CN115960205A - 原发性肝癌相关抗原的胸腺依赖性淋巴细胞抗原表位肽及其应用 - Google Patents
原发性肝癌相关抗原的胸腺依赖性淋巴细胞抗原表位肽及其应用 Download PDFInfo
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Abstract
本发明属于医学免疫学和肿瘤学领域,公开了一种原发性肝癌相关抗原的胸腺依赖性淋巴细胞抗原表位肽及其应用,抗原表位肽分别是由HLA‑A0201、HLA‑A1101、HLA‑A2402、HLA‑A3101、HLA‑A0206、HLA‑A0207、HLA‑A3303、HLA‑A3001、HLA‑A0203、HLA‑A1102、HLA‑A0301、HLA‑A0101、HLA‑A2601分子限制性的抗原肽,共110种,可以特异性地与细胞毒性胸腺依赖性淋巴细胞结合,刺激后者活化、增殖和分化,从而发挥抗肿瘤的免疫效应作用;这些抗原肽可以用来制备肝癌的治疗性和预防性疫苗,也可以用来制备检测肝癌相关抗原特异性细胞毒型胸腺依赖性淋巴细胞的检测试剂,在肝癌的预防、治疗和诊断中有着潜在的应用价值。
Description
本案是针对发明专利名称为“原发性肝癌相关抗原的胸腺依赖性淋巴细胞抗原表位肽及其应用”、申请号为“201911286067.5”、申请日为“2019-12-13”的分案。
技术领域
本发明属于医学免疫学和肿瘤学领域,特别涉及三种原发性肝癌相关抗原的胸腺依赖性淋巴细胞抗原表位肽及其应用。
背景技术
原发性肝癌是我国第4位常见恶性肿瘤和第3位肿瘤致死病因,严重威胁我国人民的生命和健康。原发性肝癌的病理类型,主要是肝细胞癌(hepatocellular carcinoma,HCC),占到85%~90%;还有少数为肝内胆管癌(intrahepatic cholangiocarcinoma,ICC)和HCC-ICC混合型等,三者在发病机制、生物学行为、分子特征、临床表现、病理组织学形态、治疗方法以及预后等方面差异较大。
在我国,HCC的高危人群主要有乙型肝炎病毒(hepatitis B virus,HBV)和/或丙型肝炎病毒(hepatitis C virus,HCV)感染、长期酗酒(酒精性肝病)、非酒精脂肪性肝炎、食用黄曲霉毒素污染的食物、多种原因引起的肝硬化以及有肝癌家族史的人群,同时,年龄40岁以上的男性风险较大。近年的研究提示糖尿病、肥胖和吸烟等也是HCC的危险因素,值得关注。
AFP、GPC3、GP73是常见的几种在肝癌中存在过表达的肿瘤相关抗原,在肝癌的诊断、治疗、病程监测等发面被广泛研究。血清甲胎蛋白(alpha-fetoprotein,AFP)阳性,是指AFP≥400μg,且排除慢性或活动性肝炎、肝硬化、睾丸或卵巢胚胎源性肿瘤以及妊娠等,高度怀疑肝癌。AFP低度升高者,应进行动态观察,并与肝功能变化对比分析。约30%的肝癌患者AFP水平正常,应检测甲胎蛋白异质体。GPC3是硫酸类肝素糖蛋白之一,高表达于原发性肝癌组织中,而其他正常组织中基本不表达或呈低表达。GPC3能够促进肿瘤细胞增殖、分化,通过结合细胞外基质、蛋白酶以及生长因子等,对肝癌肿瘤的形成、生长和转移发挥了促进作用。GP73是一种高尔基体II型跨膜蛋白,生理情况下在肝细胞中表达稀少。检测原发性肝癌患者血清GP73水平发现其呈显著高表达,且GP73的表达与患者肝功能和病情呈负相关,是肝细胞破坏严重程度的有效指标之一。本专利选用此三种肝癌相关肿瘤抗原进行研究。
细胞毒性T细胞(Cytotoxic T lymphocyte,CTL)是介导适应性免疫应答的核心细胞,在抗感染、抗肿瘤以及超敏反应和自身免疫病的发生中起着至关重要的作用,其细胞膜上的T细胞受体(T cell receptor,TCR)能够特异性识别并结合抗原递呈细胞表面MHC I类分子与抗原肽的复合物,即MHC/抗原肽复合体分子。CTL表位是指与MHC I类分子结合的抗原肽,是抗原分子中能被TCR特异性识别的线性片段或空间构象性结构,是引起免疫应答反应的基本抗原单位,在CTL活化过程中扮演着关键角色。
MHC***指主要组织相容性复合体(major histocompatibility complex,MHC),是脊椎动物基因组中一组紧密连锁的基因群,编码表达MHC I类和II类蛋白分子。HLA(human leukocyte antigen)是人类的MHC***,是人体最复杂的基因群,在人群中具有高度多态性。HLA在抗原识别、抗原呈递等机体免疫过程中发挥重要作用,是影响人体免疫反应的主要因素。对于人类肝癌而言,HLAI类分子主要负责将内源性肝癌相关抗原呈递给CD8+CTL,活化的CTLs通过分泌穿孔素和颗粒酶等使表达肿瘤相关抗原的肿瘤细胞凋亡。因此,动态监测肝癌相关抗原特异性CD8+T细胞的数量和功能可以准确反映肝癌患者针对肝癌相关抗原的特异性免疫功能状态。由于不同人的HLA分子型别不同,其对肝癌不同抗原的加工、处理和提呈能力也不相同,从而引起不同强度的肝癌相关抗原特异性T细胞免疫应答反应。根据肝癌患者不同的HLA分子型别,选择其所提呈的肝癌相关抗原的抗原肽,动态监测该肝癌相关抗原特异性的CD8+T细胞的数量和反应活性,对肝癌患者的疾病进程监测、诊断与治疗方案的制定、疗效观察和预后转归的判断等都有重要意义,是实现肝癌精准医学治疗的重要技术手段。同时,利用这些HLA-A分子高亲和力结合的肝癌相关抗原的抗原肽,还可以制备多肽疫苗或基因疫苗,预防和治疗肝癌。
但是,目前已明确的被各种HLA分子所提呈并能刺激机体引起T细胞应答反应的原发性肝癌相关抗原T细胞表位仍然极少,从而限制了对携带不同HLA等位基因的肝癌患者开展特异性T细胞检测,也限制了肝癌特异性T细胞在肝癌发生发展中的作用研究,更限制了基于HLA基因个体差异性及其提呈的肝癌相关抗原肽差异性的个性化检测和精准免疫治疗。
发明内容
本发明解决现有技术中存在的上述技术问题,提供肝癌相关抗原的胸腺依赖性淋巴细胞抗原表位肽及其应用。
为解决上述问题,本发明的技术方案如下:肝癌相关抗原的胸腺依赖性淋巴细胞抗原表位肽,其氨基酸序列为如下所示表位肽序列中的任一种:
由上述的表格可看出,上述序列分别为甲胎蛋白(AFP)、磷脂酰肌醇蛋白聚糖-3(GPC3)、高尔基体跨膜糖蛋白73(GP73)的抗原肽序列,上述序列分别与HLA-A0201、HLA-A1101、HLA-A2402、HLA-A3101、HLA-A0206、HLA-A0207、HLA-A3303、HLA-A3001、HLA-A0203、HLA-A1102、HLA-A0301、HLA-A0101、HLA-A2601分子呈高亲和力结合,在抗原提呈细胞表面组成“HLA/抗原肽”复合体分子,与抗原肽特异性CD8+T细胞克隆结合,刺激其活化、增殖和分化,发挥抗肝癌免疫效应作用。
原发性肝癌相关抗原的胸腺依赖性淋巴细胞抗原表位肽序列可用于制备肝癌多肽疫苗或基因疫苗:多肽疫苗制备:按照本发明的多肽序列人工合成一种或多种多肽,与佐剂混合制成可溶性制剂,或者由生物纳米材料装载制成纳米多肽疫苗,注入肝癌患者体内,激发患者肝癌相关抗原特异性T细胞活化、增殖,并增强其杀瘤细胞活性,从而制备成肝癌多肽疫苗。基因疫苗制备:按照本分明的多肽序列,构建一种多肽或多种多肽的重组DNA基因片段、重组质粒或者重组病毒载体,注入肝癌患者体内,使该重组基因在体内表达一种或多种多肽,激发患者肝癌相关抗原特异性T细胞活化、增殖,并增强其杀瘤细胞活性,从而制备成肝癌基因疫苗。
原发性肝癌相关抗原的胸腺依赖性淋巴细胞抗原表位肽序列可用于制备检测肝癌相关抗原特异性T细胞的检测制剂或试剂盒:按照本发明的多肽序列,人工合成一种或多种多肽,在酶联免疫斑点法、胞内细胞因子荧光染色法和酶联免疫吸附试验中,作为抗原制剂,与患者外周血单个核细胞(PBMC)混合培养,刺激肝癌相关抗原特异性T细胞活化、增殖和分泌细胞因子,进而通过其他组合试剂检测该细胞因子的合成量,从而反应特异性T细胞的数量和反应活性;也可以利用这些多肽通过基因工程技术和蛋白工程技术制备人类白细胞抗原与多肽的复合体(peptide-HLA complex)及其多聚体,再进一步制备成荧光素标记的制剂,用流式细胞分析法检测患者外周血单个核细胞群中肝癌相关抗原特异性T细胞的数量。相关试剂盒则是指由上述制剂与不同检测方法中常用的其他试剂组装而成的肝癌相关抗原特异性T细胞检测试剂盒。
原发性肝癌相关抗原的胸腺依赖性淋巴细胞抗原表位肽序列可用于制备治疗肝癌的药物:将上述基于本发明多肽序列的多肽疫苗或基因疫苗与其他免疫治疗制剂或化学治疗制剂组合,制备成治疗肝癌的临床药物。
本发明利用六种在线表位预测数据库虚拟预测13种HLA-A分子限制性的肝癌相关抗原特异性抗原表位肽序列,获得一组能分别与HLA-A0201、HLA-A1101、HLA-A2402、HLA-A3101、HLA-A0206、HLA-A0207、HLA-A3303、HLA-A3001、HLA-A0203、HLA-A1102、HLA-A0301、HLA-A0101、HLA-A2601分子呈高亲和力结合的肝癌相关抗原表位肽序列,然后通过ELISPOT功能性实验验证了其免疫原性,为制备肝癌治疗和预防性疫苗以及研制肝癌相关抗原特异性T细胞检测试剂等提供了特异性抗原肽。
1.选择甲胎蛋白(AFP)、磷脂酰肌醇蛋白聚糖-3(GPC3)、高尔基体跨膜糖蛋白73(GP73)氨基酸序列为靶向序列;
2.选择预测结果获得研究者公认的、具有较高准确性的、常用的六种表位预测数据库:SYFPEITHI、BIMAS、SVMHC、IEDB、NETMHC和EPIJEN预测上述13种HLA-A分子限制性的肝癌相关抗原表位肽序列;
3.根据一定的预测标准,对六个在线表位预测网站的预测结果进行整合分析,获得六个网站预测结果较一致的候选抗原肽序列。
4.通过IFN-γELISPOT细胞功能性实验验证肝癌相关抗原肽的免疫原性。
本发明为甲胎蛋白(AFP)、磷脂酰肌醇蛋白聚糖-3(GPC3)、高尔基体跨膜糖蛋白73(GP73)中能分别与HLA-A0201、HLA-A1101、HLA-A2402、HLA-A3101、HLA-A0206、HLA-A0207、HLA-A3303、HLA-A3001、HLA-A0203、HLA-A1102、HLA-A0301、HLA-A0101、HLA-A2601分子呈高亲和力结合并且具有免疫原性的抗原肽序列;还涉及到以上述抗原肽为基础的肝癌多肽疫苗、基因疫苗,以及以上述抗原肽为基础的检测肝癌相关抗原特异性T细胞的试剂和方法。
相对于现有技术,本发明的优点如下,
经在线虚拟预测和功能性实验验证获得的HLA-A0201、HLA-A1101、HLA-A2402、HLA-A3101、HLA-A0206、HLA-A0207、HLA-A3303、HLA-A3001、HLA-A0203、HLA-A1102、HLA-A0301、HLA-A0101、HLA-A2601分子限制性的原发性肝癌相关抗原特异性抗原表位肽以前没有被报道过。这些HLA-A分子以前也没有被报道有限制性的肝癌相关抗原肽。因此这些新的抗原表位肽序列将为研制针对肝癌治疗性和预防性多肽疫苗和基因疫苗、设计检测肝癌相关抗原特异性T细胞的试剂和方法等提供所需的关键抗原组分,即抗原表位肽序列;同时这些抗原表位肽也为针对这些特定HLA-A等位基因的肝癌患者进行个体化检测和精准医疗提供了关键的抗原组分。
附图说明:
图1为HLA-A0201分子限制的肝癌相关抗原T细胞表位肽;
A)IFN-γELISPOT方法鉴定HLA-A0201分子限制性肝癌相关抗原T细胞表位肽的检测孔、阴性对照孔和阳性对照孔斑点图;
B)检测孔与阴性对照孔斑点数统计图;
C)检测孔与阴性对照孔斑点数比值(P/N)统计图。
图2为HLA-A1101分子限制的肝癌相关抗原T细胞表位肽;
A)IFN-γELISPOT方法鉴定HLA-A1101分子限制性肝癌相关抗原T细胞表位肽的检测孔、阴性对照孔和阳性对照孔斑点图;
B)检测孔与阴性对照孔斑点数统计图;
C)检测孔与阴性对照孔斑点数比值(P/N)统计图。
图3为HLA-A2402分子限制的肝癌相关抗原T细胞表位肽;
A)IFN-γELISPOT方法鉴定HLA-A2402分子限制性肝癌相关抗原T细胞表位肽的检测孔、阴性对照孔和阳性对照孔斑点图;
B)检测孔与阴性对照孔斑点数统计图;
C)检测孔与阴性对照孔斑点数比值(P/N)统计图。
图4为HLA-A3101分子限制的肝癌相关抗原T细胞表位肽;
A)IFN-γELISPOT方法鉴定HLA-A3101分子限制性肝癌相关抗原T细胞表位肽的检测孔、阴性对照孔和阳性对照孔斑点图;
B)检测孔与阴性对照孔斑点数统计图;
C)检测孔与阴性对照孔斑点数比值(P/N)统计图。
图5为HLA-A0206分子限制的肝癌相关抗原T细胞表位肽;
A)IFN-γELISPOT方法鉴定HLA-A0206分子限制性肝癌相关抗原T细胞表位肽的检测孔、阴性对照孔和阳性对照孔斑点图;
B)检测孔与阴性对照孔斑点数统计图;
C)检测孔与阴性对照孔斑点数比值(P/N)统计图。
图6为HLA-A0207分子限制的肝癌相关抗原T细胞表位肽;
A)IFN-γELISPOT方法鉴定HLA-A0207分子限制性肝癌相关抗原T细胞表位肽的检测孔、阴性对照孔和阳性对照孔斑点图;
B)检测孔与阴性对照孔斑点数统计图;
C)检测孔与阴性对照孔斑点数比值(P/N)统计图。
图7为HLA-A3303分子限制的肝癌相关抗原T细胞表位肽;
A)IFN-γELISPOT方法鉴定HLA-A3303分子限制性肝癌相关抗原T细胞表位肽的检测孔、阴性对照孔和阳性对照孔斑点图;
B)检测孔与阴性对照孔斑点数统计图;
C)检测孔与阴性对照孔斑点数比值(P/N)统计图。
图8为HLA-A3001分子限制的肝癌相关抗原T细胞表位肽;
A)IFN-γELISPOT方法鉴定HLA-A3001分子限制性肝癌相关抗原T细胞表位肽的检测孔、阴性对照孔和阳性对照孔斑点图;
B)检测孔与阴性对照孔斑点数统计图;
C)检测孔与阴性对照孔斑点数比值(P/N)统计图。
图9为HLA-A0203分子限制的肝癌相关抗原T细胞表位肽;
A)IFN-γELISPOT方法鉴定HLA-A0203分子限制性肝癌相关抗原T细胞表位肽的检测孔、阴性对照孔和阳性对照孔斑点图;
B)检测孔与阴性对照孔斑点数统计图;
C)检测孔与阴性对照孔斑点数比值(P/N)统计图。
图10为HLA-A1102分子限制的肝癌相关抗原T细胞表位肽;
A)IFN-γELISPOT方法鉴定HLA-A1102分子限制性肝癌相关抗原T细胞表位肽的检测孔、阴性对照孔和阳性对照孔斑点图;
B)检测孔与阴性对照孔斑点数统计图;
C)检测孔与阴性对照孔斑点数比值(P/N)统计图。
图11为HLA-A0301分子限制的肝癌相关抗原T细胞表位肽;
A)IFN-γELISPOT方法鉴定HLA-A0301分子限制性肝癌相关抗原T细胞表位肽的检测孔、阴性对照孔和阳性对照孔斑点图;
B)检测孔与阴性对照孔斑点数统计图;
C)检测孔与阴性对照孔斑点数比值(P/N)统计图。
图12为HLA-A0101分子限制的肝癌相关抗原T细胞表位肽;
A)IFN-γELISPOT方法鉴定HLA-A0101分子限制性肝癌相关抗原T细胞表位肽的检测孔、阴性对照孔和阳性对照孔斑点图;
B)检测孔与阴性对照孔斑点数统计图;
C)检测孔与阴性对照孔斑点数比值(P/N)统计图。
图13为HLA-A2601分子限制的肝癌相关抗原T细胞表位肽;
A)IFN-γELISPOT方法鉴定HLA-A2601分子限制性肝癌相关抗原T细胞表位肽的检测孔、阴性对照孔和阳性对照孔斑点图;
B)检测孔与阴性对照孔斑点数统计图;
C)检测孔与阴性对照孔斑点数比值(P/N)统计图。
图14为实施例1中待鉴定表位肽免疫原性验证的实验技术路线图。
具体实施方式
实施例1:本发明HLA-A0201、HLA-A1101、HLA-A2402、HLA-A3101、HLA-A0206、HLA-A0207、HLA-A3303、HLA-A3001、HLA-A0203、HLA-A1102、HLA-A0301、HLA-A0101、HLA-A2601分子限制性的肝癌相关抗原T细胞表位,序列通过以下步骤筛选和鉴定:
1在线虚拟预测13种HLA-A分子限制的三种肝癌相关抗原的优势T细胞表位肽
选取三种肝癌相关抗原蛋白:甲胎蛋白(AFP)、磷脂酰肌醇蛋白聚糖-3(GPC3)、高尔基体跨膜糖蛋白73(GP73),通过UniProt全球蛋白资源数据库检索获得其氨基酸序列,选择研究最多的标准序列;再通过SYFPEITHI、BIMAS、SVMHC、IEDB、NETMHC、EPIJEN等六种常用的表位肽预测数据库对HLA-A0201、HLA-A1101、HLA-A2402、HLA-A3101、HLA-A0206、HLA-A0207、HLA-A3303、HLA-A3001、HLA-A0203、HLA-A1102、HLA-A0301、HLA-A0101、HLA-A2601分子限制的针对上述每种肝癌相关抗原蛋白的T细胞表位肽进行虚拟预测,其中SVMHC数据库包括MHCPEP model和SYFEPITHI model两种预测算法;IEDB数据库包括了ANN、SMM和ARB三种方法,本实验选择其中具有更好统计学意义的ANN和SMM方法。这些表位肽预测数据库网站见表1。
HLA I类分子的抗原结合凹槽两端封闭,接纳的抗原肽长度为8-11个氨基酸残基,其中以9和10个氨基酸最为常见,因此,本实验主要选择9和10个氨基酸长度的多肽作为研究对象。分别将每种肝癌相关抗原蛋白的氨基酸序列输入到预测数据库网站的相应氨基酸序列输入框,再分别选择表位肽的长度为9和10个氨基酸,然后选择特定的HLA-A分子,对肝癌相关抗原的T细胞表位肽进行在线虚拟预测。
表1表位肽预测数据库网站
针对每种HLA-A分子和每种肝癌相关抗原蛋白,将不同数据库预测出的多肽分别按评分从高到低进行排列,再从中选择至少满足两种以上预测方法评分标准的表位肽作为候选表位肽。每种HLA-A分子,针对每种肝癌相关抗原蛋白,再从候选表位肽中选出评分最高(亲和力最高)的1-5种多肽作为待鉴定表位肽。针对上述13种HLA-A分子,总计筛选预测出待鉴定的优势T细胞表位肽160种。
2分离肝癌患者外周血PBMC
取室温保存的新鲜抗凝全血,用无菌PBS适当稀释;将1倍原血体积的人淋巴细胞分离液(达科为生物,深圳)加入15mL离心管;将稀释后的血液缓缓平铺于分离液上,室温离心20min,2500rpm;吸取单个核细胞(PBMCs)层,离心洗涤2次;用无血清培养基(达科为生物,深圳)重悬,细胞计数,调整细胞浓度为2×106/mL备用。
3用IFN-γELISPOT法鉴定肝癌相关抗原T细胞表位肽的免疫原性
技术路线如图14所示:
首先筛选出对混合肽组有阳性反应的肝癌患者:将每种HLA-A分子限制的针对三种肝癌相关蛋白的待鉴定表位肽混合为一组,共13组,每组8-9种表位肽;取预包被抗人IFN-γ抗体的ELISPOT板(达科为生物,深圳),用无血清培养基(200μL/well)活化8min,每孔加入肝癌患者PBMC悬液(100μL/well);然后在检测孔中补加每种HLA-A分子限制的混合肽,混合肽中单种多肽为15μg/mL,在阳性对照孔补加PHA(2.5μg/mL),在阴性对照孔补加与检测孔相同浓度的DMSO多肽溶解液;置37℃,5%CO2培养箱孵育24h;按照人IFN-γELISPOT试剂盒说明书裂解并洗去细胞;每孔加入生物素标记的抗人IFN-γ抗体工作液(100μL/well),于37℃孵育1h;洗板后加入酶联亲和素工作液(100μL/well),继续于37℃孵育1h;洗板后加入现配的AEC显色液(100μL/well),室温避光显色20min;用去离子水洗板4-5次以终止显色;将ELISPOT板避光放置,晾干后送深圳达科为生物技术公司自动化扫描计数斑点。若阴性孔斑点数为0-5时,检测孔斑点数≧6判定为CTL反应阳性;若阴性对照孔斑点数≧6时,检测孔≧2倍阴性孔斑点数判定为CTL反应阳性。
鉴定单种表位肽的免疫原性:重新收集对混合肽有CTL阳性反应的肝癌患者外周血PBMC,加入上述ELISPOT板中,每个检测孔中补加阳性混合肽中的单种肝癌相关抗原多肽(15μg/mL),同上设置阳性对照孔和阴性对照孔,置37℃,5%CO2培养箱孵育24h,同上做ELISPOT检测。CTL反应阳性孔即表示该孔内的待鉴定表位肽具有免疫原性。
HLA-A限制性的确定:对每位肝癌患者进行HLA-A等位基因分型,结合该表位肽的虚拟亲和力,初步确定其被哪种HLA-A分子所限制和提呈。从肝癌患者中分别选取这13种HLA-A等位基因的纯合子感染者,取其PBMC,与上述所有已被验证具有免疫原性的且该HLA-A分子限制的表位肽,重新进行ELISPOT检测,进一步确定每种表位肽的HLA-A分子限制性。
4HLA-A等位基因分型
选择在表位肽鉴定实验中呈CTL阳性反应的肝癌患者,取200μL抗凝血,利用人全血基因组DNA提取试剂盒(天根生物,北京)提取基因组DNA;采用HLA-A位点特异性引物A1和A3进行PCR,扩增A位点的外显子2、内含子2、外显子3以及部分内含子l和3的DNA序列,产物大小为985bp。扩增条件为:95℃预变性3min;95℃变性15s;62℃退火15s;72℃延伸90s;35个循环;72℃延伸5min。扩增产物经1%琼脂糖凝胶电泳鉴定大小,并送上海桑尼生物科技公司进行纯化和双向测序。PCR试剂购自南京诺唯赞生物科技公司。
表2 HLA-A位点特异性的PCR扩增引物
通过Lasergene程序的Seqman软件把外显子2和外显子3的测序结果拼成一条完整的HLA-A重叠序列(Contig),仔细检查双向测序的碱基是否完整一致,找出杂合子的碱基并用兼并碱基代替,例如M表示A和C,R表示A和G,W表示A和T,S表示C和G,Y表示C和T,K表示G和T,最终确定扩增出来的HLA-A等位基因的序列片段。利用Nucleotide BLAST工具,将拼接好的HLA-A碱基序列与数据库中所有HLA-A等位基因的外显子2和外显子3序列进行比对,直到获得完全匹配的基因组合,从而确定HLA-A的等位基因。
通过IFN-γELISPOT方法从500例肝癌患者中筛选出150例对肝癌混合肽有CTL阳性反应的患者。再重新收集这些患者的PBMC,用ELISPOT法进行单种表位肽免疫原性的验证,然后结合患者的HLA-A等位基因以及表位肽与这些等位基因的虚拟预测亲和力进行综合分析。
结果显示:有9种肝癌相关抗原肽(P1-P9)能刺激HLA-A*02:01阳性患者的PBMC呈现CTL阳性反应(图1);
有15种肝癌相关抗原肽(P10-P24)能刺激HLA-A*11:01阳性患者的PBMC呈现CTL阳性反应(图2);
有10种肝癌相关抗原肽(P25-P34)能刺激HLA-A*24:02阳性患者的PBMC呈现CTL阳性反应(图3);
有9种肝癌相关抗原肽(P35-P43)能刺激HLA-A*31:01阳性患者的PBMC呈现CTL阳性反应(图4);
有9种肝癌相关抗原肽(P4、P44-P51)能刺激HLA-A*02:06阳性患者的PBMC呈现CTL阳性反应(图5);
有9种肝癌相关抗原肽(P1、P4、P7-P8、P52-56)能刺激HLA-A*02:07阳性患者的PBMC呈现CTL阳性反应(图6);
有9种肝癌相关抗原肽(P41、P57-64)能刺激HLA-A*33:03阳性患者的PBMC呈现CTL阳性反应(图7);
有10种肝癌相关抗原肽(P10、P65-P73)能刺激HLA-A*30:01阳性患者的PBMC呈现CTL阳性反应(图8);
有8种肝癌相关抗原肽(P2、P74-P80)能刺激HLA-A*02:03阳性患者的PBMC呈现CTL阳性反应(图9);
有10种肝癌相关抗原肽(P10-P12、P15、P41、P81-P85)能刺激HLA-A*11:02阳性患者的PBMC呈现CTL阳性反应(图10);
有12种肝癌相关抗原肽(P10、P16、P68、P86-94)能刺激HLA-A*03:01阳性患者的PBMC呈现CTL阳性反应(图11);
有9种肝癌相关抗原肽(P95-P103)能刺激HLA-A*01:01阳性患者的PBMC呈现CTL阳性反应(图12);
有8种肝癌相关抗原肽(P96、P104-P110)能刺激HLA-A*26:01阳性患者的PBMC呈现CTL阳性反应(图13)。
需要说明的是,上述仅仅是本发明的较佳实施例,并非用来限定本发明的保护范围,在上述实施例的基础上所做出的任意组合或等同变换均属于本发明的保护范围。
Claims (6)
1.原发性肝癌相关抗原的胸腺依赖性淋巴细胞抗原表位肽,其特征在于,所述抗原表位肽的氨基酸序列为HLA-A分子限制性的表位肽序列中的任意一种:
FYSALPGYI、NYGRLQQDVL、LYQDEKAVL。
2.一种利用要求1所述的原发性肝癌相关抗原的胸腺依赖性淋巴细胞抗原表位肽进行单个氨基酸的去除或者更换后所得抗原表位肽。
3.如权利要求1所述的原发性肝癌相关抗原的胸腺依赖性淋巴细胞抗原表位肽和权利要求2所述的抗原表位肽在制备肝癌多肽疫苗或基因疫苗中的应用。
4.如权利要求1所述的原发性肝癌相关抗原的胸腺依赖性淋巴细胞抗原表位肽和权利要求2所述的抗原表位肽在制备检测肝癌相关抗原特异性T细胞的检测制剂或试剂盒中的应用。
5.如权利要求4所述的应用,其特征在于,所述检测试剂为酶联免疫斑点法试剂、胞内细胞因子荧光染色法试剂、酶联免疫吸附试验试剂、人类白细胞抗原多聚体荧光染色或流式细胞分析法试剂。
6.如权利要求1所述的肝癌相关抗原的胸腺依赖性淋巴细胞抗原表位肽和权利要求2所述的抗原表位肽在制备治疗肝癌的药物中的应用。
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