WO2022227702A1 - 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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Definitions
- the invention belongs to the technical field of drug preparation, and in particular relates to a preparation method and application of an interfering peptide targeting SARS-CoV-2 N protein.
- SARS-CoV-2 belongs to the genus of Coronavirus (CoV), and its basic structure consists of spike (S) protein, envelope (Envelope, E) protein, membrane (membrane, M) protein, nucleocapsid (nucleocapsid) , N) protein and genomic single-stranded RNA composition.
- the N protein is the core component of the virion.
- the full-length SARS-CoV-2 N protein is 419 amino acids and is mainly composed of an N-terminal RNA-binding domain, a C-terminal dimerization domain, and other sequences connecting the two domains.
- SARS-CoV-2 N protein contains a number of relatively conserved RNA-binding positive charge distribution regions, which bind to viral genomic RNA and package RNA into ribonucleocapsid (RNP) complexes.
- RNP ribonucleocapsid
- its dimerization structure domain capable of mediating the formation of homo-oligomers of the SARS-CoV-2 N protein.
- prevention is dominated by various types of SARS-CoV-2 vaccines that are gradually being vaccinated around the world, but there is a lack of specific drugs for treatment. Small-molecule drugs generally have a long development cycle.
- the purpose of the present invention is to provide a preparation method of an interfering peptide targeting SARS-CoV-2 N protein, so as to prepare an interfering peptide drug (named as NIP-V hereinafter) for inhibiting SARS-CoV-2 in cells and animals replication and proliferation for the treatment of SARS-CoV-2 infection-related diseases.
- NIP-V interfering peptide drug
- the technical scheme of the present invention is: a preparation method of an interfering peptide targeting SARS-CoV-2 N protein, the method comprising the following steps:
- the amino acids are amino acids 346-357.
- amino acid sequence of amino acids 346-357 is FKDQVILLNKHI.
- amino acid is an L-type natural amino acid.
- step (b) the amino acid sequence of the HIV-TAT is YGRKKRRQRRR.
- step (c) the amino acid sequence of the final interfering peptide NIP-V is IHKNLLIVQDKFPPRRRQRRKKRG, and the molecular weight is 3040.69.
- Another technical solution of the present invention is: the application of an interfering peptide targeting the SARS-CoV-2 N protein in an anti-SARS-CoV-2 infection drug.
- the present invention provides a preparation method and application of an interfering peptide NIP-V targeting SARS-CoV-2 N protein, and its advantages are:
- the interfering peptide NIP-V targeting the SARS-CoV-2 N protein effectively relieves the antiviral immunosuppression mediated by the SARS-CoV-2 N protein, and significantly prevents the expression of SARS-CoV-2 in human vascular tension.
- Replication and proliferation in ACE2 (angiotensin-converting enzyme 2, ACE2) transgenic mice improved the ability of mice to resist SARS-CoV-2;
- the interaction between proteins is usually surface-surface interaction.
- the interfering peptide NIP-V targeting the SARS-CoV-2 N protein involved in the present invention can effectively block SARS-CoV -2 The interaction of the N protein itself;
- the interfering peptide NIP-V targeting the SARS-CoV-2 N protein contains the HIV-TAT sequence, which can directly make the peptide pass through the cell membrane and enter the cytoplasm to play a role without any carrier, avoiding the toxic and side effects caused by the carrier;
- the interfering peptide NIP-V targeting the N protein of SARS-CoV-2 is only a small peptide of 24 amino acids in length. According to the principle of immunology, it is not immunogenic and can avoid causing hypersensitivity reactions;
- the interfering peptide NIP-V targeting the N protein of SARS-CoV-2 can be obtained directly through the existing mature peptide synthesis technology, with high purity, controllable quality and great drug potential.
- the left picture of Figure 1 is the tertiary structure diagram of the dimerization domain (DD) dimer of the SARS-CoV-2 N protein, and the right picture is the schematic diagram of the target sequence of the interfering peptide NIP-V;
- DD dimerization domain
- Figure 2 is a schematic diagram of the molecular weight of the synthesized interfering peptide NIP-V detected by mass spectrometry (MS);
- FIG. 3 is a schematic diagram of the purity of the synthetic interfering peptide NIP-V detected by high performance liquid chromatography (HPLC);
- Figure 4 is a schematic diagram of nucleic acid detection analysis showing that treatment of ACE2 transgenic mice with interfering peptide NIP-V can significantly inhibit the proliferation of SARS-CoV-2 in lung tissue;
- Figure 5 is a schematic diagram showing that treatment of ACE2 transgenic mice with the interfering peptide NIP-V can inhibit the pulmonary lesions caused by SARS-CoV-2 infection by hematoxylin-eosin (HE) staining;
- HE hematoxylin-eosin
- Figure 6 is a schematic diagram showing that the use of interfering peptide NIP-V to treat ACE2 transgenic mice can inhibit the expression of SARS-CoV-2 N protein in the lungs after SARS-CoV-2 infection by immunofluorescence experiments;
- Figure 7 is a schematic diagram showing that the use of interfering peptide NIP-V to treat ACE2 transgenic mice can inhibit the expression of SARS-CoV-2 S protein in the lungs after SARS-CoV-2 infection by immunohistochemical experiments;
- Figure 8 is a schematic diagram showing that the interfering peptide NIP-V can enhance the secretion of IFN- ⁇ in the serum of ACE2 transgenic mice infected with SARS-CoV-2;
- Figure 9 is a schematic diagram showing that the interfering peptide NIP-V can enhance the expression of IFN- ⁇ and ISG56 mRNA in the spleen, liver and lung tissue of SARS-CoV-2 infected ACE2 transgenic mice, and reduce the load of SARS-CoV-2 genomic RNA;
- Figure 10 is a schematic diagram showing that the interfering peptide NIP-V can relieve the inhibition of the oligomerization of MAVS, a key adaptor protein of the innate immune signaling pathway, by the SARS-CoV-2 N protein.
- interfering peptide targeting SARS-CoV-2 N protein due to the large area of action of protein-protein interaction, a single small molecule may not be able to effectively interfere, and the use of macromolecular drugs such as possessing Peptides with similar action surfaces (interfering peptides) are very effective in interfering with protein-protein interactions.
- macromolecular drugs such as possessing Peptides with similar action surfaces (interfering peptides) are very effective in interfering with protein-protein interactions.
- interfering peptides According to the basic function of the SARS-CoV-2 N protein and the mechanism of inhibiting the body's innate antiviral immunity, artificially designed and synthesized interfering peptides targeting the N protein.
- the interfering peptide destroys the hydrophobic interaction by binding to the dimerization domain that mediates the oligomerization of the SARS-CoV-2 N protein, thereby releasing the inhibition of the innate immunity by the N protein, thereby inhibiting the SARS-CoV-2 virus in cells.
- the purpose of internal replication so as to achieve effective treatment of clinical conditions.
- the interfering peptide segment is first designed to target amino acids 346-357 located in the dimerization domain of SARS-CoV-2 N protein.
- the amino acid sequence of the segment is FKDQVILLNKHI, the natural amino acid is L-type, and the short peptide is designed to specifically destroy the interaction between N proteins;
- HIV-TAT is a hydrophilic sequence with the amino acid sequence YGRKKRRQRRR, which enables the peptide to cross the cell membrane in an energy-independent manner to be absorbed by the cell;
- DRI-modified peptides can improve the stability and effectiveness of peptides in cell and animal in vivo assays.
- the entire interfering peptide segment was modified into a reverse isomer, and the final amino acid sequence of the interfering peptide NIP-V was IHKNLLIVQDKFPPRRRQRRKKRG, and the molecular weight was 3040.69;
- the amino acid sequence of the interfering peptide drug NIP-V designed by the present invention is IHKNLLIVQDKFPPRRRQRRKKRG, and the targeting sequence is shown in Figure 1, using D-type amino acid as raw material to synthesize in Gill Biochemical (Shanghai) Co., Ltd.
- the synthesized interfering peptide drug NIP-V was identified by the Agilent-6125B LC/MS system (Agilent Technologies) to have a molecular weight of 3040.69, and the HPLC used an Inertsil ODS-SP liquid chromatography column (Shimadzu, 4.6 mm ⁇ 250 mm). ) as the stationary phase, using mobile phase A (100% acetonitrile, 0.1% trifluoroacetic acid) and mobile phase B (100% ultrapure water, 0.1% trifluoroacetic acid) for gradient elution, as shown in Figure 3 and Table 1 , identified by HPLC, the purity is greater than 98%.
- ACE2 transgenic mice Daan gene novel coronavirus (2019-nCoV) nucleic acid detection kit (fluorescence PCR method), SARS-CoV-2, NIP-V interfering peptide drug prepared in Example 1.
- the NIP-V interfering peptide drug was dissolved in sterile PBS to a concentration of 1 mg/mL.
- the ACE2 transgenic mice were divided into 4 groups with 8 mice in each group. The first and third groups were injected with 0.5 mL sterile PBS as control, and the second and fourth groups were injected with 0.5 mL (0.5 mg) NIP-V drug. After 1 hour, all four groups of mice were inoculated intranasally with SARS-CoV-2 after anesthesia, and each mouse was inoculated with about 1 ⁇ 10 5 TCID50 virus.
- the interfering peptide drug NIP-V can significantly reduce the load of SARS-CoV-2 in the lung tissue of ACE2 transgenic mice.
- Nucleic acid detection kit is one of the common methods to detect SARS-CoV-2 viral load.
- the SARS-CoV-2 genome in tissues was detected by absolute quantitative PCR method using Daan Gene Novel Coronavirus (2019-nCoV) nucleic acid detection kit. RNA copy number, the results are shown in Table 2.
- ACE2 transgenic mice SARS-CoV-2, NIP-V interfering peptide drug prepared in Example 1. Tissue fixation, embedding and related material reagents for HE staining (sankang).
- the NIP-V interfering peptide drug was dissolved in sterile PBS to a concentration of 1 mg/mL.
- the ACE2 transgenic mice were divided into 3 groups. The first group was not infected, the second group was injected with 0.5 mL of sterile PBS, and 1 hour later, 1 ⁇ 10 5 TCID50 of SARS-CoV-2 was intranasally inoculated, and the third group was injected with 0.5 mg of SARS-CoV-2.
- NIP-V drug, 1 x 105 TCID50 of SARS-CoV-2 was intranasally inoculated 1 hour later. Twenty-four hours after virus infection, mouse lung tissue was taken and placed in 4% paraformaldehyde/PBS for tissue fixation, paraffin sections of lung tissue were made, and the lesions of mouse lung were detected by HE staining.
- the interfering peptide drug NIP-V can significantly reduce the lung lesions of ACE2 transgenic mice caused by SARS-CoV-2 infection.
- the lung tissue of virus-uninfected mice was normal in morphology, with clear alveoli and thin septa.
- the alveolar septum was significantly thickened, and hemagglutination and inflammatory cell infiltration caused by virus infection were locally seen, which proved that the virus infection caused a significant inflammatory response.
- ACE2 transgenic mice SARS-CoV-2, NIP-V interfering peptide drug prepared in Example 1.
- Materials and reagents related to tissue fixation and embedding such as paraformaldehyde are all domestically produced.
- Rabbit anti-SARS-CoV-2 N protein antibody Abeam
- mouse anti-SARS-CoV-2 S protein antibody Abeam
- DAPI FITC-conjugated goat anti-rabbit IgG
- HRP-conjugated goat anti-mouse IgG CST
- DAB Chromogenic Kit Sanko
- mice The administration and virus stimulation of ACE2 transgenic mice were the same as in Example 3. Twenty-four hours after SARS-CoV-2 infection, mouse lung tissue was taken, tissue was fixed in 4% paraformaldehyde/PBS, and paraffin sections of lung tissue were made. The expression of SARS-CoV-2 S protein in the lungs of mice was detected by immunohistochemistry; the expression of SARS-CoV-2 N protein in the lungs of mice was detected by immunofluorescence assay.
- ACE2 transgenic mice Treatment of ACE2 transgenic mice with the interfering peptide drug NIP-V can significantly enhance the antiviral innate immune response of mice infected with SARS-CoV-2 and reduce viral proliferation in tissues.
- ACE2 transgenic mice SARS-CoV-2, NIP-V interfering peptide drug prepared in Example 1, mouse IFN- ⁇ ELISA detection kit, Trizol Japan (TAKARA company), reverse transcription kit, qPCR kit.
- Table 3 is the primers required for qPCR (synthesized by Goldwisdom)
- mice The administration and virus stimulation of ACE2 transgenic mice were the same as in Example 1. 16 and 24 hours after SARS-CoV-2 infection, blood was collected from the orbits of mice, and the blood was centrifuged at 1200 rpm for 5 min to remove blood cells and retain serum. The content of IFN- ⁇ in serum was detected by mouse IFN- ⁇ ELISA detection kit. The spleen, liver and lung tissues of mice were taken, and total RNA was extracted by Trizol method. After reverse transcription, the expression of Ifnb1 and Isg56 mRNA in spleen, liver and lung tissues and the load of SARS-CoV-2 genomic RNA were detected by qPCR. . Statistical analysis of the results was expressed as "mean ⁇ standard deviation" (mean ⁇ SEM), and compared by analysis of variance (ANOVA).
- the content of IFN- ⁇ in serum was detected by mouse IFN- ⁇ ELISA detection kit, and the results are shown in Table 4.
- the interfering peptide drug NIP-V significantly increased the content of IFN- ⁇ in the serum of ACE2 transgenic mice infected with SARS-CoV-2.
- Figure 8 As shown in Figure 8, in the PBS-treated groups (groups 1 and 3), IFN- ⁇ in the serum of mice infected with SARS-CoV-2 showed a downward trend with time, while in the NIP-V-treated group In the middle (groups 2 and 4), the content of IFN- ⁇ in the serum of mice was significantly higher than that in the PBS-treated group (Fig. 8). It can be seen that the interfering peptide drug NIP-V can significantly increase the IFN- ⁇ content in the serum of ACE2 transgenic mice infected with SARS-CoV-2.
- HEK 293T cells were plated in 6-well plates. When the cell density reached 70%, Myc-NP plasmid was transfected in 3 wells. After 24 h, cells were treated with 50 ⁇ M and 100 ⁇ M NIP-V, and 1 h later, cells were stimulated with SeV for 8 h. After 8 h, the cells were collected, and the oligomerization of MAVS was detected by semi-denaturing electrophoresis (SDD-PAGE).
- MAVS is an important adaptor protein in the innate immune signaling pathway, and oligomerization is one of the important signs of the activation of the antiviral innate immune pathway.
- SARS-CoV-2 N protein can inhibit antiviral innate immunity by acting on MAVS.
- NIP-V could rescue the innate immune signaling pathway inhibited by N protein by SDD-PAGE experiments.
- the interfering peptide drug NIP-V provided by the present invention can interact with the dimerization domain of the SARS-CoV-2 N protein, inhibit the oligomerization of the N protein, and then relieve the inhibition of the innate immunity by the N protein, thereby To achieve the purpose of inhibiting the replication of SARS-CoV-2 virus in cells and animals.
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Abstract
本发明公开了一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法,该方法包括如下步骤:设计干扰肽段靶向位于SARS-CoV-2 N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-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。
Description
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。
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-2 N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-2 N蛋白的干扰肽NIP-V可有效阻断SARS-CoV-2 N蛋白自身的相互作用;
3、靶向SARS-CoV-2 N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-2 N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-2 N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。
图1的左图为SARS-CoV-2 N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-2 N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-2S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、ISG56mRNA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。
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,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-2 N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(Agilent Technologies)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%乙腈,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1×10
5TCID50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<0.0l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1×10
5TCID50的SARS-CoV-2,第三组注射0.5mg的NIP-V药物,1小时后鼻内接种1×10
5TCID50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-2S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-2S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-2S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 Isg56 forward | AAGACAAGGCAATCACCCTCTACT |
Murine Isg56 reverse | GTCTTTCAGCCACTTTCTCCAAA |
SARS-CoV-2 forward | CTTCTCGTTCCTCATCACGTAGTC |
SARS-CoV-2 reverse | 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-βELISA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、Isg56 mRNA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<0.0l为极显著性差异。
3实验结果
通过鼠IFN-βELISA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、Isg56mRNA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、Isg56mRNA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。
Claims (7)
- 一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法,其特征在于,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。
- 根据权利要求1所述的一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法,其特征在于:在步骤(a)中,所述氨基酸为346-357位氨基酸。
- 根据权利要求2所述的一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法,其特征在于:所述346-357位氨基酸的氨基酸序列为FKDQVILLNKHI。
- 根据权利要求2所述的一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法,其特征在于:所述氨基酸为L-型天然氨基酸。
- 根据权利要求1所述的一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法,其特征在于:在步骤(b)中,所述HIV-TAT的氨基酸序列为YGRKKRRQRRR。
- 根据权利要求1所述的一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法,其特征在于: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。
- 一种靶向SARS-CoV-2 N蛋白的干扰肽在抗SARS-CoV-2感染中的应用。
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