猪链球菌2型与胶原蛋白的黏附作用
摘要
猪链球菌(Streptococcus Suis)是一种引起猪败血症、脑膜炎症、心内膜炎和关节炎的主要病原,也是一种重要的人畜共患病病原。根据荚膜多糖将其分为35个血清型,其中2型是流行最广的一种血清型。1990年首次报道在广东省发现猪链球菌2型疑似病例(黄毓茂等,1990)。1998～1999年江苏省江苏某地猪群爆发了急性败血性传染病,并感染特定人群,经鉴定其病原不是以往的C群链球菌,而是猪链球菌2型,属于兰氏分群的R群。我国对猪链球菌病的研究以往主要限于C、D、E、L等群,对2型研究的很少。
为了初步建立一种间接ELISA诊断试剂盒,制备了细菌的多克隆抗体,并检测抗体的滴度是1/512。采用包被全菌的方法,初步确定了菌体的最佳包被浓度,包被条件、封闭时间、被检血清最佳稀释度、辣根过氧化物酶标记羊抗兔lgG的最佳稀释度和反应时间等。为猪链球菌2型病的初步诊断奠定了基础。
细菌黏附于宿主细胞是黏附于粘膜的第一步,而细菌黏附于宿主细胞可以最终导致宿主菌血症或败血症。猪链球菌能侵入人源和猪源上皮和内皮细胞已经被证实。然而细菌和这些细胞如何相互作用的仍不清楚。在猪链球菌2型的致病机理中,大多数学者认为细菌粘附吞噬细胞而不被其完全吞噬,吞噬细胞带毒在宿主体内散播,因此,黏附在致病作用中有着很重要的作用。最近有的学者报道纤维连接蛋白可能是细菌定植于组织的潜在受体。
本试验初步研究了细菌与细胞外基质其中一种胶原蛋白的黏附作用。采用Elisa的方法研究了猪链球菌2型与胶原蛋白的黏附作用。结果表明:猪链球菌2型(高致病株)与猪胶原蛋白Ⅰ型的黏附力显著高于低致病菌株马链球菌兽疫亚种C7463(P＜0.01),经胰酶、蛋白激酶K和加热预处理的细菌的黏附力几乎被完全抑制(P＜0.01),而胆固醇和半乳糖预处理的细菌的黏附力部分被抑制。由此可推断细菌黏附胶原蛋白的配基很可能是蛋白质性质的,细菌的毒力因子荚膜多糖可能没有参与黏附过程,但溶血素参与了细菌与胶原蛋白的黏附过程。因此,我们认为细菌与胶原蛋白的粘附性是多种因素共同参与完成的结果,总之,其黏附机制还有待于进一步的研究。
Streptococcus suis is one major swine pathogen that cause septicemia、meningitis、endocarditis and arthritis. Of the 35 known serotypes, serotype 2 is most frequently isolated and associated with diseases. One suspected case of streptococcus suis 2 was first reported in Guang dong province in 1990. A acute infectious septicemia of swine broke out in Jiangsu province in 1998-1999, and a certain people were infected, the pathogen is streptococcus suis 2, other than C group streptococcus suis. The original classification of S.suis 2 into Lancefield groups R. However, in China, most of researches focus on Streptococcus suis in group C、D、E and L .
In order to preliminary establish one elisa of diagnostic kits, we prepared successfully the polyclonal antibody of whole bacterium, and detected the tite of antibody reach up to 1/512. We use the way of whole bacterium coat flat-bottom microtiter 96-well plates, establish the optimum concentration of bacterial coated in primary, condition of coat, blocking time, the optimum dilution of polyclonal antibody, the best dilution of horseradish peroxidase-labelled anti-rabbit IgG and the optimum reaction time etc. In a word, we set up the way of Elisa of diagnostic kits in primary.
Binding of virulent bacteria to host cells is the first step in the colonization of mucosal. surfaces. Binding may also be a first step in the invasion of host cells, a process that may lead to bacteremia and sepsis .The pathogenesis of infections caused by S. suis is not fully understood and many steps are probably involved. S.suis is able to bind to invadeendothelial and epithelial cells of human and porcine origin. Since one hypothesis of the pathogenesis of S.suis infection is that bacteria enter the bloodstream and invade the meninges and other tissues in close association with mononuclear phagocytes, but bacterium can not be phagocyted completely. Therefore, adherence plays an important role in the pathogenesis function. However, the mechanisms involved in these interactions are unknown. Recently some scholars study the adherence between streptococcus suis 2 and vascular endothelial cell、 tracheal epithelial cell、phagocytic cell.
The test study the the ability of S.suis type 2 bind to collagen, one of extracellular matrix proteins(ECM), was evaluated by ELISA. Results indicate that the virulent strain bond to collagen is obviously higher than the weakly virulent strain(p<0.01). After the live bacterial suspension were primed by trypsogen, proteinase K and heat, the bacterial adhesion can be almost blocked (p<0.01). While cholesterol and galactose just partly block bacterial adhesion. The results suggest that the adhesins for these ECM proteins are proteinaceous in nature. Capsular polysaccharide may not participate in the adherent process, but the hemolysin participate in the process of bacterial adhesion. Anyway, the mechanism are waiting for our research in further.
为了初步建立一种间接ELISA诊断试剂盒,制备了细菌的多克隆抗体,并检测抗体的滴度是1/512。采用包被全菌的方法,初步确定了菌体的最佳包被浓度,包被条件、封闭时间、被检血清最佳稀释度、辣根过氧化物酶标记羊抗兔lgG的最佳稀释度和反应时间等。为猪链球菌2型病的初步诊断奠定了基础。
细菌黏附于宿主细胞是黏附于粘膜的第一步,而细菌黏附于宿主细胞可以最终导致宿主菌血症或败血症。猪链球菌能侵入人源和猪源上皮和内皮细胞已经被证实。然而细菌和这些细胞如何相互作用的仍不清楚。在猪链球菌2型的致病机理中,大多数学者认为细菌粘附吞噬细胞而不被其完全吞噬,吞噬细胞带毒在宿主体内散播,因此,黏附在致病作用中有着很重要的作用。最近有的学者报道纤维连接蛋白可能是细菌定植于组织的潜在受体。
本试验初步研究了细菌与细胞外基质其中一种胶原蛋白的黏附作用。采用Elisa的方法研究了猪链球菌2型与胶原蛋白的黏附作用。结果表明:猪链球菌2型(高致病株)与猪胶原蛋白Ⅰ型的黏附力显著高于低致病菌株马链球菌兽疫亚种C7463(P＜0.01),经胰酶、蛋白激酶K和加热预处理的细菌的黏附力几乎被完全抑制(P＜0.01),而胆固醇和半乳糖预处理的细菌的黏附力部分被抑制。由此可推断细菌黏附胶原蛋白的配基很可能是蛋白质性质的,细菌的毒力因子荚膜多糖可能没有参与黏附过程,但溶血素参与了细菌与胶原蛋白的黏附过程。因此,我们认为细菌与胶原蛋白的粘附性是多种因素共同参与完成的结果,总之,其黏附机制还有待于进一步的研究。
Streptococcus suis is one major swine pathogen that cause septicemia、meningitis、endocarditis and arthritis. Of the 35 known serotypes, serotype 2 is most frequently isolated and associated with diseases. One suspected case of streptococcus suis 2 was first reported in Guang dong province in 1990. A acute infectious septicemia of swine broke out in Jiangsu province in 1998-1999, and a certain people were infected, the pathogen is streptococcus suis 2, other than C group streptococcus suis. The original classification of S.suis 2 into Lancefield groups R. However, in China, most of researches focus on Streptococcus suis in group C、D、E and L .
In order to preliminary establish one elisa of diagnostic kits, we prepared successfully the polyclonal antibody of whole bacterium, and detected the tite of antibody reach up to 1/512. We use the way of whole bacterium coat flat-bottom microtiter 96-well plates, establish the optimum concentration of bacterial coated in primary, condition of coat, blocking time, the optimum dilution of polyclonal antibody, the best dilution of horseradish peroxidase-labelled anti-rabbit IgG and the optimum reaction time etc. In a word, we set up the way of Elisa of diagnostic kits in primary.
Binding of virulent bacteria to host cells is the first step in the colonization of mucosal. surfaces. Binding may also be a first step in the invasion of host cells, a process that may lead to bacteremia and sepsis .The pathogenesis of infections caused by S. suis is not fully understood and many steps are probably involved. S.suis is able to bind to invadeendothelial and epithelial cells of human and porcine origin. Since one hypothesis of the pathogenesis of S.suis infection is that bacteria enter the bloodstream and invade the meninges and other tissues in close association with mononuclear phagocytes, but bacterium can not be phagocyted completely. Therefore, adherence plays an important role in the pathogenesis function. However, the mechanisms involved in these interactions are unknown. Recently some scholars study the adherence between streptococcus suis 2 and vascular endothelial cell、 tracheal epithelial cell、phagocytic cell.
The test study the the ability of S.suis type 2 bind to collagen, one of extracellular matrix proteins(ECM), was evaluated by ELISA. Results indicate that the virulent strain bond to collagen is obviously higher than the weakly virulent strain(p<0.01). After the live bacterial suspension were primed by trypsogen, proteinase K and heat, the bacterial adhesion can be almost blocked (p<0.01). While cholesterol and galactose just partly block bacterial adhesion. The results suggest that the adhesins for these ECM proteins are proteinaceous in nature. Capsular polysaccharide may not participate in the adherent process, but the hemolysin participate in the process of bacterial adhesion. Anyway, the mechanism are waiting for our research in further.
引文
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2.曹兴建,曹照明,陈相,王朔,曹季军.猪链球菌2型等细菌不同感染途径研究.中国比较医学杂志,2007,17:275-277
3.崔焕忠,张辉,钱爱东.猪链球菌2型致病机制的研究进展.畜牧与兽医,2007,39:58-60
4.黄毓茂,黄引贤,余志东,等.2型猪链球菌的初步研究.中国畜禽传染病,1990,4:1-3
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