肿瘤抗原MAGE-12新的HLA-A2限制性细胞毒性T细胞表位的预测

目的预测黑色素瘤抗原MAGE-12的HLA—A2限制性CTL表位。方法采用SYFPEITHI超基序远程预测系统与量化基序多项式法、延展基序联合应用，筛选MAGE-12抗原HLA—A0201限制性CTL表位。结果共预测出5个MAGE-12抗原HLA—A2限制性CTL表位。结论超基序法与量化基序，延展基序联合应用可以提高预测效率，为实验方法探索MAGE-12的表位提供有用线索。     

乙型脑炎病毒E蛋白HLA—A＊0201限制性细胞毒性T细胞表位的预测

目的:预测乙型脑炎病毒E蛋白(Japanese encephalitis virus envelope protein,JEV E 蛋白)的HLA-A2限制性CTL表位.方法:采用SYFPEITHI超基序法、量化基序多项式方案分析及延展基序方案联合应用,筛选JEV E 蛋白HLA-A2限制性的九肽细胞毒性T淋巴细胞(cytotoxic lymphocyte,CTL)表位.结果:通过预测获得了9个JEV E 蛋白HLA-A0201限制性的九肽CTL表位.结论:超基序法与量化基序多项式方案分析及延展基序方案的联合应用所产生的JEV E 蛋白CTL表位,为进一步研制新型乙脑肽疫苗奠定了重要的基础.     

肝癌相关肿瘤抗原AFP新的HLA—A2／A24限制性CTL表位的预测及分析

目的：预测肝癌肿瘤抗原AFP新的HLA—A2／A24限制性CTL表位,为肝癌CTL表位肽的筛选及鉴定提供依据。方法：选择与肝癌密切相关的肿瘤抗原AFP为研究目标,采用NetCTL1．2Server远程预测系统进行HLA—A2／A24限制性CTL表位预测。结果：共预测出与肝癌相关的肿瘤相关抗原AFP的8个HLA—A2限制性CTL表位,13个HLA—A24限制性CTL表位。结论：应用NetCTL1．2Server法预测CTL表位是一种高效准确的预测方法。所预测的肿瘤抗原AFP的HLA—A2／24限制性CTL表位经实验筛选、鉴定后,可为肝癌治疗性疫苗的制备选择合适的CTL表位提供依据。     

食管癌高表达抗原HLA-A2/A3 限制性细胞毒性T 淋巴细胞表位预测

 目的 预测食管癌普遍高表达蛋白COX-2和MAGE-4的HLA-A2/A3限制性CTL表位。方法运用生物信息学的方法,SYFPEITHI初步预测,结合三维构效定量关系和蛋白酶体酶切位点分析。结果 对SYFPEITHI预测〉20的九肽用MHCPred和NetChop3.0作进一步分析,并与已报道抗原肽进行比对,初步筛选出了42个潜在的CTL表位。结论 这42个九肽均未见文献报道,进一步鉴定将为CTL表位疫苗的研究提供更多的线索。其中,MAGE-422-30、202-210、286-294及COX-2479-4874个九肽具有与HLA-A2和HLA-A3超型潜在的交叉免疫活性,将为研制简约的高效广谱食管癌疫苗提供候选肽。     

肝癌相关肿瘤抗原AFP新的HLA-A2/A24 限制性CTL表位的预测及分析

目的:预测肝癌肿瘤抗原AFP新的HLA-A2/A24限制性CTL表位,为肝癌CTL表位肽的筛选及鉴定提供依据.方法:选择与肝癌密切相关的肿瘤抗原AFP为研究目标,采用NetCTL 1.2 Server远程预测系统进行HLA-A2/A24限制性CTL表位预测.结果:共预测出与肝癌相关的肿瘤相关抗原AFP的8个HLA-A2 限制性CTL表位,13个HLA-A24限制性CTL 表位.结论:应用NetCTL 1.2 Server法预测CTL表位是一种高效准确的预测方法.所预测的肿瘤抗原AFP的HLA-A2/24 限制性CTL表位经实验筛选、鉴定后,可为肝癌治疗性疫苗的制备选择合适的CTL表位提供依据.     

原癌基因Bmi-1 B细胞抗原表位及 HLA I类限制性CTL表位的生物信息学分析

目的:预测原癌基因Bmi-1(B-cell-specific Moloney murine leukemia virus insertion site 1)的B细胞抗原表位及HLA I类限制性细胞毒性T细胞（cytotoxic T cell）表位。方法:采用Ellipro(http://tools.immuneepitope.org/ellipro/)预测Bmi-1蛋白中可能存在的线性B细胞表位和构象B细胞表位;采用ProtParam、NetCTL等软件和方法预测Bmi-1中可能存在的HLA I类限制性CTL表位。结果:Bmi-1蛋白中含有6个线性B细胞表位和8个构象B细胞表位;结合HLA结合力、蛋白酶体切割效率和TAP转运效率,经过NetCTL程序预测表明:针对不同的HLA I类分子,原癌基因Bmi-1中都存在多个HLA I类分子的限制性CTL表位。结论:综合运用多个程序预测了原癌基因Bmi-1中的B细胞抗原表位及HLA I类分子的限制性CTL表位,为下一步开展针对Bmi-1的免疫靶向治疗等研究打下了基础。     

肿瘤相关抗原CT45的HLA-A2/A3限制性CTL表位的预测

目的:预测肿瘤相关抗原CT45的HLA-A2/A3限制性CTL表位,为CTL表位肽的合成和活性筛选提供依据。方法:基于新近发现的CT45抗原的氨基酸序列,用NetCTL 1.2 Server人工神经网络法远程预测系统进行HLA-A2/A3限制性CTL表位预测打分,挑选出分值较高的作为候选表位。结果:共预测出了5个潜在的HLA-A2限制性CTL表位九肽,15个潜在的HLA-A3限制性CTL表位九肽,所有这些表位均为首次报道。结论:NetCTL 1.2 Server人工神经网络法能高效的预测CTL表位肽,基于新近发现的CT45抗原,我们通过该系统成功预测出了5个潜在的HLA-A2限制性CTL表位,15个潜在的HLA-A3限制性CTL表位,可以对其进行进一步的研究。     

苦瓜蛋白MAP30的HLA—A2／A3限制性CTL表位预测

目的：预测苦瓜蛋白MAP30的HLA—A2／A3限制性CTL表位。方法：应用lasergene7．0和NetCTL-1．2Server对MAP30进行HLA—A2／A3限制性CTL表位预测。结果：MAP30的二级结构含有8个d-螺旋和9个B-折叠股;柔韧性良好氨基酸区域为19—40、99—146、166—273;亲水性指数较高区域为22—45、97—114、117～148、180—198、224—257、265—275;抗原性指数较高区域为19—28、63—71、98-114、118—123、224—246、248—260、262—274;有30个可能CTL表位,综合分析后获得2个HIJA—A2和5个HLA—A3限制性CTL表位。结论：MAP30具有潜在的7个HLA—A2／A3限制性CTL表位。     

癌-睾丸抗原SSX-3的HLA-A2/A3限制性CTL广谱表位的预测

目的:预测基于癌-睾丸抗原SSX-3的HLA-A2/A3限制性CTL广谱表位.方法: 采用MULTIPRED在线预测系统分别针对HLA-A2超型和HLA-A3超型对SSX-3序列进行CTL表位预测,其中对两种超型分值均较高的表位被初步认定为广谱表位;然后,运用NetCTL 1.2在线预测系统对初步认定的广谱表位进行蛋白酶体裂解和TAP转运效率预测进一步筛选.结果: 共预测出9个SSX-3抗原的HLA-A2/A3限制性广谱表位.结论: MULTIPRED与NetCTL 1.2在线预测系统相结合可以高效的预测广谱CTL表位;本报道为SSX-3的广谱CTL表位的实验研究提供了重要的理论依据.     

慢性粒细胞白血病相关抗原HLA-A2.1限制性CTL表位的生物信息学分析

目的:预测慢性粒细胞白血病BCR/ABL融合基因HLA-A2.1限制性细胞毒性T细胞表位.方法:运用BIMAS、SYFPEITHI、Predep、Immune Epitope Database和Analysis Resource(IEDB)软件预测BCR/ABL融合基因可能的HLA-A2.1限制性CTL表位.结果:结合BIMAS、SYFPEITHI、Predep和IEDB预测BCR/ABL融合基因的HLA-A2.1限制性CTL表位,共6条.结论:综合运用多个预测软件可提高预测效率获得目的表位肽,为下一步基础实验打下基础.     

Prediction and analysis of HLA-A2/A24-restricted cytotoxic T-lymphocyte epitopes of the tumor antigen MAGE-n using the artificial neural networks method on NetCTL1.2 Server

Cancer immunotherapy has become one of the most important therapeutic approaches to cancer in the past two decades. Tumor antigen-derived peptides have been widely used to elicit tumor-specific cytotoxic T lymphocytes (CTLs). Antigen-specific CTLs induced by MAGE-derived peptides have proven to be highly efficacious in the prevention and treatment of various types of tumor. MAGE-n is a new member of the MAGE gene family and has been shown to be closely associated with hepatocellular carcinoma. It is highly homologous to the MAGE-A gene subfamily, particularly to MAGE-3 (93%). MAGE-n-derived peptide QLVFGIEVV is a novel HLA-A2.1-restricted CTL epitope that induces MAGE-n-specific CTLs in vitro. Identification of these CTL epitopes may lead to clinical applications of these peptides as cancer vaccines for patients with MAGE-n(+)/HLA-A2(+) tumors. In the present study, HLA-A/A24-restricted CTL epitopes of antigen MAGE-n were predicted using the NetCTL1.2 Server on the web, COMB >0.85. The results showed that the NetCTL1.2 Server prediction method improved prediction efficacy and accuracy. Additionally, 8 HLA-A2- and 9 HLA-A24-restricted CTL epitope candidates (nonamers) derived from the tumor antigen MAGE-n were predicted. These nonamers, following identification via experimentation, may contribute to the development of potential antigen peptide tumor vaccines.     

Identification of HLA-A2-restricted CTL epitope encoded by the MAGE-n gene of human hepatocellular carcinoma

BACKGROUND: Identification of the cytotoxic T lymphocytes (CTL) restricted epitopes of tumor antigens opens up possibilities of developing a new cancer vaccine. For the MAGE-n has been demonstrated closely associated with hepatocellular carcinoma (HCC) and HLA-A2.1 is found in over 50% of HCC patients in China, we aim at identifying MAGE-n-encoded peptide presented by HLA-A2.1. MATERIALS: A HLA-A2.1-restricted CTL epitope was identified by using an improved "reverse immunology" strategy: (a) computer-based epitope prediction from the amino acid sequence of MAGE-n antigen; (b) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A2.1 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro; and (d) testing of the induced CTLs toward HCC cells expressing MAGE-n antigen and HLA-A2.1. RESULTS: Of the five tested peptides, effectors induced by a peptide of MAGE-n at residue position 159-167(QLVFGIEVV) lysed HCC cells expressing both MAGE-n and HLA-A2.1. Our results indicated that peptide QLVFGIEVV was a new HLA-A2.1-restricted CTL epitope capable of inducing MAGE-n specific CTLs in vitro. CONCLUSIONS: Identification of the MAGE-n /HLA-A2.1 peptide QLVFGIEVV may facilitate peptide-based specific immunotherapy for HCC. The combination of epitope prediction, epitope reconstruction method and immunological methods can improve the efficiency and accuracy of CTL epitope studies.     

联合表位肽诱导HLA-A2+人PBMC产生抗原特异性CTL及杀伤活性研究

目的:探讨MAGE-3,MAGE-n抗原表位体外联合诱导的CTL,并研究其特异性杀伤活性。方法:候选抗原表位以固相多肽合成技术合成,并用HPLC进行纯化,质谱法(MS)鉴定,以流式细胞仪筛选HLA-A2 人外周血PBMC,T2细胞负载抗原肽反复刺激活化诱导抗原特异性CTL,LDH检测其杀伤活性。结果:联合表位肽体外刺激人PBMC,能够较强地诱导抗原特异性CTL并产生特异性杀伤。结论:MAGE-3与MAGE-n的HLA-A2限制性CTL表位肽的联合应用能够产生较强的体外抗肿瘤免疫反应。     

Efficient induction of cytotoxic T lymphocytes specific to hepatocellular carcinoma using HLA-A2-restricted MAGE-n peptide in vitro

MAGE-n is a new member of MAGE gene family and has been demonstrated closely associated with hepatocellular carcinoma (HCC). In this study, MAGE-n-derived peptide-specific cytotoxic T lymphocytes (CTL) were induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with HLA-A2-restricted MAGE-n peptide-pulsed T2 cells. The induced CTLs exhibited specific lysis against T2 cells pulsed with the peptide and HLA-A2+ HCC cells expressing MAGE-n, while HLA-A2+ HCC cell lines that did not express MAGE-n could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-MHC class I monoclonal antibody. These results suggested the MAGE-n peptide could be a potential target of specific immunotherapy for HLA-A2 patients with HCC.     

树突状细胞负载MAGE-n表位肽的体外免疫学效应研究

目的:研究树突状细胞(dendritic cell,DC)负载MAGE-n表位肽QLVFGIEVV体外诱导特异性CTL的能力及其抗肿瘤效应。方法:MAGE-n表位肽以固相多肽合成技术合成,并用HPLC进行纯化,质谱法(MS)鉴定,以流式细胞仪筛选HLA-A2+人外周血PBMC,连续贴壁法分离培养人外周血来源树突状细胞,用成熟的DC负载MAGE-n表位肽QLVFGIEVV反复刺激活化诱导抗原特异性CTL,用51Cr释放法检测CTL的杀伤活性,并用抗HLA-A2分子单抗进行杀伤抑制实验。结果:用DC负载MAGE-n表位肽QLVF-GIEVV可诱导特异性CTL反应,对MAGE-n阳性表达细胞有较强的杀伤作用。结论:DC负载抗原肽QLVF-GIEVV在体外可诱发较强的特异性免疫反应。     

卵巢癌相关抗原TM4SF1 HLA-A2限制性CTL表位预测及鉴定

目的 预测并筛选免疫反应性较强的四跨膜蛋白超家族成员1（transmembrane 4 L6 family member 1,TM4SF1） 人类白细胞抗原A2（human leukocyte antigen A2,HLA-A2）限制性T淋巴细胞（cytotoxic lymphocyte,CTL）表位肽。方法 联合4种软件（BIMAS、SYFPEITHI、IEDB、PROPRED I）对TM4SF1的 HLA-A2限制性CTL表位进行预测,并采用预培养法酶联免疫斑点法（enzyme-linked immunospot assy,ELISOPT）、直接法ELISOPT对所测得表位肽的免疫反应性进行鉴定。结果 4种不同软件共预测出10条表位多肽,对其中4条（P1、P2、P8、P10）进行免疫反应性验证。多肽活化CTL能力结果显示,预培养法ELISPOT产生斑点形成细胞（spvt forming cell,SFC）的净值T明显高于直接法ELISPOT：［（阳性对照肽：322±8 vs 169±22,P<0.05）、（多肽P1：114±10 vs 39±7,P<0.05）、（多肽 P10：156±31 vs 52±8,P<0.05）］。单个活化CTL细胞分泌INF-γ因子的水平检测结果显示,预培养法 ELISPOT 产生斑点的平均大小明显大于直接法 ELISPOT［（阳性对照肽：21.91±2.45 vs 13.80±1.76,P<0.05）、（多肽P1：12.90±0.88  vs 8.31±1.40,P<0.05）、（多肽P10：17.50±3.85 vs 11.96±0.61,P<0.05）］。表位多肽P1、P10均具有免疫原性,P10的免疫活性更高。结论 预培养法ELISPOT检测多肽的免疫反应性较直接法ELISPOT灵敏性更高,多种软件联合ELISPOT预培养法可有效筛选出免疫反应性更强的卵巢癌相关抗原TM4SF1 HLA-A2限制性CTL优势表位,其中P10的免疫活性最高。