乙酰丝氨酸裂解酶和1-磷酸果糖激酶。从SS9菌株GZ0565中克隆表达这4种蛋白,用SPF微型猪SS2康复血清通过免疫转印方法进一步验证其免疫原性。上述免疫原性蛋白对应基因在SS2和SS9菌株中相当保守,可作为SS2和SS9疫苗的候选成分。4斑马鱼感染猪链球菌后转录水平的变化猪链球菌是猪的重要致病菌,然而,用普通猪判定细菌的毒力问题较多,结果不确定,阻碍了猪链球菌致病机理的研究。与猪相比,斑马鱼价廉易得、体型小、操作方便,特别适合于突变库中突变株毒力的判定。本实验室已经成功地将斑马鱼作为猪链球菌的感染模型,并且被国际兽疫局所引用。应用Affymetrix斑马鱼芯片研究斑马鱼感染SS2菌株HA9801后基因表达的变化。在总共有14900转录本的芯片中,有189个基因发生变化,其中125个基因上调表达,64个基因下调表达。在上调的基因中,若干基因的功能与免疫和炎症反应、凝血过程、补体激活及急性阶段反应及应激或防御反应有关,研究表明其中有三个基因(编码血清淀粉样蛋白A、基质金属蛋白酶9和凋亡相关半胱氨酸蛋白酶)在其它物种中与猪链球菌感染后的反应相同。下调基因有肌肉特异基因和与运动或行为的正调控有关的基因。挑选12个基因应用实时定量PCR对芯片数据进行验证,结果与芯片的数据吻合。上述发现有助于用斑马鱼模型进一步探索猪链球菌的致病机理。更多还原

【Abstract】 Streptococcus suis is a common pathogen in swine farms worldwide and is associated with a variety of diseases such as meningitis, arthritis, bronchopneumonia, and septicemia in piglets. It is also recognized as a zoonotic agent in humans especially those occupationally exposed to pigs or swine byproducts. Based on the capsular type,35 serotypes of S. suis have been described, and they are designated from 1 to 34 and 1/2. While S. suis serotype 2 (SS2) is the most widely isolated serotype, S. suis serotype 9 (SS9) is also a prevalent serotype that is frequently isolated from diseased pigs in countries such as Australia, Holland, Belgium, and Germany. Several epidemiological studies have reported that SS9 is also frequently isolated from the organs of diseased pigs in China. Furthermore, these studies have indicated that the prevalence of SS9 has increased during the last few years.1 Comparative proteome analysis of secreted proteins of ss9 isolates from diseased and healthy pigsInvestigations of virulence factors have focused on SS2. Unlike SS2, little is known about virulence factors for SS9. The two SS9 strains, GZ0565 and SH040917, were isolated from a diseased pig and a healthy pig, respectively. The virulence of these two SS9 strains was evaluated in zebrafish. The 50% lethal dose value of strain GZ0565 was 3.8×105 cfu/fish, while zebrafish injected with strain SH040917 exhibited no mortalities. For revealing proteins probably involved in different pathogenicity, a comparative proteomics approach was used to distinguish between the two-dimensional electrophoresis profiles of secreted proteins in two strains. With the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and MALDI-TOF/TOF-MS, protein spots that were unique to strain GZ0565 were identified, and led to the identification of 13 candidate proteins. The largest proportion of these proteins was metabolism-related. Five of the proteins are putative virulence-associated factors:DNA nuclease, o-acetylserine lyase, peptidoglycan-binding LysM, phosphoglycerate mutase, and putative 5’-nucleotidase. These findings contribute to the understanding of SS9 pathogenic mechanisms.2 Immunoproteomic assay of surface protein of SS9Studies on vaccine have focused on S. suis serotype 2 strains. However, little is known about immunogenic proteins for SS9. Therefore, an immunoproteomic-based approach was developed to identify immunogenic proteins of SS9. Cell wall proteins extracted from SS9 strain GZ0565 isolated from a diseased pig with meningitis were screened by two-dimensional Western blotting using anti-SS9 sera pooled from Specific Pathogen Free mice. Protein spots were excised from preparative gels and identified by MALDI-TOF-MS or MALDI-TOF-TOF-MS, which led to the identification of 8 immunogenic proteins (arginine deiminase, extracellular solute-binding protein, translation elongation factor Ts, neprilysin, peptide ABC transporter peptide-binding protein, pyruvate kinase, phosphate acetyltransferase, and fructose-bisphosphate aldolase). These immunogenic proteins, which are encoded by genes that are reasonably conserved among SS9 strains, could be developed as vaccine candidates.3 Identification of antigens common to SS2 and SS9 by immunoproteomic analysisIn addition to SS2, SS9 is another prevalent serotype, which is frequently isolated from the organs of diseased pigs in China. An immunoproteomic-based approach was developed to identify antigens common to SS2 and SS9 for vaccine development. Cell wall proteins extracted from SS2 strain HA9801 were screened by two-dimensional Western blotting using anti-SS2 sera, anti-SS9 sera, or pre-immune sera pooled from SPF mice. Protein spots on preparative gels were excised and identified by MALDI-TOF-MS, which led to the identification of four shared immunogenic proteins (arginine deiminase, translation elongation factor-Ts,1-phosphofructokinase, o-acetylserine lyase). The genes encoding these four proteins from SS9 strain GZ0565 were cloned and their proteins were overexpressed in E. coli. Western blot analysis of these recombinant proteins using the convalescent serum of an SPF mini-pig inoculated with the SS2 strain further confirmed the immunogenicity of these proteins. These immunogenic proteins, which are encoded by genes that are reasonably conserved among SS2 and SS9 strains, could be developed as vaccine candidates.4 Transcriptome profiling of zebrafish due to S. suis infectionLack of a common experimental infection model has hampered the studies on this bacterial species. Different research groups use different animal species, pigs with different ages and different immunological status, or different routes of infection. This may result in the important discrepancies regarding the virulence of even the same strain. Zebrafish as infection model for S. suis has been demonstrated by our groups, which has been cited by OIE. Here, an Affymetrix Zebrafish GeneChip was used to identify alterations in gene expression of zebrafish injected with SS2 strain HA9801. Among the 14900 transcripts in the microarray,189 genes were differentially expressed, of which 125 genes were upregulated and 64 genes were downregulated. Upregulated genes included many known components of the immune and inflammatory response, coagulation factors, complement activation and acute phase response, and stress and/or defense response. Three genes (encoding serum amyloid protein A, matrix metalloproteinase 9 and apoptosis-related cysteine protease), which have previously been implicated in the response to S. suis infection in other organisms, were also upregulated. Many of the genes we observed to be downregulated play roles in the positive regulation of locomotion or behavior, others are muscle specific. The reliability of the data obtained from the microarray was verified by performing quantitative real-time PCR on 12 representative genes. The data may provide further validation of this disease model, which will contribute to an understanding of S. suis pathogenic mechanisms.更多还原