甲型副伤寒沙门氏菌外膜蛋白免疫原性研究及其菌体抗原单克隆抗体的制备

发布时间：2018-01-31 18:59

本文关键词： 甲型副伤寒沙门氏菌 16S rRNA 外膜蛋白单克隆抗体　出处：《中国药品生物制品检定所》2006年硕士论文　论文类型：学位论文

【摘要】：近年来，我国南方一些地区以及东南亚某些国家均有甲型副伤寒沙门氏菌流行的报道。甲型副伤寒暴发流行，显示了伤寒流行菌型变迁的新特点，即由原来以伤寒沙门氏菌流行为主转为以甲型副伤寒沙门氏菌为主导菌，因此对甲型副伤寒沙门氏菌的研究非常有必要。本论文从两个方面对甲型副伤寒沙门氏菌进行了研究，即甲型副伤寒沙门氏菌外膜蛋白免疫原性的研究和甲型副伤寒沙门氏菌菌体抗原单克隆抗体的制备。论文第一部分的主要研究目的在于：探索应用单一或混合的甲型副伤寒沙门氏菌外膜蛋白作为保护性抗原，进而作为甲型副伤寒的候选疫苗的可能性。首先对本室保存的6株甲型副伤寒沙门氏菌株进行鉴定和筛选。6株菌均符合革兰氏阴性菌的形态特点，生化反应结果显示6株菌均为甲型副伤寒沙门氏菌，可信度为97％，并表现出不发酵木糖，也不产生H_2S的甲型副伤寒沙门氏菌主要生化特征。血清学检测显示6株菌与沙门氏菌诊断血清(02、012、Ha)发生了较强的凝集。建立小鼠攻毒模型对6株菌的毒力进行比较，，选择出毒力较强的50973为后续实验用菌株。借助分子生物学技术对50973的16S rDNA进行了测序，结果与GeneBank中已知的甲型副伤寒沙门氏菌的16s rRNA基因序列进行比对，两者同源性达到99％；其后建立了体外杀菌抗体检测的实验方法，确定了最佳的菌液浓度为1000CFU／ml，反应时间为16—18h，乳兔的补体作为补体来源等，并进行了验证；最后建立了甲型副伤寒沙门氏菌外膜蛋白的提取方法，在此基础上应用SDS—PAGE电泳来分离外膜蛋白的各个组分，并用电洗
[Abstract]:In recent years, some areas in southern China and some countries in Southeast Asia have reported the epidemic of Salmonella paratyphoid A. the outbreak of paratyphoid A shows the new characteristics of the change of epidemic type of typhoid. That is, from the original epidemic of Salmonella typhimurium to the predominant bacillus paratyphoid A. Therefore, it is very necessary to study the salmonella paratyphoid A. in this paper, we studied the salmonella paratyphoid A from two aspects. The immunogenicity of the outer membrane protein of Salmonella paratyphi A and the preparation of monoclonal antibody against Salmonella paratyphi A. The main purpose of the first part of the thesis is to explore the application of a single or mixed outer membrane protein of Salmonella paratyphi A as a protective antigen. At first, 6 strains of Salmonella paratyphoid A preserved in our room were identified and screened. All the 6 strains were in accordance with the morphological characteristics of Gram-negative bacteria. The results of biochemical reaction showed that all of the 6 strains were paratyphoid A Salmonella A, and the reliability was 97%. No H2S production was found in the main biochemical characteristics of Salmonella paratyphi A. serological tests showed that 6 strains of bacilli and the diagnostic serum of Salmonella spp. Ha. the virulence of 6 strains of bacteria was compared by establishing a mouse model of attacking virus. The highly virulent strain 50973 was selected for further experiment. The 16s rDNA of 50973 was sequenced by molecular biology technique. Results compared with the 16s rRNA gene sequence of Salmonella paratyphoid A in GeneBank, the homology of the two genes was 99%. After that, an experimental method for the detection of bactericidal antibody in vitro was established. The optimal concentration of bactericidal liquid was 1000 CFU / ml, the reaction time was 16-18 h, and the complement of milk rabbit was used as the source of complement. And verified; Finally, the extraction method of outer membrane protein of Salmonella paratyphi A was established. On the basis of this, SDS-PAGE electrophoresis was used to separate the components of outer membrane protein, and electrowashing was carried out.
【学位授予单位】：中国药品生物制品检定所
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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