CN116375822A - 新冠病毒特异性cd8+t细胞表位肽及其应用 - Google Patents
新冠病毒特异性cd8+t细胞表位肽及其应用 Download PDFInfo
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Abstract
本发明属于免疫学技术领域,具体公开了新冠病毒特异性CD8+T细胞表位肽,氨基酸序列为SEQ ID NO:18。本发明还公开了上述新冠病毒特异性CD8+T细胞表位肽的应用。本发明所提供的新冠病毒特异性CD8+T细胞表位肽能够产生强烈的细胞免疫应答,分泌高水平IFN‑γ,对于新型冠状病毒感染的预防、临床治疗和疫苗的研发均具有重要的科学意义和应用前景。
Description
本案属于专利申请2020109298280的分案申请。
技术领域
本发明属于免疫学技术领域,尤其涉及一种新冠病毒特异性CD8+T细胞表位肽及其应用。
背景技术
科学家在电子显微镜下,观察到引起肺炎的病原体呈现包膜,并具有类似日冕外形的典型冠状病毒形态。同时,病原基因组测序结果显示,这种冠状病毒的核酸序列与此前发现的6种冠状病毒(如SARS、MERS等)并非完全一致。世界卫生组织(WHO)将该新病毒命名为:2019新型冠状病毒(2019Novel Coronavirus,2019-nCoV)。国际病毒分类委员会(ICTV)将新型冠状病毒命名为SARS-CoV-2。
目前研究发现:冠状病毒属于套式病毒目(Nidovirales)、冠状病毒科(Coronaviridae)、冠状病毒属(Coronavirus),是目前人类已知的RNA病毒中基因组最大的病毒,其长度为27至32kb。SARS-CoV-2具有四种主要的结构蛋白,分别为刺突蛋白(S蛋白)、核衣壳蛋白(N蛋白)、膜蛋白(M蛋白)和包膜蛋白(E蛋白),其中S蛋白和N蛋白是新冠免疫检测试剂盒关键原材料,对新冠病毒的诊断和排查具有重要价值,S蛋白具有S1亚基和S2亚基,受体结合位点(RBD)位于S1亚基上,其主要功能是识别宿主细胞表面受体,介导与宿主细胞的融合。N蛋白在冠状病毒中含量丰富,是一种高度免疫原性蛋白,参与基因组复制和细胞信号通路调节。
HLA(human leucocyte antigen,人类淋巴细胞抗原)是人类主要组织相容性复合物(MHS)基因的编码产物,具有高度多态性,是人体遗传学标志,是人类发现的第一个与疾病有明确关联的遗传系统,目前已发现有70多种疾病与该系统有关。HLA主要分为HLAⅠ类抗原、HLAⅡ类抗原和HLAⅢ类抗原,HLAⅠ类抗原存在于人体所有有核细胞表面,由一个细胞膜糖蛋白A链和一个β2微球蛋白组成。HLAⅠ类抗原包括HLA-A分子、HLA-B分子和HLA-C分子,HLA-A分子分别包括多个等位基因,例如HLA-A分子包括HLA-A*2等位基因、HLA-A*11等位基因和HLA-A*24等位基因等等,HLA-A*2等位基因的分布具有明显的种族和地域特征,为疾病的研究、诊断和治疗提供了重要的参考。
根据对新型冠状病毒同源性较高的SARS等冠状病毒的研究显示:T细胞免疫应答在病毒感染后机体抗病毒防御以及机体免疫病理损伤过程中发挥了重要的作用,尤其是CD8+T细胞,其抗原特异性免疫活性11年后依然存在,说明了CD8+T细胞免疫应答在抗冠状病毒免疫防御中的重要作用及其在疫苗研发中的重要地位。
另外,新冠病毒进入体内感染宿主细胞,并在细胞内复制。中和抗体只负责细胞外的病毒清除或阻止病毒感染宿主细胞,因不能进入细胞内而对寄生在细胞内病毒无能为力。与中和抗体相比,T细胞免疫应答的第一步就是新冠病毒特异性T细胞通过其表面的细胞受体(TCR)特异性识别被病毒感染的细胞所递呈的表位肽,帮助患者或感染者彻底清除体内病毒,达到治愈新冠病毒感染的目的,所以筛选获得T细胞表位肽将成为研发疫苗材料的最好来源,为后续新冠疫苗研发提供夯实的研究基础,但是目前新冠病毒特异性CD8+T细胞表位肽至今尚未有报道。
发明内容
本发明的目的在于提供一种能够为后续新冠疫苗研发提供夯实的研究基础的新冠病毒特异性CD8+T细胞表位肽及其应用。
为了达到上述目的,本发明的技术方案如下:
本发明还提供了新冠病毒特异性CD8+T细胞表位肽,氨基酸序列如SEQ ID NO:1、SEQ ID NO:9、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19或SEQ ID NO:21所示。
本发明还提供了上述新冠CD8+T细胞表位肽在制备COVID-19疫苗中的应用。
本技术方案的原理和有益效果在于:
本发明的发明人早在2020年初就开始针对能覆盖中国人群70%以上的等位基因HLA-A*2、HLA-A*24和HLA-A*11,以及根据S蛋白的受体结合区(receptorbinding domain,RBD)的蛋白序列合成了覆盖S-RBD全长的重叠15肽(11个氨基酸残基重叠),开展COVID-19康复者恢复期外周血淋巴细胞(PBMC)中新冠病毒特异性CD8+T细胞表位肽的筛选,经过反复尝试,调整实验条件,获得新冠病毒特异性CD8+T细胞表位肽,可诱导CD8+T细胞产生强烈的细胞免疫应答,分泌高水平IFN-γ,对于新型冠状病毒感染的预防、临床治疗和疫苗的研发均具有重要的科学意义和应用前景。
具体实施方式
下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
本实施例公开了一种新冠病毒特异性CD8+T细胞表位肽,氨基酸序列如SEQ IDNO:1所示。本实施例还公开了上述新冠病毒特异性CD8+T细胞表位肽在制备COVID-19疫苗中的应用。
实施例2
本实施例与实施例1的区别仅在于:氨基酸的序列不同,本实施例提供的新冠病毒特异性CD8+T细胞表位肽,氨基酸序列如SEQ ID NO:9所示。
实施例3
本实施例与实施例1的区别仅在于:氨基酸的序列不同,本实施例提供的新冠病毒特异性CD8+T细胞表位肽,氨基酸序列如SEQ ID NO:16所示。
实施例4
本实施例与实施例1的区别仅在于:氨基酸的序列不同,本实施例提供的新冠病毒特异性CD8+T细胞表位肽,氨基酸序列如SEQ ID NO:17所示。
实施例5
本实施例与实施例1的区别仅在于:氨基酸的序列不同,本实施例提供的新冠病毒特异性CD8+T细胞表位肽,氨基酸序列如SEQ ID NO:18所示。
实施例6
本实施例与实施例1的区别仅在于:氨基酸的序列不同,本实施例提供的新冠病毒特异性CD8+T细胞表位肽,氨基酸序列如SEQ ID NO:19所示。
实施例7
本实施例与实施例1的区别仅在于:氨基酸的序列不同,本实施例提供的新冠病毒特异性CD8+T细胞表位肽,氨基酸序列如SEQ ID NO:20所示。
实施例1-7所提供的新冠病毒特异性CD8+T细胞表位肽通过以下方法获得:
首先,通过预测来源于SRAS-CoV-2(GenBank:MN908947)病毒的S蛋白和N蛋白的HLA-A*02:01、HLA-A*24:02和HLA-A*11:01限制性的CD8+9肽,以及根据S蛋白的RBD受体结合区的蛋白序列合成了覆盖S-RBD全长的重叠S-RBD 15肽(11个氨基酸重叠)。利用固相酶联免疫斑点法(enzyme linked immunospot assay,ELISPOT)、流式细胞术(flowcytometry,FCM)和酶联免疫吸附测定法(enzyme linked immunosorbent assay,ELISA)检测COVID-19康复者外周血单个核细胞(peripheral bloodmononuclear cell,PBMC),系统鉴定出SARS-CoV-2病毒的CD8+9肽表位,以及S-RBD 15肽的T细胞表位。
具体的,本实施例预测并筛选到的CD8+9肽是应用ELISPOT检测PBMC分泌IFN-γ的水平而获得鉴定。本实施例S-RBD 15肽是从N端开始,依次合成部分重叠肽,每条肽长度为15个氨基酸残基,相邻两条肽重叠11个氨基酸残基。本实施例S-RBD 15肽的T细胞表位肽:应用ELISA技术检测效应细胞分泌IFN-γ的水平而获得鉴定。
以下是具体的实验过程:
S1、从COVID-19康复者外周血中分离PBMC
PBMC应用Ficoll密度梯度离心法分离获得,具体的,抽取COVID-19康复者的外周血(EDTA抗凝剂),用RPMI 1640培养基1:1稀释,将稀释后的血样缓慢加入预先加有淋巴细胞分离液的离心管中,使分层明显(全血:RPMI1640培养基:淋巴细胞分离液的比例为1:1:1),2000rpm/min,20℃,离心20min。
结束后,用吸管轻轻吸取“云雾状”细胞层(即PBMC),加入不完全RPMI1640培养液依次(2200rpm/min,4℃,8min;1800rpm/min,4℃,5min;1800rpm/min,4℃,5min)逐渐降低离心转速来洗涤以达到减少血小板的目的,最后收集细胞重悬后,显微镜下计数。获得PBMC直接用于后续使用或液氮冻存备用。
S2、CD8+9肽的预测及合成和S-RBD 15肽的设计合成
CD8+9肽的预测及合成:利用NetMHCpan4.0软件预测了来源于SRAS-CoV-2病毒的S蛋白和N蛋白与三类HLA分子(HLA-A*02:01、HLA-A*24:02和HLA-A*11:01)结合的长度为9个氨基酸残基的肽,合成后进行下游筛选试验。如表1所示,共获得26条HLA-A*02:01限制性9肽(其中S:19条,N:7条),22条HLA-A*24:02限制性9肽(其中S:19条,N:3条),30条HLA-A*11:01限制性9肽(其中S:24条,N:6条)。
肽的分组:26条HLA-A*02:01肽被分成5组(mix-01~mix-05),每组肽混合有3~6条肽;22条HLA-A*24:02肽被分成4组(mix-06~mix-09),每组肽混合有4~6条肽;30条HLA-A*11:01肽被分成5组(mix-10~mix-14),每组肽混合有6条肽。
表1-预测的SRAS-CoV-2病毒CD8+9肽
S-RBD 15肽的设计合成:本实施例根据S-RBD(第319-541氨基酸)设计合成了15个氨基酸残基的多肽(重叠11个氨基酸),共53条,如表2所示。所有肽的合成委托南京金斯瑞公司完成。所有合成肽均经RP-HPLC测定,纯度在90%以上。肽的溶解参考说明书进行,分装后-70℃冻存备用。
表2-合成的SRAS-CoV-2病毒S-RBD 15肽
S3、体外刺激扩增肽特异性T细胞
(1)CD8+9肽刺激扩增PBMC:各选取8个HLA-A*2+、HLA-A*24+、HLA-A*11:01康复者的PBMC来筛选预测的HLA-A*02:01、HLA-A*24:02和HLA-A*11:01限制性9肽。
首先,复苏冻存的COVID-19康复者PBMC,用RPMI 1640完全培养基(10%灭活的胎牛血清,2mM L-谷氨酰胺,25mM HEPES和10μg/ml庆大霉素,100IU/1ml IL-2)培养。将PBMC以每孔2×106/2ml的浓度接种至24孔板中,加入SARS-CoV-2肽库刺激(每种肽终浓度为5μM),培养10天以扩增细胞。每3天或根据需要对半换液。培养至第10天,收集细胞并通过IFN-γ释放ELISPOT测定法检测肽特异性CD8+T细胞的存在,以筛选出单个阳性CD8+9肽。将剩余的细胞冷冻保存并保存在液氮中。
(2)S-RBD 15肽刺激扩增PBMC:将S-RBD合成的53条15肽混合,分别刺激两个COVID-19康复者的PBMC,6h后采用流式细胞术分选出IFN-γ+T细胞,随后进行大量扩增。
S4、ELISPOT筛选能够刺激COVID-19康复者PBMC产生特异性应答(分泌IFN-γ)的CD8+9肽
为了降低筛选工作量,本实施例采用“打包”的方式筛选可刺激PBMC分泌IFN-γ的单个CD8+9肽,具体的,采用商品化IFN-γELISPOT试剂盒(购自Mabtech公司)对Mix-01至Mix-14筛选可刺激PBMC分泌IFN-γ的阳性混合肽组,再在阳性混合肽组内进一步筛选可刺激PBMC分泌IFN-γ的单个CD8+9肽,按照说明书进行如下操作:
经混合肽组刺激培养的PBMC细胞在RPMI 1640培养基中静息培养12~16h。采用2μg/ml鼠抗人IFN-γmAb 50μl(1-D1K)包被96孔PVDF滤膜板,4℃过夜。PBS洗涤6遍,用RPMI1640培养基(10%胎牛血清)于37℃封闭1h后,每孔加入2×104个PBMC,依次加入混合肽组(每种肽的终浓度为5μM)。同时设阳性和阴性对照孔,阳性对照孔加入PHA(终浓度为10μg/ml),阴性对照孔加入等体积的DMSO。
37℃、5%CO2孵育14~18h后洗板,再加入1μg/ml生物素化鼠抗人IFN-γmAb 50μl(7-B6-1-Biotin),室温孵育2h,洗板后加入碱性磷酸酶(ALP)链霉亲和素50μl,室温孵育1h,洗板后加入BCIP/NBT显色液50μl,室温避光显色1h,自来水冲洗,晾干后,用ELISPOT读数仪测定斑点数,肽刺激孔的平均斑点数减去两倍阴性对照孔的斑点数,结果大于零可判为阳性。IFN-γ斑点形成细胞数以分泌IFN-γ的细胞数/2×104PBMC表示。
进一步,将筛选得到的阳性混合肽组中的每条肽依次利用商品化IFN-γELISPOT试剂盒进行鉴定,得到可刺激COVID-19康复者PBMC分泌IFN-γ的单个CD8+9肽。具体实施方式与混合肽组筛选相同,实验结果如表3所示。最终从24例COVID-19康复者中筛选到15条具有免疫原性的CD8+9肽,如表5所示。
表3ELISPOT筛选CD8+9肽实验结果
S5、ELISA筛选能够刺激COVID-19康复者PBMC产生特异性应答(分泌IFN-γ)的S-RBD 15肽
将单个S-RBD 15肽(终浓度10μM)分别与扩增后的IFN-γ+T细胞进行共孵育,筛选出可刺激COVID-19康复者的PBMC分泌IFN-γ的阳性15肽。
分别在两个COVID-19康复者的PBMC进行筛选。将PBMC以每孔2×105/200μl的浓度接种至96孔板中,分别加入10μM的S-RBD 15肽进行刺激,培养24h后收集细胞培养上清,采用ELISA检测上清中分泌IFN-γ的量,肽刺激孔值减去两倍阴性对照孔的值,结果大于零可判为阳性。实验结果如表4所示。最终从两个COVID-19康复者中筛选到5条具有免疫原性的S-RBD 15肽,如表5所示。
表4ELISA筛选S-RBD 15肽实验结果
表5筛选到的SRAS-CoV-2病毒CD8+9肽及S-RBD 15肽
序号 | pep_ID | 氨基酸序号 | 氨基酸序列 | HLA限制性 | 抗原来源 |
1 | P64 | SEQ ID NO:1 | KTFPPTEPK | A1101 | N |
2 | P63 | SEQ ID NO:2 | KLDDKDPNF | A0201 | N |
3 | P04 | SEQ ID NO:3 | RLDKVEAEV | A0201 | S |
4 | P16 | SEQ ID NO:4 | FTISVTTEI | A0201 | S |
5 | P61 | SEQ ID NO:5 | LLLDRLNQL | A0201 | N |
6 | P62 | SEQ ID NO:6 | GMSRIGMEV | A0201 | N |
7 | P77 | SEQ ID NO:7 | LALLLLDRL | A0201 | N |
8 | P74 | SEQ ID NO:8 | ALNTLVKQL | A0201 | S |
9 | P45 | SEQ ID NO:9 | NYNYLYRLF | A2402 | S |
10 | P49 | SEQ ID NO:10 | EYVSQPFLM | A2402 | S |
11 | P52 | SEQ ID NO:11 | VYDPLQPEL | A2402 | S |
12 | P53 | SEQ ID NO:12 | IYQTSNFRV | A2402 | S |
13 | P54 | SEQ ID NO:13 | VFKNIDGYF | A2402 | S |
14 | P55 | SEQ ID NO:14 | HWFVTQRNF | A2402 | S |
15 | P57 | SEQ ID NO:15 | GYLQPRTFL | A2402 | S |
16 | S-7 | SEQ ID NO:16 | NATRFASVYAWNRKR | 未知 | S |
17 | S-8 | SEQ ID NO:17 | FASVYAWNRKRISNC | 未知 | S |
18 | S-10 | SEQ ID NO:18 | RKRISNCVADYSVLY | 未知 | S |
19 | S-11 | SEQ ID NO:19 | SNCVADYSVLYNSAS | 未知 | S |
20 | S-44 | SEQ ID NO:20 | PLQSYGFQPTNGVGY | 未知 | S |
以上详细描述了本发明的较佳具体实施例。应当理解,本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此,凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案,皆应在由权利要求书所确定的保护范围内。
Claims (2)
1.新冠病毒特异性CD8+T细胞表位肽,其特征在于,氨基酸序列为SEQ ID NO:18。
2.根据权利要求1所述的新冠病毒特异性CD8+T细胞表位肽的应用,其特征在于,在制备COVID-19疫苗中的应用。
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