WO2014154134A1 - 对新型冠状病毒HCoV-EMC 2012感染具有抑制作用的多肽及其应用 - Google Patents

对新型冠状病毒HCoV-EMC 2012感染具有抑制作用的多肽及其应用 Download PDF

Info

Publication number
WO2014154134A1
WO2014154134A1 PCT/CN2014/074045 CN2014074045W WO2014154134A1 WO 2014154134 A1 WO2014154134 A1 WO 2014154134A1 CN 2014074045 W CN2014074045 W CN 2014074045W WO 2014154134 A1 WO2014154134 A1 WO 2014154134A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
emc
hcov
novel coronavirus
infection
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2014/074045
Other languages
English (en)
French (fr)
Inventor
姜世勃
陆路
刘奇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fudan University
Original Assignee
Fudan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fudan University filed Critical Fudan University
Priority to KR1020157016526A priority Critical patent/KR20150133690A/ko
Publication of WO2014154134A1 publication Critical patent/WO2014154134A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory

Definitions

  • the present invention belongs to the field of biomedicine and relates to a group of polypeptides having an inhibitory ability against a novel coronavirus HCoV-EMC 2012 infection, and also relates to a nucleic acid molecule encoding the polypeptide, a related recombinant protein, a nucleic acid molecule, an expression vector and a recombinant cell. Background technique
  • HCoV-EMC 2012 a new coronavirus from the Middle East, HCoV-EMC 2012, has caused many deaths and a mortality rate of 57%, and the rest of the patients are also suffering from serious diseases.
  • DPP4 Dipeptidylpeptidase 4
  • This virus causes a wide range of infections in human cells.
  • the new coronavirus HCoV-EMC 2012 is able to infect a variety of animals, including bats and pigs, making it a persistent source of infection, which means that the virus can spread from animals to humans.
  • Type I enveloped viruses such as HIV (HIV), influenza, SRAS coronavirus, etc., whose envelope glycoproteins and corresponding receptor-mediated viral fusion and entry are the first steps in the infection of this type of virus, and are also the most critical One of the steps. After the envelope glycoprotein binds to the receptor, the N-terminal fusion peptide of the S2 protein is exposed, allowing it to be inserted into the target cell membrane. A series of conformational changes occur in the S2 protein, which causes the protein to become a fusoge c state, bringing the virus close to the target cell membrane and promoting membrane fusion.
  • HIV HIV
  • influenza SRAS coronavirus
  • the entry inhibitor is a drug that acts on the stage of entry of the virus into the cell.
  • entry inhibitors have played an important role in the treatment of HIV.
  • Enfuvirtide (Fuzeon, also known as T20) is the first internationally recognized HIV entry inhibitor in the United States, currently used in the clinical treatment of AIDS, for those who have produced HIV reverse transcriptase. Patients with drug resistance are very effective.
  • the inventor of the present application Jiang Shibo, is the inventor of the T20 prodrug (Peptide 637-666) (U.S. Patent No.: 5, 444, 044).
  • the entry inhibitor is mostly derived from the N-terminal sequence (NHR or HR1) or C-terminal repeat (CHR or HR2) of the viral S2 protein.
  • NHR or HR1 N-terminal sequence
  • CHR or HR2 C-terminal repeat
  • the crystallographic study of gp41 shows that CHR can interact with NHR and form a a conformation of a trimer "or" six-helix bundle (6-HB) in which three parallel NHRs form a trimer coiled-coil core, and the three CHRs are packaged in anti-parallel to the height of the inner coiled spiral surface conserveed hydrophobic tracts.
  • Peptides derived from the NHR and CHR regions are called NHR-peptides and CHR-peptides, respectively.
  • Most of the CHR-peptides are potent HIV fusion inhibitors, which are mainly trimeric by internal NHR with the virus gp41. Body binding acts to interfere with 6-HB formation.
  • Another object of the present invention is to provide a recombinant protein having an inhibitory function against a novel coronavirus HCoV-EMC 2012 infection, the core region of which is a polypeptide sequence having the ability to inhibit HCov-EMC 2012 virus infection as referred to in the present application.
  • the present invention has determined the S2 protein region of the novel coronavirus HCoV-EMC 2012 S protein through a series of screening and verification, and analyzed the N-terminal sequence (HR1) and the C-terminal sequence (HR2) of the S2 protein by homology comparison.
  • the present invention provides a polypeptide comprising the sequence set forth in SEQ ID NO: 9 and having an inhibitory effect on novel coronavirus HCoV-EMC 2012 infection.
  • the polypeptide includes a polypeptide which is substituted, deleted or added with one or more amino acids as the polypeptide represented by SEQ ID NO: 9 and which is capable of inhibiting infection of HCoV-EMC virus.
  • the polypeptide has an amino acid sequence as set forth in SEQ ID NO: 6, SEQ ID NO or SEQ ID NO 8.
  • the above polypeptides can be artificially synthesized according to their amino acid sequences.
  • the invention also relates to a set of nucleic acid molecules encoding the isolated peptides of the invention. Includes codon optimized derivatives.
  • the invention also relates to a set of expression vectors comprising the isolated nucleic acid molecules of the invention.
  • the invention also relates to a set of isolated recombinant cells comprising an expression vector of the invention.
  • the invention also relates to the use of an isolated peptide, isolated nucleic acid molecule, expression vector or recombinant cell of the invention as a medicament.
  • the invention also relates to a group of pharmaceutical compositions comprising a polypeptide, a nucleic acid molecule, an expression vector, and / or recombinant cells and optionally pharmaceutically acceptable carriers or excipients.
  • the active ingredient thereof comprises the above polypeptide, recombinant protein, nucleic acid, expression vector or recombinant cell.
  • the invention further relates to the use of a polypeptide, nucleic acid molecule, expression vector or recombinant cell of the invention for the preparation of a pharmaceutical composition for inhibiting novel coronavirus HCoV-EMC 2012 infection.
  • the invention also relates to a method for inhibiting or preventing a novel coronavirus HC0-EMC2012 infection, comprising administering a subject infected with the novel coronavirus HC0V-EMC 2012 or at risk of infection with the novel coronavirus HC0V-EMC 2012, such as in vitro culture.
  • a subject infected with the novel coronavirus HC0V-EMC 2012 or at risk of infection with the novel coronavirus HC0V-EMC 2012 such as in vitro culture.
  • the cell or the like, the polypeptide, nucleic acid molecule, expression vector, recombinant cell or pharmaceutical composition of the present invention is administered.
  • the invention also relates to the use of the above polypeptide for the preparation of a medicament for the infection of a novel coronavirus HCoV-EMC 2012.
  • the above polypeptides are used to prevent novel coronavirus HCoV-EMC 2012 infected cells.
  • the new coronavirus HCoV-EMC 2012 originated in the Middle East and has caused many deaths to date, with a mortality rate of 57%.
  • HCoV-EMC 2012 is capable of infecting a variety of animals, including bats and pigs, making it a persistent source of infection, and it has now been found to spread in the home, suggesting its ability to pass humans, plus its High mortality, unknown sources and routes of transmission have attracted the attention of global public health agencies.
  • the present invention provides a set of polypeptides comprising the sequence set forth in SEQ ID NO: 9, which has been shown to inhibit the infection of the novel coronavirus HCoV-EMC 2012. African green monkey kidney cells incubated with this polypeptide have significant antiviral activity with IC50 values of even nM. Further experiments showed that HR1- 5 and HR2- 2 can bind to each other to form a fusion six-helix of hCoV-EMC. Therefore, it is speculated that the HR2 series polypeptide can bind to the HR1 region of the virus, thereby interfering with the fusion between the virus and the cell membrane, blocking the virus. infection.
  • the polypeptide of the present invention can be formulated with a medically acceptable carrier or excipient for preventing and inhibiting the infection of the novel coronavirus HCoV-EMC 2012, and can also be combined with other drugs.
  • the present invention provides a candidate polypeptide drug for the prevention or treatment of a novel coronavirus HCoV-EMC 2012 infection.
  • the present invention obtains a polypeptide inhibitor capable of inhibiting infection of the novel coronavirus HCoV-EMC 2012, and provides an important theoretical and applied basis for further research and development of a novel coronavirus HCoV-EMC 2012 preventive and therapeutic drug, and fills an international gap.
  • Figure 1 Functional domains in the HCoV-EMC2012 S2 protein molecule and the position of the polypeptide in the protein, wherein the residue number corresponds to its position in the full length of the S protein; FP, fusion peptide; HR1, heptad repeat 1 ; HR2, heptapeptide weight Complex sequence 2; TM, transmembrane domain; CP, cytoplasmic domain.
  • Figure 2 shows the results of the virus inhibition experiment of HCoV-EMC 2012 by HR2 series peptides.
  • a, b, and c are the results of virus inhibition experiments of HR2-1, HR2-2, and HR2-3 polypeptides on HCoV-EMC 2012, respectively.
  • Figure 3 shows the results of virus inhibition experiments of HCoV-EMC 2012 by HR1 series peptides. Among them, HR1_1, HR1-2, intestinal-3, HR1-4 and HR1-5 showed no significant inhibitory activity at 2-7 ⁇ .
  • FIG. 4 Shows the antiviral mechanism of HR2 polypeptide.
  • the HR2 polypeptide binds to HR1-5.
  • Figure 5. shows the establishment of the hCoV-EMC/2012 cell fusion model.
  • 293T/EGFP panel A
  • 293T/EMC/EGFP panel B
  • 293T/EGFP could not be fused to huh-7 cells, whereas 293T EMC/EGFP cells would merge with huh-7 cells; if HR2-2 was added to the final concentration of ⁇ , the fusion process was inhibited (Fig. F).
  • Figures B, D, and F are the visual fields of A, C, and E under white light, respectively.
  • IC50 which is the half-inhibitory concentration, refers to the concentration of the drug that inhibits 50% HCoV-EMC infection.
  • the inventors determined the HR1 and HR2 sequence positions of the novel coronavirus HcoV-EMC 2012 S2 protein by homologous alignment and assay of the novel coronavirus HCoV-EMC 2012 S protein with the known SARS coronavirus S2 protein. A 36-42 amino acid sized polypeptide fragment from the HR1 and HR2 fragments was selected. A total of 5 peptides from HR1 and 3 peptides from HR2 were synthesized and purified by the company (GL Biochem Shanghai Ltd) with a purity of >99%.
  • the peptides HR1- 1, HR1-2, HR1 3, HR1- 4, HR1- 5, HR2- 1, HR2-2, and HR2- 3 were diluted 4-fold; 100 ⁇ M for each sample
  • a 96-well cell culture plate pre-plated with 50,000 African green monkey kidney cells (Vero cells) per well was added and incubated at 37 ° C for 2 hours, after which the supernatant was removed, the cells were washed twice with PBS, and a new 100 ⁇ crown was added.
  • the virus was treated with HCoV-E C 2012 (10TCID50) for 1 hour at 37 ° C; the cells were then washed twice with PBS.
  • the 100 ⁇ M sample/well was then added to the 96-well plate for further action.
  • the cells were then cultured at 37 ° C for 3 days.
  • the cytopathic effect (CPE) was recorded and 10 ⁇ MTT was added to each well. Incubate for 4 hours at 37 °C; add 100 ⁇ of 0.1 ⁇ M 1 with 1% SDS per well, incubate at 37 ° C overnight and measure absorbance at 570 nm.
  • HR2-1, HR2-2, and HR2-3 in the C-terminal (HR2) polypeptide showed antiviral activity with IC50 of 1.82M, 0.62 ⁇ and 0, respectively. 43 MM, in which HR2-1 is relatively weak, while HR2-2 and HR2-3 are both active at nM.
  • HR1-1, HR1- 2, HR1- 3, HR1- 4, and HR1- 5 showed no significant inhibitory activity at 2-7 MM (Fig. 3).
  • Table 1 shows the polypeptide sequence and the activity of inhibiting HCoV-EMC infection.
  • Non-denaturing polyacrylamide gel electrophoresis N-PAGE.
  • the HR1 series peptides were mixed in HR2 series peptides (30MM final concentration) and incubated at 37'C for 30 minutes. Take 5 X high pH loading buffer and mix with 4 volumes of peptide mixture. Then, spotting (25 ⁇ M per well) in 18% non-denaturing gel (Beijing Tian Enze), electrophoresis at 125 V for 2 hours at room temperature. Coomassie blue stained. The results showed that HR1-5 produced a new peptide after mixing with the HR2 series of peptides (Fig. 4 ⁇ ). This band is the EMC virus fusion active six-helix (6- ⁇ ) band.
  • the detection temperature is 4 ° C
  • the bandwidth is 5.0 nm
  • the resolution is 0.1 nra
  • the optical path is 0.1 cm
  • the reaction time is 4.0 s
  • the scanning speed is 50 nm/min.
  • the thermal denaturation of the polypeptide was monitored at 222 nm with a temperature gradient of 5 °C/min.
  • the blank value of the buffer solution was subtracted to correct the spectral value. Smoothing the melting curve and using Jasco to calculate the midpoint temperature of the thermal dissociation transition, ie the Tm value
  • ⁇ -5 and HR2- 2 are examples (as shown in Fig. 4C), and HR1-5 alone is a random coil structure, and HR2-2 alone is a low-helix structure. When the two are mixed, the mixture is seen to be a highly helical structure. The result is consistent with the CD spectrum of the six-helix core structure.
  • the Tm value is about 9 (TC (as shown in Figure 4D).
  • HR1- 5 and HR2- 2 can be combined to form a fusion six-helix of hCoV-EMC; while HR2 series peptide can be combined with HR 1
  • the combination of 5 it is speculated that the HR2 series of polypeptides can bind to the HR1 region of the virus, thereby interfering with the fusion between the virus and the cell membrane, and blocking the viral infection.
  • Example 3 Modification of HR2-2 polypeptide
  • the inventors found that the polypeptide fragment of the novel coronavirus HCOV-EMC/2012 S2 protein HR2-2 has good biological activity. However, the requirements of the biomedical field have not yet been fully met. Therefore, the inventors adapted based on the amino acid sequence of the existing HR2-2 polypeptide, according to the literature (Otaka, A et al Remodeling of gp41-C34 peptides to highly effective inhibitors of the fusion of HIV-1 with target cells Angew Chem Int Ed Engl 41, 2937-2940 (2002)) Describes the introduction of glutamate (E), lysine (K), and arginine (R) in the interior of HR2-2 polypeptide to increase HR2-2 polypeptide The internal salt bridge, in order to increase the solubility, stability, and antiviral activity of the polypeptide. The sequence is shown in Table 2, where the lines between the amino acids represent the salt bridge formed between the two. After the polypeptide sequence is determined, synthesis and purification are carried out, and the purity is determined
  • the binary structure of the peptide was determined by circular dichroism. See Example 1 for the specific method.
  • the CD value of the newly synthesized mutant polypeptide at 222 nm was measured in accordance with 100 ° /.
  • the CD value of the ⁇ -helix is -33 ⁇ 10 3 , and the ⁇ -helix ratio of the corresponding polypeptide is calculated.
  • the ⁇ -helix ratio of the complex and the Tm value of the complex after the action of the polypeptide and HR1-5 were examined.
  • the inventors constructed an expression plasmid (pAAV-EGFP-IRES-EMC-S) capable of simultaneously expressing hCoV-EMC/2012 S protein and GFP, and transfected 293T cells (293T/EMC/EGFP).
  • pAAV-EGFP-IRES-EMC-S an expression plasmid capable of simultaneously expressing hCoV-EMC/2012 S protein and GFP
  • 293T/EMC/EGFP transfected 293T cells
  • only the plasmid pAAV-EGFP-IRES was transfected as a negative control (293T/EGFP). Since there is an IRES sequence in the middle of the protein at both ends, 293T/EMC/EGFP will achieve simultaneous, separate expression of the two proteins without interfering with each other.
  • the S protein will be presented to the cell surface, which mediates the fusion of the transfected 293T (293T/EMC/EGFP) with the target cell Huh-7, and GFP can be used to observe the transfection efficiency.
  • the fused cells will have a larger fluorescence range and a weaker fluorescence (see arrows in Figure 5-C for cells). Therefore, the fused cells can be distinguished from the unfused cells and the ratio of the fused cells can be calculated. This fusion process can be prevented by the addition of a potent peptide inhibitor such as HR2-2 in the system ( Figure 5-E).
  • test polypeptides were serially diluted in 96-well plates and mixed with 4 293T cells (293T/EMC/EGFP) that had been transfected with pAAV-hGFP-IRES-E CS, and then The 5-inch ⁇ 4 target cells huh-7 were plated in a 96-well plate 5-9 hours in advance, and incubated at 37 ° C for 4 hours to observe the inhibition of the cell fusion by the polypeptide. Calculate the half effective amount (IC50) of polypeptide inhibition of cell fusion. Results - The results are shown in Table 2. It can be seen that the introduction of the EK mutation in the HR2-2 polypeptide further stabilizes the natural structure of the polypeptide itself.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)

Abstract

本发明属于生物医药领域。本发明提供了一种多肽，该多肽含有如SEQ ID NO 9所示的序列并且对新型冠状病毒HCoV-EMC 2012感染具有抑制作用。本发明还提供了编码该多肽的核酸分子、相关重组蛋白、核酸分子、表达载体和重组细胞。本发明的多肽可与医学上常可接受的载体或赋形剂等制成药剂，用于预防和抑制新型冠状病毒HCoV-EMC 2012的感染，也可以与其他药物组合。本发明为新型冠状病毒HCoV-EMC 2012感染的预防或治疗提供候选多肽类药物。

Description

对新型冠状病毒 HCoV-EMC 2012感染具有抑制作用的多肽及其应用 技术领域
本发明属于生物医药领域， 涉及一组对新型冠状病毒 HCoV-EMC 2012感染具有抑制能 力的多肽， 还涉及编码该多肽的核酸分子、 相关重组蛋白、 核酸分子、 表达载体和重组细 胞。 背景技术
2012年， 一种源自中东的新型冠状病毒 HCoV-EMC 2012， 已经导致了多人死亡， 并且 死亡率高达 57%， 其余患者也都患上了严重疾病。 目前研究发现该新型冠状病毒 HCoV- EMC 2012与 SARS冠状病毒感染所用的受体不同，其受体为二肽基肽酶 4 (Dipeptidylpeptidase 4, DPP4), 一种分布广泛的人细胞受体， 从而导致该病毒对人体细胞均有广泛的感染能力。 另外， 新型冠状病毒 HCoV- EMC 2012 能够感染多种动物， 包括蝙蝠和猪， 使其成为持续的 感染源， 这也意味着该病毒有从动物传播给人类的可能。 并且， 目前已发现该病毒可在家 庭中进行传播， 暗示了其具有了人传人的能力， 加上其高死亡率、 未知来源及传播途径引 起了全球公共健康机构的高度关注。 同时， 针对新型冠状病毒 HCoV- EMC 2012 尚无有效的 特异性药物，现有治疗措施仅是针对患者症状进行支持治疗。因此，急需一种针对 HCoV- EMC 的特异性治疗和预防性药物。
I 型包膜病毒， 如艾滋病病毒 （HIV)、 流感、 SRAS冠状病毒等， 其包膜糖蛋白和相应 受体介导的病毒融合和进入是该类病毒感染的第一个步骤， 也是最关键的步骤之一。 包膜 糖蛋白与受体结合后， S2蛋白 N端的融合肽被暴露， 使其能够插入靶细胞膜中。之后 S2蛋 白发生一系列构象改变， 导致蛋白质成为其促进融合的状态 （fusoge c state ), 使得病 毒和靶细胞膜密切接近并促进膜融合。
进入抑制剂即是作用于病毒进入细胞阶段的药物。 目前， 进入抑制剂在艾滋病病毒 (HIV)的治疗方面已起到重要的作用。例如， 恩夫韦肽（Enfuvirtide， Fuzeon, 又称 T20) 是美国 FDA批准的国际上第一个多肽类 HIV进入抑制剂， 其目前被用于艾滋病的临床治疗， 对那些已产生 HIV逆转录酶抑制剂抗药性的病人有很好的疗效。 本申请的发明人之 姜世勃是 T20前体药物 （多肽 637-666) 的发明人 （美国专利号： 5, 444, 044)。
进入抑制剂多来源于病毒 S2蛋白的 N-端序列 （NHR或 HR1 ) 或 C-端重复序列 （CHR或 HR2 )。 以 HIV为例， 其 gp41的晶体学研究显示， CHR能够与 NHR相互作用， 形成被称作"发 夹三聚体"或"六螺旋束"（6-HB)的构象， 其中三个平行的 NHR形成三聚体卷曲螺旋核心， 三个 CHR反向平行地包装成沿着内部卷曲螺旋表面的高度保守的疏水沟。 源自 NHR和 CHR 区的肽分别被称作 NHR-肽和 CHR-肽。大部分的 CHR-肽是有效的 HIV融合抑制剂， 它们主要 是通过与病毒 gp41内部 NHR三聚体结合以干扰 6-HB形成而发挥作用。 发明内容
本发明的目的是提供一组对新型冠状病毒 HCoV- EMC 2012感染具有抑制功能的多肽片 段。
本发明的另外一个目的是提供对新型冠状病毒 HCoV- EMC 2012感染具有抑制功能的重 组蛋白， 该蛋白的核心区域是本申请中所涉及的具有抑制 HCov-EMC 2012病毒感染能力的 多肽序列。 本发明经过一系列的筛选和验证确定了新型冠状病毒 HCoV-EMC 2012 S蛋白的 S2蛋白 区域， 通过同源性比较分析了 S2蛋白的 N-端序列 （HR1 ) 和 C端序列 （HR2)。 根据该序列 设计了针对于新型冠状病毒 HCoV-EMC 2012 的进入抑制剂， 为进一歩研发新型冠状病毒 HCoV-EMC 2012的预防和治疗药物提供重要的理论及应用基础， 填补了本技术领域的空白。 本发明以此为理论基础。
本发明提供了一种多肽， 该多肽含有如 SEQ ID NO 9所示的序列并且对新型冠状病毒 HCoV-EMC 2012感染均具有抑制作用。
所述的多肽包括由如 SEQ ID NO 9所示的多肽经过取代、 缺失或添加一个或者多个氨 基酸而构成并且能够抑制 HCoV-EMC病毒感染的多肽。
在本发明的一个实施例中， 所述的多肽的氨基酸序列如 SEQ ID NO 6、 SEQ ID NO 7 或者 SEQ ID NO 8所示。
上述多肽可以根据其氨基酸序列人工合成。
本发明还涉及了一组核酸分子，其编码本发明所述分离的肽。包括密码子优化衍生物。 本发明还涉及了一组表达载体， 其包含本发明所述分离的核酸分子。
本发明还涉及了一组分离的重组细胞， 其包含本发明所述的表达载体。
本发明还涉及了本发明所述的分离的肽、 分离的核酸分子、 表达载体或重组细胞作为 药物的应用。
本发明还涉及了一组药物组合物， 其包含本发明所述的多肽、 核酸分子、 表达载体和 /或重组细胞以及任选存在的药物可接受的载体或赋形剂。 例如， 其有效成分包含上述多 肽、 重组蛋白、 核酸、 表达载体或者重组细胞。
本发明还涉及了本发明所述的多肽、 核酸分子、 表达载体或重组细胞在制备用于抑制 新型冠状病毒 HCoV-EMC 2012感染药物组合物中的应用。
本发明还涉及了一种抑制或预防新型冠状病毒 HC0V-EMC 2012感染的方法， 包括给予 被新型冠状病毒 HC0V-EMC 2012感染或有被新型冠状病毒 HC0V-EMC 2012感染风险的对象， 例如体外培养的细胞等， 施用本发明所述的多肽、 核酸分子、 表达载体、 重组细胞或药物 组合物。
本发明还涉及上述多肽在制备抗新型冠状病毒 HCoV-EMC 2012感染药物中的应用。 例 如， 将上述多肽用于预防新型冠状病毒 HCoV-EMC 2012感染细胞。 新型冠状病毒 HCoV- EMC 2012源自中东， 至今已经导致了多人死亡， 并且死亡率高达 57%。 HCoV-EMC 2012 能够感染多种动物， 包括蝙蝠和猪， 使其成为持续的感染源， 并且， 目前己发现该病毒可在家庭中进行传播， 暗示了其具有了人传人的能力， 加上其高死亡率、 未知来源及传播途径引起了全球公共健康机构的高度关注。 于新型冠状病毒 HCoV-EMC 2012尚无有效的特异性药物， 因此， 急需一种针对 HCoV- EMC的特异性治疗和预防性药物。
本发明提供了一组含有如 SEQ ID NO 9所示序列的多肽， 试验表明其对新型冠状病毒 HCoV-EMC 2012感染具有抑制作用。 与该多肽共同孵育的非洲绿猴肾细胞具有显著抗病毒活 性， 其 IC50 值甚至达到 nM 级。 进一步试验表明， HR1- 5 与 HR2- 2 能相互结合， 形成 hCoV-EMC的融合六螺旋， 因而推测 HR2系列多肽能与病毒的 HR1区域结合， 从而干扰病毒 与细胞膜之间的融合， 阻断病毒感染。 本发明的多肽可与医学上常可接受的载体或赋形剂 等制成药剂， 用于预防和抑制新型冠状病毒 HCoV-EMC 2012 的感染， 也可以与其他药物组 合。 本发明为新型冠状病毒 HCoV- EMC 2012感染的预防或治疗提供候选多肽类药物。
本发明获得了能够抑制新型冠状病毒 HCoV-EMC 2012的感染的多肽抑制剂， 为进一步 研发新型冠状病毒 HCoV-EMC 2012 的预防和治疗药物提供重要的理论及应用基础， 填补了 国际空白。 附图说明
图 1、 HCoV-EMC2012 S2蛋白分子中的功能结构域以及多肽在该蛋白中的位置， 其中， 残基编号对应于其在 S蛋白全长的位置； FP， 融合肽； HR1, 七肽重复序列 1 ; HR2,七肽重 复序列 2; ™， 跨膜结构域； CP， 胞质结构域。
图 2、 显示 HR2系列多肽对 HCoV- EMC 2012的病毒抑制实验结果，
其中， a、 b、 c分别为 HR2- 1、 HR2-2 、 HR2-3多肽对 HCoV- EMC 2012的病毒抑制实验 结果。
图 3、 显示 HR1系列多肽对 HCoV-EMC 2012的病毒抑制实验结果， 其中， HR1_1、 HR1-2, 腸- 3、 HR1- 4和 HR1-5在 2-7 μΜ均未显示明显的抑制活性。
图 4、 显示 HR2多肽的抗病毒作用机制。 HR2多肽与 HR1- 5结合。
图 5、 显示 hCoV-EMC/2012 细胞融合模型的建立。 293T/EGFP (图 A) or 293T/EMC/EGFP (图 B)与新型冠状病毒 hCoV-EMC/2012的靶细胞 huh-7共孵育 37'C， 4 h。 293T/EGFP不能与 huh-7细胞发生融合， 而 293T EMC/EGFP细胞与 huh-7细胞将会发生融 合； 如果加入终浓度 Ι ΟμΜ的 HR2-2， 则该融合过程被抑制 （图 F)。 图 B、 D、 F分别为 A、 C、 E在白光下的视野。
表 1、 多肽序列及抑制活性。 其中， IC50即半数抑制浓度， 是指抑制 50% HCoV- EMC感 染的药物浓度。 具体实施方式
本发明通过下述实施例进一步阐明， 但任何实施例或其组合不应当理解为对本发明的 范围或实施方式的限制。 本发明的范围由所附权利要求书限定， 结合本说明书和本领域一 般常识， 本领域普通技术人员可以清楚地明白权利要求书所限定的范围。 在不偏离本发明 的精神和范围的前提下， 本领域技术人员可以对本发明的技术方案进行任何修改或改变， 这种修改和改变也包含在本发明的范围内。 实验材料- 多肽：
发明人通过将新型冠状病毒 HCoV- EMC 2012 S蛋白与已知 SARS 冠状病毒 S2蛋白进行 同源性比对和试验测定， 确定了新型冠状病毒 HcoV-EMC 2012 S2蛋白的 HR1和 HR2序列位 置。 选取来自 HR1和 HR2片段的 36-42个氨基酸大小的多肽片段。 共获的来自于 HR1的 5 条多肽和来自于 HR2的 3条多肽， 通过公司 （GL Biochem Shanghai Ltd )进行合成及纯化， 其纯度〉99%。 实施例 1 多肽对新型冠状病毒 HcoV- EMC 2012进入抑制实验
将多肽 HR1- 1、 HR1-2, HR1 3、 HR1- 4、 HR1- 5、 HR2- 1、 HR2-2和 HR2- 3 (图 1所示) 进行 4倍梯度稀释； 每种样品取 100 μΐ加入预先铺有每孔 50, 000个非洲绿猴肾细胞（Vero 细胞）的 96孔细胞培养板， 37°C孵育 2小时， 之后去除上清，用 PBS洗细胞 2次，加入 100 μΐ新型冠状病毒 HCoV-E C 2012 (10TCID50)， 37°C孵育 1小时； 之后再将细胞用 PBS洗 2 次。 之后再将 100 μΐ样品 /孔加入 96孔板与细胞进一步作用。 之后细胞在 37°C培养 3天。 记录细胞病变效应 (CPE), 之后每孔加入 10 μΐ MTT。 37°C孵育 4小时； 每孔再加入 100 μΐ 含有 1%SDS 的 0. 01M HC1， 37°C孵育过夜后于波长 570nm测量吸光度。
抗病毒结果如图 2所示， C-端 (HR2) 多肽中 HR2-1、 HR2-2和 HR2- 3均显示出了抗病 毒活性，其 IC50分别为 1. 82M , 0. 62 μΜ和 0. 43 MM,其中 HR2-1的活性相对较弱，而 HR2-2 和 HR2-3的活性均在 nM级。但 N-端 (HR1 )多肽， 如 HR1- 1、 HR1- 2、 HR1- 3、 HR1- 4和 HR1- 5 在 2-7 MM均未显示明显的抑制活性 （图 3)。
表 1是多肽序列及抑制 HCoV-EMC感染的活性。
¾ j
~ . Wn ic T
名称 (^ )
HRl-l GITQQVLSENQKLIAN FNQALGAMQTGFTTTNEAFQK >6.25
(SEQ ID NO 1 )
HR1-2 AMQTGFTTTNEAFQKVQDAVN AQALSKLASELSNTF >6 25
( SEQ ID NO 2)
HR1-3 AFQKVQDAVNNNAQALSKLASELSNTFGAISASIGDII >6.25
(SEQ ID NO 3 )
HR1-4 SELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRL >6.25
(SEQ ID NO 4)
HR1-5 ANKFNQALGAMQTGFTTTNEAFQKVQDAVNNNAQALSKLASE >6.25
(SEQ ID NO 5 )
HR2-1 IPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESY 1 82
(SEQ ID NO 6)
HR2-2 SLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL 0 62
(SEQ ID NO 7) LDLTYEMLSLQQVVKALNESYIDLKELGNYTYYNKW
( SEQ ID NO 8)
实施例 2 HR1-5与 HR2- 2互作 本实验参照文献 ((Interaction between heptad repeat 1 and 2 regions in spike protein of SARS- associated coronavirus : implications for virus fusogenic mechanism and ident ification of fusion inhibitors.》 (Liu，S. W. et al. Lancet 363, 938-947， 2004 ) 操作。
1、 非变性聚丙稀酰胺凝胶电泳 （N-PAGE)。 将 HR1系列多肽分别于 HR2系列多肽混合 (终浓度各 30MM), 37'C孵育 30分钟。 取 5 X高 PH值上样缓冲液， 与 4倍体积的肽混合物 混匀。 然后点样 （每孔 25 μΐ ) 于 18%非变性胶中 （北京天恩泽）， 于室温下 125V恒压电泳 2小时。 考马斯亮蓝染色。 结果显示， HR1-5在与 HR2系列多肽混合以后， 均产生了一条新 的多肽 （图 4Α)。 该条带即为 EMC病毒融合活性六螺旋 (6- ΗΒ)条带。
2、 高效液相色谱检测分子量： 利用 GE公司 superdex 75 10/300GL柱进行分子筛 HPLC (SR-PAGE), 检测寡聚体的形成。 以 HR1的多肽 HR1- 5和 HR2的多肽 HR2-2为代表， 将其 混合 （终浓度是 50 mol/L), 37'C温育 30分钟， 100 样品注入与 HPLC系统连接的色谱 柱中。 用 ρΗ 7 2的磷酸盐缓冲液作为流动相， 流速为 0. 8 mL/mir。 GE公司蛋白纯化系统 记录 220 nm的紫外吸光值。 同时使用 HIV多肽 N36， C34以及其六螺旋产物作为本实验的 分子量参照。
结果可见（如图 4B所示）， HR1-5和 HR2-2混合之后， 发生了相互作用， 在单体主峰之 ^形成新的检测峰， 根据其位置可见， 其分子量略大于 HIV N36/C34形成的六螺旋。 符合 H 1-5/HR2-2所形成的的六螺旋大小 （25. 8kD)。 3、 圆二色谱测定多肽二级结构。 用 50議 ol/L、 pH 7. 2 的磷酸缓冲液将游离多肽或多 肽混合物稀释到终浓度为 10 Mmol/L。 使用分光偏振仪 (J-815型， Jascolnc, Japan)测定 圆二色谱。 检测温度 4°C， 带宽 5. 0 nm, 解析度 0. 1 nra, 光径 0. 1 cm, 反应时间 4. 0 s , 扫描速度 50 nm/min。 在 222 nm以 5 °C/min 的温度梯度变化监测多肽的热变性。 减去缓冲 溶液的空白对照来校正谱值。 对熔解曲线进行平滑化处理， 并用 Jasco计算热解离转变的 中点温度， 即 Tm值
结果显示， 以隠-5、 HR2- 2为例可见 （如图 4C所示）， 单独的 HR1- 5为无规则卷曲结 构， 单独的 HR2-2 为低螺旋结构。 当两者混合以后， 可见其混合物为高度的螺旋结构。 结 果符合六螺旋核心结构的 CD光谱。 而其 Tm值约为 9(TC (如图 4D所示)。 综上表明， HR1- 5与 HR2- 2 能相互结合， 形成 hCoV- EMC的融合六螺旋； 而 HR2系列多 肽则能与 HR 1 5相结合， 因而推测 HR2系列多肽能与病毒的 HR1区域结合， 从而干扰病毒 与细胞膜之间的融合， 阻断病毒感染。 实施例 3: 对 HR2-2多肽的改造
多肽：
发明人发现新型冠状病毒 HCOV-EMC/2012 S2 蛋白的多肽片段 HR2-2具有较好的生物 活性。 然而尚不能完全满足生物医药领域的要求。 因此， 发明人基于已有 HR2-2多肽的的 氨基酸序列进行改造， 根据文献 （Otaka， A et al Remodeling of gp41-C34 peptide leads to highly effective inhibitors of the fusion of HIV- 1 with target cells Angew Chem Int Ed Engl 41， 2937-2940 (2002)) 描述的方法， 在 HR2-2多肽的内部引入谷氨酸 (E)、 赖氨酸 (K)、 精氨酸 (R), 以增加 HR2-2多肽内部的盐桥， 从而达到增加多肽溶解度、 稳定性， 以及抗病毒活性 的目的。序列见表 2，其中氨基酸之间的连线代表两者之间形成的盐桥。多肽序列确定以后， 进行合成及纯化， 测定其纯度 >99%。 实验方法及结果：
1 HR2-2及突变体溶解度的检测：
将多肽加入 PBS ( ρΗ7 2 ) 以及 ¾0中至过饱和。 12000rpm离心 10分钟， 取上清， 使用 nanodrop仪测定吸光度 A280。 在 ExPASy (http.//web expasy.org protparam/) 网站 获取多肽相应的消光系数， 将 A280 与消光系数系数代入公式 (μΜ)=(Α280/消光系 数 106中， 计算多肽的摩尔数， 即为该多肽的溶解度。
2 圆二色谱测定多肽二级结构。 具体方法见实施例 1。 检测新合成的突变体多肽在 222nm的 CD值， 依照 100°/。α- 螺旋的 CD值为 -33χ 103， 计算相应多肽的 α-螺旋比例。 同时检测多肽与 HR1-5作用后， 其复合物的 α-螺旋比例， 以及 Tm值。
3细胞融合模型的构建， 以及多肽抑制活性的检测。 发明人构建一种能够同时表达 hCoV-EMC/2012 S protein以及 GFP的表达质粒 (pAAV-EGFP-IRES-EMC-S ) ， 转染 293T细胞 (293T/EMC/EGFP)。 同时仅转染 pAAV-EGFP-IRES的质粒作为阴性对照（293T/EGFP)。由于在两端蛋白中间具有 IRES 序列， 因此， 293T/EMC/EGFP将实现两个蛋白的同时的、 分别的表达， 而不会相互干 扰。其中 S蛋白将递呈到细胞表面，介导转染的 293T( 293T/EMC/EGFP)与靶细胞 Huh-7 的融合， GFP可以用于观察转染效率。同时， 由于融合后 GFP将重新分布到靶细胞中， 因此， 融合细胞的荧光范围会更大、 荧光更弱 （见图 5-C中箭头指示细胞） 。 因此， 可 以将融合细胞与未融合的细胞进行区别并计算融合细胞的比率。体系中如果加入有效的 多肽抑制剂如 HR2-2, 即可阻止该融合过程 （图 5-E) 。 实验过程中， 将受试多肽在 96孔板中进行梯度稀释， 与 Ι χΙΟ4个已经被转染 pAAV-hGFP-IRES-E C-S的 293T细胞 (293T/EMC/EGFP)进行混合， 之后再与提前 5-9 小时铺在的 96孔板的每孔 5-ΙΟχ ΙΟ4靶细胞 huh-7上， 在 37°C下孵育 4小时， 观察多肽 对细胞融合的抑制情况。 计算多肽对细胞融合抑制的半数有效量 （IC50) 。 结果- 结果如表 2。 可见， 在 HR2-2多肽中引入 EK突变后， 进一步稳定了多肽自身的天然 结构。 同时， 突变后的多肽与 HR1-5形成的复合物的 a-螺旋度得以增加、 Tm上升。 可见 突变多肽与 HR1-5所形成复合物的稳定性也得以提高。 溶解度的结果也显示， 突变后的多 肽增加了在 PBS以及 ¾0中的溶解度， 同时， 从表 2可看出突变后的多肽的 IC50值减少， 表明所述多肽提高了药效。 表 2、 HR2-2突变体的生物物理学特征及生物活性
螺旋 HI -5/HR2 溶解度 （mg/ml)
多肽名 序列 Κ50 (μΜ) 比例 ot-螺旋 Tm PBS (pH 7 2) H20
HR2-2 SLTQINTTLLDLTYEMLSLQQVV LALNESYIDLKEL 1 8 2% 71 8% 87 2 0 38±0 07 0 06±0 01 0 93±0 15
HR2-2- Ml SLTQINTTLLDLJSYEMRSLQQVVKALNESYIDLKEL 36 4% 74 0% 87 9 4 42±0 08 4 16±0 15 0 85±0 08
( SEQ ID NO 10 ) 1 1
HR2-2-M2 SLTQINTTLLOLEYEMKKLEEVVKKLEESYIOLKEL 42 4% 71±0 22 107 21±5 57 0 55±0 04
( SEQ ID NO 11 ) I 1 1———— I
注- 通过在 HR2-2多肽序列中引入谷氨酸 (E)、 赖氨酸 (K)、 精氨酸 (R)突变， 以增加 HR2-2多肽内部的盐桥， 从而达到增加溶解度、 多肽 定性， 以及抗病毒活性的目的。 序列中突变的氨基酸加黑并用斜体标注。 连线代表相应氨基酸位置的 E-K，E-R，K-E形成的盐桥。 IC50是 用指多肽抑制病毒新型冠状病毒 S蛋白介导的细胞融合的半数有效量。

Claims

1. 一种对新型冠状病毒 HCoV-EMC 2012感染均具有抑制作用的多肽， 其特征在于， 该多肽含有如 SEQ ID NO 9所示的序列。
2. 如权利要求 1所述的多肽， 其特征在于， 所述的多肽能够抑制 HCoV-EMC病毒感 染并且该多肽的氨基酸序列由如 SEQ ID NO 9所示的多肽经过取代、 缺失或添加一个或者多 个氨基酸而构成。
3. 如权利要求 2所述的多肽，其特征在于，所述的多肽的氨基酸序列如 SEQ ID NO 6、 SEQ ID NO 7或者 SEQ ID NO 8所示。
4. 含有权利要求 1所述的多肽的重组蛋白。
5. 一种核酸， 其特征在于， 所述的核酸编码权利要求 1所述的多肽。
6. 一种表达载体， 其特征在于， 所述的表达载体包含权利要求 5所述的核酸。
7. —种重组细胞， 其特征在于， 所述的重组细胞含有权利要求 6所述的表达载体。
8. 一种药物组合物， 其特征在于， 其有效成分包含权利要求 1所述的多肽、 权利要 求 4所述的重组蛋白、 权利要求 5所述的核酸、 权利要求 6所述的表达载体或者权利要求 7 所述的重组细胞。
9. 权利要求 1所述的多肽在制备抗新型冠状病毒 HCoV-EMC 2012感染药物中的应用。
10. 如权利要求 9所述的应用， 其特征在于， 将权利要求 1所述的多肽加入可能受到 新型冠状病毒 HCoV-EMC 2012感染的体外培养物。
PCT/CN2014/074045 2013-03-26 2014-03-25 对新型冠状病毒HCoV-EMC 2012感染具有抑制作用的多肽及其应用 Ceased WO2014154134A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020157016526A KR20150133690A (ko) 2013-03-26 2014-03-25 신종 코로나바이러스 HCoV-EMC 2012 감염에 대해 억제 작용이 있는 폴리펩티드 및 이의 응용

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201310099025.7 2013-03-26
CN201310099025.7A CN104072592B (zh) 2013-03-26 2013-03-26 对新型冠状病毒HCoV-EMC 2012感染具有抑制作用的多肽及其应用

Publications (1)

Publication Number Publication Date
WO2014154134A1 true WO2014154134A1 (zh) 2014-10-02

Family

ID=51594230

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2014/074045 Ceased WO2014154134A1 (zh) 2013-03-26 2014-03-25 对新型冠状病毒HCoV-EMC 2012感染具有抑制作用的多肽及其应用

Country Status (3)

Country Link
KR (1) KR20150133690A (zh)
CN (1) CN104072592B (zh)
WO (1) WO2014154134A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111363847A (zh) * 2020-02-12 2020-07-03 广州微远基因科技有限公司 基于CRISPR技术的2019-nCoV快速检测引物组及其用途
US11479582B2 (en) 2020-10-16 2022-10-25 King Faisal University Anti-SARS-CoV-2 fusion peptides
WO2023050661A1 (zh) * 2021-09-28 2023-04-06 北京亦科信息菌素研究院有限公司 一种抗新冠病毒的信息菌素及其应用
CN118496318A (zh) * 2024-07-18 2024-08-16 华南理工大学 一种香菇柄蛋白源dpp-iv和ace双重抑制肽lp-6及其应用

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107022008B (zh) * 2016-01-30 2021-04-23 山西锦波生物医药股份有限公司 广谱地抑制人类冠状病毒感染的多肽及其应用
WO2019136824A1 (zh) * 2018-01-15 2019-07-18 中国人民解放军军事科学院军事医学研究院 抑制MERS-CoV感染的多肽
CN111592594B (zh) * 2020-03-13 2022-05-10 北京大学 一种抗新型冠状病毒的单克隆抗体及其应用
CN111349150B (zh) * 2020-03-24 2021-02-09 北京中科微盾生物科技有限责任公司 一种抑制新型冠状病毒的多肽及其用途
CN112410465A (zh) * 2020-03-27 2021-02-26 大连民族大学 新型冠状病毒SARS-CoV-2 ORF1ab和N基因恒温扩增引物组及试剂盒
US10975126B1 (en) 2020-04-23 2021-04-13 King Faisal University MERS-CoV inhibitor peptides
CN111732637B (zh) * 2020-05-25 2022-03-01 上海交通大学 抑制新型冠状病毒SARS-CoV-2感染宿主细胞的多肽及其应用
CN111499692B (zh) * 2020-06-16 2020-12-04 国家纳米科学中心 靶向新型冠状病毒covid-19的多肽及其应用
CN111647048B (zh) * 2020-06-22 2021-04-02 中国科学院昆明动物研究所 干扰多肽在制备抗SARS-CoV-2药物中的应用
WO2021258250A1 (zh) 2020-06-22 2021-12-30 中国科学院昆明动物研究所 干扰多肽在制备抗SARS-CoV-2药物中的应用
CN112138160A (zh) * 2020-08-24 2020-12-29 复旦大学 griffithsin在制备新型冠状病毒感染药物中的用途
CN113563433B (zh) * 2021-06-04 2023-03-31 山西锦波生物医药股份有限公司 多肽、其制备方法和用途
CN119613503B (zh) * 2024-07-22 2026-02-06 山西锦波生物医药股份有限公司 可抑制类mers冠状病毒感染的多肽及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1784422A (zh) * 2003-04-08 2006-06-07 科罗诺瓦蒂夫股份有限公司 引起严重急性呼吸综合症(sars)的冠状病毒
CN101076591A (zh) * 2004-07-21 2007-11-21 香港大学 一种引起呼吸道感染的新型人病毒及其用途
WO2011004146A1 (en) * 2009-07-07 2011-01-13 Institute For Animal Health Chimaeric protein

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1784422A (zh) * 2003-04-08 2006-06-07 科罗诺瓦蒂夫股份有限公司 引起严重急性呼吸综合症(sars)的冠状病毒
CN101076591A (zh) * 2004-07-21 2007-11-21 香港大学 一种引起呼吸道感染的新型人病毒及其用途
WO2011004146A1 (en) * 2009-07-07 2011-01-13 Institute For Animal Health Chimaeric protein

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GAO, J. ET AL.: "Structure of the Fusion Core and Inhibition of Fusion by a Heptad Repeat Peptide Derived from the S Protein of Middle East Respiratory Syndrome Coronavirus", JOURNAL OF VIROLOGY, vol. 87, no. 24, 31 December 2013 (2013-12-31) *
LU , L. ET AL.: "Structure-based Discovery of Middle East Respiratory Syndrome Coronavirus Fusion Inhibitor", NATURE COMMUNICATIONS, 28 January 2014 (2014-01-28) *
VAN, B.S. ET AL.: "Genomic Characterization of a Newly Discovered Coronavirus Associated with Acute Respiratory Distress Syndrome in Humans", MBIO, vol. 3, no. 6, 20 November 2012 (2012-11-20), XP055128733, DOI: doi:10.1128/mBio.00473-12 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111363847A (zh) * 2020-02-12 2020-07-03 广州微远基因科技有限公司 基于CRISPR技术的2019-nCoV快速检测引物组及其用途
CN111363847B (zh) * 2020-02-12 2021-06-29 广州微远医疗器械有限公司 基于CRISPR技术的2019-nCoV快速检测引物组及其用途
US11479582B2 (en) 2020-10-16 2022-10-25 King Faisal University Anti-SARS-CoV-2 fusion peptides
WO2023050661A1 (zh) * 2021-09-28 2023-04-06 北京亦科信息菌素研究院有限公司 一种抗新冠病毒的信息菌素及其应用
CN118496318A (zh) * 2024-07-18 2024-08-16 华南理工大学 一种香菇柄蛋白源dpp-iv和ace双重抑制肽lp-6及其应用

Also Published As

Publication number Publication date
CN104072592A (zh) 2014-10-01
CN104072592B (zh) 2016-12-28
KR20150133690A (ko) 2015-11-30

Similar Documents

Publication Publication Date Title
WO2014154134A1 (zh) 对新型冠状病毒HCoV-EMC 2012感染具有抑制作用的多肽及其应用
KR102848852B1 (ko) 폴리펩티드, 이의 제조방법 및 용도
CN107022008B (zh) 广谱地抑制人类冠状病毒感染的多肽及其应用
CN102883740B (zh) 用于使hiv失活并阻断hiv的双功能分子
US7456251B2 (en) HIV fusion inhibitor peptides with improved biological properties
Eggink et al. Selection of T1249-resistant human immunodeficiency virus type 1 variants
Chong et al. Two MT hook residues greatly improve the antiviral activity and resistance profile of the HIV-1 fusion inhibitor SC29EK
WO2025087234A1 (zh) 灭活冠状病毒的融合蛋白及其应用
CN105646717B (zh) 一种长效hiv融合抑制剂及其应用
WO2005002500A2 (en) Inhibitors of coronavirus
WO2026021236A1 (zh) 可抑制类mers冠状病毒感染的多肽及其应用
CN102816215A (zh) 抑制hiv的短小多肽及药物用途
JP2011521648A (ja) Ｈｉｖの侵入を阻害するための二機能性分子
CN106397548A (zh) 用于抑制hiv感染的多肽及其药物用途
Pan et al. A novel chimeric protein-based HIV-1 fusion inhibitor targeting gp41 glycoprotein with high potency and stability
KR20240166574A (ko) 점액점착 및 바이러스 결합 특성을 가진 2중 기능성 펩타이드
WO2013127300A1 (zh) 用于抑制hiv的多肽、其药物组合物及其用途
CN104151402B (zh) 病毒性心肌炎环肽疫苗及其制备方法
US20030219451A1 (en) Stable helical C peptides and uses therefor
CN120098150B (zh) 一种广谱抗新型冠状病毒的双靶点多肽及其应用
US20050221288A1 (en) Variant tat proteins and methods for use thereof
Abancó et al. Optimized ACE2‐Fc fusion proteins with picomolar neutralization activity against highly evolved SARS‐CoV‐2 variants
CN106543273A (zh) 用于抑制hiv感染的多肽及其药物用途
HK40118032A (zh) 可抑制类mers冠状病毒感染的多肽及其应用
Zhu Respiratory Syncytial Virus Fusion Protein Interaction with its Cellular Receptor, Nucleolin

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14772801

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 20157016526

Country of ref document: KR

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14772801

Country of ref document: EP

Kind code of ref document: A1