甲型副伤寒沙门氏菌CMCC 50973外膜蛋白的免疫蛋白质组学研究
发布时间：2018-08-14 13:22
【摘要】： 伤寒是一种急性全身性感染，包括伤寒沙门氏菌(Salmonella typhi)引起的伤寒和甲、乙、丙型副伤寒沙门氏菌(Salmonella paratyphi A，B and C)引起的副伤寒。甲型副伤寒沙门氏菌是甲型副伤寒的病原菌，通过粪—口途径传播，感染剂量为10~3～10~9，人是唯一宿主。人群对甲型副伤寒沙门氏菌普遍易感，但儿童和青壮年发病最高。现在据估计每年全球伤寒(包括副伤寒)患者约2200万，死亡超过20万人。自1996年以来，我国的甲型副伤寒的发病率也在逐年升高。更为严重的是，甲型副伤寒沙门氏菌临床耐药株以及多重耐药株(MDR)已经相当广泛，使临床治疗变得更加困难，一些患者由于治疗不彻底而成为带菌者。因此，需要研究出安全、高效、简便、经济的疫苗来预防甲型副伤寒。 免疫蛋白质组学是通过双向电泳分离病原蛋白质组，再用不同病人的超免血清鉴定出有强免疫性的蛋白，这一蛋白质组的分支，很有希望成为寻找药物和疫苗靶位的有效途径(因为证明这些蛋白能引起宿主的免疫应答)。甲型副伤寒沙门氏菌是无荚膜的革兰氏阴性菌，其外膜蛋白在致病和刺激机体免疫应答方面有非常重要的作用。因为细菌的膜蛋白(尤其是外膜蛋白)抗原，往往是一些反应性抗原，它们与机体免疫系统有十分密切的相互作用。因此，在疫苗研究中，病原体的表面蛋白具有很重要的地位。 本实验提取了甲型副伤寒沙门氏菌的外膜蛋白，建立了甲型副伤寒沙门氏菌的外膜蛋白双向电泳参考图谱。在考马斯亮兰染色的胶上共取了80个外膜蛋白点，鉴定出61个，代表30种蛋白。结合根据基因组预测的外膜蛋白对鉴定的所有蛋白质点的等电点/分子量吻合程度、分布以及功能分类等进行了初步的分析。进一步采用免疫蛋白质组的方法，对提取细菌的外膜蛋白进行双向电泳分离后，再转移到PVDF膜上，与甲型副伤寒病人恢复期的血清进行免疫印迹反应，记录具有免疫反应的外膜蛋白点。在考马斯亮兰染色的胶上取外膜蛋白点以及有免疫反应的蛋白点，酶切后进行MALDI-TOF质谱鉴定。在PVDF膜上共有45个免疫反应点，，鉴定到42个点，代表23种蛋白质抗原，采用psort(www.psort.org)软件分析，外膜蛋白13种，胞质蛋白3种，定位不明确(可能是多处定位)1种，未知定位6种。这些蛋白有希望成为药靶或疫苗的候选成分。
[Abstract]:Typhoid fever is an acute systemic infection, including typhoid fever caused by (Salmonella typhi) and paratyphoid fever caused by (Salmonella paratyphi A B and C) of Salmonella typhimurium. Salmonella paratyphoid A is the pathogen of paratyphoid A. Paratyphoid A is most susceptible to salmonella A in the population, but it is the highest in children and young adults. It is now estimated that about 22 million people worldwide suffer from typhoid fever (including paratyphoid fever) each year, with more than 200000 deaths. Since 1996, the incidence of paratyphoid A in China has also been increasing year by year. More seriously, the clinical resistant strains and multidrug resistant strains of Salmonella paratyphi A (MDR) have been widely used, which makes clinical treatment more difficult, and some patients become carriers because of incomplete treatment. Therefore, a safe, efficient, simple and economical vaccine is needed to prevent paratyphoid A (paratyphoid A). Immunoproteomics is the separation of pathogenic proteome by two-dimensional electrophoresis, and then the identification of strongly immunized proteins with the superimmune serum of different patients, a branch of the proteome. It is promising to be an effective way to target drugs and vaccines (as these proteins have been shown to elicit host immune responses). Salmonella paratyphoid A is a gram-negative bacterium without capsule, and its outer membrane protein plays an important role in pathogenic and stimulating immune response. Because bacterial membrane proteins (especially outer membrane proteins) antigens are often reactive antigens, they interact very closely with the immune system. Therefore, surface proteins of pathogens play an important role in vaccine research. The outer membrane protein of Salmonella paratyphi A was extracted and the reference map of the outer membrane protein of Salmonella paratyphi A was established. A total of 80 outer membrane protein spots were extracted from Coomassie brilliant blue staining glue and 61 proteins representing 30 proteins were identified. The isoelectric point / molecular weight coincidence degree, distribution and functional classification of all the identified protein spots were preliminarily analyzed in combination with the outer membrane protein predicted by the genome. The outer membrane protein of the extracted bacteria was separated by two dimensional electrophoresis and then transferred to the PVDF membrane. The protein was imprinted with the sera of paratyphoid A patients during the convalescence period. The outer membrane protein spots with immune response were recorded. The outer membrane protein spots and immunoreactive protein spots were extracted from Coomassie brilliant blue stained glue and identified by MALDI-TOF mass spectrometry after enzyme digestion. A total of 45 immunoreactive sites were identified on PVDF membrane, representing 23 protein antigens. Psort (www.psort.org) software was used to analyze 13 kinds of outer membrane proteins, 3 kinds of cytoplasmic proteins, 1 species of unclear localization (possibly multiple loci) and 6 kinds of unknown localization. These proteins are expected to be candidates for drug targets or vaccines.
【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】：2006
【分类号】：R392
本文编号：2182987
[Abstract]:Typhoid fever is an acute systemic infection, including typhoid fever caused by (Salmonella typhi) and paratyphoid fever caused by (Salmonella paratyphi A B and C) of Salmonella typhimurium. Salmonella paratyphoid A is the pathogen of paratyphoid A. Paratyphoid A is most susceptible to salmonella A in the population, but it is the highest in children and young adults. It is now estimated that about 22 million people worldwide suffer from typhoid fever (including paratyphoid fever) each year, with more than 200000 deaths. Since 1996, the incidence of paratyphoid A in China has also been increasing year by year. More seriously, the clinical resistant strains and multidrug resistant strains of Salmonella paratyphi A (MDR) have been widely used, which makes clinical treatment more difficult, and some patients become carriers because of incomplete treatment. Therefore, a safe, efficient, simple and economical vaccine is needed to prevent paratyphoid A (paratyphoid A). Immunoproteomics is the separation of pathogenic proteome by two-dimensional electrophoresis, and then the identification of strongly immunized proteins with the superimmune serum of different patients, a branch of the proteome. It is promising to be an effective way to target drugs and vaccines (as these proteins have been shown to elicit host immune responses). Salmonella paratyphoid A is a gram-negative bacterium without capsule, and its outer membrane protein plays an important role in pathogenic and stimulating immune response. Because bacterial membrane proteins (especially outer membrane proteins) antigens are often reactive antigens, they interact very closely with the immune system. Therefore, surface proteins of pathogens play an important role in vaccine research. The outer membrane protein of Salmonella paratyphi A was extracted and the reference map of the outer membrane protein of Salmonella paratyphi A was established. A total of 80 outer membrane protein spots were extracted from Coomassie brilliant blue staining glue and 61 proteins representing 30 proteins were identified. The isoelectric point / molecular weight coincidence degree, distribution and functional classification of all the identified protein spots were preliminarily analyzed in combination with the outer membrane protein predicted by the genome. The outer membrane protein of the extracted bacteria was separated by two dimensional electrophoresis and then transferred to the PVDF membrane. The protein was imprinted with the sera of paratyphoid A patients during the convalescence period. The outer membrane protein spots with immune response were recorded. The outer membrane protein spots and immunoreactive protein spots were extracted from Coomassie brilliant blue stained glue and identified by MALDI-TOF mass spectrometry after enzyme digestion. A total of 45 immunoreactive sites were identified on PVDF membrane, representing 23 protein antigens. Psort (www.psort.org) software was used to analyze 13 kinds of outer membrane proteins, 3 kinds of cytoplasmic proteins, 1 species of unclear localization (possibly multiple loci) and 6 kinds of unknown localization. These proteins are expected to be candidates for drug targets or vaccines.
【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】：2006
【分类号】：R392
【引证文献】
相关期刊论文 前2条
1 高翔;闫梅英;阚飙;;甲型副伤寒沙门菌疫苗靶标蛋白筛选技术及应用[J];疾病监测;2011年07期
2 王斌;李娜;董晓宇;梁昊宇;陈翠萍;陈薇;曾明;;甲型副伤寒沙门菌外膜BtuB蛋白的原核表达、纯化及其免疫保护性[J];中国生物制品学杂志;2012年06期
相关博士学位论文 前1条
1 杨艳玲;羊布鲁氏菌蛋白质组学分析及免疫候选抗原的筛选与鉴定[D];吉林大学;2011年
本文编号：2182987
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