副猪嗜血杆菌HbpA蛋白功能及其致病中作用的初步研究

发布时间：2018-05-20 07:09

本文选题：副猪嗜血杆菌 + 毒力因子　；参考：《东北林业大学》2015年硕士论文

【摘要】：近年来副猪嗜血杆菌病呈爆发流行趋势,死亡率高达50%,已逐渐成为世界范围内的重要细菌性疾病,给养猪业造成了巨大的经济损失。为了探讨副猪嗜血杆菌(Haemophilus parasuis, HPS) HbpA蛋白的功能及其在致病中的作用,从新的角度诠释HPS的毒力特性和感染致病机制,本研究应用免疫蛋白质组学技术,以新发现的具有免疫原性的HbpA蛋白为切入点,以功能基因组学为理论基础,从多方面多层次探索了HbpA蛋白生物学功能,研究结果如下：1、利用蛋白质组学技术和技术筛选并鉴定得到HPS的一个具有免疫原性的周质蛋白HbpA。2、利用基因重组技术构建重组质粒pET28a-HbpA并诱导表达出可溶性的目的蛋白,大小为59.4kD, PI=7.06。3、利用技术验证HbpA蛋白能和鼠源HbpA单抗发生特异性结合,证明了HbpA蛋白的特异性。4、利用Heme-agarose法验证了HbpA蛋白在体外环境下能Heme结合,当HbpA的量一定时,HbpA-Heme复合体的分子量在一定的范围内(0mM-4mM)随Heme浓度的升高而增大；当Heme浓度达到一定限度(4mM)后,HbpA-Heme复合体的分子量将不再增大,而是维持在一个恒定的数值。5、利用IEM技术确定野生株HbpA蛋白和Heme的复合体主要存在于细胞外膜,内膜和细胞核。6、利用免疫组化技术直观的证明了HPS在猪肺脏内的确表达出HbpA蛋白。7、通过测定HPS的生长曲线证明了Fe离子是HPS生存、繁殖的重要元素。8、利用抗体体外杀菌技术证明了在体外环境下HbpA抗体能杀死近半数的HPS菌体。9、利用RT-PCR技术验证了HPS在Fe离子缺乏的环境下体内HbpA基因表达量是正常培养时的2倍。本研究从分子水平阐明了HbpA蛋白在HPS的存活、繁殖和致病机制方面的功能,并从新的角度诠释HPS的毒力特性和感染致病机制,对有效的疫苗和药物阻断剂的研究具有一定的理论和实践意义。
[Abstract]:In recent years, Haemophilus parasuis disease has become an important bacterial disease in the world, which has caused huge economic losses to the pig industry. The mortality rate of Haemophilus paratosus is as high as 50%, and it has gradually become an important bacterial disease in the world. In order to investigate the function of Haemophilus parasuis, HPS) HbpA protein and its role in pathogenicity, the virulence of HPS and the pathogenesis of infection were interpreted from a new point of view. Based on the newly discovered immunogenicity HbpA protein and functional genomics, the biological function of HbpA protein was explored from many aspects and levels. The results are as follows: 1. A immunogenicity protein HbpA.2of HPS was screened and identified by proteomics techniques. The recombinant plasmid pET28a-HbpA was constructed by gene recombination technique and the soluble protein was induced to express. The size of HbpA protein was 59.4 kD and Pi was 7.06.3. The specific binding ability of HbpA protein to murine HbpA monoclonal antibody was verified by the technique, and the specificity of HbpA protein was proved by using Heme-agarose method. Heme-agarose method was used to verify that HbpA protein could bind to Heme in vitro. When the amount of HbpA is constant, the molecular weight of HbpA-Heme complex increases with the increase of Heme concentration in a certain range, and the molecular weight of HbpA-Heme complex will not increase when the concentration of Heme reaches a certain limit of 4mM). The IEM technique was used to determine that the complex of HbpA protein and Heme mainly existed in the extracellular membrane. The expression of HbpA protein. 7 in pig lung was proved by immunohistochemical technique. The growth curve of HPS showed that Fe ~ (2 +) was the survival of HPS. The important element of reproduction. 8. Using antibody bactericidal technique in vitro, it was proved that HbpA antibody could kill nearly half of HPS cell. RT-PCR technique was used to verify that the expression of HbpA gene in the environment of Fe ion deficiency was positive. Twice as much as in normal culture. The purpose of this study was to elucidate the function of HbpA protein in the survival, reproduction and pathogenesis of HPS at the molecular level, and to interpret the virulence and pathogenicity of HPS from a new perspective. It has certain theoretical and practical significance for the study of effective vaccines and drug blockers.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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