CN114685628B - 一种SARS-CoV-2的RBD的抗原表位肽及其应用 - Google Patents
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Abstract
本发明提供了一种SARS‑CoV‑2的RBD的抗原表位肽,包含氨基酸序列为SEQ ID NO:11和/或SEQ ID NO:17的抗原优势表位。本发明还提供了其在制备用于诊断、预防或治疗SARS‑CoV‑2感染的药物中应用。本发明提供的抗原势表位肽具有抑制SARS‑CoV‑2的RBD和人血管紧张素转换酶2(ACE2)结合的效应,可以用于制备预防SARS‑CoV‑2感染的高效、低毒、高安全性的基于RBD的药物。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及病毒抗原技术领域,尤其涉及一种SARS-CoV-2的RBD的抗原表位肽及其应用。
背景技术
新型冠状病毒(Severe Acute Respiratory Syndrome Coronavirus 2,SARS-CoV-2,简称新冠)感染危害严重,且疫情仍呈全球性持续性发展,给我国的疫情防控带来严峻挑战。
由于目前尚无特效的新冠治疗药物,疫苗是控制新冠感染的理想选择。尽管 当前已有数种新冠疫苗上市,但新冠病毒变异新毒株如Delta株和Omicron株 等不断出现,使现有疫苗接种的有效性和持久性面临挑战。新冠感染主要初始部 位是呼吸道粘膜,而目前已有的新冠疫苗大多数是通过肌肉注射(简称肌注)免 疫,其诱导的呼吸道粘膜免疫应答有限,且肌注免疫可能无法及时阻止宿主粘膜 部位的病毒传播。选择合适的佐剂及免疫途径同时诱导呼吸道粘膜及全身性免 疫应答是新冠疫苗急需解决的关键问题。解析SARS-CoV-2重要抗原在不同佐剂 及免疫途径下诱发保护性应答的有效成分及其免疫应答机制,是解决该科学问 题的基础和前提。
刺突蛋白(Spike protein,简称S蛋白或Spike蛋白)是SARS-CoV-2的 重要结构蛋白,是现有新冠疫苗中除灭活疫苗外的其他疫苗的抗原成分。Spike 蛋白含有S1和S2两个亚基,其中S1亚基的C端含有受体结合结构域(Receptor binding domain,简称RBD结构域),Spike蛋白通过其RBD结构域识别宿主细 胞表面的受体血管紧张素转化酶E2(ACE2),介导病毒进入宿主细胞,RBD结构 域的氨基酸变异会导致病毒的种属特异性和感染特性的变化。RBD含有整个 SARS-CoV-2病毒中最具免疫显性的中和表位,COVID-19恢复者血清中90%的中 和性抗体都由RBD诱导产生。靶向RBD的中和性抗体具有预防和治疗SARS-CoV- 2感染的潜在价值。RBD可规避S1亚基的NTD结构域抗体引起ADE效应。此外, RBD性质稳定、易于重组表达和纯化制备,因此是新冠疫苗的理想抗原。
研究证实,具有中和能力的抗体在新冠病毒的防护中发挥保护作用。SARS- CoV-2的中和抗体的滴度与临床结局相关。由于蛋白抗原发挥其功能主要通过其 中的表位来体现特异性,其中发挥主要作用的是“免疫优势”表位。因此,RBD 抗原中免疫优势应答的保护性表位的鉴定,是提升和优化基于RBD抗原的SARS- CoV-2疫苗设计的重要前提。筛选RBD的免疫优势表位,是激发更有效的RBD免 疫应答的前提。目前的已知的RBD的B细胞表位,或通过生物信息学软件推测, 或通过单克隆抗体鉴定,或使用人或动物免疫模型中鉴定,尚未有关于在不同免 疫途径中全面筛选参与免疫应答的RBD的“优势表位”的报道。由于新冠病毒的 初始感染部位主要是呼吸道黏膜,因此,亟需建立一种准确有效的筛选及鉴定在 “滴鼻途径”下参与免疫应答的RBD的B细胞“优势表位”的方法。
发明内容
本发明针对现有技术存在的上述问题,提供了一种SARS-CoV-2的RBD的抗 体优势表位肽的鉴定方法,以及该表位肽在制备用于诊断、预防和/或治疗SARS- CoV-2感染的药物中的应用。
本发明首先提供了一种SARS-CoV-2的RBD的抗原表位肽,包含氨基酸序列 为SEQID NO:11和/或SEQ ID NO:17的抗原优势表位。
在根据本发明的一个实施方案中,所述抗原表位肽N端或C端偶联有多肽 标记;优选地,所述多肽标记为生物素标记或荧光标记。
本发明进一步提供了上述的抗原表位肽在制备SARS-CoV-2感染的特异抗体 诊断试剂中的应用。
本发明还提供了上述的抗原表位肽在制备用于预防或治疗SARS-CoV-2感染 的疫苗中的应用。
本发明进一步提供了上述的抗原表位肽作为抗SARS-CoV-2感染药物筛选靶 标的应用。
在根据本发明的一个实施方案中,还提供了一种用于预防或治疗SARS-CoV- 2感染的药物组合物,其包含上述的抗原表位肽和药学上可接受的辅料。
在根据本发明的一个实施方案中,还包含佐剂,所述佐剂优选为AddaVax。
在根据本发明的一个实施方案中,所述药物组合物为经鼻施用的剂型;优选 为喷雾剂、滴鼻剂、粉末剂、凝胶制剂或微球制剂。
本发明进一步提供了一种用于SARS-CoV-2感染的诊断试剂,其包含上述的 抗原表位肽,所述抗原表位肽包被于检测载体上,所述检测载体选自聚苯乙烯微 量反应板、胶体金试剂条、磁珠和微流控芯片中的任一种。
在根据本发明的一个实施方案中,还包含特异性识别鼠IgG抗体的第二抗 体;
优选地,所述第二抗体选自羊抗鼠单克隆抗体、兔抗鼠多克隆抗体中的一种;
优选地,所述第二抗体偶联有激活或猝灭所述特异性荧光基团的配合基团。
本发明的上述技术方案的有益效果如下:
本发明方法所得到的RBD抗体免疫优势表位肽具有抑制RBD和人血管紧张 素转换酶2(ACE2)结合的效应,可以用于制备预防SARS-CoV-2感染的高效、 低毒、高安全性的基于RBD的药物,如预防型疫苗。
本发明提供的SARS-CoV-2的RBD的抗体优势表位肽有抑制RBD和人血管紧 张素转换酶2(ACE2)结合的效应,用该优势表位免疫动物,免疫制剂无无关成 分或有害成分,以该抗体优势表位肽制备的特异性单克隆抗体可以较好地的预 防SARS-CoV-2感染。
经序列比对分析,本发明提供的SARS-CoV-2的RBD的抗体优势表位肽在多 种SARS-CoV-2毒株中序列保守,因此,其也可用作SARS-CoV-2的诊断试剂或 用于对其他SARS-CoV-2感染的预防和治疗。
附图说明
图1为使用RBD滴鼻免疫BALB/c小鼠作为一抗对本发明筛选到的重叠肽进 行ELISA检测的结果图谱;
图2为使用RBD滴鼻免疫BALB/c小鼠的血清特异性IgG抗体效价图谱;
图3为使用RBD滴鼻免疫BALB/c小鼠的血清特异性IgG抗体亚型图谱;
图4为本发明筛选到的RBD免疫BALB/c小鼠的抗血清抑制RBD和人血管紧 张素转换酶2(ACE2)结合的效应能力分析中和抗体滴度结果图谱;
图5为本发明筛选到的RBD免疫BALB/c小鼠的抗血清抑制RBD和人血管紧 张素转换酶2(ACE2)结合的效应能力分析血清稀释倒数图结果图谱;
图6为本发明筛选到的抗体免疫优势表位肽RBD61-78、RBD97-114在RBD三维结 构中的位置分布分析结果图谱;
图7为本发明筛选到的抗体免疫优势表位肽RBD61-78、RBD97-114的氨基酸序列 保守性分析结果图谱。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合附图 及具体实施例进行详细描述。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例 中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1重叠肽的获得
以RBD蛋白序列(Sequence ID:6XDG_E)为基础,相对于预期的重叠肽的长 度,每次向下游移动小于重叠肽的长度的氨基酸个数,再次获得另一条预期的重 叠肽。其中,预期的重叠肽的长度可以为15-30个氨基酸,每次步移的氨基酸个 数可以为4-8个,本实施例中从第1号氨基酸开始,每次步移6个氨基酸,合 成步移重叠的18个氨基酸多肽(上海吉尔生化科技有限公司),共得到35条 重叠肽。纯度均大于95%。合成的步移重叠肽信息如表1所示。将合成肽段用二 甲亚砜(DMSO)溶解至1mg/mL的储存浓度,分装后-70℃冻存,临用时用PBS稀 释成1mM。
表1步移重叠肽信息
实施例2 RBD免疫血清的收集和保存
突变SEB三个毒素位点的重组SEB全蛋白由基于参考文献所公开的方法构 建获得(参考文献:JintaoZou et al.a-Hemolysin-Aided Oligomerization of the SpikeProtein RBD Resulted in Improved Immunogenicity and Neutralization AgainstSARS-CoV-2 Variants ariants.Front Immunol.2021 Sep 24;12:757691.),免疫小鼠的RBD蛋白剂量调整至20ug/只,佐剂AddaVax 剂量为50μL,免疫途径为滴鼻免疫,经0、14、21天免疫三次。测抗血清效价, 选择RBD效价大于1:64000的小鼠免疫血清用于后续检测。
实施例3 RBD的B细胞免疫优势表位的筛选
调整重叠肽包被浓度5μg/孔(以全蛋白RBD(SSequence ID:6XDG_E)为阳 性对照),对孔板进行包被-洗涤-封闭-再次洗涤后,加入实施例2获得的RBD 免疫抗血清,稀释度为1:50,孵育1.5h-洗涤后,加入HRP-羊抗小鼠IgG(购自 恩晶生物,货号E1WP319),稀释度为1:5000,洗涤后加入TMB底物显色液(购 自Beyotime/碧云天,货号P0209-100ml),终止反应后在450nm处读OD值,按 照公式18氨基酸重叠肽OD检测值-空白对照检测值)/(阴性肽OD检测值-空白 对照检测值)≥2.1为阳性重叠肽。经SPSS16.0数据检验,获得相对于其他阳 性重叠肽读数具有显著统计学意义的阳性重叠肽,定义为B细胞的免疫优势表 位肽,也即免疫优势表位肽。无关肽OVA192–201(SEQ ID NO:36EDTQAMPFRV)为阴 性对照肽。
结果:如图1所示,有2个阳性多肽RBD61-78(SEQ ID NO:11 CYGVSPTKLNDLCFTNVY)、RBD97-114(SEQ ID NO:17TGKIADYNYKLPDDFTGC)相对于 其他优势肽读数具有显著统计学意义,定义为优势表位,该图中OVA192-201为阴性 无关肽;通过该方法不仅筛选获得了RBD的所有B细胞表位,而且明确了RBD的 B细胞优势表位。
实施例4 RBD免疫小鼠的抗血清抑制RBD和人血管紧张素转换酶2(ACE2) 结合的效应能力分析;
RBD的蛋白在AddaVax佐剂(购自InvivoGen)辅助下于第0,14,21天经 三次肌注免疫6-8周BALB/c小鼠,末次免疫后第7天,尾静脉收集抗血清。
为评价RBD的免疫原性,将实例2中所述RBD蛋白辅以AddaVax佐剂后滴 鼻途经免疫小鼠,三免后小鼠尾静脉血样采集,进行RBD特异性抗体水平检测。 免疫小鼠血清的RBD特异性IgG抗体效价结果如图2所示,免疫组血清RBD特 异性IgG抗体几何平均效价增加至约106。其中,RBD特异性IgG抗体效价log10 对数值为5.95±0.2375(P<0.05)。上述结果表明:RBD辅以AddaVax佐剂滴鼻 免疫,可以诱导高水平的RBD特异性抗体。
免疫小鼠血清的RBD特异性IgG抗体亚型分析结果如图3所示,RBD特异性 IgG抗体主要以IgG1亚型为主。
首先通过竞争ELISA方法检测了中和抗体滴度,以评价RBD诱导的功能性 抗体水平。竞争抑制试验使用试剂盒操作(Anti-SARS-CoV-2 Neutralizing Antibody TiterSerologic Assay Kit,购自Acrobiosystems,Beijing, China)。免疫小鼠的血清样本以1:10稀释,并与0.3μg/ml HRP-SARS-CoV-2 刺突蛋白的RBD混合,进行重复分析。酶标仪在450nm处检测光密度值(OD值), 根据公式(1-样品平均OD值/阴性对照平均OD值)×100%计算中和抑制率。结 果如图4所示,20μg RBD 3次免疫后诱导的抗体NT50(Titers of 50%Neutralization,50%中和滴度)达到1:3412(P<0.05),血清稀释倒数图如图 5所示。上述结果说明:RBD辅以AddaVax佐剂滴鼻免疫,所诱导的高水平的RBD 特异性抗体具有竞争抑制RBD结合人血管紧张素转换酶2的中和能力。
实施例5免疫优势表位肽在RBD全蛋白三维结构中的位置
本实施例用于说明抗体免疫优势表位肽在RBD全蛋白三维结构中的位置分 布分析结果。
在PubMed蛋白公开数据库下载已报道RBD蛋白的3D结构图,用PyMOL 1.1 program软件标注实验中筛选出的免疫优势表位肽的序列位置。
结果如图6所示,该优势肽RBD61-78、RBD97-114分别位于RBD三维晶体结构的 不同loop区,可见,该优势表位肽序列(抗体优势表位)是RBD表位疫苗的可 靠候选分子。
实施例6免疫优势表位肽在SARS-CoV-2各毒株中的氨基酸序列保守性分 析
本实施例用于本发明筛选到的抗体免疫优势表位肽RBD61-78、RBD97-114的氨基 酸序列保守性分析结果。
在Genbank数据库检索30株SARS-CoV-2各毒株的RBD蛋白的氨基酸序列, 用NCBI的Basic Local Alignment Search Tool(BLAST)软件进行氨基酸序列 比对分析,随机选择30株SARS-CoV-2毒株进行多序列比对(Multiple Alignment),网站地址为https://blast.ncbi.nlm.nih.gov/Blast.cgi。
结果如图7所示,该优势肽RBD61-78、RBD97-114在30种SARS-CoV-2各毒株中 的氨基酸序列保守,因此具有良好的应用前景。
以上所述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,在不脱离本发明所述原理的前提下,还可以做出若干改进和润饰,这 些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国人民解放军陆军军医大学
<120> 一种SARS-CoV-2的RBD的抗原表位肽及其应用
<141> 2022-03-24
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Claims (14)
1. 一种SARS-CoV-2的RBD的抗原表位肽,其特征在于,所述抗原表位肽的氨基酸序列为SEQ ID NO:11或SEQ ID NO:17。
2.如权利要求1所述的抗原表位肽,其特征在于,所述抗原表位肽N端或C端偶联有多肽标记。
3.如权利要求2所述的抗原表位肽,其特征在于,所述多肽标记为生物素标记或荧光标记。
4.如权利要求1-3任一所述的抗原表位肽在制备SARS-CoV-2感染的特异抗体诊断试剂中的应用。
5.如权利要求1-3任一所述的抗原表位肽在制备用于预防或治疗SARS-CoV-2感染的疫苗中的应用。
6.一种用于预防或治疗SARS-CoV-2感染的药物组合物,其特征在于,包含如权利要求1-3任一所述的抗原表位肽和药学上可接受的辅料。
7.如权利要求6所述的药物组合物,其特征在于,还包含佐剂。
8.如权利要求7所述的药物组合物,其特征在于,所述佐剂为AddaVax。
9.如权利要求6-8任一所述的药物组合物,其特征在于,所述药物组合物为经鼻施用的剂型。
10.如权利要求9所述的药物组合物,其特征在于,剂型为喷雾剂、滴鼻剂、粉末剂、凝胶制剂或微球制剂。
11.一种用于SARS-CoV-2感染的诊断试剂,其特征在于,包含如权利要求1-3任一所述的抗原表位肽,所述抗原表位肽包被于检测载体上,所述检测载体选自聚苯乙烯微量反应板、胶体金试剂条、磁珠和微流控芯片中的任一种。
12.如权利要求11所述诊断试剂,其特征在于,还包含特异性识别鼠IgG抗体的第二抗体。
13.如权利要求12所述诊断试剂,其特征在于,所述第二抗体选自羊抗鼠单克隆抗体、兔抗鼠多克隆抗体中的一种。
14.如权利要求12所述诊断试剂,其特征在于,所述第二抗体偶联有激活或猝灭特异性荧光基团的配合基团。
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