CN114057852A - 一种预防新型冠状病毒肺炎covid-19的多肽及其用途 - Google Patents
一种预防新型冠状病毒肺炎covid-19的多肽及其用途 Download PDFInfo
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Abstract
本发明涉及免疫学技术、生物技术及生物医药领域,具体涉及一种预防新型冠状病毒肺炎COVID‑19的多肽及其用途。所述抗原性多肽的氨基酸序列选自以下任意一种:1)氨基酸序列是SEQ ID NO.1‑SEQ NO.11所示的多肽;2)与SEQ ID NO.1‑SEQ ID NO.11所示氨基酸序列具有85%以上相同氨基酸序列的多肽;3)将SEQ ID NO.1‑SEQ ID NO.11所示氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到具有相同功能的多肽。本发明提供的预防新型冠状病毒肺炎COVID‑19的多肽是基于新型冠状病毒S蛋白序列中筛选出来特异性抗原性的片段通过化学合成得到的,通过免疫原实验证明其具有较强的抗原性,可在人体内诱导出中和抗体,有望成为多肽疫苗抗原,为预防新型冠状病毒肺炎COVID‑19提供新的疫苗。
Description
技术领域
本发明涉及免疫学技术、生物技术及生物医药领域,具体涉及一种预防新型冠状病毒肺炎COVID-19的多肽及其用途。
背景技术
冠状病毒是一种带有RNA的正向包膜病毒,其基因组大小约为26~32kb,是已知基因组最大的RNA病毒。基因组RNA和磷酸化核衣壳(N)蛋白被埋在磷脂双层中并被刺突糖蛋白三聚体(S)覆盖,膜(M)蛋白(III型跨膜糖蛋白)和包膜(E)蛋白位于病毒包膜的S蛋白之间。冠状病毒具有多种宿主,包括禽类和哺乳动物,特别是蝙蝠。冠状病毒是在自然界中广泛存在的一类病毒,可引起包括呼吸道、消化道及神经系统在内的多系统疾病,高致病性冠状病毒感染已成为近10年来广受关注的公共卫生问题。2002年11月,首例严重急性呼吸系统综合症(SARS)发生在中国佛山。2012年,中东呼吸综合征冠状病毒(MERS-CoV)是21世纪发现的第二种高致病性冠状病毒。MERS-CoV的死亡率很高,多达40%的患者死亡。2019新型冠状病毒(SARS-CoV-2)于2019年12月首次发现,其感染导致的新型冠状病毒肺炎(COVID-19)正在影响全球上百万的患者。
COVID-19患者会出现不同程度的症状,从发烧或轻度咳嗽到肺炎,更严重者甚至死亡。目前COVID-19的致死率约为2%至4%,尽管死亡率低于SARS和MERS,但新型冠状病毒(SARS-CoV-2)具有潜伏期长、传染性强和重症率较高的特点,与引发非典型肺炎的SARS病毒不同,部分病例潜伏期具有传染性,另有一些病毒携带者没有表现出任何明显症状,这增加了疫情的防控难度。因此,快速研制出能够提升群体免疫水平并阻断病毒传播的预防疫苗已成为全球当前最为迫切的重大需求。
自18世纪末期人类首次应用生物制品牛痘疫苗以来,疫苗已在消除多种传染病中发挥了不可替代的作用。在COVID-19被确认后,国内外多家机构开展了COVID-19疫苗的研究,目前有至少5条技术路线在同步开展,包括核酸疫苗、病毒载体疫苗、灭活疫苗、重组蛋白疫苗、减毒流感病毒载体疫苗等,但鉴于疫苗的自身特点,疫苗要严格按照国家规定的研发生产流程进行,在动物实验通过后,还必须完成中试研究、临床申报及I~III期的临床试验研究,获得批准后才有可能正式进入生产阶段。SARS疫苗被搁置至今,正是因为 II期临床试验后,疫情已完全控制,无法再进行Ⅲ期试验。目前对COVID-19疫苗的后期临床实试验设计还不明确,需要制定其多国参与方案,以确定候选疫苗的安全有效。因此,全部在研疫苗尚未得到上市许可。
SARS-CoV-2的核衣壳由核衣壳蛋白(Nucleocapsid protein,N)包裹单股正链RNA形成;外有包膜,嵌有三种糖蛋白:刺突蛋白(spikeprotein,S)、包膜蛋白(envelopeprotein,E)和膜蛋白(membrane protein,M)。S蛋白位于病毒最外层,在膜上规则排列成冠状结构,参与病毒与宿主细胞表面的病毒受体结合并介导病毒通过膜融合进入细胞的过程,在诱导宿主产生中和抗体过程中起重要作用。
目前国内外COVID-19疫苗的研发均是以S蛋白作为首要目标抗原,但有报道称同属冠状病毒的SARS-CoV的S蛋白也可能诱发抗体依赖性免疫增强反应(ADE),因此筛选 S蛋白中特异性片段(即多肽)作为抗原,可以有效的预防COVID-19同时避免潜在ADE 的发生。
采用组合化学技术对蛋白抗原决定簇谱进行了研究(“交叉重叠”多肽化合物),快速发现了最小的抗原决定簇,并构建了决定簇谱。该方法的特点是以一定数目的氨基酸残基肽(比如1到50个氨基酸残基)合成“交叉重叠”片段,将蛋白序列通过逐个错位(或者间隔错位,包括从2到合成肽片段的氨基酸总位数)的方式全部合成出来,然后进行抗原- 抗体反应(或者其它生物学目的的筛选反应等,如筛选发现T-细胞免疫抗原决定簇,以及 SARS-CoV的受体配基等),一次便可以得到所有最短的多肽抗原决定簇,进而绘成抗原决定簇谱。再将这些活性短肽经过适当延长、或者有序线性连接后进行抗SARS-CoV人阳性血清筛选反应,从而确认了可用于制备SARS-CoV诊断试剂、药物以及疫苗的B-细胞多肽化合物以及它们的图谱。
SARS-CoV-2与SARS-CoV有很高的同源性。采用构建SARS-CoV的“交叉重叠”化学库筛选得到抗原决定簇,将其置换为同位置的SARS-CoV-2序列是快速发现新型冠状病毒抗原的有效途径。
合成肽疫苗是按照病原体抗原基因中已知或者预测的某段抗原表位氨基酸序列,通过化学技术合成具有抗原性的短肽,与载体结合后加入佐剂所制成的疫苗,是最为理想和安全的新型疫苗,也是目前在研的用于预防、控制感染性疾病和治疗恶性肿瘤的新型疫苗。
发明内容
本发明的目的是提供一种预防新型冠状病毒肺炎COVID-19的多肽及其用途。本发明基于SARS-CoV相关S蛋白的全部“交叉重叠”多肽抗原决定簇谱,通过构建SARS- CoV-2B-细胞抗原同样位置的多肽以及设计合成新的潜在的多肽,使用新冠病毒感染者的阳性血清对其进行筛选,成功快速发现了新型冠状病毒的B-细胞抗原多肽。
为实现上述目的,本发明采用以下技术方案。
一种抗原性多肽,所述抗原性多肽的氨基酸序列选自以下任意一种。
1)氨基酸序列是SEQ ID NO.1-SEQ NO.11所示的多肽。
2)与SEQ ID NO.1-SEQ ID NO.11所示氨基酸序列具有85%以上相同氨基酸序列的多肽。
3)将SEQ ID NO.1-SEQ ID NO.11所示氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到具有相同功能的多肽。
一种免疫原性组合物,包含所述的抗原性多肽的一种或多种和至少一种药物载体或赋形剂。
所述的抗原性多肽一种或多种和所述的疫苗组合物在制备用于预防新型冠状病毒肺炎COVID-19的疫苗中的应用。
进一步地,所述疫苗为任何免疫学上可接受的剂型,包括肌肉注射、皮内注射、皮下注射、微针注射的给药方式。
进一步地,所述用于预防新型冠状病毒肺炎COVID-19感染的疫苗为任何免疫学上可接受的剂量。
一种诊断组合物,所述诊断组合物包含上述抗原性多肽的一种或多种。
进一步地,所述诊断组合物用于制备检测患者中的新型冠状病毒感染的生物制品。
所述生物制品为诊断试剂、试剂盒或探针。
一种药物组合物,所述药物组合物包含上述至少一种抗原性多肽以及药学上可接受的运载体。
如上述所述的抗原性多肽一种或多种或所述的药物组合物在制备用于预防或治疗新型冠状病毒肺炎COVID-19的药物中的应用。
所述药物为任何药学上任何可接受的剂型。
所述药物为任何药学上任何可接受的剂量。
与现有技术比,本发明的有益效果如下。
本发明提供的预防新型冠状病毒肺炎COVID-19的多肽是基于新型冠状病毒S蛋白序列中筛选出来特异性抗原性的片段通过化学合成得到的,通过免疫原实验证明其具有较强的抗原性,可在人体内诱导出中和抗体,有望成为多肽疫苗抗原。
本发明提供的预防新型冠状病毒肺炎COVID-19的合成短肽,将多种短肽组合,与全序列相比可以降低副作用发生风险。现有报道表明SARS-CoV-2已发生变异,与本发明提供的多肽序列对比,未发生所有序列同时变异现象,有效的降低变异导致的抗原性损失,同时可以快速筛选出序列变异部分,增加新的短肽,合成制备新疫苗,能够快速应对病毒变异,减少研发成本及研发周期。
本发明提供的预防新型冠状病毒肺炎COVID-19的合成多肽,降低疫苗生产所需的场地要求,易于大规模批量生产;用合成的目标多肽通过与载体蛋白结合制备成多肽偶联物,加入佐剂制备成为合成肽疫苗,用于COVID-19的预防,相对于传统的弱毒疫苗和灭活疫苗以及其他几种新型的疫苗,合成肽类疫苗安全、无毒、稳定,由于其分子结构小而简单,很少会出现严重的并发症及医源性感染问题。
具体实施方式
下面结合实施例对本发明作进一步的阐述,以下实施例仅为本发明的优选实施例,并不限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
实施例1候选多肽序列的选择方法、合成多肽的方法。
一、候选多肽的选择。
SARS-CoV-2与SARS-CoV的功能蛋白同源性很高,依据已发现的SARS-CoV抗原及抗原决定簇谱,对位换取为SARS-CoV-2多肽序列,并依据SARS-CoV抗原决定簇位置进行适当延长、增加抗原决定簇设计了一系列候选多肽。
依据SARS-CoV-2的RBD/ACE2结合特点设计了一系列候选多肽,其氨基酸序列如SEQ ID NO.1-SEQ NO.11所示。
二、多肽合成。
1、采用常规的固相合成多肽的方法,制备了氨基酸序列是SEQ ID NO.1-SEQNO.11 所示的多肽,具体步骤如下。
(1)脱Fmoc保护。
将市售的Rink Amide AM resin装入一个有过滤器且耐有机溶剂的反应管中,并用一个耐有机溶剂的盖子盖紧。用DMF(二甲基甲酰胺)洗涤1分钟后,加入过量的20%哌啶/DMF(体积比)溶液,盖紧后轻轻摇晃反应管,使之混合均匀并保持脱除Fmoc保护15分钟。抽干后再用DMF洗涤三次。
(2)肽键缩合。
将经过预活化(预活化2-3分钟后)的3倍树脂氨基摩尔量的Fmoc保护的氨基酸、3倍树脂氨基摩尔等量的活化剂HBTU、以及6倍树脂氨基摩尔等量的DIPEA(N,N-二异丙基乙基胺)加入反应管中,使用DMF作为溶剂,并保证以上的试剂完全溶解,可完全覆盖树脂。每隔2-3分钟摇晃混匀树脂一次,反应20-30分钟。
然后,室温下加入50倍摩尔过量的乙酸酐DMF溶液后10分钟抽干,再在室温下加入过量的20%哌啶/DMF(体积比)溶液反应30分钟。过滤掉溶液,树脂用DMF洗涤5 次。
以上步骤循环反复执行,直到拟合成多肽的全部Fmoc保护氨基酸按线性方式连接到树脂上。
(3)多肽裂解。
多肽裂解液选用强酸处理,比如TFA(三氟乙酸)。室温下用TFA\水\EDT二巯基乙醇\苯酚(体积比:92.5:2.5:2.5:2.5)处理树脂2小时。然后仔细收集裂解液到一个玻璃收集器中,并加入用冰预冷的乙醚,收集沉淀多肽,并继续用冷乙醚洗涤5-6次,得到粗品肽。
(4)多肽纯化。
粗品肽经过HPLC(高效液相色谱)纯化,收集,冻干,最终再经HPLC检验纯度(214nm波长)大于85%及质谱检验正确的分子量。
2.鉴定结果。
合成多肽化合物的纯度以及分子量结果见表1。
表1.氨基酸序列是SEQ ID NO.1-SEQ NO.11所示的多肽结果。
实施例二SEQ ID NO.1-SEQ NO.11所示的多肽特异性抗原性研究实验。
将实施例1获得的多肽分别与16份新冠病人血清反应。由于新冠状病毒感染人血清中含有抗新冠状病毒的抗体,并可与来自于新冠状病毒蛋白序列的多肽抗原发生免疫反应,经过酶联免疫反应放大并显色为阳性,测定其与多肽抗原的结合,其具体步骤如下。
1)包被:包被液为0.07M NaHCO3,0.03M Na2CO3(pH=9.6),包被浓度10μg/孔, 4℃过夜。
2)清洗:PBST(pH=7.4,含0.05%的Tween 20)清洗5遍,拍干。
3)封闭:封闭液为1~3%的BSA,100μL/孔,37℃2小时。
4)清洗:PBST(pH=7.4,含0.05%的Tween 20)清洗5遍,拍干。
5)加入一抗:病人抗血清用生理盐水1:20稀释,100μL/孔,37℃孵育1.5小时。
6)清洗:PBST(pH=7.4,含0.05%的Tween 20)清洗5遍,拍干。
7)加入二抗:HRP-兔抗人IgG抗体用PBS1:7500稀释,100μL/孔,37℃孵育2小时。
8)清洗:PBST(pH=7.4,含0.05%的Tween 20)清洗5遍,拍干。
9)加入酶反应底物:酶反应底物为TMB,100μL/孔,37℃孵育10分钟。
10)加终止液:2MH2SO4,50μL/孔。
11)读数:在酶标仪中测量450nm的OD值。
12)对照及数据处理:设无酶反应底物孔为阴性对照孔。抗新冠状病毒感染人血清反应孔OD值的平均值3阴性对照孔的OD平均值的两倍以上即为阳性结果,具体结果见表2。
13)结论:由表2可知,实施例1合成的如氨基酸序列SEQ ID NO.1-SEQ NO.11所示的多肽均具有较强的抗原性,患者血清中的抗新冠病毒的抗体可与SEQ ID NO.1-SEQNO.11所示的多肽抗原结合,有望成为多肽疫苗抗原。
表2合成多肽抗原与新型冠状病毒感染人血清的免疫反应原性结果。
序列表
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Claims (12)
1.一种抗原性多肽,其特征在于,所述抗原性多肽的氨基酸序列选自以下任意一种:
1)氨基酸序列是SEQ ID NO .1-SEQ NO.11所示的多肽;
2)与SEQ ID NO.1-SEQ ID NO.11所示氨基酸序列具有85%以上相同氨基酸序列的多肽;
3)将SEQ ID NO.1-SEQ ID NO.11所示氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到具有相同功能的多肽。
2.一种免疫原性组合物,其特征在于,包含如权利要求1所述的抗原性多肽的一种或多种和至少一种药物载体或赋形剂。
3.如权利要求1所述的抗原性多肽一种或多种和权利要求2所述的疫苗组合物在制备用于预防新型冠状病毒肺炎COVID-19的疫苗中的应用。
4.如权利要求3所述的应用,其特征在于,所述疫苗为任何药学上可接受的剂型,包括肌肉注射、皮内注射、皮下注射、微针注射的给药方式。
5.如权利要求3所述的应用,其特征在于,所述疫苗为任何药学上可接受的剂量。
6.一种诊断组合物,其特征在于,所述诊断组合物包含如权利要求1所述的抗原性多肽的一种或多种。
7.如权利要求6所述的诊断组合物在制备用于检测患者中的新型冠状病毒感染的生物制品中的应用。
8.如权利要求7所述的应用,其特征在于,所述生物制品是试剂、试剂盒或探针。
9.一种药物组合物,其特征在于,所述药物组合物包含上述抗原性多肽的一种或多种以及药学上可接受的载体。
10.如权利要求1所述的抗原性多肽的一种或多种或如权利要求9所述的药物组合物在制备用于预防或治疗新型冠状病毒肺炎COVID-19的药物中的应用。
11.如权利要求10所述的应用,其特征在于,所述药物为任何药学上可接受的剂型。
12.如权利要求10所述的应用,其特征在于,所述药物为任何药学上可接受的剂量。
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