CN113493497B - 2019-nCoV新冠病毒ORF10蛋白的抗原表位多肽及其应用 - Google Patents
2019-nCoV新冠病毒ORF10蛋白的抗原表位多肽及其应用 Download PDFInfo
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Abstract
本发明公开了一组2019‑nCoV新冠病毒ORF10蛋白的抗原表位多肽及其应用。所述抗原表位多肽的氨基酸序列为SEQ ID NO.1所示的氨基酸序列,或者与SEQ ID NO.1所示的氨基酸序列具有90%以上同源性且具有相同功能的氨基酸序列。本发明筛选的高免疫原性多肽能够有效诱导机体产生免疫反应,同时又避免了无关抗原的干扰,减少了疫苗的副反应。另一方面,本发明的多肽疫苗成分清晰,对人体无危害性,保障了安全性。
Description
技术领域
本发明属于多肽疫苗技术领域,具体涉及2019-nCoV新冠病毒ORF10蛋白的抗原表位多肽及其应用。
背景技术
目前针对新型冠状病毒(2019-nCoV)疫苗的研发,与过去20年开发SARS-CoV疫苗的策略基本相同,包括①灭活病毒疫苗,②病毒载体疫苗以及③亚单位疫苗。本发明所设计的多肽疫苗属于一类新型的亚单位疫苗。亚单位疫苗,指的是通过化学分解或有控制性的蛋白质水解方法,提取细菌、病毒的特殊蛋白质结构,筛选出具有免疫活性的片段制成的疫苗。在病原体与机体的免疫反应过程中,仅有少量的抗原拥有免疫原性从而刺激机体产生免疫应答,因此筛选出这些具有免疫活性的抗原所制备的亚单位疫苗能够有效发挥疫苗作用,同时避免无关抗原诱发的抗体,减少疫苗的副反应。目前我国在亚单位疫苗的研制方面还处于较为落后的地位。
新型冠状病毒2019-nCoV颗粒包含4种结构蛋白,分别是刺突蛋白,膜蛋白,包膜蛋白和核衣壳蛋白。ORF10是一个核衣壳磷蛋白,该蛋白将病毒基因组包装到螺旋核糖核衣壳(RNP)中,并在病毒自组装过程中起基本作用。它是一种具有多种活性的蛋白质,与近缘病毒的同源基因相似度高达90%,这可能是因为该基因负责着病毒基因组包装的看家功能。另外有研究表明ORF10能够抑制宿主血红素代谢从而导致进一步的临床症状。因此,高保守性的ORF10是制备新型冠状病毒药物和多肽的理想靶点之一。
发明内容
本发明的目的是,提供一组2019-nCoV新冠病毒ORF10蛋白的抗原表位多肽及其应用。
本发明对病人MHC分子与病毒蛋白分子进行免疫亲和力分析,预测高免疫原性的抗原表位,作为刺激T细胞免疫反应的多肽疫苗。同时,筛选具有B细胞表位高亲和力的抗原表位,作为诱导B细胞体液免疫反应的多肽疫苗。MHC分子结合的肽段长8~11,分子上有一个沟,是抗原结合的部位,叫做肽结合槽(peptide-binding groove or cleft),槽底有6个口袋:Pocket A~F,Pocket A和Pocket F负责固定肽段,Pocket B~E负责结合不同的肽段,保持特异性。MHC分子抗原结合凹槽与抗原肽结合的特点与MHC结合成复合物的抗原肽往往带有两个或两个以上的关键氨基酸(锚着残基,anchor residue)专司和MHC分子肽结合槽中的多肽结合基序相结合,二者具有一定的特异性。本发明利用机器学习模型,使用已知的MHC分子和抗原的结合情况进行训练,得到亲和力模型,从而预测新型冠状病毒的抗原肽与MHC分子的亲和力。使用随机森林算法根据蛋白质序列预测B细胞抗原决定簇,该算法对根据晶体结构确定的表位和非表位氨基酸进行训练,筛选打分高于设定阈值的肽段作为潜在的BCR识别疫苗分子。
本发明通过生物信息算法,模拟病毒抗原与宿主免疫系统的识别模式,从2019-nCoV新冠病毒ORF10蛋白中筛选并预测出免疫原性高的多肽序列作为候选抗原,然后进一步通过多肽疫苗的有效性实验验证,获得具有疫苗作用的抗原表位多肽。
本发明第一方面提供一组2019-nCoV新冠病毒ORF10蛋白的抗原表位多肽,所述抗原表位多肽的氨基酸序列为SEQ ID NO.1所示的氨基酸序列;或者与SEQ ID NO.1所示的氨基酸序列具有90%以上同源性且具有相同功能的氨基酸序列。
本发明第二方面提供编码所述2019-nCoV新冠病毒ORF10蛋白的抗原表位多肽的多核苷酸序列。
本发明第三方面提供所述的抗原表位多肽中的一种或多种在制备人类冠状病毒疫苗中的应用。优选地,所述人类冠状病毒为2019-nCoV新冠病毒。
本发明第四方面提供一种抗体,所述抗体特异性地与所述的抗原表位多肽结合。
本发明第五方面提供一种检测试剂盒,该试剂盒包含所述的任一种抗原表位多肽,或所述的抗体,或抗原表位多肽与抗体的组合。
本发明提供的检测试剂盒用于体外检测样品中是否存在人类冠状病毒或抗人类冠状病毒抗体。优选地,所述人类冠状病毒为2019-nCoV新冠病毒。
与现有技术相比,本发明的有益效果如下:
1,本发明基于已知的病毒序列特征和计算机算法来筛选设计多肽疫苗,多肽分子的序列清晰,便于生产和制备。越过了使用复杂的实验手段分离特定病毒组分的技术难题。
2,本发明的疫苗设计考虑主要组织相容性复合体(MHC)与多肽分子的亲和力,因此能够有效地被T细胞识别,从而诱导机体的细胞免疫反应。相比常规的亚单位疫苗或灭活病毒疫苗仅能激活体液免疫反应,多肽疫苗的免疫效力更强。
3,本发明通过人工筛选得到的高免疫原性多肽能够有效诱导机体产生免疫反应,同时又避免了无关抗原的干扰,减少了疫苗的副反应。另一方面,多肽疫苗成分清晰,对人体无危害性,保障了安全性。
4,本发明提供的多肽疫苗能够有效的与新冠肺炎恢复期病人的血清中的IgG和IgM抗体发生较强的中和反应。恢复期病人血清中,较健康人的特异性抗体具有抵抗新冠病毒的作用,因此,本发明的多肽疫苗具有在人体中诱导抵抗新冠病毒的中和抗体的能力,从而提高机体对新冠病毒的免疫力。
附图说明
图1为本发明设计筛选抗原表位多肽的流程图。
图2A为SEQ ID NO.1的IgG抗体检测中,每个患者样本在IgG抗体反应测试中的最大反应强度与健康组的比较图。
图2B为SEQ ID NO.1的IgG抗体检测中,同一个患者治愈后不同恢复期的IgG抗体反应比较图。
图2C为SEQ ID NO.1的IgG抗体检测中,按照恢复期不同阶段分组,各患者样本的IgG抗体反应与健康组的比较图。
图3A为SEQ ID NO.1的IgM抗体检测中,每个患者样本在IgM抗体反应测试中的最大反应强度与健康组的比较图。
图3B为SEQ ID NO.1的IgM抗体检测中,同一个患者治愈后不同恢复期的IgM抗体反应比较图。
图3C为SEQ ID NO.1的IgM抗体检测中,按照恢复期不同阶段分组,各患者样本的IgM抗体反应与健康组的比较图。
具体实施方式
下面结合实施例对本发明的技术方案进行详细说明。以下采用的试剂和生物材料如未特别说明,均为商业化产品。
实施例1抗原表位多肽的设计筛选
参见图1,为本发明设计筛选抗原表位多肽的流程图。利用机器学习模型,使用已知的MHC分子和抗原的结合情况进行训练,得到亲和力模型,从而预测新型冠状病毒的抗原肽与MHC分子的亲和力。使用随机森林算法根据蛋白质序列预测B细胞抗原决定簇,该算法对根据晶体结构确定的表位和非表位氨基酸进行训练,筛选打分高于设定阈值的肽段作为潜在的BCR识别疫苗分子。对于计算机模型筛选的多肽序列,利用固相合成法获得多肽片段,然后通过中和抗体试验和刺激细胞因子实验评估多肽作为疫苗的有效性,得到最终的抗原表位多肽。
针对2019-nCoV新冠病毒ORF10蛋白的序列特征和空间构象,利用上述算法设计筛选出一种抗原表位多肽序列,序列信息如下:
SEQ ID NO.1:FAFPFTIYSLLLCRMNSRNYIAQVDVVN
实施例2抗原表位多肽的合成
采用固相合成方法合成设计筛选的抗原表位多肽序列(SEQ ID NO.1)。固相合成多肽的具体过程如下:
氯甲基聚苯乙烯树脂作为不溶性的固相载体,首先将一个氨基被封闭基团保护的氨基酸共价连接在固相载体上。在三氟乙酸的作用下,脱掉氨基的保护基,这样第一个氨基酸就接到了固相载体上了。然后氨基被封闭的第二个氨基酸的羧基通过N,Nˊ-二环己基碳二亚胺(DCC,Dicyclohexylcarbodiimide)活化,羧基被DCC活化的第二个氨基酸再与已接在固相载体的第一个氨基酸的氨基反应形成肽键,这样在固相载体上就生成了一个带有保护基的二肽。重复上述肽键形成反应,使肽链从C端向N端生长,直至达到所需要的肽链长度。最后脱去保护基,用HF水解肽链和固相载体之间的酯键,就得到了合成好的肽。
实施例3抗原表位多肽疫苗的有效性评估
实验方法:将合成的抗原表位多肽均采用C端生物素基团标记,并使用生物素-链霉亲和素化学方法打印到裁片上,制成多肽芯片。制备的多肽芯片用于检测血清中IgG和IgM抗体与抗原表位多肽的中和反应程度。
从医院收集19例新冠肺炎治愈恢复期患者的血清样本和10例健康人的血清样本。恢复期的患者在接受治疗前确诊为新冠肺炎,因此他们都曾经接触过新冠病毒,则在治愈后血清中仍然保留抗新冠病毒的抗体。通过在这些样本中检测针对每个抗原表位多肽的抗体反应强度,并与健康人进行比较,可以评估本发明中制备的抗原表位多肽在人体中诱导中和抗体的能力。
1、SEQ ID NO.1的IgG抗体检测结果
SEQ ID NO.1所示的抗原表位多肽与IgG抗体反应测试中,19例患者样本中有15例(78.9%)患者的最强抗体反应高于健康组。参见图2A,为每个患者样本在IgG抗体反应测试中的最大反应强度与健康组的比较图,图中灰色为健康组,红色为抗体反应强度大于健康组的患者即阳性组,蓝色为抗体反应强度低于健康组的患者即阴性组。通过图2A可以看出:15例患者的抗体反应强度大于健康组,4例患者的抗体反应强度低于健康组。参见图2B,为同一个患者治愈后不同恢复期的IgG抗体反应比较图,几个阶段都高于健康组的标为红色,若某一阶段低于健康组则标为蓝色。通过图2B可以看出:有5例(26.3%)患者在恢复期的所有阶段(1-7个月中)的抗体反应都高于健康组,其余14例则出现某一个阶段低于健康组。参见图2C,为按照恢复期不同阶段分组,各患者样本的IgG抗体反应与健康组的比较图。通过图2C可以看出:在第一个月中有9个(50%)血清样本的抗体反应高于健康组,在第4个月中有8个(44.4%)血清样本的抗体反应高于健康组,在第7个月中有9个(60%)血清样本的抗体反应高于健康组。
2、SEQ ID NO.1的IgM抗体检测结果
SEQ ID NO.1所示的抗原表位多肽与IgM抗体反应测试中,19例患者样本中有18例(94.7%)患者的最强抗体反应高于健康组。参见图3A,为每个患者样本在IgM抗体反应测试中的最大反应强度与健康组的比较图,图中灰色为健康组,红色为抗体反应强度大于健康组的患者即阳性组,蓝色为抗体反应强度低于健康组的患者即阴性组。通过图3A可以看出:18例患者的抗体反应强度大于健康组,1例患者的抗体反应强度低于健康组。参见图3B,为同一个患者治愈后不同恢复期的IgM抗体反应比较图,几个阶段都高于健康组的标为红色,若某一阶段低于健康组则标为蓝色。通过图3B可以看出:有11例(57.9%)患者在恢复期的所有阶段(1-7个月中)的抗体反应都高于健康组,其余8例则出现某一个阶段低于健康组。参见图3C,为按照恢复期不同阶段分组,各患者样本的IgM抗体反应与健康组的比较图。通过图3C可以看出:在第一个月中有13个(72.2%)血清样本的抗体反应高于健康组,在第4个月中有13个(72.2%)血清样本的抗体反应高于健康组,在第7个月中有12个(80%)血清样本的抗体反应高于健康组。
上述仅为本发明的部分优选实施例,本发明并不仅限于实施例的内容。对于本领域中的技术人员来说,在本发明技术方案的构思范围内可以有各种变化和更改,所作的任何变化和更改,均在本发明保护范围之内。
序列表
<110> 安达生物药物开发(深圳)有限公司
<120> 2019-nCoV新冠病毒ORF10蛋白的抗原表位多肽及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Phe Ala Phe Pro Phe Thr Ile Tyr Ser Leu Leu Leu Cys Arg Met Asn
1 5 10 15
Ser Arg Asn Tyr Ile Ala Gln Val Asp Val Val Asn
20 25
Claims (3)
1.一种2019-nCoV新冠病毒ORF10蛋白的抗原表位多肽,其特征在于:所述抗原表位多肽的氨基酸序列为SEQ ID NO.1所示的氨基酸序列。
2.一种多核苷酸,其特征在于:编码权利要求1所述的抗原表位多肽。
3.一种检测试剂盒,其特征在于:包含权利要求1所述的抗原表位多肽。
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