猪链球菌2型分子伴侣trigger factor调控机制研究

Streptococcus suis serotype 2 is an important zoonotic pathogen that causes serious diseases such as meningitis, septicemia, endocarditis, arthritis and septic shock in pigs and humans.It brought serious losses to pig breeding industry and posed a serious threat to people at same time. Reasearch about the mechenism and prevention of streptococcus suis 2 has became a research focus in the fields from 2005 an unprecedented epidemic in Sichuan province of China. Signal transduction system which can regulat its growth、reproduction and virulence has been payed close attention. Trigger factor is an important molecular chaperone which involved in protein export and signal transduction. Deletion of the trigger factor gene can cause lower virulence and tolerance of streptococcus suis 2 in our preliminary study, but the regulative mechenism is still unclear.　In the research many kinds of technologies and methods such as chromatin immunoprecipitation sequencing、gene chips and protemics were used.System studied and expounded the molecular mechanism of trigger factor , which control the gene transcription and expression lead to the depression of virulence and tolerance of streptococcus suis 2. The research will lay the foundation for the pathogenesis research and disease prevention of streptococcus suis 2.

猪链球菌是能够引起如心内膜炎、关节炎、脑膜炎和急性出血性败血症等多种症状的一类重要的人猪共患病病原菌，它对养猪业带来严重的损失，同时给人带来威胁。自2005年我国四川发生人猪共患链球菌病以来，链球菌致病机制及防控的研究已经成为研究热点，特别是其生长、繁殖及毒力调控等起着重要作用的信号转导系统备受关注。本实验室前期研究发现具有信号转导作用的猪链球菌2型分子伴侣trigger factor基因缺失后能够引起细菌的毒力和耐受性均显著降低，但是其对细菌毒力及耐受性调控的分子机制仍不清楚。本研究拟通过染色质免疫共沉淀测序、基因芯片以及蛋白质组学等体内、体外多种技术和手段，从分子水平上系统的研究阐述trigger factor 调控猪链球菌2型基因转录与表达，影响致病性和耐受性的分子机制，为猪链球菌2型致病机理的研究和疾病的预防奠定基础。

本研究PCR扩增获得大小为519bp的trigger factor基因tig，并利用原核表达系统对该基因进行表达，得到大小约为21kd的蛋白，将纯化的蛋白质用弗氏佐剂乳化后免疫兔子， 二次加强免疫后10天采血分离收集trigger factor蛋白质抗体。用甲醛分别固定培养至对数生长期的猪链球菌2型野生菌株SC21、trigger factor基因缺失菌株Δtig和互补菌株CΔtig，然后用溶菌酶处理固定好的菌株以破碎细菌的细胞壁，之后用超声波破碎溶菌酶处理过的细菌。用所制备的trigger factor蛋白的抗体与上述处理过的三个菌株进行免疫共沉淀，捕获亲本菌株和互补菌株中trigger factor蛋白质，将未结合的杂质洗掉后，再用洗脱液洗脱捕获的trigger factor蛋白质结合的DNA，通过高通量测序分析所捕获的DNA序列，一共捕获18个目的基因片段，通过与trigger factor基因缺失菌株Δtig作为对照，互补菌株CΔtig作为辅佐参考，除去非特异性结合的DNA序列，最后初步确认NisK/R等几个基因是能够与trigger factor结合的片段， 进一步运用基因芯片技术和凝胶电泳迁移实验（EMSA）体外验证了与trigger factor结合的蛋白质，确定了NisK/R等几个基因是受trigger factor调控的。并通过构建NisK/R双组份系统基因缺失菌株和互补菌株，通过体内外试验对NisK/R双组份系统功能进行了验证，结果表明NisK/R双组份系统缺失后与trigger factor基因缺失后，猪链球菌2型的致病性及对环境的耐受性改变基本一致，但是在对NisK/R双组份系统进行功能验证时发现，NisK/R双组份系统除了调控猪链球菌2型的毒力及耐受性之外，还调控了猪链球菌2型对抗生素的敏感性，这对猪链球菌2型耐药性产生提供了一种新的解释。本研究阐明trigger factor通过调控NisK/R双组份系统基因表达导致细菌毒力和耐受性降低，改变细菌致病性的部分机制，剖析了猪链球菌2型的分子致病机理。同时所构建的基因缺失菌株对猪具有很好的免疫保护作用，这对猪链球菌2型新型基因缺失疫苗的研发提供了依据候选菌株。该研究从理论上剖析了猪链球菌2型的分子致病机理，同时对猪链球菌2型的防控奠定了基础。