猪链球菌2型表面精氨酸肽酶的鉴定与其功能的研究

发布时间：2018-03-25 04:36

本文选题：猪链球菌　切入点：精氨酸蛋白酶　出处：《华中农业大学》2015年硕士论文

【摘要】：猪链球菌是新生的重要的人畜共患病原菌,感染人类和猪后会出现败血症、关节炎、心内膜炎和脑膜炎等症状。至今为止,据有文献记录报道,猪链球菌已造成全球20多个国家超过1584例人类感染。根据荚膜多糖抗原的不同,猪链球菌共计可分为33种血清型,而其中2型是临床病例分离中最为常见的血清型,其次是14型,其他类型血清型如1型、4型、5型、16型和24型也均有报道。近年来虽然学术界对S.suis的感染关注度日益加大,然而对于猪链球菌致病机理,尤其是诱发脑膜炎以及中毒样休克综合征的潜在机制,知之甚少。鉴于病原菌蛋白酶在降解宿主组织、逃避和干扰宿主免疫系统等多方面的重要效能,在本研究中,我们重点关注猪链球菌精氨酸蛋白酶的鉴定工作,以及探讨其在猪链球菌感染过程中所起的作用。淀粉酶结合蛋白B(Abpb)是本室早期鉴定到的重要的免疫原性蛋白,通过生物信息分析,显示其假想的精氨酸蛋白酶功能。在本研究中,利用大肠杆菌表达系统,重组表达并纯化了该目的蛋白。并利用明胶酶谱试验确立了Abpb蛋白酶功能,和p NA偶联的合成肽实验确立了Abpb能高效分解精氨酸肽底物特性。另外重组表达的Abpb蛋白,能够体外促进RAW264.7分泌高水平炎性因子。对于Abpb蛋白的免疫原性验证和研究,我们利用小鼠模型,腹腔免疫小鼠,ELISA检测血清特异性抗体效价结果显示Abpb能刺激小鼠产生高滴度特异性抗体,在被动攻毒实验中Abpb能给予小鼠很好的的免疫保护效果。对于Abpb基因表达定位,本研究通过了系列Western blot和流式实验确认了Abpb表达于猪链球菌细胞壁上以及少量分泌形式的表达。为了探讨Abpb在猪链球菌2型感染中所起的作用,我们利用大肠杆菌和猪链球菌温敏型穿梭载体p SET4s在SC19亲本株下构建了Abpb失活突变株△Abpb。在Balb/C小鼠腹腔感染模型上,突变株△Abpb相比较于野生菌株表现出明显的毒力下降。由于猪链球菌早期的成功粘附和侵入表皮细胞和随后的有效逃避和抵抗免疫细胞清除是猪链球菌成功感染的关键步骤,鉴于△Abpb下降的毒力,于是我们比较△Abpb突变株和野生菌株在粘附人喉表皮细胞Hep-2细胞能力的差异,结果显示Abpb基因突变后,猪链球菌对Hep-2粘附能力下降明显。与此同时,本研究分离了小鼠腹腔原代巨噬细胞,在将突变株和野生菌株与其孵育时发现,巨噬细胞能够有效的将△Abpb突变株吞噬杀伤,效果为35%左右,然而对亲本株几乎检测不到任何吞噬效果。为了进一步验证突变株△Abpb能够更易清除,体内共感染实验被采取,在将等剂量的△Abpb突变株和野生菌株SC19混合后感染小鼠,并在不同时间点分别分离小鼠脾、肝、肺、脑以及血液中△Abpb突变株和亲本株SC19含菌量的差异,结果显示在感染过程中尤其是早期和晚期中,亲本株SC19菌量明显多于△Abpb突变株,在不同时间和器官中含量约为△Abpb突变株的2-8倍,这一结果同样暗示着Abpb突变后猪链球菌削弱的感染能力以及更易清除。通过以上实验,在此我们鉴定了猪链球菌2型细胞壁精氨酸蛋白酶,并发现该蛋白酶能够降解宿主相关组织和促进猪链球菌体内克隆繁殖,并且与S.suis抵抗宿主巨噬细胞清除相关,是猪链球菌新型的重要的毒力相关因子。另外,该蛋白能够给予小鼠很好的免疫保护效果是理想的亚单位疫苗候选蛋白。
[Abstract]:Streptococcus suis is a new important zoonotic pathogens, human and swine infection occurs after septicemia, arthritis, endocarditis, meningitis and other symptoms. So far, according to records reported, Streptococcus suis has resulted in more than more than 20 countries around the world in 1584 cases of human infection. According to the capsular polysaccharide antigen is different, can be divided into a total of Streptococcus suis for the 33 serotypes, of which 2 is the most common type of serum clinical separation, followed by type 14, other types of serum type such as type 1, type 4, type 5, type 16 and type 24 were also reported in recent years. Although the academia of S.suis infection increasing attention however, the pathogenic mechanism of Streptococcus suis, especially induced meningitis and the potential mechanism of poisoning like shock syndrome, poorly understood. In view of the pathogen in the host tissue protease degradation, and avoid interference with the host immune system and so on important Efficiency, in this study, we focus on the identification of Streptococcus suis arginine protease, and explore its role in Streptococcus suis infection. Amylase binding protein B (Abpb) is the early identification to an immunogenic protein by bioinformatics analysis, the hypothetical arginine acid protease function. In this study, the E.coli Expression System, recombinant expression and purification of the target protein. Using zymography experiment established Abpb protease, peptide synthesis experiments and P coupled NA established Abpb can efficiently decompose arginine peptide substrate. In addition, recombinant Abpb protein, can in vitro RAW264.7 secreting high levels of inflammatory factors. The immunogenicity of Abpb protein and verification, we use the mouse model, intraperitoneal immunization of mice, ELISA detection of serum specific antibody titer results Abpb can stimulate mice to produce high titer of specific antibodies in the passive poison attack experiment Abpb mice can give good immune protection effect. The Abpb gene expression and localization, this study through a series of Western blot and flow cytometry experiments confirmed the expression of Abpb in Streptococcus suis cell walls and a few secretory expression in order to form. To investigate the effects of Abpb on Streptococcus suis type 2 infection in the role, we use the temperature sensitive Escherichia coli and Streptococcus suis shuttle vector p SET4s SC19 in the parental strain were constructed by inactivation of Abpb mutant in Abpb. delta model Balb/C mice infected with the mutant Abpb, compared to the wild strains showed significantly decreased virulence due to the success of the early adhesion of Streptococcus suis. And invasion of epidermal cells and effectively escape and subsequent immune cell clearance is a key step in the success of Streptococcus suis infection, in view of Abpb The decline of virulence, we compared differences between Abpb mutant and wild-type strain on the adhesion ability of Hep-2 cells in human laryngeal epithelial cells, the results showed that Abpb gene mutation, Hep-2 of Streptococcus suis adhesion ability decreased significantly. At the same time, the separation of the mouse peritoneal primary macrophages found in the mutant and wild strains were incubated with when macrophages could be Abpb mutant phagocytic rate is about 35%, however, the parent strain is almost not detected any phagocytic effect. In order to further verify the mutant Abpb can be more easily removed in vivo, CO infection experiment was taken, the dose of Abpb and mutant strains and wild strains of hybrid SC19 after the infection of mice, and at different time points were isolated from mouse spleen, liver, lung, brain and the differences in blood Abpb mutant and parent strains of SC19 bacteria, results in infection In the process of especially early and late, parental strains of SC19 were obviously higher than in the Abpb mutant, in different time and organs was about 2-8 times of the Abpb mutant strains, the results also imply that the Abpb mutant of Streptococcus suis infection ability weakened and easier to remove. By the experiment, we identification of Streptococcus suis type 2 cell wall arginine protease, and found that the protease can degrade host tissue and promote in vivo cloning and breeding of Streptococcus suis, S.suis resistance of host macrophages to clear, is the important model of Streptococcus suis virulence factors. In addition, the protein can give the mice immune protective effect of good subunit the ideal vaccine candidate protein.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.611

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