CN107022006A - 乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列 - Google Patents

乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列 Download PDF

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CN107022006A
CN107022006A CN201710191364.6A CN201710191364A CN107022006A CN 107022006 A CN107022006 A CN 107022006A CN 201710191364 A CN201710191364 A CN 201710191364A CN 107022006 A CN107022006 A CN 107022006A
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Abstract

本发明公开了一组乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列，分别是由HLA‑A*0101、A*0301、A*3001、A*3101、A*3201、A*3303、A*6801等分子限制性的抗原肽，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，从而发挥抗乙型肝炎病毒的免疫效应作用。这些抗原肽可以用来制备乙型肝炎病毒感染的治疗性和预防性疫苗，也可以用来制备检测乙型肝炎病毒特异性细胞毒型胸腺依赖性淋巴细胞的检测试剂等，在乙型肝炎的预防、治疗和诊断中有着潜在的应用价值。

Description

乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列
技术领域
本发明属于医学免疫学和感染病学领域，涉及多种乙型肝炎病毒的抗原表位肽序列。
背景技术
乙型肝炎病毒(Hepatitis B virus,HBV)感染是最常见感染性疾病之一，严重危及人类的健康。世界卫生组织2015年7月统计数据显示，预计全球1/3的人口感染过HBV，约2.48亿HBV携带者，后期可发展为肝硬化和原发性肝细胞癌。每年约78万HBV感染的患者因疾病进展为肝硬化、肝衰竭或是肝癌而导致死亡。流行病学调查显示在中国大陆地区约8亿人感染过HBV，乙肝表面抗原(hepatitis B surface antigen,HBsAg)携带率高达10.34％，即我国目前约有1.2亿人为HBV携带者，占全球慢性感染人数的一半。另外，我国也是一个肝癌大国，约占全球肝癌人数的55％以上。
HBV基因组由不完全双链环状DNA组成，长的为负链，含3020-3320个碱基，短的为正链，含1700-2800个碱基。包括四个开放阅读框(ORF)，分别称为C、X、P和S编码区。C区由pre-C和C基因组成，分别由不同的起始密码子调控，在相同的终止密码子处结束，pre-C和C基因共同编码pre-C蛋白，Pre-C蛋白再经过切割加工后形成核心抗原(HBeAg)。S区由S基因、PreS1和PreS2基因组成，由3个不同的起始密码子启动翻译，在同一个终止密码子处终止翻译，分别编码表面抗原(HBsAg)蛋白、PreS1蛋白和PreS2蛋白。P区基因编码的HBV的聚合酶蛋白(HBpol)。X区的基因编码X蛋白(HBx)，包含154个氨基酸，是最小的一个开放阅读框。
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系统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系统，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。HLAI类分子主要负责将内源性HBV抗原呈递给CD8+CTL，活化的CTLs通过分泌穿孔素和颗粒酶等使病毒感染的肝细胞凋亡，同时分泌特定的细胞因子抑制HBV的复制。因此，动态监测HBV抗原特异性CD8+T细胞的数量和功能可以准确反映乙肝感染者针对HBV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HBV不同抗原的加工、处理和提呈能力也不相同，从而引起不同程度的HBV抗原特异性免疫应答反应。根据乙肝患者不同的HLA分子型别，选择其所提呈的HBV特异性抗原肽，动态监测该HBV抗原肽特异性的CD8+T细胞的数量和功能，对HBV感染者的疾病进程监测、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，是实现乙肝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HBV特异性抗原肽，还可以制备多肽疫苗或基因疫苗，预防和治疗HBV感染。
目前，国内外报道的与HLA-A分子呈高亲和力结合的HBV特异性抗原肽中，HLA-A*0201、A*0203、A*0206、A*1101和A*2402等分子限制性的抗原肽序列被报道和鉴定的较多。而中国人群中频率相对较高的HLA-A*0101、A*0301、A*3001、A*3101、A*3201、A*3303和A*6801等分子的限制性HBV抗原肽则较少报道。本专利通过生物信息学方法，利用六种在线表位预测数据库对HLA-A等位基因型限制的HBV特异性抗原表位肽进行预测，获得一组分别与HLA-A*0101、A*0301、A*3001、A*3101、A*3201、A*3303和A*6801等HLA-A分子呈高亲和力结合的HBV特异性抗原肽序列，为制备HBV感染的治疗和预防性疫苗以及检测HBV抗原特异性T细胞的试剂等提供候选抗原表位肽。
发明内容
技术问题：本发明提供一种能制备HBV感染的治疗和预防性疫苗以及研制HBV抗原特异性T细胞检测试剂的乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列。
技术方案：本发明的乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列，为如下所示序列中的任一种：
进一步的，本发明中，该序列为乙肝病毒核心抗原HBcAg、表面抗原HBsAg、聚合酶蛋白HBpol或X蛋白HBx的抗原肽序列。
进一步的，本发明中，该序列能分别HLA-A*0101、A*0301、A*3001、A*3101、A*3201、A*3303、A*6801分子呈高亲和力结合。
进一步的，本发明中，序列通过以下步骤制备：
1.1通过GeneBank数据库或UniProt全球蛋白资源数据库检索获得乙肝病毒蛋白氨基酸序列；
1.2利用六种表位预测数据库SYFPEITHI、BIMAS、SVMHC、IEDB、NETMHC和EPIJEN分别预测步骤1.1所得乙型肝炎病毒抗原中能分别与HLA-A*0101、A*0301、A*3001、A*3101、A*3201、A*3303、A*6801分子呈高亲和力结合的多肽序列；
1.3对步骤1.2所得六种在线表位预测网站的预测结果，选择同一HLA-A分子至少有两种及以上方法都预测获得高亲和力结果的某一多肽序列作为乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列。
本发明的一种将上述的乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列在制备多肽疫苗或基因疫苗中的应用，是将与HLA-A分子呈高亲和力结合的乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽制备成多肽疫苗或基因疫苗。
本发明的一种将上述乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列在检测乙型肝炎病毒抗原特异性T细胞中的应用，是以与HLA-A分子呈高亲和力结合的乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽作为试剂，检测乙型肝炎病毒抗原表位肽特异性胸腺依赖性淋巴细胞。
本发明的上述乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列在检测乙型肝炎病毒抗原特异性T细胞中的应用，所述试剂为酶联免疫斑点法试剂、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。
本发明利用六种在线表位预测数据库预测6种HLA-A等位基因型限制性的HBV特异性抗原表位肽序列，获得一组能分别与HLA-A*0101、A*0301、A*3001、A*3101、A*3201、A*3303和A*6801等HLA-A分子呈高亲和力结合的HBV特异性抗原表位肽序列，为制备HBV感染的治疗和预防性疫苗以及研制HBV抗原特异性T细胞的检测试剂等提供了候选抗原表位肽。
1.选择乙肝病毒核心抗原(HBcAg)、表面抗原(HBsAg)、聚合酶蛋白(HBpol)和X蛋白(HBx)氨基酸序列为靶向序列；
2.选择预测结果获得研究者的公认、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6种HLA-A分子限制性的HBV特异性抗原表位肽序列；
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。
本发明为乙肝病毒核心抗原(HBcAg)、表面抗原(HBsAg)、聚合酶蛋白(HBpol)和X蛋白(HBx)中能分别与HLA-A*0101、A*0301、A*3001、A*3101、A*3201、A*3303和A*6801等蛋白分子呈高亲和力结合的抗原肽序列；还涉及到以上述抗原肽为基础的乙型肝炎多肽疫苗、基因疫苗、乙型肝炎的治疗与预防方法，以及以上述抗原肽为基础的检测乙型肝炎病毒抗原特异性T细胞的试剂和方法。
有益效果：本发明与现有技术相比，具有以下优点：
预测获得的HLA-A*0101、A*0301、A*3001、A*3101、A*3201、A*3303和A*6801等HLA-A分子限制性的HBV特异性抗原表位肽以前也没有被报道过。这些HLA-A分子以前也没有被报道有限制性的HBV抗原肽。因此这些新的抗原表位肽序列将为研制针对乙型肝炎治疗性和预防性多肽疫苗和基因疫苗、设计检测乙型肝炎病毒抗原特异性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等位基因的乙型肝炎患者进行个体化检测和精准医疗提供了关键的抗原组分。
具体实施方式
本发明的HLA-A分子限制性HBV特异性抗原肽，包括以下步骤：
1.通过GeneBank数据库和UniProt全球蛋白资源数据库检索获得HBV蛋白氨基酸序列，选择研究最多的蛋白序列，最终选定的蛋白序列为HBsAg(P03138)、HBcAg(P03146)、HBpol(P03156)和HBx(P03165)。
2.利用六种常用表位预测数据库SYFPEITHI、BIMAS、SVMHC、IEDB、NETMHC和EPIJEN(http://www.syfpeithi.de/、http://www-bimas.cit.nih.gov/molbio/hla_bind/、ttp://www.sbc.su.se/～pierre/svmhc/、http://www.immuneepitope.org/tools.do/、http://www.cbs.dtu.dk/services/NetMHC/、http://www.ddg-pharmfac.net/epijen/EpiJen/EpiJen.htm/)分别将上述乙肝病毒抗原HBsAg(P03138)、HBcAg(P03146)、HBpol(P03156)和HBx(P03165)的氨基酸序列与HLA-A*0101、A*0301、A*3001、A*3101、A*3201、A*3303和A*6801等HLA-A分子中的每一种进行亲和力和空间构象的吻合度分析，根据一定的标准预测筛选每种HLA-A分子限制性的抗原表位肽序列，分别选择长度为9和10个氨基酸的结合肽作为候选抗原表位肽。具体的筛选标准如下：SYFPEITHI预测选取评分>20或者排名在前10的肽序列；BIMAS预测选取评分>10或者排名在前10的肽序列；SVMHC预测选取评分>0.5或者排名在前10的肽序列；IEDB预测选取评IC50<500nM或者排名在前10的肽序列；NETMHC预测选取Rank Threshold<2.0Rank Threshold<2.0或者排名在前10的肽序列；EPIJEN预测选取IC50<500nM或者排名在前10的肽序列。
3.针对每一种HLA-A分子，经不同数据库预测出的表位肽再根据亲和力从高到低进行排列，选取高亲和力的肽序列作为该种HLA-A分子限制性的HBV候选抗原表位肽。同时对六种在线表位预测网站的预测结果进行整合分析，对每一种HLA-A分子，筛选至少达到两种及以上方法筛选标准的某一表位肽作为该HLA-A分子限制性的候选抗原表位肽。
表1是通过上述六个在线表位预测网站预测并最终筛选出的HLA-A限制性HBV特异性抗原表位肽序列。
四种被用来预测抗原表位的乙型肝炎病毒抗原的氨基酸序列如下：
SEQ ID NO1：
HBsAg(P03138)：
SEQ ID NO2：
HBcAg(P03146)：
表1 HLA-A限制性HBV相关抗原表位的预测结果
---：该预测网站没有针对该MHC分子的预测方法；
NS：没有获得该表位的计算数据。
SEQ ID NO3：
HBpol(P03156)：
SEQ ID NO4：
HBx(P03165)：
上述实施例仅是本发明的优选实施方式，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和等同替换，这些对本发明权利要求进行改进和等同替换后的技术方案，均落入本发明的保护范围。
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Claims (7)

1.一种乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列，其特征在于，该序列为如下所示序列中的任一种：
2.根据权利要求1所述的乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列，其特征在于，该序列为乙肝病毒核心抗原HBcAg、表面抗原HBsAg、聚合酶蛋白HBpol或X蛋白HBx的抗原肽序列。
3.根据权利要求1所述的乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列，其特征在于，该序列能分别与HLA-A*0101、A*0301、A*3001、A*3101、A*3201、A*3303、A*6801分子呈高亲和力结合。
4.根据权利要求1、2或3所述的乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列，其特征在于，所述序列通过以下步骤制备：
1.1通过GeneBank数据库或UniProt全球蛋白资源数据库检索获得乙肝病毒蛋白氨基酸序列；
1.2利用六种表位预测数据库SYFPEITHI、BIMAS、SVMHC、IEDB、NETMHC和EPIJEN分别预测步骤1.1所得乙型肝炎病毒抗原中能分别与HLA-A*0101、A*0301、A*3001、A*3101、A*3201、A*3303、A*6801分子呈高亲和力结合的多肽序列；
1.3对步骤1.2所得六种在线表位预测网站的预测结果，选择同一HLA-A分子至少有两种及以上方法都预测获得高亲和力结果的某一多肽序列作为乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列。
5.一种将权利要求1、2、3或4所述的乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列在制备多肽疫苗或基因疫苗中的应用，其特征在于，该应用是将与HLA-A分子呈高亲和力结合的乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽制备成多肽疫苗或基因疫苗。
6.一种将权利要求1、2、3或4所述的乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列在检测乙型肝炎病毒抗原特异性T细胞中的应用，其特征在于，该应用是以与HLA-A分子呈高亲和力结合的乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽作为试剂，检测乙型肝炎病毒抗原表位肽特异性胸腺依赖性淋巴细胞。
7.根据权利要求6所述的乙型肝炎病毒抗原的胸腺依赖性淋巴细胞抗原表位肽序列在检测乙型肝炎病毒抗原特异性T细胞中的应用，其特征在于，所述试剂为酶联免疫斑点法试剂、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。
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CN111393504A (zh) * 2020-03-18 2020-07-10 北京鼎成肽源生物技术有限公司 一种肝癌抗原组合及其应用、细胞毒性t淋巴细胞
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CN114573705B (zh) * 2022-03-17 2024-05-14 杭州师范大学 特异性启动抗乙型肝炎病毒t细胞免疫的双特异性抗体及其应用
CN114591404A (zh) * 2022-03-23 2022-06-07 成都朗谷生物科技股份有限公司 适用于白细胞抗原单倍型为hla-a2个体的乙型肝炎病毒抗原肽及其应用
CN116751262A (zh) * 2023-07-31 2023-09-15 重庆医科大学国际体外诊断研究院 靶向乙型肝炎病毒核心蛋白的多肽及其应用

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