CN113072623B - 一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法及应用 - Google Patents
一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种靶向SARS‑CoV‑2N蛋白的干扰肽的制备方法,该方法包括如下步骤:设计干扰肽段靶向位于SARS‑CoV‑2N蛋白的二聚化结构域内的氨基酸;将所述干扰肽段与HIV‑TAT融合;将与HIV‑TAT融合的干扰肽段修饰为逆向异构体,获得最终干扰肽NIP‑V的氨基酸序列;使用D‑型氨基酸为原料合成干扰肽NIP‑V。上述干扰肽药物NIP‑V能够与SARS‑CoV‑2N蛋白的二聚结构域相互作用,抑制N蛋白寡聚化,进而解除N蛋白对先天免疫的抑制,从而达到抑制SARS‑CoV‑2病毒在细胞和动物体内复制的目的。
Description
技术领域
本发明属于药物制备技术领域,具体涉及一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法及应用。
背景技术
SARS-CoV-2属冠状病毒属(Coronavirus,CoV),其基本结构由刺突(spike,S)蛋白、包膜(envelope,E)蛋白、膜(membrane,M)蛋白、核衣壳(nucleocapsid,N)蛋白以及基因组单链RNA组成。N蛋白是病毒粒子的核心成分,SARS-CoV-2 N蛋白全长419个氨基酸,主要由N端RNA结合结构域、C端二聚结构域以及连接两个结构域的其他序列构成。SARS-CoV-2 N蛋白全序列含有多个较为保守的结合RNA的正电荷分布区域,其与病毒基因组RNA结合,将RNA包装成核糖核蛋白(ribonucleocapsid,RNP)复合体,此外其二聚结构域能够介导SARS-CoV-2 N蛋白形成同源寡聚体。目前在新型冠状病毒肺炎防治相关工作中,预防以全球逐渐开始接种的多类SARS-CoV-2疫苗为主流,但治疗则缺乏特效药。小分子药物一般研制周期很长,目前都是旧药新用,且药效饱受质疑,比如羟氯喹、洛匹那韦/利托那韦以及瑞德西韦等。除小分子药物外,血浆疗法虽好,但有一定风险。因为血浆因人而异,是一个复杂的混合物,而且血浆来源有限,不能大规模使用。
因此,针对上述问题,有必要提出进一步的解决方案。
发明内容
本发明目的是提供一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法,以制备一种干扰肽药物(以下命名为NIP-V)用于抑制细胞和动物体内SARS-CoV-2的复制和增殖,治疗SARS-CoV-2感染相关的疾病。
本发明的技术方案是:一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法,该方法包括如下步骤:
(a)设计干扰肽段靶向位于SARS-CoV-2 N蛋白二聚结构域内的氨基酸;
(b)将所述干扰肽段与HIV-TAT融合;
(c)将与HIV-TAT融合的干扰肽段修饰为逆向异构体,获得最终干扰肽NIP-V的氨基酸序列;
(d)使用D-型氨基酸为原料合成干扰肽NIP-V。
进一步的,在步骤(a)中,所述氨基酸为346-357位氨基酸。
进一步的,所述346-357位氨基酸的氨基酸序列为FKDQVILLNKHI。
进一步的,所述氨基酸为L-型天然氨基酸。
进一步的,在步骤(b)中,所述HIV-TAT的氨基酸序列为YGRKKRRQRRR。
进一步的,在步骤(c)中,所述最终干扰肽NIP-V的氨基酸序列为IHKNLLIVQDKFPPRRRQRRKKRG,分子量为3040.69。
本发明的另一技术方案是:一种靶向SARS-CoV-2N蛋白的干扰肽在抗SARS-CoV-2感染药物中的应用。
本发明提供了一种靶向SARS-CoV-2 N蛋白的干扰肽NIP-V的制备方法及应用,其优点是:
1、靶向SARS-CoV-2 N蛋白的干扰肽NIP-V有效解除了SARS-CoV-2 N蛋白介导的抗病毒免疫抑制,显著地阻止了SARS-CoV-2在表达人源血管紧张素转化酶2(angiotensin-converting enzyme 2,ACE2)转基因小鼠中的复制和增殖,提升了小鼠抗SARS-CoV-2的能力;
2、蛋白间的相互作用通常为面-面互作,相比于传统的小分子药物,本发明涉及的靶向SARS-CoV-2N蛋白的干扰肽NIP-V可有效阻断SARS-CoV-2N蛋白自身的相互作用;
3、靶向SARS-CoV-2N蛋白的干扰肽NIP-V包含HIV-TAT序列,可直接使肽段穿过细胞膜进入细胞质内发挥作用,不需要任何的载体,避免载体引起的毒副作用;
4、D-型氨基酸较天然L-型氨基酸在动物体内的降解较为缓慢,将干扰肽修饰为D-型氨基酸逆向(D-retroinverso,DRI)异构体在以往的临床试验中被证明具有良好的耐受性和治疗效果,所以靶向SARS-CoV-2N蛋白的DRI修饰干扰肽NIP-V具备进行临床试验的可行性;
5、靶向SARS-CoV-2N蛋白的干扰肽NIP-V长度仅为24氨基酸小肽,根据免疫学原理本身不具备免疫原性,可避免引起超敏反应;
6、靶向SARS-CoV-2 N蛋白的干扰肽NIP-V可直接通过现有成熟的多肽合成技术获得,具有高纯度,质量可控,成药潜力较大。
附图说明
图1的左图为SARS-CoV-2N蛋白的二聚结构域(dimerization domain,DD)二聚体的三级结构图,右图为干扰肽NIP-V的靶序列示意图;
图2为用质谱法(mass spectrometry,MS)检测合成的干扰肽NIP-V的分子量示意图;
图3为用高效液相色谱法(high performance liquid chromatography,HPLC)检测合成的干扰肽NIP-V的纯度示意图;
图4为核酸检测分析发现使用干扰肽NIP-V处理ACE2转基因小鼠可显著抑制SARS-CoV-2在肺组织中的增殖示意图;
图5为通过苏木精-伊红(hematoxylin-eosin,HE)染色发现使用干扰肽NIP-V处理ACE2转基因小鼠可抑制SARS-CoV-2感染导致的肺部的病变的示意图;
图6为通过免疫荧光实验发现使用干扰肽NIP-V处理ACE2转基因小鼠可抑制SARS-CoV-2感染后肺部SARS-CoV-2N蛋白的表达的示意图;
图7为通过免疫组化实验发现使用干扰肽NIP-V处理ACE2转基因小鼠可抑制SARS-CoV-2感染后肺部SARS-CoV-2 S蛋白的表达的示意图;
图8为干扰肽NIP-V可增强SARS-CoV-2感染ACE2转基因小鼠后血清中的IFN-β分泌的示意图;
图9为干扰肽NIP-V可增强SARS-CoV-2感染ACE2转基因小鼠后脾、肝、肺组织中的IFN-β、ISG56 mRNA表达,降低SARS-CoV-2基因组RNA的载量的示意图;
图10为干扰肽NIP-V可解除SARS-CoV-2 N蛋白对固有免疫信号通路关键接头蛋白MAVS的寡聚化的抑制的示意图。
具体实施方式
本发明所述的一种靶向SARS-CoV-2N蛋白的干扰肽的制备方法,介于蛋白质-蛋白质相互作用由于作用面积较大,单个小分子可能无法有效干扰,利用大分子药物比如拥有类似作用面的多肽(干扰肽)来干扰蛋白间的相互作用十分有效。根据SARS-CoV-2 N蛋白的基本功能以及抑制机体先天抗病毒免疫的机制,人工设计并合成靶向N蛋白的干扰肽。该干扰肽通过结合介导SARS-CoV-2N蛋白寡聚的二聚结构域的作用面,破坏疏水相互作用,进而解除N蛋白对先天免疫的抑制,达到抑制SARS-CoV-2病毒在细胞内复制的目的,从而实现临床病症的有效治疗。
本发明所述的一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法,首先设计干扰肽段靶向位于SARS-CoV-2 N蛋白二聚结构域内的346-357位氨基酸,该段氨基酸序列为FKDQVILLNKHI,天然氨基酸为L-型,设计短肽用于特异性破坏N蛋白间的互作;
其次,为了促进细胞对NIP-V的吸收,干扰肽段设计为与HIV-TAT融合。HIV-TAT是一段亲水的序列,氨基酸序列为YGRKKRRQRRR,能够通过不依赖能量的方式使肽段跨过细胞膜从而被细胞吸收;
然后,DRI修饰肽段可以提高肽段在细胞和动物体内试验的稳定性和有效性。将整条干扰肽段修饰为逆向异构体,最终干扰肽NIP-V的氨基酸序列为IHKNLLIVQDKFPPRRRQRRKKRG,分子量为3040.69;
最后,使用D-型氨基酸为原料合成干扰肽NIP-V,肽段的纯度在98%以上。
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合实施例进一步说明本发明的技术方案。但是本发明不限于所列出的实施例,还应包括在本发明所要求的权利范围内其他任何公知的改变。
此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例,也不是单独的或选择性的与其他实施例互相排斥的实施例。
实施例1
干扰肽药物NIP-V的合成和检测
本发明设计的干扰肽药物NIP-V氨基酸序列为IHKNLLIVQDKFPPRRRQRRKKRG,靶向序列如图1所示,使用D-型氨基酸为原料于吉尔生化(上海)有限公司合成。
如图2所示,合成的干扰肽药物NIP-V经Agilent-6125B液质联用系统(AgilentTechnologies)鉴定分子量为3040.69,HPLC采用Inertsil ODS-SP液相色谱柱(岛津,4.6mm×250mm)为固定相,使用流动相A(100%腈,0.1%三氟乙酸)和流动相B(100%超纯水,0.1%三氟乙酸)进行梯度洗脱,如图3及表1所示,经HPLC鉴定,纯度大于98%。
保留时间 | 含量(%) | 峰面积 | 峰高度 |
11.891 | 98.03 | 6613214 | 514625 |
12.236 | 1.966 | 132591 | 20430 |
表1
实施例2
使用干扰肽药物NIP-V处理ACE2转基因小鼠可显著降低SARS-CoV-2在小鼠体内的增殖。
1实验材料
ACE2转基因小鼠,达安基因新型冠状病毒(2019-nCoV)核酸检测试剂盒(荧光PCR法),SARS-CoV-2,实施例1中制备的NIP-V干扰肽药物。
2实验方法
使用灭菌PBS溶解NIP-V干扰肽药物,使其浓度为1mg/mL。ACE2转基因小鼠分为4组,每组8只,第一三组注射0.5mL灭菌PBS作为对照,第二四组注射0.5mL(0.5mg)NIP-V药物。1小时后,所有四组小鼠麻醉后鼻内接种SARS-CoV-2,每只小鼠接种约1×105TCID50病毒。感染病毒16小时、24小时后,分别取第一二组、第三四组小鼠肺组织,通过达安基因公司的新型冠状病毒(2019-nCoV)核酸检测试剂盒对小鼠肺部SARS-CoV-2的核酸含量进行检测。结果的统计分析以“均值±标准偏差”(mean±SEM)表示,采用方差分析(ANOVA)进行比较,p<0.05为显著性差异,p<0.01为极显著性差异。
3实验结果
请参阅图4,如图4所示,干扰肽药物NIP-V可显著降低SARS-CoV-2在ACE2转基因小鼠肺组织中的载量。
核酸检测试剂盒是检测SARS-CoV-2病毒载量的常用手段之一,通过达安基因新型冠状病毒(2019-nCoV)核酸检测试剂盒通过绝对定量PCR方法检测组织中SARS-CoV-2基因组RNA拷贝数,结果见表2。
表2
表2结果表明在PBS处理组中(1、3组),随着SARS-CoV-2病毒载量随时间出现上升趋势,而在NIP-V处理组中(2、4组),SARS-CoV-2病毒载量大大降低,且多数小鼠肺组织中无法检测到SARS-CoV-2。由此可知,干扰肽药物NIP-V可显著降低SARS-CoV-2在ACE2转基因小鼠肺组织中的载量。
实施例3
使用干扰肽药物NIP-V处理ACE2转基因小鼠可显著抑制SARS-CoV-2感染导致的小鼠肺部的病变。
1实验材料
ACE2转基因小鼠,SARS-CoV-2,实施例1中制备的NIP-V干扰肽药物。组织固定、包埋以及HE染色相关材料试剂(生工)。
2实验方法
使用灭菌PBS溶解NIP-V干扰肽药物,使其浓度为1mg/mL。ACE2转基因小鼠分为3组,第一组不感染,第二组注射0.5mL灭菌PBS,1小时后鼻内接种1×105TCID50的SARS-CoV-2,第三组注射0.5mg的NIP-V药物,1小时后鼻内接种1×105TCID50的SARS-CoV-2。病毒感染24小时后,取小鼠肺组织,置于4%多聚甲醛/PBS中进行组织固定,制作肺组织的石蜡切片,通过HE染色检测小鼠肺部的病变。
3实验结果
请参阅图5,如图5所示,干扰肽药物NIP-V可显著降低SARS-CoV-2感染导致的ACE2转基因小鼠肺部病变。未感染病毒的小鼠的肺组织形态正常,肺泡清晰,间隔纤细。而PBS+SARS-CoV-2处理组肺泡间隔显著增厚,局部可见病毒感染引起的血细胞凝集以及炎性细胞浸润,证明病毒感染引起了明显的炎症反应。而NIP-V+SARS-CoV-2处理组肺泡间隔增厚不明显,且血细胞凝集和炎性细胞浸润现象显著低于PBS+SARS-CoV-2组。
实施例4
使用干扰肽药物NIP-V处理ACE2转基因小鼠可显著抑制小鼠肺组织中SARS-CoV-2的N蛋白、S蛋白的表达。
1实验材料
ACE2转基因小鼠,SARS-CoV-2,实施例1中制备的NIP-V干扰肽药物。多聚甲醛等组织固定、包埋相关材料试剂均为国产。兔抗SARS-CoV-2 N蛋白抗体(Abcam),鼠抗SARS-CoV-2 S蛋白抗体(Abcam),DAPI,FITC偶连的羊抗兔IgG,HRP偶连的羊抗鼠IgG(CST),DAB显色试剂盒(生工)
2实验方法
ACE2转基因小鼠的给药和病毒刺激同实施例3。SARS-CoV-2感染24小时后,取小鼠肺组织,使用4%多聚甲醛/PBS中进行组织固定,制作肺组织的石蜡切片。通过免疫组化检测小鼠肺部SARS-CoV-2 S蛋白的表达情况;通过免疫荧光实验检测小鼠肺部SARS-CoV-2 N蛋白的表达情况。
3实验结果
请参阅图6和图7,如图6中所示,我们通过偶连FITC的荧光二抗检测SARS-CoV-2 N蛋白的表达,在未感染病毒的小鼠的肺组织中几乎无荧光信号,而在PBS+SARS-CoV-2处理组小鼠的肺组织中具有较强的荧光信号,在NIP-V+SARS-CoV-2处理组小鼠的肺组织中只能检测到较弱的N蛋白的信号。同样地,如图7所示,通过免疫组化检测SARS-CoV-2 S蛋白在ACE2转基因小鼠肺部的表达,通过DAB显色法在存在S蛋白的部位出现棕红色染色。在未感染病毒的小鼠的肺组织中无S蛋白信号,在PBS+SARS-CoV-2处理组小鼠出现强S蛋白信号和炎性细胞浸润,而在NIP-V+SARS-CoV-2处理组小鼠的肺组织中的S蛋白信号远低于PBS+SARS-CoV-2处理组小鼠。由此可知,干扰肽药物NIP-V可明显降低SARS-CoV-2感染ACE2转基因小鼠后肺部的SARS-CoV-2 N蛋白和S蛋白表达。
实施例5
使用干扰肽药物NIP-V处理ACE2转基因小鼠可显著提升小鼠感染SARS-CoV-2的抗病毒固有免疫反应,降低组织中病毒增殖。
1实验材料
ACE2转基因小鼠,SARS-CoV-2,实施例一中制备的NIP-V干扰肽药物,鼠IFN-βELISA检测试剂盒,Trizol日本(TAKARA公司),反转录试剂盒,qPCR试剂盒。表3为进行qPCR所需要的引物(金唯智合成)
Primers | Sequence(5′-3′) |
Murine 18S forward | CGCGGTTCTATTTTGTTGGT |
Murine 18S reverse | AGTCGGCATCGTTTATGGTC |
Murine Ifnb1 forward | TCCTGCTGTGCTTCTCCACCACA |
Murine Ifnb1 reverse | AAGTCCGCCCTGTAGGTGAGGTT |
Murine Isg56forward | AAGACAAGGCAATCACCCTCTACT |
Murine Isg56reverse | GTCTTTCAGCCACTTTCTCCAAA |
SARS-CoV-2forward | CTTCTCGTTCCTCATCACGTAGTC |
SARS-CoV-2reverse | TTGCTCTCAAGCTGGTTCAATC |
表3
2实验方法
ACE2转基因小鼠的给药和病毒刺激同实施例1。SARS-CoV-2感染16、24小时后,从小鼠眼眶取血,将血液通过1200rpm 5min离心去除血细胞,保留血清。使用鼠IFN-β ELISA检测试剂盒检测血清中的IFN-β含量。取小鼠的脾、肝、肺组织,使用Trizol法提取总RNA,反转录后通过qPCR检测脾、肝、肺组织中的Ifnb1、Isg56mRNA的表达以及SARS-CoV-2基因组RNA的载量。结果的统计分析以“均值±标准偏差”(mean±SEM)表示,采用方差分析(ANOVA)进行比较,p<0.05为显著性差异,p<0.01为极显著性差异。
3实验结果
通过鼠IFN-β ELISA检测试剂盒检测血清中的IFN-β含量,结果见表4。
表4
如表4所示,干扰肽药物NIP-V显著提升SARS-CoV-2感染ACE2转基因小鼠血清中IFN-β的含量。请参阅图8,如图8所示,在PBS处理组中(1、3组),小鼠感染SARS-CoV-2后血清中IFN-β随时间出现下降趋势,而在NIP-V处理组中(2、4组),小鼠血清中IFN-β含量显著高于PBS处理组(图8)。由此可知,干扰肽药物NIP-V可明显提升SARS-CoV-2感染ACE2转基因小鼠后血清中的IFN-β含量。
如图9所示,使用干扰肽药物NIP-V预处理ACE2转基因小鼠后,感染SARS-CoV-2所诱导的Ifnb1、Isg56 mRNA的表达相对PBS处理组均有所提升,证明NIP-V处理增强了小鼠对SARS-CoV-2的抗病毒固有免疫反应;从而降低组织内SARS-CoV-2的增殖。相对于未刺激小鼠,mRNA增加倍数如下表5所示。
表5
如表5所示,qPCR结果表明NIP-V处理提升SARS-CoV-2感染ACE2转基因小鼠脾、肝、肺组织中Ifnb1、Isg56 mRNA的表达,抑制SARS-CoV-2增殖。
实施例6
使用干扰肽药物NIP-V处理细胞可解除SARS-CoV-2 N蛋白对MAVS寡聚化的抑制。
1实验材料
SARS-CoV-2 N蛋白表达质粒Myc-NP,按照实施例一中制备的NIP-V干扰肽药物,仙台病毒(SeV),胎牛血清、DMEM培养基、青霉素/链霉素溶液(Gibco);HEK 293T细胞系(ATCC来源),MAVS抗体、Myc抗体及相关二抗(CST公司)。
2实验方法
将HEK 293T细胞铺6孔板,待细胞密度达到70%时,在3个孔中转染Myc-NP质粒,24h后使用50μM、100μM NIP-V处理细胞,1h后使用SeV刺激细胞8h。8h后收集细胞,通过半变性电泳(SDD-PAGE)的方法检测MAVS寡聚化的情况。
3实验结果
MAVS是固有免疫信号通路中重要的接头蛋白,寡聚化是抗病毒固有免疫通路激活的重要标志之一。而SARS-CoV-2 N蛋白可通过作用于MAVS抑制抗病毒固有免疫。我们通过SDD-PAGE实验研究NIP-V是否可拯救被N蛋白抑制的固有免疫信号通路。如图10所示,在静息状态下,MAVS无寡聚化发生;SeV刺激激活固有免疫信号通路,可诱导MAVS发生寡聚化;在细胞中转染N蛋白可抑制SeV诱导的MAVS寡聚化;而使用NIP-V预先处理细胞,可呈剂量依赖性地恢复MAVS的寡聚化,证明NIP-V可解除SARS-CoV-2 N蛋白对MAVS的抑制,增强固有免疫信号传递。
综上所述,本发明提供的干扰肽药物NIP-V能够与SARS-CoV-2 N蛋白的二聚结构域相互作用,抑制N蛋白寡聚化,进而解除N蛋白对先天免疫的抑制,从而达到抑制SARS-CoV-2病毒在细胞和动物体内复制的目的。
应说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围当中。
SEQUENCE LISTING
<110>苏州大学
<120>一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法及应用
<160> 3
<210>1
<211>12
<212> PRT
<213>人工序列
<400> 1
FKDQVILLNKHI 12
<210> 2
<211>11
<212> PRT
<213>人工序列
<400>2
YGRKKRRQRRR 11
<210>3
<211>24
<212> PRT
<213>人工序列
<400>3
IHKNLLIVQDKFPPRRRQRRKKRG 24
Claims (2)
1.一种靶向SARS-CoV-2的N蛋白的干扰肽,其特征在于:干扰肽NIP-V的氨基酸序列为IHKNLLIVQDKFPPRRRQRRKKRG,分子量为3040.69Da。
2.如权利要求1所述的一种靶向SARS-CoV-2的N蛋白的干扰肽在制备抗SARS-CoV-2感染药物中的应用。
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