基于非天然氨基酸进行TRAG-3抗原表位的改造及亲和力分析
发布时间：2018-08-13 12:33
【摘要】：目的:利用非蛋白氨基酸对TRAG-3(58-66) ( ILLRDAGLV) CTL表位肽进行结构修饰,以期筛选到既能克服肽酶水解,又能在体激发较强CTL反应的高亲和性CTL模拟表位,为进一步探索基于非蛋白氨基酸CTL模拟表位设计高效拟肽疫苗奠定基础。 方法:首先,利用非天然氨基酸对TRAG-3(58-66) ( ILLRDAGLV) CTL表位的各个天然氨基酸位点进行替换,即氨基酸由L型替换为D型,得到修饰的肽配体(Altered peptide ligands. APLs);然后,借助本实验室的SCI O2工作站及并行计算系统,并对APLs与HLA-A*0201分子的三维结构进行分子模拟研究与实验相结合,进行CTL模拟表位的高通量筛选,从中筛选出2条MHC亲和力较高的APLs合成、纯化和鉴定;最后,通过体外经典的T2肽结合实验和多肽在人血浆中稳定性实验分析各肽在体内抗酶解的能力以及其半衰期。 结果:利用非天然氨基酸对TRAG-3(58-66) ( ILLRDAGLV) CTL表位的各个天然氨基酸位点进行替换,得到了8条CTL模拟表位肽(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);利用计算机分子模拟研究,我们得到2条CTL模拟表位肽P5 TRAG-3(58-66) ILL RD(D) AG LV、P6 TRAG-3(58-66) ILLRDA(D)GLV,与HLA-A*0201分子相互作用,其氢键数目分别为12、8,P5比天然表位肽(氢键数目为11)要多,P6较天然表位肽略少,且锚定残基(P2—P9)的距离分别为18.38,16.29,均满足大部分HLA-A*0201限制性CTL表位的锚定残基在15—20 ?之间的要求,其溶剂可及表面积P5中的P2为0,P9为0.193?2,P6中P2为0.386?2,P9为0.902?2,均分别小于天然表位肽中P2为1.401?2,P9为3.035?2 ,故综上所述,我们得到2条CTL模拟表位肽P5 TRAG-3(58-66) ILL RD(D) AG LV、P6 TRAG-3(58-66) ILLRDA(D)GLV,与HLA-A*0201分子相互作用,其亲和力较天然表位肽要更好;通过体外经典的T2肽结合实验,也验证了与计算机分子模拟研究同样的结果,FI值分别为1.50,2.09,均较天然表位肽(FI值为1.49)要更好。 结论:利用非蛋白氨基酸替换相对应位点的天然氨基酸进行结构改造得到的8个APLs,通过计算机分子模拟研究筛选得到两条亲和力较天然表位更好的模拟表位肽,然后通过体外的亲和力实验分析,与分子模拟结果分析实验的一致性较好,验证了计算机分子筛选的准确性,在后续的研究中进一步验证P5: ILLRD(D)AGLV、P6: ILLRDA(D)GLV在体外血浆中的抗酶解能力以及计算其半衰期,以及其对TRAG-3+肿瘤细胞的杀伤效应和应用于非小细胞肺癌病人的临床试验,为进一步探索基于非蛋白氨基酸CTL模拟表位设计高效拟肽疫苗奠定基础。
[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. 缁撴灉:鍒╃敤闈炲ぉ鐒舵皑鍩洪吀瀵筎RAG-3(58-66) (ILLRDAGLV) CTL琛ㄤ綅鐨勫悇涓ぉ鐒舵皑鍩洪吀浣嶇偣杩涜鏇挎崲,寰楀埌浜，
本文编号：2181007
[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. 缁撴灉:鍒╃敤闈炲ぉ鐒舵皑鍩洪吀瀵筎RAG-3(58-66) (ILLRDAGLV) CTL琛ㄤ綅鐨勫悇涓ぉ鐒舵皑鍩洪吀浣嶇偣杩涜鏇挎崲,寰楀埌浜，
本文编号：2181007
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