鼠疫耶尔森氏菌毒力相关蛋白蛋白芯片的研制与血清抗体谱的研究

发布时间：2018-03-13 20:01

本文选题：鼠疫耶尔森氏菌　切入点：蛋白芯片　出处：《中国人民解放军军事医学科学院》2005年硕士论文　论文类型：学位论文

【摘要】：背景鼠疫是一种烈性传染病,到目前为止人们对鼠疫的致病因素、流行机制还没有完全了解。虽然现在已经有多种鼠疫疫苗,但这些疫苗对肺型鼠疫的免疫保护作用并不理想。基于F1和V抗原基础上,加入其它具有免疫保护作用成分的多亚单位疫苗将是未来鼠疫疫苗研究的一个方向。对感染患者与免疫动物体内血清抗体谱的研究将为更好的了解鼠疫的致病机制,寻找新的亚单位疫苗成分提供线索。蛋白芯片技术是近年来发展起来的能够同时研究多个抗原-抗体、蛋白-蛋白相互作用的高通量技术,它具有大规模、高通量、所需样品少等特点。方法挑选出202条鼠疫菌已知的或推测的与毒力相关蛋白的基因与表达载体pET32a连接在大肠肝菌BL21(DE3)中表达,利用表达的目的蛋白氨基端带有6×His标签与Ni-NTA具有亲和的原理,通过96孔板对蛋白进行高通量纯化。纯化的蛋白点在醛基化玻片上制备蛋白芯片。通过与鼠疫患者血清、EV76疫苗株免疫兔血清、鼠疫菌感染兔血清及自然疫源地黄鼠血清反应,检测不同血清中抗体升高的种类及趋势。结果通过与不同来源的鼠疫菌感染血清作用,共有15个蛋白在所有的血清中均检测到相应抗体,其中YPMT1.84,YPMT1.24c,YPMT1.48c,YPCD1.31c,YPCD1.28,YPCD1.32c,YPCD1.26c,YPCD1.06c,YPO1435,YPO1303,YPO2118,YPO3319,YPO2113,YPO2090,YPO2394抗体升高幅度大,持续时间长,这些蛋白可以作为下一阶段鼠疫疫苗研究的重点,研究其保护性。除了在所有的血清抗体谱中均升高的蛋白外,在各不同血清的抗体谱中均存在特异性抗体升高的蛋白,这些蛋白可能与鼠疫菌在机体内的致病性相关。分析不同感染状态下的抗体谱,将为更深入的了解鼠疫菌的致病机制提供线索。
[Abstract]:Background Yersinia pestis is a severe infectious disease. So far, people have not fully understood the pathogenic factors and epidemic mechanism of plague. Although there are many kinds of Yersinia pestis vaccine, But the immune protection of these vaccines against pulmonary plague is not ideal. Based on F1 and V antigens, Adding other multi-subunit vaccines with immuno-protective components will be a direction of future plague vaccine research. The study of serum antibody spectrum in infected patients and immunized animals will provide a better understanding of the pathogenic mechanism of plague. Protein chip technology, developed in recent years, is a high-throughput technology that can simultaneously study multiple antigen-antibody, protein-protein interactions, which has a large scale and high throughput. Few samples are needed. Methods A total of 202 Yersinia pestis virulence related protein genes and expression vectors (pET32a) were selected and expressed in E. coli BL21DE3. The amino terminal of the expressed target protein was labeled with 6 脳 His to be compatible with Ni-NTA. The purified protein was purified by high throughput with 96 well plate. The protein chip was prepared on the aldehydated glass slide. The rabbit serum was immunized with the serum of Yersinia pestis EV76 vaccine strain, the rabbit serum infected with Yersinia pestis and the serum of the natural foci of yellow rat. The types and trends of antibody elevation in different serum were detected. Results A total of 15 proteins were detected in all of the sera infected with Yersinia pestis from different sources. Among them, YPMT1.84mYPMT1.24cPMT1.48cPMT1.48cHN YPCD1.31cHYPCD1.32cPD1.26cYPCD1.26cYPCD1.06cD1.06cPPO1303nYPO2118YPO3319 YPO2113An YPO2090YPO2394 increased significantly and lasted for a long time. These proteins can be used as the focus of the next stage of plague vaccine research and study their protection. In addition to the proteins that are elevated in all serum antibody profiles, there is a specific antibody elevation protein in each serum antibody profile. These proteins may be related to the pathogenicity of Yersinia pestis in the organism. The analysis of antibody profiles under different infection states will provide clues for a deeper understanding of the pathogenic mechanism of Yersinia pestis.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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