猪链球菌2型在猪脑内及热和氧应激条件下表达上调基因的筛选与功能研究
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摘要
猪链球菌2型(Streptococcus suis serotype 2,SS2)是一种重要的人畜共患病原菌,可引起人的中毒性休克综合症和死亡,引起猪的脑膜炎、心内膜炎、败血症、关节炎、浆膜炎和肺炎,是猪的一种重要细菌性传染病,给养猪业造成重大的经济损失。荚膜多糖(CPS)、溶菌酶释放蛋白(MRP)、细胞外蛋白因子(EF)、溶血素和黏附素等是SS2的重要的毒力因子,但这些毒力因子并不能完全解释SS2的致病机理。广义的毒力因子应该包括那些与体内外环境应答有关的所有因子。所谓的环境应答即病原菌在感染过程中受到温度、渗透压、氧、pH及营养物质利用率等条件影响时所产生的变化。这些感染相关因子的鉴定与表达调控的研究,不仅有利于病原菌致病机理的解析,而且对疫苗、药物和诊断技术的设计与研制具有重要的指导意义。本研究运用转录序列选择性捕获(selective capture oftranscribed sequences,SCOTS)技术鉴定出SS2在高温、厌氧和感染猪脑组织中上调表达的基因,并对其中几种SS2细胞壁蛋白的免疫原性进行了研究,现将主要研究结果报告如下:
1.猪链球菌2型在感染猪脑组织中上调表达基因的鉴定以1.5×10~5 CFUSS2 SC-19株鼻内接种5头健康仔猪,72h后采集感染猪的脑组织,提取总RNA,同时提取体外培养的SS2的总RNA,运用SCOTS技术进行六轮筛选,获得SS2在感染猪脑组织中的上调表达的基因71个。DNA序列分析结果表明,这些包括如下6类:与新陈代谢有关的有30个基因;与细胞壁有关的有12个基因;与复制有关的有9个基因;与蛋白有关的有3个基因;与转录有关的有9个基因;另外有8个功能尚不清楚。
2.热应激和厌氧对猪链球菌2型的基因表达调控热应激与厌氧是两种很重要的环境信号,对病原菌的基因表达发挥重要的调控作用。本实验中,以常规体外培养条件为对照,用SCOTS技术筛选出SS2在高温条件下上调表达的基因33个、在厌氧条件下上调表达的基因21个,为SS2在应激条件下的基因表达调控的深入研究奠定了基础。
3.体内诱导的猪链球菌2型胞壁蛋白的免疫原性研究从感染猪脑组织中筛选出的71个上调表达的基因中筛选出4个编码细胞壁相关蛋白的基因,进行了克隆表达和蛋白质纯化。将纯化的重组蛋白与氢氧化铝佐剂混合后免疫小鼠2次后,分别用2×LD_(50)和5×LD_(50)的SS2强毒株攻击。试验结果表明,4种重组蛋白均能诱导小鼠产生高滴度的抗体;在使用2×LD_(50)的攻毒剂量时,4种重组蛋白免疫的小鼠都可以保护,但在使用5×LD_(50)的攻毒剂量时,只有Hp0254和Hp1311N具有较好的保护力,可作为候选疫苗抗原。
Streptococcus suis serotype 2 (SS2) is an important and world-wide pathogen that causes meningitis, endocarditis, septicemia, arthritis, polyserositis and pneumonia in swine. It is also an important zoonotic agent for humans in contact with diseased pigs or their products. S. suis has become a major problem in swine producing countries due to intensive management practices. Although several virulence associated factors, including capsular polysaccharide (CPS), muramidase released protein (MRP), extracellular protein factor (EF), suilysin, and adhesins, have been recognized, the pathogenesis mechanism of SS2 is not fully understood. A more expansive definition of virulence functions includes factors involved in the adaptive response to environmental stresses sinces pathogens are exposed to alterations in temperature, osmolarity, oxygen tension, pH and nutrient availability during infection. Extensive identification of these infection associated factors and their regulation will not only help us to understand the pathogenesis, but also contribute to design and develop novel vaccines, drugs and diagnostic techniques. In this study, selective capture of transcribed sequences (SCOTS) approach was used to identify the up-regulated genes of SS2 in response to heat stress, oxygen restriction and infection in porcine brain, respectively. The immunogenecity of several cell wall-associated proteins, encoded by the in vovo-induced genes, were evaluated as well. The principal results were described as the following:
Identification of genes preferentially expressed by SS2 in porcine brain. Five 35-day-old healthy piglets were infected with 1.5×10~5 CFU of S. suis strain SC-19 intranasally. At the 72 hours, the affected piglets were sacrificed. The infected brain tissues, as well as in vitro cultured SC-19, were subjected for RNA extraction. 71 genes of SS2 were identified to be upregulated during infection in the brain. They were classified into six groups according to DNA sequenceing and bioinformatic analysis: 30 metabolism-related genes, 12 cell wall-related genes, 9 reproduction-related genes, 3 protein-related genes, 9 transcription-related genes, and 8 functionally unknow genes.
Identification of genes preferentially expressed under heat stress and anaerobic condition by SS2. Heat stress and oxygen restriction are important environmental signals to regulate gene expression of pathogenic bacteria. In this study, 33 and 21 genes of SS2 were identified to be preferentially expressed under high-temperature (42 C) and anaerobic conditions respectively. .The process that might be essential for the pathogenesis of the meningitis caused by this pathogen.
Immunogenecity of cell wall-held proteins of SS2. Four genes encoding cell wall-associated proteins were chosen from the 71 in vivo-induced genes. Recombinant proteins were prepared and purified. Their immunogencity was evaluated using a mouse infection model. After twice vaccination using each of the recombinant proteins mixed with Al(0H)3 adjuvant. The results showed that all four recombinant proteins could induce high levels of antibodies, which could protect mice from challenge with a dose of 2×LD_(50) high virulent SS2 strain. The mice vaccinated with recombinant Hp0254 and Hp1311N were even protected from challenge with a dose of 5×LD_(50) SS2. These indicated that Hp2054 and Hp1311N could sever as potential vaccine candidates against SS2.
1.猪链球菌2型在感染猪脑组织中上调表达基因的鉴定以1.5×10~5 CFUSS2 SC-19株鼻内接种5头健康仔猪,72h后采集感染猪的脑组织,提取总RNA,同时提取体外培养的SS2的总RNA,运用SCOTS技术进行六轮筛选,获得SS2在感染猪脑组织中的上调表达的基因71个。DNA序列分析结果表明,这些包括如下6类:与新陈代谢有关的有30个基因;与细胞壁有关的有12个基因;与复制有关的有9个基因;与蛋白有关的有3个基因;与转录有关的有9个基因;另外有8个功能尚不清楚。
2.热应激和厌氧对猪链球菌2型的基因表达调控热应激与厌氧是两种很重要的环境信号,对病原菌的基因表达发挥重要的调控作用。本实验中,以常规体外培养条件为对照,用SCOTS技术筛选出SS2在高温条件下上调表达的基因33个、在厌氧条件下上调表达的基因21个,为SS2在应激条件下的基因表达调控的深入研究奠定了基础。
3.体内诱导的猪链球菌2型胞壁蛋白的免疫原性研究从感染猪脑组织中筛选出的71个上调表达的基因中筛选出4个编码细胞壁相关蛋白的基因,进行了克隆表达和蛋白质纯化。将纯化的重组蛋白与氢氧化铝佐剂混合后免疫小鼠2次后,分别用2×LD_(50)和5×LD_(50)的SS2强毒株攻击。试验结果表明,4种重组蛋白均能诱导小鼠产生高滴度的抗体;在使用2×LD_(50)的攻毒剂量时,4种重组蛋白免疫的小鼠都可以保护,但在使用5×LD_(50)的攻毒剂量时,只有Hp0254和Hp1311N具有较好的保护力,可作为候选疫苗抗原。
Streptococcus suis serotype 2 (SS2) is an important and world-wide pathogen that causes meningitis, endocarditis, septicemia, arthritis, polyserositis and pneumonia in swine. It is also an important zoonotic agent for humans in contact with diseased pigs or their products. S. suis has become a major problem in swine producing countries due to intensive management practices. Although several virulence associated factors, including capsular polysaccharide (CPS), muramidase released protein (MRP), extracellular protein factor (EF), suilysin, and adhesins, have been recognized, the pathogenesis mechanism of SS2 is not fully understood. A more expansive definition of virulence functions includes factors involved in the adaptive response to environmental stresses sinces pathogens are exposed to alterations in temperature, osmolarity, oxygen tension, pH and nutrient availability during infection. Extensive identification of these infection associated factors and their regulation will not only help us to understand the pathogenesis, but also contribute to design and develop novel vaccines, drugs and diagnostic techniques. In this study, selective capture of transcribed sequences (SCOTS) approach was used to identify the up-regulated genes of SS2 in response to heat stress, oxygen restriction and infection in porcine brain, respectively. The immunogenecity of several cell wall-associated proteins, encoded by the in vovo-induced genes, were evaluated as well. The principal results were described as the following:
Identification of genes preferentially expressed by SS2 in porcine brain. Five 35-day-old healthy piglets were infected with 1.5×10~5 CFU of S. suis strain SC-19 intranasally. At the 72 hours, the affected piglets were sacrificed. The infected brain tissues, as well as in vitro cultured SC-19, were subjected for RNA extraction. 71 genes of SS2 were identified to be upregulated during infection in the brain. They were classified into six groups according to DNA sequenceing and bioinformatic analysis: 30 metabolism-related genes, 12 cell wall-related genes, 9 reproduction-related genes, 3 protein-related genes, 9 transcription-related genes, and 8 functionally unknow genes.
Identification of genes preferentially expressed under heat stress and anaerobic condition by SS2. Heat stress and oxygen restriction are important environmental signals to regulate gene expression of pathogenic bacteria. In this study, 33 and 21 genes of SS2 were identified to be preferentially expressed under high-temperature (42 C) and anaerobic conditions respectively. .The process that might be essential for the pathogenesis of the meningitis caused by this pathogen.
Immunogenecity of cell wall-held proteins of SS2. Four genes encoding cell wall-associated proteins were chosen from the 71 in vivo-induced genes. Recombinant proteins were prepared and purified. Their immunogencity was evaluated using a mouse infection model. After twice vaccination using each of the recombinant proteins mixed with Al(0H)3 adjuvant. The results showed that all four recombinant proteins could induce high levels of antibodies, which could protect mice from challenge with a dose of 2×LD_(50) high virulent SS2 strain. The mice vaccinated with recombinant Hp0254 and Hp1311N were even protected from challenge with a dose of 5×LD_(50) SS2. These indicated that Hp2054 and Hp1311N could sever as potential vaccine candidates against SS2.
引文
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