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  本文關鍵詞：幽門螺桿菌尿素酶B亞單位Th表位鑒定及表位疫苗的設計與免疫原性研究　出處：《第三軍醫(yī)大學》2006年碩士論文　論文類型：學位論文

  更多相關文章： 幽門螺桿菌 尿素酶B亞單位 Th表位 BALB/c(H-2d)小鼠 表位疫苗

【摘要】： 目的 表位疫苗是近年來發(fā)展起來的一種獨特的疫苗設計思路,是目前研制感染性疾病、惡性腫瘤以及自身免疫性疾病等疫苗設計的新方向�；诒砦坏囊呙缭O計可以特異性的增強保護性免疫反應的強度,而排除非保護性以及抑制性免疫反應的影響。因此,在篩選抗原的保護性B細胞和T細胞表位基礎上,合理組合設計表位疫苗,可以避免常規(guī)抗原結構中可能出現(xiàn)的非必要以及抑制性表位的作用。 幽門螺桿菌(Hp)自然感染時,激發(fā)的免疫應答不能產(chǎn)生有效的保護作用,因而有必要在抗原選擇以及表位水平對抗原進行改造,激發(fā)更有效的免疫應答。目前對Hp感染的免疫保護機制研究表明:機體的CD4+T細胞而非CD8+T細胞反應對Hp感染的免疫保護是必需的。因此,鑒定Hp抗原的Th表位并明確各表位特異性的T細胞免疫應答特征,成為以表位為基礎的幽門螺桿菌疫苗設計的前提。在篩選Hp抗原的特異性B細胞和Th細胞表位的基礎上,合理組合設計表位疫苗,有可能激發(fā)有效的免疫應答。本課題以BALB/c小鼠(H-2d)為動物模型,采用生物信息學預測,淋巴細胞功能實驗鑒定Hp保護性抗原UreB的H-2d限制性的Th細胞表位,并利用篩選出的Th表位和文獻報道的一個UreB的中和性B細胞表位設計、構建表位疫苗,并對其免疫原性進行初步研究。 方法 1.RANKPEP軟件預測全長UreB蛋白上可能的H-2d限制性Th表位,合成多肽,淋巴細胞增殖實驗、流式細胞分析及CD4+T淋巴細胞增殖實驗進行鑒定;MHC限制性分析表位識別的遺傳限制性;ELISA方法分析表位刺激CD4+T淋巴細胞分泌的細胞因子;淋巴細胞增殖實驗分析表位之間的協(xié)同效應;ELISA檢測表位肽與鼠抗rUreB及鼠抗Hp血清的免疫反應性。 2.初步鑒定的表位肽免疫BALB/c小鼠,淋巴細胞增殖實驗檢測細胞免疫應答,RT-PCR檢測脾淋巴細胞的細胞因子mRNA,流式細胞分析多肽免疫小鼠的脾CD4+T淋巴細胞相對數(shù)量。 3.將鑒定的Hp的UreB的三個Th表位及一個中和性B細胞表位串聯(lián)起來,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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R392

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本文編號：1360242