本文選題：結(jié)核桿菌 + CFP21　； 參考：《鄭州大學(xué)》2010年碩士論文

【摘要】： 結(jié)核病(Tuberculosis, TB)是由結(jié)核桿菌(Mycobacterium tuberculosis,Mtb)引起的一種慢性傳染病,目前仍然是導(dǎo)致傳染病發(fā)病率和死亡率較高的疾病之一。尤其是近年來(lái),由于人體免疫缺陷病毒雙重感染的出現(xiàn)而造成個(gè)體機(jī)會(huì)性感染愈發(fā)嚴(yán)重。目前唯一可用的用于結(jié)核病預(yù)防的疫苗是卡介苗(Bacillus Calmette-Guerin, BCG),但其在預(yù)防成年人患肺結(jié)核的有效性方面受到了限制,并且伴隨著結(jié)核桿菌多重耐藥菌株的出現(xiàn),人們迫切需要研制新型的抗結(jié)核疫苗。 由CD4+輔助性T(Th1)細(xì)胞和CD8+毒性T淋巴細(xì)胞(CTL)所介導(dǎo)的細(xì)胞免疫反應(yīng)在抗結(jié)核感染保護(hù)性免疫中發(fā)揮著主導(dǎo)作用。結(jié)核新型疫苗的研究依賴(lài)于能被CD8+T細(xì)胞識(shí)別從而產(chǎn)生IFN-γ而發(fā)揮殺傷效應(yīng)的結(jié)核抗原及其相應(yīng)表位的鑒定。分泌蛋白以及細(xì)胞壁蛋白是結(jié)核桿菌主要的免疫保護(hù)抗原,并且位于Mtb基因組和BCG差別區(qū)(RD區(qū))內(nèi)的分泌蛋白由于其在BCG中的缺失,逐漸成為疫苗候選抗原的熱點(diǎn)。優(yōu)勢(shì)抗原表位的鑒定,對(duì)發(fā)展更有效的結(jié)核病治療性多表位肽疫苗具有重要的意義,并且能夠?yàn)榛A(chǔ)研究提供理論基礎(chǔ)。 結(jié)核桿菌分泌蛋白CFP21 (culture filtrate proteins 21,培養(yǎng)濾液蛋白21)位于RD2區(qū),具有高度的免疫原性。CFP21能夠誘導(dǎo)結(jié)核感染結(jié)核患者產(chǎn)生T細(xì)胞增殖反應(yīng)以及釋放高水平IFN-γ,發(fā)揮細(xì)胞殺傷作用,是抗結(jié)核疫苗設(shè)計(jì)的理想候選抗原。 CFP21 HLA-A*0201/03限制性表位的鑒定與改造主要通過(guò)以下工作來(lái)完成：我們首先運(yùn)用在線(xiàn)數(shù)據(jù)庫(kù),以SYFPEITHI初步預(yù)測(cè),結(jié)合BIMAS和NetCTL 1.2在線(xiàn)分析,初步篩選出一系列的天然表位肽。已有相關(guān)文獻(xiàn)報(bào)道,通過(guò)對(duì)抗原表位錨定位點(diǎn)的氨基酸替換,能夠增強(qiáng)表位與HLA-Ⅰ分子的結(jié)合力。因此,我們通過(guò)將天然表位,按1Y、2L和/或9L進(jìn)行氨基酸置換,獲得改造肽,利用數(shù)據(jù)庫(kù)再次分析,以期尋求結(jié)合力更高的表位。綜合分析預(yù)測(cè)結(jié)果,選取在至少兩種數(shù)據(jù)庫(kù)中評(píng)分結(jié)果位于前10的表位肽,分別是4條母體肽p5 (SLVRIVGVV)、p13 (VVATTLALV)、p134 (AVADHVAAV)、p189 (NIMAHVSYV)以及3條改造肽p189-1Y2L9L(YLMAHVSYL)、p134-1Y2L (YLADHVAAV)、p134-1Y2L9L (YLADHVAAL)。對(duì)已經(jīng)篩選出來(lái)的表位,我們采用標(biāo)準(zhǔn)Fmoc方案合成多肽,產(chǎn)物經(jīng)反向高效液相色譜(RP-HPLC)分析、純化,獲得了純度高于95%的九肽產(chǎn)物。經(jīng)質(zhì)譜測(cè)定,分子量所得測(cè)定值與理論值相符合。 通過(guò)內(nèi)源性抗原提呈途徑中所必需的抗原多肽轉(zhuǎn)運(yùn)蛋白(TAP)缺陷的T2細(xì)胞結(jié)合力以及肽/HLA穩(wěn)定性實(shí)驗(yàn),對(duì)初步篩選的表位進(jìn)行驗(yàn)證。結(jié)果顯示,相對(duì)于母體肽p134,改造肽p134-1Y2L、p134-1Y2L9L與HLA分子具有更高的結(jié)合力與穩(wěn)定性。通過(guò)結(jié)合力及穩(wěn)定性的初步篩選,在7條候選肽中,只有母體肽p134以及相應(yīng)的兩條改造肽,用于后續(xù)ELISPOT以及細(xì)胞毒殺傷作用的T細(xì)胞免疫活性檢測(cè)。通過(guò)HLA-A*0201+PPD+/HLA-A*03+PPD+健康供者外周血單核細(xì)胞(PBMCs)誘導(dǎo)出能夠分泌IFN-γ的細(xì)胞數(shù)的分析以及特異性CTL的LDH殺傷活性檢測(cè)顯示,母體肽p134以及兩條改造肽p134-1Y2L和p134-1Y2L9L都能夠誘導(dǎo)T細(xì)胞反應(yīng),但只有p134誘導(dǎo)出的CTL對(duì)靶細(xì)胞具有明顯的殺傷效應(yīng)。通過(guò)上述的工作,我們證明,CFP21134-142 (AVADHVAAV),是CFP21潛在的免疫優(yōu)勢(shì)HLA-A*0201/03限制性表位。 本課題鑒定出一個(gè)源于結(jié)核桿菌分泌蛋白CFP21的HLA-A*0201/03限制性表位—p134 (AVADHVAAV)。該表位能有效激發(fā)HLA-A*0201和HLA-A*03限制性CTL的免疫應(yīng)答,且為一種多等位基因廣譜CTL表位,能夠?yàn)榻Y(jié)核病的疫苗設(shè)計(jì)及免疫治療奠定基礎(chǔ)。
[Abstract]:Tuberculosis ( TB ) is a chronic infectious disease caused by Mycobacterium tuberculosis ( Mtb ) , which is still one of the diseases that result in higher incidence and mortality of infectious diseases . Especially in recent years , because of the double infection of human immunodeficiency virus , opportunistic infections have become more and more serious . The only vaccine available for tuberculosis prevention is BCG , but it has been limited in the prevention of adult tuberculosis , and there is an urgent need to develop a new anti - tuberculosis vaccine .



The cell immune response mediated by CD4 + helper T ( Th1 ) cells and CD8 + cytotoxic T lymphocytes ( CTL ) plays a leading role in the protective immunity of anti - tuberculosis infection . The research of new vaccines relies on the identification of TB antigens which can be recognized by CD8 + T cells to produce IFN - 緯 and their corresponding epitopes . The secretory proteins and the cell wall proteins are the main immune protective antigens of mycobacterium tuberculosis .



Mycobacterium tuberculosis secretory protein CFP21 ( culture filtrate proteins 21 , culture filtrate protein 21 ) is located in RD2 region , and has high immunogenicity . CFP21 can induce T cell proliferation reaction in patients with tuberculosis infection and release high levels of IFN - gamma and exert cytotoxic effect , and is an ideal candidate antigen for anti - tuberculosis vaccine design .



A series of epitope peptides were obtained by substitution of amino acids with BIMAS and NetCTL 1.2 . The results were analyzed by reversed high performance liquid chromatography ( RP - HPLC ) .



The results showed that , compared with the parent peptide p134 , the modified peptide p134 - 1Y2L and p134 - 1Y2L9L had higher binding force and stability . The results showed that the parent peptide p134 and two modified peptides p134 - 1Y2L and p134 - 1Y2L9L were able to induce T cell reaction .



This study identified a restricted epitope - p134 ( AVADHVAAV ) derived from Mycobacterium tuberculosis secretory protein CFP21 . This epitope can effectively stimulate the immune response of HLA - A * 0201 and HLA - A * 03 restricted CTL , and provide a basis for vaccine design and immunotherapy for tuberculosis .

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 孫偉紅;人磷脂酰乙醇胺結(jié)合蛋白4（hPEBP4）的HLA-A～*0201限制性CD8～+ CTL表位的鑒定及其功能研究[D];第二軍醫(yī)大學(xué);2006年

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本文編號(hào)：1746891