豬鏈球菌PCR檢測方法的建立及纖連蛋白結(jié)合蛋白的克隆和表達(dá)
發(fā)布時間：2018-10-24 18:03
【摘要】：豬鏈球菌(Streptococcus suis，SS)是重要的人獸共患病的病原，能引起豬和人類的腦膜炎、敗血癥、關(guān)節(jié)炎、心內(nèi)膜炎、肺炎以及突發(fā)性死亡，對養(yǎng)豬業(yè)造成巨大的經(jīng)濟損失。迄今為止，根據(jù)SS莢膜多糖(capsular polysaccharide，CPS)抗原性的不同，可將其分為35個血清型(1～34，1／2)，主要致病血清型為SS1、SS2、SS1／2、SS7、SS9和SS14，其中以SS2流行最廣、致病性最強。研究發(fā)現(xiàn)，并不是所有的SS2都有致病性，菌株致病性強弱與其是否表達(dá)毒力相關(guān)因子有很大關(guān)系。目前，已知的與SS致病性相關(guān)的毒力因子主要有莢膜多糖、溶菌酶釋放蛋白(muraminidase released protein，MRP)、胞外因子(extracellular factor，EF)、溶血素(suilysin，Sly)、和IgG結(jié)合蛋白、44000的蛋白等，在SS致病方面一般都是協(xié)同發(fā)揮作用。對于SS檢測和分型，國內(nèi)研究主要針對SS2，所以本試驗第一部分中，根據(jù)Genbank中g(shù)dh、cps1、cps2、cps7、cps9和mrp、epf(epf~*)、sly設(shè)計引物建立兩個多重PCR，希望實現(xiàn)對SS系統(tǒng)的檢測。 此外，近年來發(fā)現(xiàn)SS一種新的毒力因子—纖連蛋白結(jié)合蛋白。作為一種值得重視的細(xì)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。有研究說它可以作為交叉保護性抗原，有望用于SS的檢測和免疫等方面。本試驗第二部分是以四川資陽人源分離株為模板進行的表達(dá)純化等工作。 1 SS及其主要致病血清型多重PCR檢測方法的建立 根據(jù)SS谷氨酸脫氫酶基因和血清型1型、2型、1／2型、7型、9型和14型莢膜多糖編碼基因核酸序列，分別設(shè)計SS型通用引物和型特異性引物，建立、優(yōu)化多重PCR檢測方法，并檢測分析種屬背景明確的菌株73株(其中豬鏈球菌49株、其他對照菌株24株)及臨床分離樣本94株(包括四川資陽人源和豬源臨床分離樣本45株)。其中73株種屬背景明確菌株多重PCR種檢測結(jié)果符合率為87.5％，6種主要致病血清型檢出率可達(dá)率為100％。24株對照菌株在種和血清型水平均檢測為陰性。對45株四川豬鏈球菌病暴發(fā)現(xiàn)場分離菌株進行檢測，其中41株為SS2。上述結(jié)果提示本試驗建立的多重PCR方法，可確定被檢測SS是否屬于主要致病血清型，特異性和敏感性好，可用于豬鏈球菌病的快速診斷和流行病學(xué)調(diào)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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：S852.61;R378.12
本文編號：2292137
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：S852.61;R378.12
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