【作者】 陈明水；

【导师】 叶韵斌； 陈强；

【作者基本信息】 福建医科大学 ， 免疫学， 2009， 硕士

【摘要】 【目的】表位是抗原中能被免疫细胞特异性识别的线性片段或空间构象性结构,是引起免疫应答和免疫反应的基本单位,鉴定合适的抗原表位是诱导产生有效肿瘤免疫应答的关键。本研究利用三种计算机算法筛选Survivin抗原HLA-A2+限制性CTL表位;利用不同亲和力的表位肽分别诱导CTL,探讨其免疫学效应和杀瘤特性。【方法】采用超基序、量化基序法和蛋白酶体裂解法相结合对靶抗原Survivin HLA-A2+限制性CTL表位进行预测筛选;用流式细胞仪检测侯选表位肽与T2细胞结合后荧光强度变化,应用HLA-A2结合实验和流式细胞术检测候选表位肽的结合亲和力,以荧光系数FI(fluorescence index)表示;根据HLA-A2解离实验和流式细胞术检测候选肽的稳定性,以复合物解离50%的时间DC50(dissociation complex 50%)表示。分别用筛选出的高亲和力、中等亲和力及低亲和力表位肽体外诱导CTL并用酶联免疫斑点(ELISPOT)法检测CTL产生分泌IFN-γ的能力;用乳酸脱氢酶(LDH)释放法检测CTL的杀瘤能力。【结果】1.用超基序法、量化基序法和蛋白酶体裂解法筛选了九条Survivin九肽CTL表位,分别是:20STFKNWPFL28 (SV20-28)、23KNWPFLEGC31 (SV23-31)、96LTLGEFLKL104 (SV96-104)、6LPPAWQPFL14 (SV6-14)、33CTPERMAEA41 (SV33-41)、46CPTENEPDL54 (SV46-54)、130KVRRAIEQL138 (SV130-138)、37RMAEAGFIH45 (SV37-45)、88SVKKQFEEL96 (SV88-96)。2.亲和力分析显示: SV20-28、SV96-104、SV130-138、SV23-31的荧光指数(Fluorescence Index, FI)分别为8.61、6.88、5.89、3.81;SV33-41、SV6-14、SV46-54、SV37-45、SV88-96的FI分别为0.31、-0.29、-0.4、-0.16和-0.03;稳定性分析结果为:SV20-28、SV96-104、SV130-138, DC50( dissociation complex 50)>8h;SV23-31 DC50为4-6h;其余表位肽DC50小于4小时。结果提示:SV20-28、SV96-104、SV130-138为高亲和力表位肽;SV23-31为中等亲和力表位肽;SV33-41、SV6-14、SV46-54、SV37-45、SV88-96为低亲和力表位肽。3.Survivin高亲和性表位SV20-28和中等亲和力表位SV23-31负载DC诱导的CTL能产生分泌IFN-γ细胞,在ELISPOT试验中效靶比为1:1时产生的斑点数分别为183.42±16.07、76.08±8.42,显著高于阴性对照组斑点数,P﹤0.05;而低亲和力表位SV88-96诱导的CTL未能产生明显的IFN-γ分泌细胞。高亲和力及中等亲和力表位肽诱导的CTL能以MHC-I限制方式杀伤Survivin阳性细胞,在效靶比为:50:1、25:1、12.5:1时的杀伤率分别分别为(62.40±5.16)%、(44.0±2.32)%、(26.53±1.07)%和(33.42±4.76)%、(25.16±2.64)%、(15.83±1.57)%,显著高于阴性对照组:(5.59±0.16)%、(4.76.0±0.32)%、(2.93±0.07)%;而低亲和力表位肽SV88-96诱导的免疫细胞对靶细胞没有明显的杀伤作用。【结论】超基序法、量化基序法和蛋白酶体裂解法相结合可快速有效地预测抗原表位;Survivin表位肽SV20-28、SV23-31可有效诱导CTL产生免疫反应,并能以MHC-I限制方式杀伤Survivin阳性细胞。更多还原

【Abstract】 【Objective】Epitope can be defined as linear fragment or conformation structure derived from a protein antigen that is recognized by immunocyte and raising immune response.Identification of the appropriate and effective Epitope is essential to develop of efficient protocols for tumor immuno response. We use three computer algorithms to screen and identify of HLA-A2+ -restricted survivin-derived CTL epotide and evaluate the immune response and test the ability of CTL generated in vitro with different kinds of survivin-derived peptide.【Methods】HLA-A2+ -restricted survivin-derived CTL epotides were predicted by using a combination of three computer algorithms that is supermotif, quantitative motif and proteasomal cleavages methods. The binding capacity and complex stability of candidate epitope peptides were measured by using flowcytometry.【Results】We have screened and identified of nine HLA-A2 survivin nonamers that is 20STFKNWPFL28(SV20-28)、23KNWPFLEGC31(SV23-31)、96LTLGEFLKL104 (SV96-104)、6LPPAWQPFL14(SV6-14)、33CTPERMAEA41(SV33-41)、46CPTENEPDL54(SV46-54)、130KVRRAIEQL138(SV130-138)、37RMAEAGFIH45(SV37-45)、88SVKKQFEEL96(SV88-96);The binding affinity analysis showed that candidate peptides SV20-28、SV96-104、SV130-138 and SV23-31 have the fluoresence index(FI) of 8.61、6.88、5.89、3.81 respectively, peptides SV33-41、SV6-14、SV46-54、SV37-45 and SV88-96 have the FI 0.31、-0.29、-0.4、-0.16 and -0.03 respectively. The peptide/HLA-A2 complexes stablity assay indicated that the dissociation complex 50(DC50) for peptides SV20-28、SV96-104、SV130-138 were longer than 8h,for SV23-31 was 4-6h and for the rest candidate peptides were less than 4h. Based on their ability to binding to HLA-A2 and to form stable peptide/HLA-A2 complexes, we separated nine candidate survivin peptides into three groups: High-affinity peptide: SV20-28、SV96-104、SV130-138 ; Intermediate-affinity peptide: SV23-31and Low-affinity peptide: SV33-41、SV6-14、SV46-54、SV37-45、SV88-96. Survivin-specific CTL could be generated from high-affinity and intermediate-affinity survivin peptide SV20-28 and SV23-31 loaded dendritic cell and secreted IFN-γ-producing cells. The number of the spots for CTL/SV20-28 cell and CTL/SV23-31 cell was 183.42±16.07 and 76.08±8.42 respectively which is markedly higher than that of negative control with P﹤0.05. While no obvious immuno-responses was observed for CTL/SV88-96 cell; Survivin-specific CTL induced from high-affinity and intermediate-affinity survivin peptide SV20-28 and SV23-31 can kill survivin positive cancer cells in MHC class I-restricted fashion. The specific lysis rate for CTL/SV20-28 and CTL/SV23-31 were (62.40±5.16)%,(44.0±2.32)%,(26.53±1.07)% and (33.42±4.76)%,(25.16±2.64)%,(15.83±1.57)% respectively with the effector to targe was 50 to1,25 to1 and 12.5 to1.【Conclusions】Antigen epitope could be quickly and efficiently predicted by using supermotif、quantitative motif and proteasomal cleavages methods. Survivin-derived peptides SV20-28 and SV23-31 can efficiently induce survivin-specific CTLs which can recongnize and kill survivin positive cancer cells in MHC class I-restricted fashion.更多还原