【摘要】：目的:利用非蛋白氨基酸對TRAG-3(58-66) ( ILLRDAGLV) CTL表位肽進行結(jié)構(gòu)修飾,以期篩選到既能克服肽酶水解,又能在體激發(fā)較強CTL反應(yīng)的高親和性CTL模擬表位,為進一步探索基于非蛋白氨基酸CTL模擬表位設(shè)計高效擬肽疫苗奠定基礎(chǔ)。 方法:首先,利用非天然氨基酸對TRAG-3(58-66) ( ILLRDAGLV) CTL表位的各個天然氨基酸位點進行替換,即氨基酸由L型替換為D型,得到修飾的肽配體(Altered peptide ligands. APLs);然后,借助本實驗室的SCI O2工作站及并行計算系統(tǒng),并對APLs與HLA-A*0201分子的三維結(jié)構(gòu)進行分子模擬研究與實驗相結(jié)合,進行CTL模擬表位的高通量篩選,從中篩選出2條MHC親和力較高的APLs合成、純化和鑒定;最后,通過體外經(jīng)典的T2肽結(jié)合實驗和多肽在人血漿中穩(wěn)定性實驗分析各肽在體內(nèi)抗酶解的能力以及其半衰期。 結(jié)果:利用非天然氨基酸對TRAG-3(58-66) ( ILLRDAGLV) CTL表位的各個天然氨基酸位點進行替換,得到了8條CTL模擬表位肽(APLs)——P1 TRAG-3(58-66) I(D)LLRDAGLV、P2 TRAG-3(58-66) IL(D)LRDAGLV、P3 TRAG-3(58-66 ILL(D)RDAGLV、P4 TRAG-3(58-66) ILLR(D)DAGLV、P5 TRAG-3(58-66)ILLRD(D)AGLV、P6 TRAG-3(58-66) ILLRDA(D)GLV、P8 TRAG-3(58-66)ILLRDAGL(D)V、P9 TRAG-3(58-66)ILLRDAGLV(D);利用計算機分子模擬研究,我們得到2條CTL模擬表位肽P5 TRAG-3(58-66) ILL RD(D) AG LV、P6 TRAG-3(58-66) ILLRDA(D)GLV,與HLA-A*0201分子相互作用,其氫鍵數(shù)目分別為12、8,P5比天然表位肽(氫鍵數(shù)目為11)要多,P6較天然表位肽略少,且錨定殘基(P2—P9)的距離分別為18.38,16.29,均滿足大部分HLA-A*0201限制性CTL表位的錨定殘基在15—20 ?之間的要求,其溶劑可及表面積P5中的P2為0,P9為0.193?2,P6中P2為0.386?2,P9為0.902?2,均分別小于天然表位肽中P2為1.401?2,P9為3.035?2 ,故綜上所述,我們得到2條CTL模擬表位肽P5 TRAG-3(58-66) ILL RD(D) AG LV、P6 TRAG-3(58-66) ILLRDA(D)GLV,與HLA-A*0201分子相互作用,其親和力較天然表位肽要更好;通過體外經(jīng)典的T2肽結(jié)合實驗,也驗證了與計算機分子模擬研究同樣的結(jié)果,FI值分別為1.50,2.09,均較天然表位肽(FI值為1.49)要更好。 結(jié)論:利用非蛋白氨基酸替換相對應(yīng)位點的天然氨基酸進行結(jié)構(gòu)改造得到的8個APLs,通過計算機分子模擬研究篩選得到兩條親和力較天然表位更好的模擬表位肽,然后通過體外的親和力實驗分析,與分子模擬結(jié)果分析實驗的一致性較好,驗證了計算機分子篩選的準(zhǔn)確性,在后續(xù)的研究中進一步驗證P5: ILLRD(D)AGLV、P6: ILLRDA(D)GLV在體外血漿中的抗酶解能力以及計算其半衰期,以及其對TRAG-3+腫瘤細(xì)胞的殺傷效應(yīng)和應(yīng)用于非小細(xì)胞肺癌病人的臨床試驗,為進一步探索基于非蛋白氨基酸CTL模擬表位設(shè)計高效擬肽疫苗奠定基礎(chǔ)。
[Abstract]:Aim: to study the structural modification of TRAG-3 (58-66) (ILLRDAGLV) CTL epitope peptide) by non-protein amino acids in order to obtain a high affinity CTL mimic epitope which can not only overcome peptidase hydrolysis but also excite strong CTL reaction in vivo. It lays a foundation for further exploring the design of highly efficient peptide vaccine based on CTL mimic epitopes of nonprotein amino acids. Methods: firstly, the natural amino acid sites of TRAG-3 (58-66) (ILLRDAGLV) CTL epitope) were replaced by non-natural amino acids. The modified peptide ligand (Altered peptide ligands. was obtained by replacing amino acids from L-type to D-type. APLs); then, with the help of SCI O2 workstation and parallel computing system in our laboratory, and combining the molecular simulation of APLs and HLA-A*0201 molecules with experiments, the high throughput screening of CTL mimic epitopes was carried out. Two APLs bands with high affinity for MHC were selected for synthesis, purification and identification. Finally, the anti-enzymatic ability and half-life of the peptides in vivo were analyzed by the classical T2 peptide binding assay in vitro and the stability experiment of peptides in human plasma. 緇撴灉:鍒╃敤闈炲ぉ鐒舵皚鍩洪吀瀵筎RAG-3(58-66) (ILLRDAGLV) CTL琛ㄤ綅鐨勫悇涓ぉ鐒舵皚鍩洪吀浣嶇偣榪涜鏇挎崲,寰楀埌浜，

本文編號：2181007