CN111499692B - 靶向新型冠状病毒covid-19的多肽及其应用 - Google Patents
靶向新型冠状病毒covid-19的多肽及其应用 Download PDFInfo
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Abstract
本发明提供了靶向新型冠状病毒COVID‑19的多肽及其应用,所述多肽包括如SEQ ID NO:1~28所示的氨基酸序列;或所述多肽为将SEQ ID NO:1~28在N端和/或C端添加1~20个氨基酸多肽序列和/或共轭连接碳链而获得的具有靶向冠状病毒COVID‑19的活性的分子。本发明采用一珠一化合物(one‑bead‑one‑compound,OBOC)的组合化学方法合成多肽库,利用多肽库与新型冠状病毒表面的S蛋白,筛选得到的多肽与冠状病毒COVID‑19的S蛋白具有强结合能力,为新型冠状病毒检测试剂和治疗药物的研发提供了依据。
Description
技术领域
本发明属于生物医学技术领域,涉及靶向新型冠状病毒COVID-19的多肽及其应用。
背景技术
病毒(virus)是形态最微小、结构最简单的一类微生物,属于非细胞型微生物,没有典型的细胞结构和产生能量的酶***,只能寄生在活细胞内生长繁殖。病毒在医学微生物中占有十分重要的地位,在微生物引起的各种疾病中,病毒性疾病的占比高达75%。病毒性疾病不仅传染性强、流行范围广,而且有效药少。近年来发生的大型冠状病毒传染性肺炎,引起了社会各界的广泛关注。而新型冠状病毒肺炎更是成为关注和讨论的焦点,然而尚未研发出针对新型冠状病毒的特效药,因此,迫切需要开发一种非传统的抗病毒策略。
S蛋白是冠状病毒入侵细胞的武器,具有非常明确的目标特异性,在介导病毒与宿主细胞表面的受体结合以及膜融合的过程中起着关键作用;由于冠状病毒是单链RNA结构,且基因组庞大,具有极强的突变性,使得冠状病毒极易逃避宿主免疫***的清除作用。冠状病毒的目标特异性和高度突变性,使得对于冠状病毒的有效治疗相当棘手。
因此,有必要研究与S蛋白具有特异性结合作用的多肽的特征,为进一步研发抗病毒药物提供依据。
发明内容
针对现有技术的不足和实际需求,本发明提供了靶向新型冠状病毒COVID-19的多肽及其应用,所述多肽能够特异性结合新型冠状病毒表面的S蛋白,在新型冠状病毒的药物研发等方面具有重要意义。
为达此目的,本发明采用以下技术方案:
第一方面,本发明提供了一种靶向冠状病毒COVID-19的多肽,所述多肽包括如SEQID NO: 1~28所示的氨基酸序列;
或所述多肽为将SEQ ID NO: 1~28在N端和/或C端添加1~20个氨基酸多肽序列和/或共轭连接碳链而获得的具有靶向冠状病毒COVID-19的活性的分子;
SEQ ID NO: 1:NFRHTHR;
SEQ ID NO: 2:FNRHTHR;
SEQ ID NO: 3:IGVRGGW;
SEQ ID NO: 4:KWPYKKS;
SEQ ID NO: 5:KTYHTHR;
SEQ ID NO: 6:IRQFFKK;
SEQ ID NO: 7:FYIKINH;
SEQ ID NO: 8:KSPPYHK;
SEQ ID NO: 9:WVHFYHF;
SEQ ID NO: 10:KPYIWKS;
SEQ ID NO: 11:KWPFKKS;
SEQ ID NO: 12:IGHVPGT;
SEQ ID NO: 13:WIGVRNW;
SEQ ID NO: 14:QGTHTAH;
SEQ ID NO: 15:NSGGSVH;
SEQ ID NO: 16:AAARFST;
SEQ ID NO: 17:PIGVPHT;
SEQ ID NO: 18:QVAVLYQ;
SEQ ID NO: 19:FLGVYYH;
SEQ ID NO: 20:ALTGIAV;
SEQ ID NO: 21:FVFLVLL;
SEQ ID NO: 22:VVIGIVN;
SEQ ID NO: 23:NFTISVTT;
SEQ ID NO: 24:RVVVLSFE;
SEQ ID NO: 25:AAYYVGYL;
SEQ ID NO: 26:SIIAYTMSLG;
SEQ ID NO: 27:GVVFLHVTYV;
SEQ ID NO: 28:FTGCVIAWNR。
本发明中,采用一珠一化合物(one-bead-one-compound,OBOC)的组合化学方法合成多肽库,利用多肽库与新型冠状病毒表面的S蛋白,筛选得到如SEQ ID NO: 1~28所示的具有靶向冠状病毒COVID-19的活性的多肽。
优选地,所述多肽靶向冠状病毒COVID-19的S蛋白。
本发明筛选的多肽中,如SEQ ID NO: 1~28所示的氨基酸序列的中间若干氨基酸为主要功能结构域,用于靶向新型冠状病毒表面的S蛋白,在SEQ ID NO: 1~28的N端和/或C端基础上,连接含有n个氨基酸的多肽,其中n为1~20之间的整数,和/或共轭连接碳链CH3(CH2)nCOOH,n为6~18之间的整数,将不会影响活性多肽的空间构象、仍然保持对S蛋白的靶向性功能(参考文献:H. Wei et al., A tumour-selective cascade activatableselfdetained system for drug delivery and cancer imaging, NatureCommunication (2019) 10:4861)。
第二方面,本发明提供了一种第一方面所述的多肽的筛选方法,所述方法包括以下步骤:
(1)利用一珠一化合物法合成多肽库;
(2)向步骤(1)的多肽库中加入S蛋白进行孵育,筛选得到具有特异性结合S蛋白作用的多肽。
根据本发明,一珠一化合物(one-bead-one-compound,OBOC)法是一种组合化学方法,基于OBOC方法建立的多肽库主要用于筛选具有蛋白靶向功能的多肽,具有通量高、筛选效率高等优势;本发明利用多肽库与新型冠状病毒表面的S蛋白进行筛选,最终得到能够与S蛋白特异性结合的多肽树脂,经过测序得到多肽的氨基酸序列;本发明的方法建立了蛋白与多肽之间的联系,为发现与新型冠状病毒结合的多肽提供了简便易行的方法,为进一步研发抗病毒类药物提供了依据。
优选地,步骤(1)所述多肽库的合成方法包括:
(1’)在树脂微珠上首先合成前导序列;
(2’)将树脂微珠均分后,将一个Fmoc保护的氨基酸进行活化并加入树脂微珠中,与脱保护的前一个氨基酸进行缩合反应;
(3’)将树脂微珠混合,进行Fmoc脱保护,重复步骤(2’)和(3’),直至最后一个氨基酸合成完成,得到所述多肽库。
本发明中,在固相合成过程中,通过对树脂微珠的多次混合与均分,使每个树脂微珠上带有唯一一种随机多肽序列,构建得到多肽库。
优选地,步骤(1’)所述前导序列的长度为2~4 aa。
优选地,所述前导序列包括甲硫氨酸(M)。
本发明中,首先在树脂微珠上合成含有甲硫氨酸的前导序列,主要作用是CNBr3乙醇溶液将甲硫氨酸裂解生成高丝氨酸,使得功能性多肽从树脂微珠上裂解下来。
根据本发明,前导序列中还含有其他19种氨基酸中的任意一种或多种,优选为含有甘氨酸(G),用于将树脂微珠与功能性多肽相间隔。
优选地,步骤(2’)所述活化的试剂包括N,N-二甲基甲酰胺(DMF)、甲醇、N-甲基吗啉、N,N'-二异丙基碳二亚胺(DIC)或1-羟基苯并***(HOBT)中的任意一种或至少两种的组合,优选为N,N'-二异丙基碳二亚胺(DIC)和1-羟基苯并***(HOBT)的混合液。
优选地,步骤(3’)所述脱保护的试剂包括含有六氢吡啶的DMF溶液或含有哌嗪和1,8-二氮杂二环十一碳-7-烯的DMF溶液,优选为含有20%六氢吡啶的DMF溶液,或含有5%哌嗪和2% 1,8-二氮杂二环十一碳-7-烯的DMF溶液。
优选地,步骤(1)所述多肽库中多肽的长度为7~10 aa。
本发明中,合成长度为7~10 aa的多肽构建多肽库,不仅丰富了多肽的序列多样性,提高了多肽库中的多肽数量,理论上多肽库中含有197~1910种多肽(合成的多肽中不含有甲硫氨酸),而且保证了筛选功能性多肽的效率,有助于高效筛选得到具有结合S蛋白功能的多肽;同时,多肽的长度较短,分子量小,具有免疫原性弱、活性高等优点,在体内外抗病毒方面具有广阔的应用前景。
优选地,步骤(2)所述S蛋白包括如SEQ ID NO: 29所示的氨基酸序列;
SEQ ID NO: 29:
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT。
优选地,步骤(2)所述S蛋白标记有荧光基团。
优选地,步骤(2)所述孵育的时间为2~4 h,例如可以是2 h、3 h或4 h,优选为2 h。
优选地,步骤(2)所述筛选为根据荧光信号进行筛选。
作为优选技术方案,本发明提供了一种第一方面所述的多肽的筛选方法,所述方法包括以下步骤:
(1)在树脂微珠上首先合成含有甲硫氨酸的长度为2~4 aa的前导序列;
(2)将树脂微珠均分后,将一个Fmoc保护的氨基酸进行活化并加入树脂微珠中,与脱保护的前一个氨基酸进行缩合反应;
(3)将树脂微珠混合,采用含有六氢吡啶的DMF溶液进行Fmoc脱保护,重复步骤(2)和(3),直至最后一个氨基酸合成完成,得到所述多肽库,所述多肽库中多肽的长度为7~10aa;
(4)向多肽库中加入如SEQ ID NO: 29所示的荧光标记S蛋白,孵育2~4 h,根据荧光信号筛选得到具有特异性结合S蛋白作用的树脂;
(5)将树脂经过裂解去除侧链保护基团,获得脱离树脂的多肽,进一步测序确认多肽包括如SEQ ID NO: 1~28所示的氨基酸序列。
第三方面,本发明提供了一种冠状病毒COVID-19检测试剂盒,所述试剂盒包括包被多肽,所述包被多肽为第一方面所述的多肽。
优选地,所述包被多肽直接和/或间接包被在固相载体上。
优选地,所述固相载体包括酶标板、磁珠、微球、亲和膜或液相芯片中的任意一种或至少两种的组合。
优选地,所述试剂盒还包括S蛋白的特异性抗体。
优选地,所述S蛋白的特异性抗体上标记有信号基团。
优选地,所述试剂盒还包括显色液和/终止液。
第四方面,本发明提供了一种第一方面所述的多肽和/或第三方面所述的试剂盒在制备冠状病毒COVID-19检测试剂和/或治疗药物中的应用。
与现有技术相比,本发明具有如下有益效果:
(1)本发明采用OBOC法合成多肽库,利用多肽库与新型冠状病毒表面的S蛋白,筛选得到如SEQ ID NO: 1~28所示的具有靶向冠状病毒COVID-19的活性的多肽;
(2)采用本发明筛选的多肽与S蛋白结合能力强、特异性好、分子量小、免疫原性弱、活性高,构建了冠状病毒COVID-19的免疫检测试剂盒,为疑似患者的快速确诊提供了技术支持;
(3)本发明的多肽筛选方法具有通量高、筛选效率高等优势,建立了蛋白与多肽之间的联系,为发现与新型冠状病毒结合的多肽提供了简便易行的方法,为进一步研发抗病毒类药物提供了依据。
附图说明
图1为结合有FITC标记S蛋白的OBOC多肽库的荧光显微镜图;
图2(A)为多肽序列NFRHTHR的二级质谱图,图2(B)为多肽序列KTYHTHR的二级质谱图,图2(C)为多肽序列KPYIWKS的二级质谱图,图2(D)为多肽序列KWPFKKS的二级质谱图;
图3为酶联免疫实验对多肽的二次筛选分析;
图4(A)为KWPFKKS与S蛋白的结合力分析,图4(B)为PIGVPHT与S蛋白的结合力分析,图4(C)为NSGGSVH与S蛋白的结合力分析;
图5(A)为KWPFKKS的抗病毒研究结果,图5(B)为PIGVPHT的抗病毒研究结果,图5(C)为NSGGSVH的抗病毒研究结果;
图6(A)为在PIGVPHT的N端添加多肽序列KGAG后得到的新肽的质谱图,图6(B)为在PIGVPHT的C端添加多肽序列KGAG后得到的新肽的质谱图,图6(C)为在PIGVPHT的N端添加月桂酸(C12)后得到的新肽的质谱图;
图7为新肽与S蛋白结合的酶联免疫结果,其中,1’-KGAGPIGVPHT、2’-PIGVPHTKGAG、3’-C12-PIGVPHT、4’-KAAGGKWPFKKS、5’-C12-KWPFKKS、6’-NSGGSVHKNSG、7’-GGAANSGGSVH;
图8(A)为KGAGPIGVPHT与S蛋白的结合力分析,图8(B)为PIGVPHTKGAG与S蛋白的结合力分析,图8(C)为C12-PIGVPHT与S蛋白的结合力分析,图8(D)为KAAGGKWPFKKS与S蛋白的结合力分析,图8(E)为C12-KWPFKKS与S蛋白的结合力分析,图8(F)为NSGGSVHKNSG与S蛋白的结合力分析,图8(G)为GGAANSGGSVH与S蛋白的结合力分析;
图9为KWPFKKS及经过衍生后的新肽二级结构变化图。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果,以下结合实施例和附图对本发明作进一步地说明。可以理解的是,此处所描述的具体实施方式仅仅用于解释本发明,而非对本发明的限定。
实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道商购获得的常规产品。
实施例1 S蛋白的生物素或FITC标记
本实施例首先对S蛋白(SEQ ID NO: 29)进行生物素或FITC标记,步骤如下:
将0.15 mol/L NaCl溶液和0.15 mol/L pH=9.0的NaHCO3-Na2CO3缓冲液按摩尔比为9:1混合,向混合液加入S蛋白进行蛋白溶解,使S蛋白的终浓度为10 mg/mL;
向S蛋白溶液中加入生物素或FITC,进行冰浴,避光条件下磁力搅拌过夜;
第二天,将混合液置于截留分子量为200的透析袋中,在去离子水条件下透析两天,并多次换水;
最后收集透析后的溶液,冷冻干燥机冻干,得到生物素或FITC标记的S蛋白。
实施例2 OBOC多肽库的建立
本实施例利用OBOC法在树脂微珠(beads)上合成多肽库,步骤如下:
(1)取1.8 g树脂beads置于合成管中,加入一定量的DMF浸没树脂进行树脂溶胀,浸泡2 h以上;
(2)向合成管中加入Fmoc保护的甲硫氨酸(M),并加入HOBT(1-羟基苯并***)和DIC(N,N'-二异丙基碳二亚胺)的混合液,摇床反应2 h以上;用甲醇和DMF反复交替冲洗三次,冲洗过程中用吸管反复将beads吹起;之后对beads进行茚三酮沸水浴检验,beads显色为无色;
(3)加入脱保护剂(20%六氢吡啶+80%DMF)进行Fmoc脱保护10 min,用甲醇和DMF反复交替冲洗三次,冲洗过程中用吸管反复将beads吹起;之后对beads进行茚三酮沸水浴检验,beads显色为深蓝紫色;
(4)向合成管中加入Fmoc保护的甘氨酸(G),重复步骤(2)和(3);
(5)将合成管中的树脂平均分为19份,向每一支合成管中加入相应剂量的Fmoc保护的氨基酸(除甲硫氨酸之外的19种天然氨基酸)和HOBT(1-羟基苯并***)/DIC(N, N'-二异丙基碳二亚胺)混合液,摇床反应合成6 h以上;
(6)用甲醇和DMF反复交替冲洗三次,冲洗过程中用吸管反复将beads吹起,确保充分混合冲洗;
(7)抽检其中3种多肽的合成效率,取出beads用乙醇浸泡后进行茚三酮沸水浴检验,重复步骤(5)-(7),直至beads变为无色,将所有合成管中的树脂混合,进行Fmoc脱保护;
(8)重复步骤(5)-(8),直至最后一个氨基酸合成完成;
(9)用脱保护剂(20%六氢吡啶+80%DMF)脱去侧链Fmoc保护基后,依次用DMF、甲醇、DMF冲洗三次,室温下避光干抽15 min,得到较为干燥的树脂;将干燥的树脂转移至小反应瓶(提前烘干)中,加入磁子,用酸性脱保护剂(TFA 82.5%、苯酚5%、水10%、TRIS2.5%)脱去所有的酸响应侧链保护基,室温反应4小时,构建得到多肽库。
实施例3 筛选靶向S蛋白的多肽
将FITC标记的S蛋白与多肽库在PBS缓冲液中培养2小时,荧光显微镜下观察发光情况(激发波长405 nm),将观察到的有明显绿色荧光的多肽树脂挑选出来。
如图1所示为FITC标记的S蛋白与OBOC多肽库作用后的荧光显微镜图片,结合有S蛋白的beads可观察到绿色荧光,根据荧光挑选出具有结合S蛋白作用的多肽。
挑选出的多肽树脂用超纯水洗涤数次并干燥,采用30%的CNBr3乙醇溶液裂解过夜,离心处理取上清液,经过脱盐处理后进行多肽测序。
示例性地,如图2(A)、图2(B)、图2(C)和图2(D)所示为多肽序列的二级质谱图,通过二级质谱中显示的多肽碎片峰分析,多肽序列分别为NFRHTHR(SEQ ID NO: 1)、KTYHTHR(SEQ ID NO: 5)、KPYIWKS(SEQ ID NO: 10)和KWPFKKS(SEQ ID NO: 11)。通过二级质谱测序分析所有筛选得到的多肽,得到如SEQ ID NO: 1~28所示的具有靶向S蛋白功能的多肽。
实施例4 多肽与S蛋白的酶联免疫反应
将筛选的如SEQ ID NO: 1~28所示的多肽用1×PBS包被缓冲液稀释至浓度分别为1 μM、5 μM、10 μM或20 μM,随后加入到ELISA板中,加入量为100 μL/孔,4℃过夜包被;
去除包被液,加入300 μL含有0.05% Tween20的1×PBST洗涤缓冲液,静置20 s,洗板3次,拍板;加入封闭液100 μL/孔,室温封闭2 h,37℃孵育1 h;
封闭结束后去除封闭液,将ELISA板拍干;
向酶标板中加入生物素标记S蛋白100 μL/孔,调控浓度比例,设置重复对照,封板后37℃培养90 min,拍板洗涤数次;
加入100 μL酶结合物工作液至反应孔中,封板后37℃培养30 min,拍板洗涤数次;
加入100 μL显色底物至反应孔中,封板后37℃培养15 min;
加入50 μL终止液,即刻用酶标仪测量450 nm波长下的OD值。
如图3所示,采用ELISA方法定性考察多肽与S蛋白的结合能力,结果显示,28条多肽与S蛋白均具有结合能力,但是结合能力存在差异,比如相比于其他多肽,KWPFKKS(SEQID NO: 11)、PIGVPHT(SEQ ID NO: 17)、GVVFLHVTYV(SEQ ID NO: 27)、AAARFST(SEQ IDNO: 16)和FVFLVLL(SEQ ID NO: 21)与S蛋白具有更强的结合能力,因此通过ELISA的结果进一步可以筛选出结合力相对更强的多肽。
为定量考察多肽与蛋白的结合,通过SPRi的方法测试多肽与蛋白的结合常数Kd。示例性地,如图4(A)、图4(B)和图4(C)所示,多肽KWPFKKS(SEQ ID NO: 11)、PIGVPHT(SEQID NO: 17)、NSGGSVH(SEQ ID NO: 15)的Kd值在10-7~10-9,说明多肽与S蛋白能很好的结合,具有很好的靶向作用。
实施例5 多肽的抗病毒效果
本实施例考察多肽抗病毒效果。示例性地,如图5(A)、图5(B)和图5(C)所示分别为多肽KWPFKKS(SEQ ID NO: 11)、PIGVPHT(SEQ ID NO: 17)、NSGGSVH(SEQ ID NO: 15)的抗病毒效果,随着浓度的增高,多肽表现出明显的抗病毒效果。纵坐标为荧光强度,横坐标为浓度,荧光强度越高表明抗病毒效果越好。
实施例6
本实施例对多肽PIGVPHT(SEQ ID NO: 17)、KWPFKKS(SEQ ID NO: 11)和NSGGSVH(SEQ ID NO: 15)作为活性肽进行了扩充,以验证在SEQ ID NO: 1~28的N端和C端添加序列后,得到新肽仍然具有结合S蛋白的活性。
图6(A)为在PIGVPHT的N端添加KGAG后得到的新肽的质谱图,图6(B)为在PIGVPHT的C端添加KGAG后得到的新肽的质谱图,图6(C)为在PIGVPHT的N端添加C12后得到的新肽的质谱图。
如图7所示,KGAGPIGVPHT(1’)、PIGVPHTKGAG(2’)、C12-PIGVPHT(3’)、KAAGGKWPFKKS(4’)、C12-KWPFKKS(5’)、NSGGSVHKNSG(6’)和GGAANSGGSVH(7’)与S蛋白均具有结合能力,说明以筛选的多肽SEQ ID NO: 1~28为功能性序列、在N端和/或C端添加序列后并不影响原多肽的S蛋白结合活性,获得的新肽同样具有结合S蛋白的能力。
如图8(A)、图8(B)、图8(C)、图8(D)、图8(E)、图8(F)和图8(G)所示分别为新肽1’、2’、3’、4’、5’、6’和7’与蛋白的结合常数Kd,Kd值在10-7~10-9,说明新肽与S蛋白能很好的结合,具有很好的靶向作用。
如图9所示为多肽KWPFKKS(SEQ ID NO: 11)及经过衍生后的新肽二级结构变化图,说明新肽并没有影响活性多肽的二级结构的变化。
综上所述,本发明采用OBOC法合成多肽库,筛选得到靶向冠状病毒COVID-19的S蛋白的多肽,所述多肽与S蛋白结合能力强,由此构建的免疫检测试剂盒为快速诊断病人提供了支持;本发明的多肽筛选方法具有通量高、筛选效率高等优势,为发现与新型冠状病毒结合的多肽提供了简便易行的方法。
申请人声明,本发明通过上述实施例来说明本发明的详细方法,但本发明并不局限于上述详细方法,即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
SEQUENCE LISTING
<110> 国家纳米科学中心
<120> 靶向新型冠状病毒COVID-19的多肽及其应用
<130> 20200520
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Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val
1115 1120 1125
Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro
1130 1135 1140
Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn
1145 1150 1155
His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn
1160 1165 1170
Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu
1175 1180 1185
Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu
1190 1195 1200
Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu
1205 1210 1215
Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met
1220 1225 1230
Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys
1235 1240 1245
Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro
1250 1255 1260
Val Leu Lys Gly Val Lys Leu His Tyr Thr
1265 1270
Claims (9)
1.靶向冠状病毒COVID-19的多肽,其特征在于,所述多肽为如SEQ ID NO: 11、15或17所示的氨基酸序列。
2.靶向冠状病毒COVID-19的分子,其特征在于,所述分子为KGAGPIGVPHT、PIGVPHTKGAG、月桂酸-PIGVPHT、KAAGGKWPFKKS、月桂酸-KWPFKKS、NSGGSVHKNSG和GGAANSGGSVH。
3.一种冠状病毒COVID-19检测试剂盒,其特征在于,所述试剂盒包括包被多肽或包被分子;其中,
所述多肽为权利要求1所述的多肽,所述分子为权利要求2所述的分子。
4.根据权利要求3所述的试剂盒,其特征在于,所述包被多肽或包被分子直接和/或间接包被在固相载体上。
5.根据权利要求4所述的试剂盒,其特征在于,所述固相载体包括酶标板、磁珠、微球、亲和膜或液相芯片中的任意一种或至少两种的组合。
6.根据权利要求3所述的试剂盒,其特征在于,所述试剂盒还包括S蛋白的特异性抗体。
7.根据权利要求6所述的试剂盒,其特征在于,所述S蛋白的特异性抗体上标记有信号基团。
8.根据权利要求3所述的试剂盒,其特征在于,所述试剂盒还包括显色液和/或终止液。
9.一种如权利要求1所述的多肽和/或如权利要求2所述的分子在制备冠状病毒COVID-19检测试剂和/或治疗药物中的应用。
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US10787501B1 (en) | 2020-04-02 | 2020-09-29 | Regeneron Pharmaceuticals, Inc. | Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments |
BR112022024662A2 (pt) | 2020-06-03 | 2023-04-04 | Regeneron Pharma | Métodos para tratamento ou prevenção de infecções por sars-cov-2 e covid-19 com anticorpos de glicoproteína spike anti-sars-cov-2 |
CN113769121B (zh) * | 2020-09-18 | 2022-10-28 | 中国原子能科学研究院 | 针对冠状病毒或流感病毒引发疾病的放射性治疗药物及其制备方法 |
CN112358533B (zh) * | 2020-10-30 | 2023-07-14 | 上海泽润生物科技有限公司 | 重组刺突蛋白及其制备方法和用途 |
CN112521452B (zh) * | 2020-12-08 | 2022-04-01 | 中山大学 | 靶向干扰素γ的多肽及其应用 |
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CN112763628A (zh) * | 2021-01-15 | 2021-05-07 | 北京生物制品研究所有限责任公司 | 新型冠状病毒灭活疫苗中新型冠状病毒n蛋白含量的检测方法 |
CN112684060A (zh) * | 2021-01-15 | 2021-04-20 | 北京生物制品研究所有限责任公司 | 新型冠状病毒灭活疫苗中新型冠状病毒s蛋白含量的检测方法 |
CN114858906A (zh) * | 2021-02-04 | 2022-08-05 | 北京毅新博创生物科技有限公司 | 用于诊断新冠肺炎的试剂盒 |
CN112876540B (zh) * | 2021-02-09 | 2022-07-15 | 中国医学科学院基础医学研究所 | 一种亲和肽在制备抗冠状病毒药物中的应用 |
WO2022202816A1 (ja) * | 2021-03-22 | 2022-09-29 | ペプチエイド株式会社 | ペプチド及びペプチドを含む組成物 |
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