WO2022205822A1 - 一种抑制新型冠状病毒感染的抗菌肽及其用途 - Google Patents

一种抑制新型冠状病毒感染的抗菌肽及其用途 Download PDF

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WO2022205822A1
WO2022205822A1 PCT/CN2021/122042 CN2021122042W WO2022205822A1 WO 2022205822 A1 WO2022205822 A1 WO 2022205822A1 CN 2021122042 W CN2021122042 W CN 2021122042W WO 2022205822 A1 WO2022205822 A1 WO 2022205822A1
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novel coronavirus
infection
cov
sars
inhibiting
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PCT/CN2021/122042
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French (fr)
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凌建群
王益行
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杭州先端生物科技有限公司
江苏吉锐生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K4/00—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • the invention relates to the technical field of biomedicine, in particular to an antibacterial peptide for inhibiting novel coronavirus infection and use thereof.
  • the new coronavirus SARS-CoV-2 is mainly transmitted through respiratory droplets and close contact. It has a strong infectious ability. Some patients are asymptomatic but have the ability to infect others. The susceptibility of the crowd is high, and the infectivity is significantly increased. The difficulty of preventing and controlling the source is greatly increased.
  • Vaccines mainly include: inactivated or attenuated virus vaccines, protein-based vaccines, vector vaccines, DNA and RNA vaccines, etc. (Chen Huijuan, Liu Qiqi, Wang Shengqi. Novel Coronavirus Pneumonia: Laboratory Testing, Treatment and Vaccines (English) [J] . Chinese Journal of Biochemistry and Molecular Biology, 2021, 37(01):11-19.).
  • drugs and vaccines have more or less certain problems in the process of treating the new coronavirus, such as poor treatment effect, adverse reactions, and poor stability.
  • Antibacterial peptides as new biologically active ingredients, have attracted widespread attention because of their broad-spectrum antibacterial properties, inhibiting or killing bacteria, fungi, viruses, parasites, etc., and being mild and non-irritating.
  • Antimicrobial peptide AMPs have been reported to be excellent candidates for viral infection (Abid, Qureshi, Nishant, et al. AVPdb: a database of experimentally validated antiviral peptides targeting medically important viruses [J]. Nucleic Acids Research, 2013, 42(D1): 1147-53.).
  • Zhao H disclosed that among 11 peptides derived from mouse ⁇ -defensin 4, only the short peptide P9 exhibited potent broad-spectrum antiviral effects against a variety of respiratory viruses, including influenza A viruses H1N1, H3N2, H5N1, H7N7, H7N9, SARS-CoV and MERS-CoV, while the other 10 peptides derived from mouse ⁇ -defensin 4 had no anti-SARS-CoV effect (Zhao H, Zhou J, Zhang K, et al. al.A novel peptide with potent and broad-spectrum antiviral activities again multiple respiratory viruses[J].Rep,2016,6(1):22008.).
  • the inventors unexpectedly found that the antimicrobial peptide of the present invention has the effect of inhibiting the new coronavirus, has no cytotoxicity, high safety, no hormones, and no antibiotics, which is of great significance for the prevention and treatment of the new coronavirus , to further provide new ideas and scientific basis for clinical prevention and treatment of SARS-CoV-2 infection.
  • one object of the present invention is to provide an antimicrobial peptide that inhibits novel coronavirus infection
  • the second object is to provide an antimicrobial peptide composition that inhibits novel coronavirus infection
  • the third object is to provide A kind of antibacterial peptide for inhibiting novel coronavirus infection and purposes thereof, characterized in that: the amino acid sequence of the antibacterial peptide of the present invention is SEQ ID: NO.1.
  • a kind of antibacterial peptide for inhibiting novel coronavirus infection and purposes thereof characterized in that the amino acid sequence of the antibacterial peptide is SEQ ID NO.1.
  • the novel coronavirus is SARS-CoV-2.
  • the uses of the antimicrobial peptides and compositions thereof include, but are not limited to, inhibition of novel coronavirus, treatment or prevention of diseases caused by novel coronavirus infection, and improvement of disease symptoms caused by novel coronavirus infection.
  • compositions for inhibiting novel coronavirus infection characterized in that the composition comprises the antibacterial peptide, and the amino acid sequence of the antibacterial peptide is SEQ ID NO.1.
  • the antimicrobial peptide composition includes other pharmaceutically acceptable adjuvants, carriers or auxiliary components.
  • the other pharmaceutically acceptable adjuvants, carriers or auxiliary components include drugs for the treatment of novel coronavirus infection.
  • the present invention provides an antibacterial peptide for inhibiting novel coronavirus infection and use thereof.
  • the antibacterial peptide of the present invention has the effect of inhibiting novel coronavirus, and at 80 ⁇ M, the inhibition rate of SARS-CoV-2 is 87.1%, and With the increase of the concentration of antimicrobial peptides, the inhibitory effect of antimicrobial peptides on the expression of viral genes is continuously enhanced, and at the same time, it has no cytotoxicity, and can be used for COVID-19 after compounding with other pharmaceutically acceptable excipients, carriers or auxiliary components. Treatment and related uses of -19. Compared with the prior art, the antimicrobial peptides and their compositions can effectively inhibit the new coronavirus, and have high safety, no hormones, and no antibiotics. Provide new ideas and scientific basis for infection and treatment of SARS-CoV-2 infection.
  • FIG. 1 Immunofluorescence detection of infection of Vero-E6 cells by SARS-CoV-2.
  • Figure 2 RT-qPCR detection of the effect of different concentrations of antimicrobial peptides on Vero-E6 cells infected by SARS-CoV-2.
  • One object of the present invention is to provide an antibacterial peptide for inhibiting novel coronavirus infection
  • the second object is to provide an antibacterial peptide composition for inhibiting novel coronavirus infection
  • the third object is to provide an antibacterial peptide composition for inhibiting novel coronavirus infection
  • the antibacterial peptide and the purposes of the composition thereof are characterized in that: the amino acid sequence of the antibacterial peptide of the present invention is SEQ ID: NO.1.
  • the novel coronavirus is SARS-CoV-2.
  • the uses of the antimicrobial peptides and compositions thereof include, but are not limited to, inhibition of novel coronavirus, treatment or prevention of diseases caused by novel coronavirus infection, and improvement of disease symptoms caused by novel coronavirus infection.
  • compositions for inhibiting novel coronavirus infection characterized in that the composition comprises the antibacterial peptide, and the amino acid sequence of the antibacterial peptide is SEQ ID NO.1.
  • the antimicrobial peptide composition includes other pharmaceutically acceptable adjuvants, carriers or auxiliary components.
  • the other pharmaceutically acceptable excipients, carriers or auxiliary components include drugs for treating novel coronavirus infection.
  • the antimicrobial peptide or composition of the present invention is administered to humans or animals in a safe and effective amount.
  • Those skilled in the art can determine the preferred dosage suitable for certain situations according to conventional methods. The specific dosage should consider the route of administration, The patient's health and other factors are within the skill of the skilled physician.
  • the peptides and compositions of the present invention can be administered by spray or inhalation.
  • antibacterial peptides and compositions thereof of the present invention other pharmaceutically acceptable adjuvants, carriers or auxiliary components can be used in an amount within a common range, and the appropriate amount can be determined by those of ordinary skill in the art according to practical applications .
  • antimicrobial peptides described in the present invention can be obtained by chemical synthesis, expression, separation and purification by genetic engineering technology (for specific methods, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).
  • composition and “antibacterial peptide composition” are not specified and can be used interchangeably, and refer to a product containing the antibacterial peptide of the present invention as an active ingredient.
  • the "new coronavirus” mentioned is a new type of virus, which is SARS-CoV-2, and "2019 novel coronavirus” and “2019-nCoV” are both aliases of this virus.
  • SARS-CoV-2 infection is an infectious disease caused by a novel coronavirus (SARS-CoV-2), including but not limited to "new coronavirus pneumonia”.
  • preventing SARS-CoV-2 infection includes the use of preventing novel coronavirus from infecting healthy people, and refers to preventive measures to protect or prevent individuals who have not yet been infected with the virus.
  • pharmaceutically acceptable means that a substance or composition must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the mammal to be treated with it.
  • adjuvants include non-toxic solid fillers, diluents, adjuvants, wrapping materials or other formulation adjuvants.
  • pharmaceutically acceptable carrier refers to any one of various carriers or carriers known to those skilled in the art. Additionally, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions and emulsions. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, inert gases, and the like.
  • the technical means used in the examples are conventional means known to those skilled in the art, the unmentioned experimental methods are conventional experimental methods, various raw materials, reagents, instruments and equipment used in the examples, etc. Unless otherwise specified, they can be purchased from the market or can be prepared by existing methods.
  • SARS-CoV-2 virus strain The strain isolated by Naval Military Medical University in February 2020, genome information: GenBank: MT627325.1.
  • Vero-E6 cells were co-infected and detected by immunofluorescence to verify whether the antimicrobial peptides had inhibitory effects on the new coronavirus.
  • Vero-E6 cells Resuscitate and activate Vero-E6 cells.
  • the cell activation is complete and the viability is good, inoculate it in a 96-well cell culture plate with a seeding density of about 80%; the medium used for cell culture is: DMEM + 10% fetal bovine serum (FBS). )+DA;
  • a to e are respectively the use of PBS containing antimicrobial peptides with concentrations of 0, 10 ⁇ M, 20 ⁇ M, 40 ⁇ M, and 80 ⁇ M to infect Vero-E6 cells after incubation with SARS-CoV-2 virus for 20 minutes, and immunofluorescence detection after 24 hours;
  • f For the control group, that is, normal cells without virus infection under the same conditions. Green fluorescence is positive for SARS-CoV-2.
  • the antimicrobial peptide has an inhibitory effect on the new coronavirus, and with the increase of the concentration, its inhibitory effect is continuously enhanced in a dose-dependent manner.
  • the antimicrobial peptide After co-incubating the antimicrobial peptide with the new coronavirus, it was inoculated into a cell culture plate containing Vero-E6 cells, and the expression intensity of the viral gene was measured.
  • Resuscitate and activate Vero-E6 cells Resuscitate and activate Vero-E6 cells.
  • the medium used for cell culture is: DMEM+10%FBS+DA;
  • the internal reference primers used in use are:
  • GAPDH-F CAAGGTCATCCATGACAACTTTG
  • GAPDH-R GTCCACCACCCTGTTGCTGTAG
  • the primers for detecting SARS-CoV-2 gene are:
  • nCov-F GGGGAACTTCTCCTGCTAGAAT
  • nCoV-R CAGACATTTTGCTCTCAAGCTG
  • succinate dehydrogenase in the mitochondria of living cells can reduce exogenous thiazolyl blue (MTT) to water-insoluble blue-purple crystalline formazan (Formazan) and deposit in cells, but dead cells have no such function.
  • Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value can be measured at a suitable wavelength with an enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells. Within a certain cell number range, the amount of MTT crystal formation is proportional to the cell number.
  • Collect log-phase cells adjust the concentration of cell suspension, add 100 ⁇ L to each well, and plate the cells to adjust the density to 1000-10000 cells/well. 5% CO 2 , incubate at 37°C until the cell monolayer covers the bottom of the well (96-well flat bottom plate), add antimicrobial peptides with different concentration gradients of 1 ⁇ M, 6.25 ⁇ M, 12.5 ⁇ M, 25 ⁇ M, 50 ⁇ M, 100 ⁇ M, and set up 5 duplicate wells .
  • control wells cells, the same concentration of antimicrobial peptide dissolution medium, culture medium, MTT, DMSO.
  • the results are shown in Figure 3.
  • the relative cell proliferation rate was greater than 80% at the concentrations of 1 ⁇ M, 6.25 ⁇ M, 12.5 ⁇ M, 25 ⁇ M, 50 ⁇ M and 100 ⁇ M of antimicrobial peptides.
  • the antimicrobial peptide of the present invention can effectively inhibit the infection of SARS-COV-2 without causing toxic and side effects of cells.
  • antibacterial peptide of the present invention is composed of 19 amino acids, and the degradation products are natural amino acids, which do not produce drug residues, have no biological toxicity, and have no irritation.
  • the antimicrobial peptide of the present invention can effectively inhibit the infection of SARS-COV-2, and does not cause the biological toxicity of cells, and the antimicrobial peptide and its composition are in the inhibition of novel coronavirus, treatment or prevention caused by novel coronavirus infection. It is of great significance in disease and improvement of disease symptoms caused by new coronavirus infection, and provides new ideas and scientific basis for further clinical prevention and treatment of SARS-CoV-2 infection, and has application prospects.

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Abstract

本发明属于生物医药技术领域，提供了一种抑制新型冠状病毒感染的抗菌肽、其组合物及相关用途。所述组合物以抗菌肽为主要活性成分，添加药学上可接受的其他辅料、载体或辅助性成分。所述抗菌肽及其组合物具有抑制新型冠状病毒的作用，可用于预防和抑制由新型冠状病毒感染引起的疾病，安全性好、无激素、无抗生素。

Description

一种抑制新型冠状病毒感染的抗菌肽及其用途 技术领域
本发明涉及生物医药技术领域，具体涉及抑制新型冠状病毒感染的抗菌肽及其用途。
背景技术
新型冠状病毒SARS-CoV-2主要通过呼吸道飞沫和密切接触传播，传染能力极强，部分患者无症状却具备感染他人的能力，人群的易感性较高，传染性明显增大，对行走传染源的防控难度大大提高。
被新型冠状病毒SARS-CoV-2感染引起的急性呼吸道传染病(COVID-19)患者主要症状为发热、乏力、咳嗽，偶有鼻塞、流涕、咽痛及腹泻等不适(张诗琬,喻雪琴,陈芳,敬雪明,梅怡晗,梅小平.新型冠状病毒肺炎的研究进展[J].山东医药,2020,60(13):110-114.)。
研究表明，COVID-19部分患者存在无症状或仅有轻微的症状，但其传染病毒的能力并不低，这些隐性感染者也很有可能直接引发新一轮的疫情。
由于冠状病毒有广泛宿主特征，以及其自身的复杂基因组进化结构，在生物进化的过程中很容易发生重组和遗传变异，呈现出生物遗传上的的多样性，使该冠状病毒预防和早期治疗困难较大(陈运春,柯海霞,邱聪颖,黄燕,蒙夏玲,胡建东.新型冠状病毒的流行现状与研究进展[J].检验医学与临床,2021,18(01):131-133+141.)。
故，有效地控制COVID-19疫情、治愈感染的患者并减少病毒感染人数、减少由疫情所带来的巨大经济损失是当前世界面临的重要问题。
目前多种药物正在进入临床试验，有望对疫情得到有效控制。除抗新冠病毒药物外，疫苗、抗体类药物都备受关注，通过综合性中西医结合防治措施，达到早预防、早发现、早隔离、早治疗，阻断病情进展，取得了一定的临床疗效或改善(新型冠状病毒肺炎中西医结合防治专家共识[J].中国中西医结合杂志,2020,40(12):1413-1423.)。
现有抗病毒药物、抗菌药物中，部分抗SARS-CoV-2的效果不理想或者有不良反应，所以当下防控新冠病毒的方法大多聚焦在疫苗的研发上，目前在研究中针对新冠病毒的疫苗主要有：灭活或减毒病毒疫苗、基于蛋白质的疫苗、载体疫苗及DNA和RNA疫苗等(陈慧娟,刘琪琦,王升启.新型冠状病毒肺炎:实验室检测、治疗及疫苗(英文)[J].中国生物化学与分子生物学报,2021,37(01):11-19.)。
WHO显示，截止2021年2月26日，在全球，已有184个疫苗进入临床前开发阶段，其中83个疫苗处于临床开发阶段，多数具有一定的局限性，如免疫原性降低、致癌等等。(HTTPS://WWW.WHO.INT/PUBLICATIONS/M/ITEM/DRAFT-LANDSCAPE-OF-COVID-19-CANDIDATE-VACCINES)。并且，生产厂家的不同，疫苗引发的不良反应事件及程度略有差异。
综上所述，部分药物以及疫苗在治疗新型冠状病毒的过程中或多或少存在一定问题，如治疗效果不佳、存在不良反应、稳定性差等。 而抗菌肽作为新型生物活性成分，因其具有广谱抗菌性，对细菌、真菌、病毒、寄生虫等有抑制或杀灭作用，同时温和无刺激等特点得到了广泛的关注。
许多肽抑制剂已显示出对病毒的活性(Tincho M B,Gabere M N,Pretorius A.In Silico Identification and Molecular Validation of Putative AntimicrobialPeptides for HIV Therapy[J].Journal of AIDS&Clinical Research,2016,7(9).)。据报道抗菌肽AMPs是病毒感染的优秀候选者(Abid,Qureshi,Nishant,et al.AVPdb:a database of experimentally validated antiviral peptides targeting medically important viruses[J].Nucleic Acids Research,2013,42(D1):1147-53.)。但是氨基酸序列和结构的不同，不同的AMP具有抗病毒作用也不同(Biplab K.Maiti，Potential Role of Peptide-Based Antiviral Therapy against SARS-CoV-2Infection，ACS Pharmacology&Translational Science,2020,3(4),783-785)。
并且，Zhao H公开了在11种衍生自小鼠β-防御素4的肽中，仅短肽P9对多种呼吸道病毒表现出有效的广谱抗病毒作用，包括甲型流感病毒H1N1，H3N2，H5N1，H7N7，H7N9，SARS-CoV和MERS-CoV，而其他的10种衍生自小鼠β-防御素4的肽均无抗SARS-CoV病毒的作用(Zhao H,Zhou J,Zhang K,et al.A novel peptide with potent and broad-spectrum antiviral activities against multiple respiratory viruses[J].Rep,2016,6(1):22008.)。
由此可见，不同抗菌肽间，即使同源，其抗病毒特性也不相同。
而本发明人在研究过程中，意外的发现，本发明所述抗菌肽具有抑制新型冠状病毒的作用，无细胞毒性，安全性高，无激素，无抗生素，对新型冠状病毒的防治具有重要意义，进一步为临床预防SARS-CoV-2感染和治疗SARS-CoV-2感染提供新的思路和科学依据。
发明内容
针对现有技术的不足，本发明的一个目的在于提供一种抑制新型冠状病毒感染的抗菌肽，第二个目的在于提供一种抑制新型冠状病毒感染的抗菌肽组合物，第三个目的在于提供一种抑制新型冠状病毒感染的抗菌肽及其组合物的用途，其特征在于：本发明所述抗菌肽的氨基酸序列为SEQ ID:NO.1。
一种抑制新型冠状病毒感染的抗菌肽及其组合物的用途，其特征在于所述抗菌肽的氨基酸序列为SEQ ID NO.1。
优选的，所述新型冠状病毒为SARS-CoV-2。
优选的，所述该抗菌肽及其组合物的用途包括但不限于抑制新型冠状病毒、治疗或预防由新型冠状病毒感染引起的疾病、改善由新型冠状病毒感染引起的疾病症状。
一种抑制新型冠状病毒感染的抗菌肽组合物，其特征在于所述组合物包括所述抗菌肽，所述抗菌肽的氨基酸序列为SEQ ID NO.1。
优选的，所述抗菌肽组合物包括药学上可接受的其他辅料、载体或辅助性成分。
所述药学上可接受的其他辅料、载体或辅助性成分包括治疗新型 冠状病毒感染的药物。
本发明提供了一种抑制新型冠状病毒感染的抗菌肽及其用途，本发明所述抗菌肽具有抑制新型冠状病毒的作用，在80μM时，对SARS-CoV-2的抑制率为87.1％，并且随着抗菌肽浓度的增加，抗菌肽对病毒基因的表达的抑制作用不断增强，同时不具有细胞毒性，可与其他药学上可接受的其他辅料、载体或辅助性成分复配后，用于COVID-19的治疗及相关用途。与现有技术相比，抗菌肽及其组合物可有效抑制新型冠状病毒，并且安全性高，无激素，无抗生素，对新型冠状病毒的防治具有重要意义，进一步为临床预防SARS-CoV-2感染和治疗SARS-CoV-2感染提供新的思路和科学依据。
附图说明
图1免疫荧光检测SARS-CoV-2对Vero-E6细胞的感染情况。
图2 RT-qPCR检测不同浓度抗菌肽对SARS-CoV-2感染Vero-E6细胞的影响情况。
图3不同浓度抗菌肽的细胞毒性试验结果。
具体实施方式
本发明的一个目的在于提供一种抑制新型冠状病毒感染的抗菌肽，第二个目的在于提供一种抑制新型冠状病毒感染的抗菌肽组合物，第三个目的在于提供一种抑制新型冠状病毒感染的抗菌肽及其组合物的用途，其特征在于：本发明所述抗菌肽的氨基酸序列为SEQ ID:NO.1。
一种抑制新型冠状病毒感染的抗菌肽及其组合物的用途，其特征 在于所述抗菌肽的氨基酸序列为SEQ ID NO.1。
优选的，所述新型冠状病毒为SARS-CoV-2。
优选的，所述该抗菌肽及其组合物的用途包括但不限于抑制新型冠状病毒、治疗或预防由新型冠状病毒感染引起的疾病、改善由新型冠状病毒感染引起的疾病症状。
一种抑制新型冠状病毒感染的抗菌肽组合物，其特征在于所述组合物包括所述抗菌肽，所述抗菌肽的氨基酸序列为SEQ ID NO.1。
优选的，所述抗菌肽组合物包括药学上可接受的其他辅料、载体或辅助性成分。
所述药学上可接受的其他辅料、载体或辅助性成分包括治疗新型冠状病毒感染的药物。
使用时，是将安全有效量的本发明所述抗菌肽或组合物施用于人或动物，本领域技术人员可按常规方法确定适于某种情况的优选剂量，具体剂量应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
优选的，本发明所述肽和组合物可以通过喷雾或者吸入的方式给药。
在本发明的抗菌肽及其组合物中，药学上可接受的其他辅料、载体或辅助性成分可以在共同范围内的量使用，并且其合适的量可以由本领域普通技术人员根据实际应用来确定。
本发明中所述抗菌肽可通过化学合成，也可以通过基因工程技术表达、分离纯化得到(具体方法可参见Sambrook et al.，Molecular  Cloning：A Laboratory Manual，Cold Spring Harbor Laboratory Press，Cold Spring Harbor，NY，2001)。
本文中术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
本文中，“组合物”、“抗菌肽组合物”未特别说明，可以互换使用，指的是包含本发明所述抗菌肽为有效成分的制品。
本文中，所述的“新型冠状病毒”是一类新型病毒，为SARS-CoV-2，“2019新型冠状病毒”、“2019-nCoV”均为该病毒的别名。
本文中，所述的“SARS-CoV-2感染”是由新型冠状病毒(SARS-CoV-2)引发的感染性疾病，包括但不限于“新型冠状病毒肺炎”。
本文中，所述“预防SARS-CoV-2感染”的用途包括预防新型冠状病毒感染健康人群的用途，指保护或防止尚未感染病毒的个体所做的预防性措施。
本文中，“药学上可接受的”是指物质或组合物必须与包含制剂的其它成分和/或用其治疗的哺乳动物化学上和/或毒理学上相容。
本文中，“药学上可接受的其他辅料”包括无毒固填充剂、稀释剂、佐剂、包裹材料或其他制剂辅料。
本文中，“药学上可接受的载体”是指本领域技术人员已知的各种载体或载体中的任何一种。另外，这样的药学上可接受的载体可以 是水性或非水性溶液，悬浮液和乳液。也可以存在防腐剂和其他添加剂，例如抗微生物剂，抗氧化剂，螯合剂，惰性气体等。
在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾，本说明书优先。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，未提及的实验方法为常规实验方法，实施例中用到的各种原材料、试剂、仪器和设备等除特别说明外，均可通过市场购买得到或者可通过现有方法制备得到。
本部分实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
实施例1 抗菌肽体外抗SARS-CoV-2活性检测
抗菌肽体外抗SARS-CoV-2活性效果评估实验涉及病毒培养，按照国家卫健委《新型冠状病毒感染的肺炎防控方案(第四版)》要求，所有病毒培养均在生物安全三级实验室(BSL-3)的生物安全柜内进行。使用病毒培养物提取核酸，裂解剂或灭活剂的加入在与病毒培养等同级别的实验室和防护条件下进行。活病毒在采用可靠的方法灭活后进行的核酸检测等操作在生物安全二级实验室(BSL-2)进行。
SARS-CoV-2病毒株：海军军医大学于2020年2月分离获得的毒株，基因组信息：GenBank:MT627325.1。
1.1荧光免疫组化检测
将不同浓度的抗菌肽与SARS-CoV-2病毒共同孵育后，共同感染Vero-E6细胞，用免疫荧光检测，以验证抗菌肽是否对新型冠状病毒有抑制作用。
复苏并活化Vero-E6细胞，在细胞活化完成、活力较好时，接种于96孔细胞培养板中，接种密度为80％左右；细胞培养所用培养基为：DMEM+10％胎牛血清(FBS)+DA；
准备SARS-CoV-2病毒悬液，并控制其滴度为：1×10 7pfu/mL；使用PBS(磷酸缓冲液，pH7.4)溶解抗菌肽固体，制备成浓度为4mM的抗菌肽溶液母液；以MOI(感染复数)＝0.01的比例计算感染细胞所需的SARS-CoV-2病毒数量，将SARS-CoV-2活病毒与不同浓度、相同体积的抗菌肽溶液于37℃共孵育20min(使用PBS稀释抗菌肽溶液母液，使孵育时抗菌肽终浓度分别为：0、10μM、20μM、40μM、80μM)。
将孵育后的上清100μl分别加入已经接种有Vero-E6的细胞培养板中，每孔含培养基100μl，混匀后于二氧化碳培养箱中培养24h；将培养完成的Vero-E6细胞吸除培养基上清，并使用无菌PBS溶液轻柔洗涤两次；使用预冷的甲醇于-20℃固定20min，弃上清；使用含3％BSA的PBS溶液(PBS溶液配制)于室温条件封闭1h，弃上清；使用SARS-CoV-2病毒S蛋白抗体作为一抗，于4℃孵育过夜；使用PBS清洗孵育后的培养孔3次，每次5min；使用荧光二抗488室温孵育1h，使用PBS清洗培养孔3次，每次5min。
进行免疫荧光检测，SARS-CoV-2对Vero-E6细胞的感染情况见图1。
其中，a～e分别是使用含抗菌肽浓度为0、10μM、20μM、40μM、80μM的PBS与SARS-CoV-2病毒共孵育20min后侵染Vero-E6细胞，24h后进行免疫荧光检测；f为对照组，即同条件下未感染病毒的正常细胞。绿色荧光为SARS-CoV-2阳性。
由图1可知，抗菌肽对新型冠状病毒有抑制作用，并且随着浓度的增加，其抑制作用不断增强，存在剂量依赖性。
1.2 RT-qPCR检测
将抗菌肽与新型冠状病毒共同孵育后，接种到含Vero-E6细胞的细胞培养板中，对病毒基因的表达强度进行测定。
复苏并活化Vero-E6细胞，在细胞活化完成、活力较好时，接种于24孔细胞培养板中，接种密度为80％左右，细胞培养所用培养基为：DMEM+10％FBS+DA；
准备SARS-CoV-2病毒悬液，并控制其滴度为：1×10 7pfu/mL；使用PBS(磷酸缓冲液，pH7.4)溶解抗菌肽固体，制备成浓度为4mM的抗菌肽溶液母液；以MOI(感染复数)＝0.01的比例计算感染细胞所需的SARS-CoV-2病毒数量，将SARS-CoV-2活病毒与不同浓度、相同体积的抗菌肽溶液于37℃共孵育40min(使用PBS稀释抗菌肽溶液母液，使孵育时抗菌肽终浓度分别为：0、10μM、20μM、40μM、80μM)。
将孵育后的上清200μl分别加入已经接种有Vero-E6的细胞培 养板中，每孔含培养基300μl，混匀后于冰上孵育2h用于后续检测；感染完成的Vero-E6细胞，吸除培养基上清，并使用无菌PBS溶液轻柔洗涤两次；使用0.4mL/孔的Trizol，于室温下孵育10min，收集裂解液与2mL离心管中；后续按照标准Trizol法提取RNA、mRNA的逆转录、qPCR实验方法完成实验。
使用中所用内参引物为：
GAPDH-F：CAAGGTCATCCATGACAACTTTG
GAPDH-R：GTCCACCACCCTGTTGCTGTAG
检测SARS-CoV-2基因引物为：
nCov-F：GGGGAACTTCTCCTGCTAGAAT
nCoV-R：CAGACATTTTGCTCTCAAGCTG
RT-qPCR检测不同浓度抗菌肽对SARS-CoV-2感染Vero-E6细胞的病毒基因表达相对强度见图2。
由图2可知，低浓度的样品表现出较低的抑制新型冠状病毒活性，并且随着抗菌肽浓度的增加，抗菌肽对病毒基因的表达的抑制作用不断增强，80μM的抗菌肽可将病毒基因的表达抑制至12.9％，抑制率达87.1％。故本发明所述抗菌肽可以有效抑制SARS-COV-2的感染。
实施例2 抗菌肽对Vero细胞的细胞毒性
由于活细胞线粒体中的琥珀酸脱氢酶能使外源性噻唑蓝(MTT)还原为水不溶性的蓝紫色结晶甲臜(Formazan)并沉积在细胞中，而死细胞无此功能。二甲基亚砜(DMSO)能溶解细胞中的甲臜，用酶联免疫 检测仪在合适波长处测定其光吸收值，可间接反映活细胞数量。在一定细胞数范围内，MTT结晶形成的量与细胞数成正比。
收集对数期细胞，调整细胞悬液浓度，每孔加入100μL，铺板使待测细胞调密度至1000-10000个/孔。5％CO 2，37℃孵育，至细胞单层铺满孔底(96孔平底板)，加入1μM、6.25μM、12.5μM、25μM、50μM、100μM不同浓度梯度的抗菌肽，设5个复孔。
5％CO 2，37℃孵育24小时，倒置显微镜下观察。每孔加入10μL MTT溶液(5mg/mL，即0.5％(w/v)MTT)，继续培养4h后，小心吸去孔内培养液。每孔加入150μL DMSO，置振荡器上低速振荡10min，使结晶物充分溶解。在酶联免疫检测仪OD 490nm处测量各孔的吸光值。
同时设置调零孔(培养基、MTT和DMSO)，对照孔(细胞、相同浓度的抗菌肽溶解介质、培养液、MTT、DMSO)。
结果如图3所示，抗菌肽在1μM、6.25μM、12.5μM、25μM、50μM、100μM的浓度下，细胞相对增值率均大于80％。
参考王琛等(王琛,廖馨,王妍婷,范媛,梁睿贞.四种消毒剂应用于DUWLs微生物污染控制的生物安全性评价[J].口腔医学,2019,39(07):596-600.)公开的文献，5级毒性评价标准分级表(表1)，可知，抗菌肽在100μM浓度下对Vero细胞没有细胞毒性。
表1 细胞毒性评价标准表
细胞相对增值率/％(RGR/％) 细胞毒性评分
80-100 0
60-80 1
40-60 2
20-40 3
0-20 4
综上所述，本发明的抗菌肽可以有效抑制SARS-COV-2的感染，且不引起细胞的毒副作用。
且本发明所述抗菌肽为19个氨基酸组成，降解产物为天然氨基酸，不产生药物残留，无生物毒性，无刺激。
综合考虑，本发明所述抗菌肽可以有效抑制SARS-COV-2的感染，且不引起细胞的生物毒性，抗菌肽及其组合物在抑制新型冠状病毒、治疗或预防由新型冠状病毒感染引起的疾病、改善由新型冠状病毒感染引起的疾病症状中均具有重要意义，为进一步为临床预防和治疗SARS-CoV-2感染提供新的思路和科学依据，具有应用前景。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。

Claims (8)

  1. 一种抑制新型冠状病毒感染的抗菌肽及其组合物的用途，其特征在于所述抗菌肽的氨基酸序列为SEQ ID NO.1。
  2. 根据权利要求1所述的用途，其特征在于所述新型冠状病毒为SARS-CoV-2。
  3. 根据权利要求1或2所述的用途，其特征在于该抗菌肽及其组合物的用途包括但不限于抑制新型冠状病毒、治疗或预防由新型冠状病毒感染引起的疾病、改善由新型冠状病毒感染引起的疾病症状。
  4. 一种抑制新型冠状病毒感染的抗菌肽组合物，其特征在于所述抗菌肽的氨基酸序列为SEQ ID NO.1。
  5. 根据权利要求4所述的抗菌肽组合物，其特征在于所述组合物包括药学上可接受的其他辅料、载体或辅助性成分。
  6. 根据权利要求4或5所述的抗菌肽组合物，其特征在于所述新型冠状病毒为SARS-CoV-2。
  7. 根据权利要求4或5所述的抗菌肽组合物，其特征在于所述药学上可接受的其他辅料、载体或辅助性成分包括治疗新型冠状病毒感染的药物。
  8. 根据权利要求6所述的抗菌肽组合物，其特征在于所述药学上可接受的其他辅料、载体或辅助性成分包括治疗新型冠状病毒感染的药物。
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