幽门螺杆菌尿素酶B亚单位Th表位鉴定及表位疫苗的设计与免疫原性研究

发布时间：2017-12-31 15:00

本文关键词：幽门螺杆菌尿素酶B亚单位Th表位鉴定及表位疫苗的设计与免疫原性研究　出处：《第三军医大学》2006年硕士论文　论文类型：学位论文

【摘要】： 目的表位疫苗是近年来发展起来的一种独特的疫苗设计思路,是目前研制感染性疾病、恶性肿瘤以及自身免疫性疾病等疫苗设计的新方向。基于表位的疫苗设计可以特异性的增强保护性免疫反应的强度,而排除非保护性以及抑制性免疫反应的影响。因此,在筛选抗原的保护性B细胞和T细胞表位基础上,合理组合设计表位疫苗,可以避免常规抗原结构中可能出现的非必要以及抑制性表位的作用。幽门螺杆菌(Hp)自然感染时,激发的免疫应答不能产生有效的保护作用,因而有必要在抗原选择以及表位水平对抗原进行改造,激发更有效的免疫应答。目前对Hp感染的免疫保护机制研究表明:机体的CD4+T细胞而非CD8+T细胞反应对Hp感染的免疫保护是必需的。因此,鉴定Hp抗原的Th表位并明确各表位特异性的T细胞免疫应答特征,成为以表位为基础的幽门螺杆菌疫苗设计的前提。在筛选Hp抗原的特异性B细胞和Th细胞表位的基础上,合理组合设计表位疫苗,有可能激发有效的免疫应答。本课题以BALB/c小鼠(H-2d)为动物模型,采用生物信息学预测,淋巴细胞功能实验鉴定Hp保护性抗原UreB的H-2d限制性的Th细胞表位,并利用筛选出的Th表位和文献报道的一个UreB的中和性B细胞表位设计、构建表位疫苗,并对其免疫原性进行初步研究。方法 1.RANKPEP软件预测全长UreB蛋白上可能的H-2d限制性Th表位,合成多肽,淋巴细胞增殖实验、流式细胞分析及CD4+T淋巴细胞增殖实验进行鉴定;MHC限制性分析表位识别的遗传限制性;ELISA方法分析表位刺激CD4+T淋巴细胞分泌的细胞因子;淋巴细胞增殖实验分析表位之间的协同效应;ELISA检测表位肽与鼠抗rUreB及鼠抗Hp血清的免疫反应性。 2.初步鉴定的表位肽免疫BALB/c小鼠,淋巴细胞增殖实验检测细胞免疫应答,RT-PCR检测脾淋巴细胞的细胞因子mRNA,流式细胞分析多肽免疫小鼠的脾CD4+T淋巴细胞相对数量。 3.将鉴定的Hp的UreB的三个Th表位及一个中和性B细胞表位串联起来,表
[Abstract]:Purpose Epitope vaccine is a unique vaccine design idea developed in recent years, which is currently developing infectious diseases. New directions in vaccine design such as malignant tumors and autoimmune diseases. Epitope based vaccine design can specifically enhance the intensity of protective immune response. Therefore, on the basis of screening protective B cells and T cell epitopes of antigens, the epitope vaccine was designed. It can avoid the nonessential and inhibitory epitopes that may appear in the normal antigen structure. When Helicobacter pylori (HP) is infected naturally, the immune response can not produce effective protective effect, so it is necessary to modify the antigen at antigen selection and epitope level. At present, the immune protective mechanism of HP infection shows that the immune protection of CD4 T cells rather than CD8 T cells is necessary for HP infection. To identify the Th epitopes of HP antigen and to identify the specific T cell immune response characteristics of each epitope. On the basis of screening specific B cells and Th cell epitopes of HP antigen, a reasonable combination of epitope vaccines was designed. It is possible to stimulate an effective immune response. In this study, BALB/c mice were used as animal models and bioinformatics was used to predict the immune response. The H-2d-restricted Th epitopes of HP protective antigen UreB were identified by lymphocyte function assay. Using the screened Th epitopes and a UreB neutralizing B cell epitope design, the epitope vaccine was constructed and its immunogenicity was preliminarily studied. Method 1. RANKPEP software predicted the possible H-2d-restricted Th epitopes on the full-length UreB protein, synthesized polypeptides and proliferated lymphocytes. Flow cytometry (FCM) and CD4 T lymphocyte proliferation assay were used for identification. Genetic restriction of epitope recognition by MHC restriction analysis; The cytokines secreted by CD4 T lymphocytes stimulated by epitopes were analyzed by ELISA. Lymphocyte proliferation assay was used to analyze the synergistic effect between epitopes. ELISA was used to detect the immunoreactivity of epitope peptide with mouse anti rUreB and anti HP serum. 2. The BALB/c mice were immunized with epitope peptides, and the cytokines mRNA of spleen lymphocytes were detected by lymphocyte proliferation assay and reverse transcription-polymerase chain reaction (RT-PCR). The relative number of CD4 T lymphocytes in spleen of polypeptide immunized mice was analyzed by flow cytometry. 3. The three Th epitopes and a neutralizing B cell epitope of the identified HP UreB are connected together.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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