本文選題：結核桿菌 + CFP21　； 參考：《鄭州大學》2010年碩士論文

【摘要】： 結核病(Tuberculosis, TB)是由結核桿菌(Mycobacterium tuberculosis,Mtb)引起的一種慢性傳染病,目前仍然是導致傳染病發(fā)病率和死亡率較高的疾病之一。尤其是近年來,由于人體免疫缺陷病毒雙重感染的出現而造成個體機會性感染愈發(fā)嚴重。目前唯一可用的用于結核病預防的疫苗是卡介苗(Bacillus Calmette-Guerin, BCG),但其在預防成年人患肺結核的有效性方面受到了限制,并且伴隨著結核桿菌多重耐藥菌株的出現,人們迫切需要研制新型的抗結核疫苗。 由CD4+輔助性T(Th1)細胞和CD8+毒性T淋巴細胞(CTL)所介導的細胞免疫反應在抗結核感染保護性免疫中發(fā)揮著主導作用。結核新型疫苗的研究依賴于能被CD8+T細胞識別從而產生IFN-γ而發(fā)揮殺傷效應的結核抗原及其相應表位的鑒定。分泌蛋白以及細胞壁蛋白是結核桿菌主要的免疫保護抗原,并且位于Mtb基因組和BCG差別區(qū)(RD區(qū))內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。優(yōu)勢抗原表位的鑒定,對發(fā)展更有效的結核病治療性多表位肽疫苗具有重要的意義,并且能夠為基礎研究提供理論基礎。 結核桿菌分泌蛋白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能夠誘導結核感染結核患者產生T細胞增殖反應以及釋放高水平IFN-γ,發(fā)揮細胞殺傷作用,是抗結核疫苗設計的理想候選抗原。 CFP21 HLA-A*0201/03限制性表位的鑒定與改造主要通過以下工作來完成：我們首先運用在線數據庫,以SYFPEITHI初步預測,結合BIMAS和NetCTL 1.2在線分析,初步篩選出一系列的天然表位肽。已有相關文獻報道,通過對抗原表位錨定位點的氨基酸替換,能夠增強表位與HLA-Ⅰ分子的結合力。因此,我們通過將天然表位,按1Y、2L和/或9L進行氨基酸置換,獲得改造肽,利用數據庫再次分析,以期尋求結合力更高的表位。綜合分析預測結果,選取在至少兩種數據庫中評分結果位于前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)。對已經篩選出來的表位,我們采用標準Fmoc方案合成多肽,產物經反向高效液相色譜(RP-HPLC)分析、純化,獲得了純度高于95%的九肽產物。經質譜測定,分子量所得測定值與理論值相符合。 通過內源性抗原提呈途徑中所必需的抗原多肽轉運蛋白(TAP)缺陷的T2細胞結合力以及肽/HLA穩(wěn)定性實驗,對初步篩選的表位進行驗證。結果顯示,相對于母體肽p134,改造肽p134-1Y2L、p134-1Y2L9L與HLA分子具有更高的結合力與穩(wěn)定性。通過結合力及穩(wěn)定性的初步篩選,在7條候選肽中,只有母體肽p134以及相應的兩條改造肽,用于后續(xù)ELISPOT以及細胞毒殺傷作用的T細胞免疫活性檢測。通過HLA-A*0201+PPD+/HLA-A*03+PPD+健康供者外周血單核細胞(PBMCs)誘導出能夠分泌IFN-γ的細胞數的分析以及特異性CTL的LDH殺傷活性檢測顯示,母體肽p134以及兩條改造肽p134-1Y2L和p134-1Y2L9L都能夠誘導T細胞反應,但只有p134誘導出的CTL對靶細胞具有明顯的殺傷效應。通過上述的工作,我們證明,CFP21134-142 (AVADHVAAV),是CFP21潛在的免疫優(yōu)勢HLA-A*0201/03限制性表位。 本課題鑒定出一個源于結核桿菌分泌蛋白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的免疫應答,且為一種多等位基因廣譜CTL表位,能夠為結核病的疫苗設計及免疫治療奠定基礎。
[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 .

【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392

【參考文獻】

相關博士學位論文 前1條

1 孫偉紅;人磷脂酰乙醇胺結合蛋白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;2006年

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本文編號：1746891