本文關(guān)鍵詞：幽門(mén)螺桿菌尿素酶B亞單位Th表位鑒定及表位疫苗的設(shè)計(jì)與免疫原性研究　出處：《第三軍醫(yī)大學(xué)》2006年碩士論文　論文類(lèi)型：學(xué)位論文

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【摘要】： 目的 表位疫苗是近年來(lái)發(fā)展起來(lái)的一種獨(dú)特的疫苗設(shè)計(jì)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(shè)計(jì)的新方向�；诒砦坏囊呙缭O(shè)計(jì)可以特異性的增強(qiáng)保護(hù)性免疫反應(yīng)的強(qiáng)度,而排除非保護(hù)性以及抑制性免疫反應(yīng)的影響。因此,在篩選抗原的保護(hù)性B細(xì)胞和T細(xì)胞表位基礎(chǔ)上,合理組合設(shè)計(jì)表位疫苗,可以避免常規(guī)抗原結(jié)構(gòu)中可能出現(xiàn)的非必要以及抑制性表位的作用。 幽門(mén)螺桿菌(Hp)自然感染時(shí),激發(fā)的免疫應(yīng)答不能產(chǎn)生有效的保護(hù)作用,因而有必要在抗原選擇以及表位水平對(duì)抗原進(jìn)行改造,激發(fā)更有效的免疫應(yīng)答。目前對(duì)Hp感染的免疫保護(hù)機(jī)制研究表明:機(jī)體的CD4+T細(xì)胞而非CD8+T細(xì)胞反應(yīng)對(duì)Hp感染的免疫保護(hù)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(xì)胞免疫應(yīng)答特征,成為以表位為基礎(chǔ)的幽門(mén)螺桿菌疫苗設(shè)計(jì)的前提。在篩選Hp抗原的特異性B細(xì)胞和Th細(xì)胞表位的基礎(chǔ)上,合理組合設(shè)計(jì)表位疫苗,有可能激發(fā)有效的免疫應(yīng)答。本課題以BALB/c小鼠(H-2d)為動(dòng)物模型,采用生物信息學(xué)預(yù)測(cè),淋巴細(xì)胞功能實(shí)驗(yàn)鑒定Hp保護(hù)性抗原UreB的H-2d限制性的Th細(xì)胞表位,并利用篩選出的Th表位和文獻(xiàn)報(bào)道的一個(gè)UreB的中和性B細(xì)胞表位設(shè)計(jì)、構(gòu)建表位疫苗,并對(duì)其免疫原性進(jìn)行初步研究。 方法 1.RANKPEP軟件預(yù)測(cè)全長(zhǎng)UreB蛋白上可能的H-2d限制性Th表位,合成多肽,淋巴細(xì)胞增殖實(shí)驗(yàn)、流式細(xì)胞分析及CD4+T淋巴細(xì)胞增殖實(shí)驗(yàn)進(jìn)行鑒定;MHC限制性分析表位識(shí)別的遺傳限制性;ELISA方法分析表位刺激CD4+T淋巴細(xì)胞分泌的細(xì)胞因子;淋巴細(xì)胞增殖實(shí)驗(yàn)分析表位之間的協(xié)同效應(yīng);ELISA檢測(cè)表位肽與鼠抗rUreB及鼠抗Hp血清的免疫反應(yīng)性。 2.初步鑒定的表位肽免疫BALB/c小鼠,淋巴細(xì)胞增殖實(shí)驗(yàn)檢測(cè)細(xì)胞免疫應(yīng)答,RT-PCR檢測(cè)脾淋巴細(xì)胞的細(xì)胞因子mRNA,流式細(xì)胞分析多肽免疫小鼠的脾CD4+T淋巴細(xì)胞相對(duì)數(shù)量。 3.將鑒定的Hp的UreB的三個(gè)Th表位及一個(gè)中和性B細(xì)胞表位串聯(lián)起來(lái),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(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R392

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本文編號(hào)：1360242