猪链球菌PCR检测方法的建立及纤连蛋白结合蛋白的克隆和表达

发布时间：2018-10-24 18:03

【摘要】：猪链球菌(Streptococcus suis，SS)是重要的人兽共患病的病原，能引起猪和人类的脑膜炎、败血症、关节炎、心内膜炎、肺炎以及突发性死亡，对养猪业造成巨大的经济损失。迄今为止，根据SS荚膜多糖(capsular polysaccharide，CPS)抗原性的不同，可将其分为35个血清型(1～34，1／2)，主要致病血清型为SS1、SS2、SS1／2、SS7、SS9和SS14，其中以SS2流行最广、致病性最强。研究发现，并不是所有的SS2都有致病性，菌株致病性强弱与其是否表达毒力相关因子有很大关系。目前，已知的与SS致病性相关的毒力因子主要有荚膜多糖、溶菌酶释放蛋白(muraminidase released protein，MRP)、胞外因子(extracellular factor，EF)、溶血素(suilysin，Sly)、和IgG结合蛋白、44000的蛋白等，在SS致病方面一般都是协同发挥作用。对于SS检测和分型，国内研究主要针对SS2，所以本试验第一部分中，根据Genbank中gdh、cps1、cps2、cps7、cps9和mrp、epf(epf~*)、sly设计引物建立两个多重PCR，希望实现对SS系统的检测。此外，近年来发现SS一种新的毒力因子—纤连蛋白结合蛋白。作为一种值得重视的细菌非菌毛黏附素，它普遍存在于SS的除32和34型以外所有血清型中。有研究说它可以作为交叉保护性抗原，有望用于SS的检测和免疫等方面。本试验第二部分是以四川资阳人源分离株为模板进行的表达纯化等工作。 1 SS及其主要致病血清型多重PCR检测方法的建立根据SS谷氨酸脱氢酶基因和血清型1型、2型、1／2型、7型、9型和14型荚膜多糖编码基因核酸序列，分别设计SS型通用引物和型特异性引物，建立、优化多重PCR检测方法，并检测分析种属背景明确的菌株73株(其中猪链球菌49株、其他对照菌株24株)及临床分离样本94株(包括四川资阳人源和猪源临床分离样本45株)。其中73株种属背景明确菌株多重PCR种检测结果符合率为87.5％，6种主要致病血清型检出率可达率为100％。24株对照菌株在种和血清型水平均检测为阴性。对45株四川猪链球菌病暴发现场分离菌株进行检测，其中41株为SS2。上述结果提示本试验建立的多重PCR方法，可确定被检测SS是否属于主要致病血清型，特异性和敏感性好，可用于猪链球菌病的快速诊断和流行病学调查。
[Abstract]:Streptococcus suis (Streptococcus suis,SS) is an important zoonotic pathogen, which can cause meningitis, septicemia, arthritis, endocarditis, pneumonia and sudden death in pigs and humans. Up to now, according to the antigenicity of SS capsule polysaccharide (capsular polysaccharide,CPS), it can be divided into 35 serotypes (1: 34 / 1 / 2). The main pathogenic serotypes are SS1,SS2,SS1/2,SS7,SS9 and SS14, among which SS2 is the most prevalent and pathogenicity. It was found that not all SS2 had pathogenicity, and the pathogenicity of the strain was closely related to the expression of virulence factors. At present, the main virulence factors associated with the pathogenicity of SS are capsular polysaccharides. Lysozyme releasing protein (muraminidase released protein,MRP), extracellular factor (extracellular factor,EF), hemolysin (suilysin,Sly), IgG binding protein, and protein 44000 are generally synergistic in the pathogenesis of SS. For SS detection and typing, the domestic research is mainly focused on SS2,. In the first part of this experiment, according to gdh,cps1,cps2,cps7,cps9 and mrp,epf (epf~*), sly) in Genbank, two multiplex PCR, are designed to realize the detection of SS system. In addition, fibronectin binding protein (fibronectin binding protein), a new virulence factor of SS, has been found in recent years. As a kind of bacterial actinomycetes adhesion, it is widely found in all serotypes of SS except 32 and 34. It has been reported that it can be used as a cross-protective antigen and could be used in SS detection and immunity. The second part of this experiment is about the expression and purification of SS and its main pathogenic serotypes based on the template of Ziyang human isolate in Sichuan province. 1 SS and its main pathogenic serotypes are heavy. PCR assay was established according to the nucleic acid sequences of SS glutamate dehydrogenase gene and serotype 1, 2, 1 / 2, 7, 9 and 14 capsule polysaccharides. SS universal primers and type-specific primers were designed to establish and optimize multiple PCR detection methods, and 73 strains (49 strains of Streptococcus suis) were detected and analyzed. Other control strains were 24 and clinical isolates were 94 (including 45 clinical samples from Ziyang and Pig). Among the 73 strains with definite species background, the coincidence rate of multiple PCR strains was 87.5%. The detectable rate of 6 main pathogenic serotypes was 100. 24 control strains were negative in both species and serotype levels. 45 strains isolated from Sichuan swine streptococcus disease outbreak site were detected, of which 41 strains were SS2.. These results suggest that the multiplex PCR method established in this study can determine whether the detected SS belongs to the main pathogenic serotype, and has good specificity and sensitivity. It can be used for the rapid diagnosis and epidemiological investigation of swine streptococcosis.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：S852.61;R378.12

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