WO2021237932A1 - Tcr富集克隆型及其获取方法与应用 - Google Patents
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Abstract
TCR富集克隆型及其获取方法与应用。所述TCR富集克隆型的氨基酸序列分别为:CAANRGSGYSTLTF_CSARGERGEKLFF、CASSSGGSYIPTF_CASSLAGGHETQYF、CAVNSYNTDKLIF_CATSREEDNTYEQYF、CAVGGNEKLTF_CASSQGTGRSSPLHF和CAASAVGGAQKLVF_CATSRGTLYGYTF。所述TCR富集克隆型可用于疫苗开发。
Description
本申请要求于2020年05月23日提交中国专利局的中国专利申请的优先权,其中国专利申请包括:申请号为202010444542.3,发明名称为“一种TCR富集克隆型及其获取方法与应用”;申请号为202010444541.9,发明名称为“一种TCR富集克隆型及其获取方法与应用”;申请号为202010445718.7,发明名称为“一种TCR富集克隆型及其获取方法与应用”;申请号为202010444548.0,发明名称为“一种TCR富集克隆型及其获取方法与应用”;以及申请号为202010444546.1,发明名称为“一种TCR富集克隆型及其获取方法与应用”,其全部或部分内容通过引用结合在本申请中。
本发明涉及生物医药技术领域,特别涉及TCR富集克隆型及其获取方法与应用。
新型冠状病毒(2019-novel coronavirus,2019-nCoV)感染暴发流行对全球公众健康构成了严重威胁。由2019-nCoV引起的疾病被世界卫生组织(World Health Organization,WHO)正式命名为COVID-19(coronavirus disease 2019)。COVID-19在临床上表现为肺炎、发烧、咳嗽、肌肉疼痛、疲劳、腹泻、严重时导致死亡。WHO报告显示,2019-nCoV已造成世界大流行。接种疫苗是有效预防病毒感染流行的手段,但是目前尚无新型冠状病毒疫苗(以下简称新冠疫苗)。针对公共快速传播的COVID-19病毒,目前 治疗手段仍以支持护理为主,针对COVID-19的抗病毒药物、抗体、疫苗等仍在临床实验阶段,均未研发成功。大都采用与当年SARS相同的方法对症治疗,用激素压制免疫系统引发的炎症反应,但这同时也会让人体会非常脆弱,难免受到损伤。迫切需要有效的疫苗开发遏制病毒在全球爆发。
T细胞(抗原)受体(T cell receptor,TCR),TCR为所有T细胞表面的特征性标志,以非共价键与CD3结合,形成TCR—CD3复合物。TCR的作用是识别抗原。TCR是由两条不同肽链构成的异二聚体,由α、β两条肽链组成,每条肽链又可分为可变区(V区),恒定区(C区),跨膜区和胞质区等几部分;其特点是胞质区很短。TCR分子属于免疫球蛋白超家族,其抗原特异性存在于V区;V区(Vα、Vβ)又各有三个高变区CDR1、CDR2、CDR3,其中以CDR3变异最大,直接决定了TCR的抗原结合特异性。在TCR识别MHC-抗原肽复合体时,CDR1,CDR2识别和结合MHC分子抗原结合槽的侧壁,而CDR3直接与抗原肽相结合。本发明中所提供的TCR富集克隆型通过共有的CDR3可变区对特异性的识别抗原表位具有积极作用,TCR富集克隆型能够用于指导疫苗开发,并为疫苗开发研究提供方向和理论依据。
发明内容
为了实现上述目的,本发明公开了TCR富集克隆型,其氨基酸序列分别为:CAANRGSGYSTLTF_CSARGERGEKLFF、CASSSGGSYIPTF_CASSLAGGHETQYF、CAVNSYNTDKLIF_CATSREEDNTYEQYF、CAVGGNEKLTF_CASSQGTGRSSPLHF和CAASAVGGAQKLVF_CATSRGTLYGYTF。
本发明还公开了上述TCR富集克隆型的获取方法,包括以下步骤:
S1、采集治愈后病毒性感染患者的外周血单个核细胞样本;
S2、对所采集的样本进行TCR/BCR V(D)J免疫组库测序分析,找到共有的CDR3可变区;
S3、对治愈后病毒性感染患者富集程度高的细胞克隆型分布进行检测,从而可获得TCR富集克隆型。
其中,所述氨基酸序列为CAANRGSGYSTLTF_CSARGERGEKLFF和CAVGGNEKLTF_CASSQGTGRSSPLHF的TCR富集克隆型富集在效应性CD4+T记忆细胞的细胞簇中,氨基酸序列为CASSSGGSYIPTF_CASSLAGGHETQYF、CAVNSYNTDKLIF_CATSREEDNTYEQYF和CAASAVGGAQKLVF_CATSRGTLYGYTFT的CR富集克隆型富集在效应CD8+T细胞的细胞簇中。
本发明还公开了上述TCR富集克隆型在指导疫苗开发中的应用。
其中,所述疫苗为2019-nCoV疫苗。
有益技术效果:本发明TCR富集克隆型及其获取方法与应用,本发明中的TCR富集克隆型的氨基酸序列分别为:CAANRGSGYSTLTF_CSARGERGEKLFF、CASSSGGSYIPTF_CASSLAGGHETQYF、CAVNSYNTDKLIF_CATSREEDNTYEQYF、CAVGGNEKLTF_CASSQGTGRSSPLHF和CAASAVGGAQKLVF_CATSRGTLYGYTF,且上述TCR富集克隆型均能够通过共有CDR3可变区对特异性识别的抗原表位具有积极意义,因此它们均能够用于指导疫苗开发,为疫苗开发研究提供方向和理论依据。
图1是本发明中TCR富集克隆型获取方法的流程图,
图2是本发明中TCR富集克隆型在一患者中所处位置及其在TCR细胞中富集程度的示意图,
图3是本发明中TCR富集克隆型在另一患者中所处位置及其在TCR细胞中富集程度的示意图。
为了使本技术领域的人员更好地理解本发明的技术方案,下面结合附图对本发明作进一步的详细说明。
本发明提供了TCR富集克隆型,其氨基酸序列分别为:CAANRGSGYSTLTF_CSARGERGEKLFF、CASSSGGSYIPTF_CASSLAGGHETQYF、CAVNSYNTDKLIF_CATSREEDNTYEQYF、CAVGGNEKLTF_CASSQGTGRSSPLHF和CAASAVGGAQKLVF_CATSRGTLYGYTF。
如图1、图2、图3所示,在本实施例中,上述TCR富集克隆型的获取方法具体包括以下步骤:
S1、采集治愈后病毒性感染患者的外周血单个核细胞样本;
S2、对所采集的样本进行TCR/BCR V(D)J免疫组库测序分析,找到共有的CDR3可变区;
S3、对治愈后病毒性感染患者富集程度高的细胞克隆型分布进行检测,从而可获得所述TCR富集克隆型。
本实施例中,选取四个2019-nCoV病毒性感染患者的外周血单个核细胞作为采样样本,然后对采集样本进行TCR/BCR V(D)J免疫组库测序分析,尽管测序分析结果没有共有的细胞克隆型,但是存在一个共有CDR3可变区, 通过对病毒性感染患者富集程度高的细胞克隆型分布进行检测,获取到富集在效应CD8+T细胞细胞簇中且氨基酸序列为CASSSGGSYIPTF_CASSLAGGHETQYF、CAVNSYNTDKLIF_CATSREEDNTYEQYF和CAASAVGGAQKLVF_CATSRGTLYGYTF的TCR富集克隆型,以及富集在效应性CD4记忆细胞的细胞簇中氨基酸序列为CAANRGSGYSTLTF_CSARGERGEKL和CAVGGNEKLTF_CASSQGTGRSSPLHF的TCR富集克隆型,因此,所述TCR富集克隆型通过共有的CDR3可变区对特异性的识别抗原表位具有积极作用。
在本实施例中,将上述TCR富集克隆型应用于指导疫苗开发,特别是应用于指导2019-nCoV疫苗开发,由于上述TCR富集克隆型通过共有的CDR3可变区对特异性的识别抗原表位具有积极作用,因此,上述TCR富集克隆型能够用于指导疫苗开发,并为疫苗开发研究提供方向和理论依据。
在本实施例中,为了深入了解对SARS-Cov2病毒感染的特异性免疫反应,我们进行了可将受体克隆型多样性与每个细胞的基因表达谱联系起来的配对TCR/BCR V(D)J免疫组库测序,在此过程中,我们并没有在治愈后的患者中找到共享的T细胞和B细胞克隆型,但是在扫描治愈后患者T细胞和B细胞克隆型频次时发现五种高频T细胞克隆型,而且,每一位愈后患者的总检出细胞中的T细胞均超过3%,从而可以判定T细胞在病毒的清除过程中具有关键作用,在所发现的五种T细胞克隆型中,其中有两种富集在效应性CD4+T记忆细胞的细胞簇中,另外三种富集在效应性CD8+T细胞的细胞簇中,进一步证实了这两个T细胞亚群在抵抗SARS-Cov2再次感染的特异性免疫反应,因此,本发明所公开的TCR富集克隆型能够用于指导疫苗开发,并为疫苗开发研究提供方向和理论依据。
以上对本发明所提供的TCR富集克隆型及其获取方法与应用进行了详细介绍。本文中应用了具体个例对本发明的原理及实施方式进行了阐述,以上实施例的说明只是用于帮助理解本发明的核心思想。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,还可以对本发明进行若干改进和修饰,这些改进和修饰也落入本发明权利要求的保护范围内。
Claims (5)
- TCR富集克隆型,其特征在于,所述TCR富集克隆型的氨基酸序列分别为:CAANRGSGYSTLTF_CSARGERGEKLFF、CASSSGGSYIPTF_CASSLAGGHETQYF、CAVNSYNTDKLIF_CATSREEDNTYEQYF、CAVGGNEKLTF_CASSQGTGRSSPLHF和CAASAVGGAQKLVF_CATSRGTLYGYTF。
- 如权利要求1所述的TCR富集克隆型的获取方法,其特征在于,所述获取方法包括以下步骤:S1、采集治愈后病毒性感染患者的外周血单个核细胞样本;S2、对所采集的样本进行TCR/BCRV(D)J免疫组库测序分析,找到共有的CDR3可变区;S3、对治愈后病毒性感染患者富集程度高的细胞克隆型分布进行检测,进而可获得所述TCR富集克隆型。
- 如权利要求2所述的TCR富集克隆型的获取方法,其特征在于,所述氨基酸序列为CAANRGSGYSTLTF_CSARGERGEKLFF和CAVGGNEKLTF_CASSQGTGRSSPLHF的TCR富集克隆型富集在效应性CD4+T记忆细胞的细胞簇中,氨基酸序列为CASSSGGSYIPTF_CASSLAGGHETQYF、CAVNSYNTDKLIF_CATSREEDNTYEQYF和CAASAVGGAQKLVF_CATSRGTLYGYTF的TCR富集克隆型富集在效应CD8+T细胞的细胞簇中。
- 如权利要求1所述的TCR富集克隆型或采用权利要求2或3所述的TCR富集克隆型的获取方法获取的TCR富集克隆型在指导疫苗开发中的应用。
- 如权利要求4所述的TCR富集克隆型在指导疫苗开发中的应用,其特征在于,所述疫苗为2019-nCoV疫苗。
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