副猪嗜血杆菌HbpA蛋白功能及其在致病中的作用研究

Haemophilus parasuis(Hps) is the most important pathogenic bacteria which is severely harmful to pig farming industry. However, the pathogenic mechanism and its ability to acquire heme iron have not been explored. In vitro study has proved that HbpA from Hps is a secreted, immunogenic protein. Nevertheless, the in vivo function is still unknown. This project will be carried out with following approaches based on the new identified HbpA protein by our team. First, the accurate function of HbpA protein will be identified using Heme-Agarose binding assay Cellular localization of binding between heme and Hps will be studied by immunoelectron microscopy. Construction of HbpA gene mutation, performing cell infection test, pathological observation and distribution and quantification of the bacteria could clarify the effect of HbpA protein in regarding to the survival and reproduction; and the Pathogenicity of Hps. Finally, the function of HbpA protein in the process of Pathogenicity will be illustrated with above study combined with analysis on the differentially expressed genes by transcriptomic sequencing. The research data will not only enrich molecular basis of the pathogenesis of specific adaption of Hps to the host, but also provide theoretical foundation for designing drugs against Hps.

副猪嗜血杆菌（Hps）是严重危害全球养猪业的重要病原菌，但其摄取血红素铁的能力与致病机制的关系不清楚。研究发现血红素结合蛋白（HbpA）是铁摄取系统的重要组成部分，具有结合宿主体内血红素功能并且是主要毒力因子。体外试验鉴定Hps的一个具有免疫原性的HbpA蛋白，但其在体内的作用仍然不详。本项目拟以本课题组在Hps中新发现的HbpA蛋白为研究对象，先采用Heme-agarose结合方法验证HbpA蛋白的精确功能，结合免疫电镜确定Hps结合血红素的细胞定位；再构建hbpA基因缺失突变株，进行细胞感染存活试验、病理学观察和组织细菌分布和定量分析，明确缺失hbpA基因突变株与Hps增殖水平的关系以及对Hps致病力的影响，最终结合转录组测序分析差异表达基因阐明HbpA蛋白在致病过程中的作用。研究结果将丰富Hps与宿主特异适应性的分子致病机理，也为Hps阻断性药物治疗设计提供理论依据。

副猪嗜血杆菌是严重危害全球养猪业的重要的动物细菌病原，具有严格的宿主特异性，因此利用宿主体内血红素建立快速感染成为细菌致病性的一种重要途径。本研究利用蛋白质组学技术鉴定得到HbpA蛋白，进行了表达和纯化，获得目的产物；利用Heme-agarose法验证了重组HbpA蛋白在体外环境下能具有结合Heme的特异性生物学功能；利用免疫电镜（IEM）技术，确定HbpA蛋白和猪血红素结合位点进行亚细胞定位，确定该蛋白发挥生物学功能的细胞位置在细胞膜上和细胞周质间；利用免疫组化技术验证HPS在猪肺脏内HbpA蛋白表达情况，验证HbpA蛋白能和鼠源HbpA单克隆抗体发生特异性结合，证明了HbpA蛋白的特异性和免疫原性；利用RT-PCR技术验证了HPS在Fe离子缺乏的环境下体内hbpA基因表达量，评价HbpA蛋白的表达受到Fe离子调控的机制。本研究以HbpA蛋白的生物学功能为切入点，对HbpA蛋白结合血红素功能受到细胞内Fe离子的调控，最终阐明HPS利用这一机制快速建立感染途径从而导致细菌的持续性感染，为探索新的细菌感染机制奠定基础。