WO2022073234A1 - Peptide for inhibiting eb virus, dna encoding peptide, and use thereof - Google Patents

Peptide for inhibiting eb virus, dna encoding peptide, and use thereof Download PDF

Info

Publication number
WO2022073234A1
WO2022073234A1 PCT/CN2020/120203 CN2020120203W WO2022073234A1 WO 2022073234 A1 WO2022073234 A1 WO 2022073234A1 CN 2020120203 W CN2020120203 W CN 2020120203W WO 2022073234 A1 WO2022073234 A1 WO 2022073234A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
viral
acid sequence
epstein
barr virus
Prior art date
Application number
PCT/CN2020/120203
Other languages
French (fr)
Chinese (zh)
Inventor
高嵩
何会萍
罗梦
曹雨露
欧均颖
于冰
Original Assignee
中山大学肿瘤防治中心
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 中山大学肿瘤防治中心 filed Critical 中山大学肿瘤防治中心
Priority to PCT/CN2020/120203 priority Critical patent/WO2022073234A1/en
Publication of WO2022073234A1 publication Critical patent/WO2022073234A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to a peptide for inhibiting Epstein-Barr virus, DNA encoding the peptide and application thereof.
  • Epstein-Barr virus is a gamma-herpes virus, also known as a double-stranded DNA virus, associated with a variety of human malignancies. According to statistics, about 95% of the world's population carries the virus. EBV is an oncogenic virus that is closely associated with the development of a variety of specific human tumors, including nasopharyngeal carcinoma, Hodgkin's lymphoma, Burkitt's lymphoma, and gastric cancer. According to the World Cancer Report released by the International Agency for Research on Cancer (IARC) in 2008, EBV causes 1% of global cancers and 5.6% of all infectious cancers. According to the IARC classification criteria for carcinogens, EBV is listed in the first group of carcinogens. Currently, there is no effective clinical approach to prevent or eliminate EBV infection.
  • IARC International Agency for Research on Cancer
  • the present invention provides a peptide comprising the amino acid sequence shown in SEQ ID NO:1.
  • the invention provides a nucleic acid sequence encoding the peptide of the first aspect of the invention.
  • the present invention provides an expression vector comprising the nucleic acid sequence of the second aspect of the present invention.
  • the present invention provides the peptide of the first aspect of the present invention, the nucleic acid sequence of the second aspect of the present invention, or the expression vector of the third aspect of the present invention in the preparation for the treatment of Epstein-Barr virus-related diseases Use in medicine.
  • the present invention provides a method for inhibiting Epstein-Barr virus replication, the method comprising administering the peptide of the first aspect of the present invention or the expression vector of the third aspect of the present invention.
  • the present invention provides a method of treating an Epstein-Barr virus-related disease, the method comprising administering to a patient the peptide of the first aspect of the present invention or the expression vector of the third aspect of the present invention.
  • the present invention provides a kit comprising the peptide of the first aspect of the present invention, the nucleic acid sequence of the second aspect of the present invention, or the expression vector of the third aspect of the present invention.
  • the peptide of the present invention can effectively reduce the copy number of the virus genome, so the design of small molecule inhibitors according to the peptide can potentially alleviate and treat Epstein-Barr virus infection or human diseases associated with Epstein-Barr virus.
  • Figure 1 shows details of the BBRF2 ⁇ -BSRF1 ⁇ interface, where: Panel a schematically shows the recognizable residues (colored regions) of BBRF2 ⁇ (green) and BSRF1 ⁇ (blue) in the BBRF2 ⁇ -BSRF1 ⁇ crystal complex with full Comparison of long BBRF2 and BSRF1; panel b shows two views of the heterodimer in the same colors as panel a, the N- and C-termini of BSRF1 ⁇ are annotated; panel c shows one of the two antiparallel alpha helices of BSRF1 ⁇
  • Figures d-f show the hydrophobic interface (d), the polar interface (e), and the BSRF1 ⁇ N-terminal loop-mediated interface (f) at the BBRF2 ⁇ -BSRF1 ⁇ interface, respectively, with secondary structural elements annotated,
  • the amino acid residues involved are colored in the color of the molecule to which they belong; panel g shows a surface conservation map of BBRF2 ⁇ (
  • Figure 2 shows BSRF1 ⁇ -derived peptides and their binding to BBRF2 ⁇ , wherein: Panel a shows a schematic representation of the amino acid lengths of five BSRF1 ⁇ -derived peptides (P1-P5), where N represents the N-loop region in the resolved structure; Panel b shows BLI analysis of five peptides bound to 10 ⁇ g ml -1 His6 - BBRF2 ⁇ , only P1 was shown to bind His6 - BBRF2 ⁇ ; panel c shows peptide 1 (P1) and BBRF2 ⁇ measured by BLI Binding affinity of: different concentrations of P1 were combined with 10 ⁇ g ml -1 His-tagged BBRF2 ⁇ ; panel d shows that P1 competes with BSRF1 ⁇ for binding to BBRF2 ⁇ : 200 nM BBRF2 ⁇ was mixed with different concentrations of P1, with 10 ⁇ g ml -1 immobilized BSRF1 ⁇ binding; panel e shows that TAT-P1 reduce
  • BBRF2 and BSRF1 (or its homologs) are believed to be critical for the life cycle of Epstein-Barr virus, and targeting the binding surface of BBRF2 and BSRF1 has the potential to be a potential clinical therapeutic strategy against EBV.
  • the inventors designed five BSRF1-derived peptides (P1-P5) as shown in Fig. 2a, which cover the BBRF2-binding peptides on the N-loop, ⁇ A or ⁇ B of BSRF1 site.
  • the present invention provides a peptide comprising the amino acid sequence shown in SEQ ID NO:1.
  • the target peptide can be linked with other peptides with specific functions to achieve the corresponding functions.
  • the peptide of interest can be linked to a cell penetrating peptide, thereby allowing the peptide of interest to readily penetrate the cell membrane and enter the cell.
  • the peptide further comprises a cell penetrating peptide such as a TAT sequence and a linker peptide.
  • the invention provides a nucleic acid sequence encoding the peptide of the first aspect of the invention.
  • nucleotide sequence encoding the peptide in the case of knowing the amino acid sequence of the peptide.
  • the nucleic acid sequence is the sequence shown in SEQ ID NO:2.
  • SEQ ID NO:2 The sequence of SEQ ID NO:2 is shown below:
  • nucleotide sequence shown in SEQ ID NO: 2 can encode the peptide of the present invention, but its degenerate sequence can also encode the peptide of the present invention.
  • the present invention provides an expression vector comprising the nucleic acid sequence of the second aspect of the present invention.
  • the so-called "expression vector” refers to a vector that adds expression elements (such as promoter, RBS, terminator, etc.) on the basis of the basic skeleton of the cloning vector, so that the target gene can be expressed.
  • the expression vector can be a plasmid vector, a phage vector, a virus vector, etc., which are not particularly limited, as long as the target protein can be expressed.
  • the present invention provides the peptide of the first aspect of the present invention, the nucleic acid sequence of the second aspect of the present invention, or the expression vector of the third aspect of the present invention in the preparation for the treatment of Epstein-Barr virus-related diseases Use in medicine.
  • the Epstein-Barr virus-related disease is selected from the group consisting of infectious mononucleosis, linked lymphoproliferative syndrome, viral hemophagocytic syndrome, oral hairy leukoplakia, viral Meningitis, peripheral neuritis, viral pneumonia, viral myocarditis, nasopharyngeal carcinoma, Hodgkin lymphoma, Burkitt lymphoma, gastric cancer, hepatocellular carcinoma, lymphoepithelioid sarcoma, salivary gland tumor, breast cancer, thymoma, Primary effusion lymphoma or B/T/NK cell lymphoma.
  • the present invention provides a method for inhibiting Epstein-Barr virus replication, the method comprising administering the peptide of the first aspect of the present invention or the expression vector of the third aspect of the present invention.
  • the present invention provides a method of treating an Epstein-Barr virus-related disease, the method comprising administering to a patient the peptide of the first aspect of the present invention or the expression vector of the third aspect of the present invention.
  • the Epstein-Barr virus-related disease is selected from the group consisting of infectious mononucleosis, linked lymphoproliferative syndrome, viral hemophagocytic syndrome, oral hairy leukoplakia, viral Meningitis, peripheral neuritis, viral pneumonia, viral myocarditis, nasopharyngeal carcinoma, Hodgkin lymphoma, Burkitt lymphoma, gastric cancer, hepatocellular carcinoma, lymphoepithelioid sarcoma, salivary gland tumor, breast cancer, thymoma, Primary effusion lymphoma or B/T/NK cell lymphoma.
  • the present invention provides a kit comprising the peptide of the first aspect of the present invention, the nucleic acid sequence of the second aspect of the present invention, or the expression vector of the third aspect of the present invention.
  • the peptides of the present invention can effectively reduce the viral genome copy number, and thus can alleviate and treat Epstein-Barr virus infection and Epstein-Barr virus-related human diseases.
  • test methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, were purchased from conventional chemical reagent stores. It should be noted that the above summary section and the following detailed description are only for the purpose of specifically illustrating the present invention and are not intended to limit the present invention in any way. The scope of the present invention is to be determined by the appended claims without departing from the spirit and spirit of the present invention.
  • the cDNAs of BBRF2 and BSRF1 were amplified from the genome of human herpesvirus 4 strain M81.
  • the cDNAs of full-length BBRF2 and BBRF2 17-278 (BBRF2 ⁇ ) were cloned into the engineered pET28 vector (pSKB) and expressed in E. coli Rosetta (DE3) cells with a 6 ⁇ His-tag at the N-terminus followed by PreScission cleavage site fusion protein.
  • Transformed bacteria were grown in Terrific Broth (TB) medium at 37°C with the addition of 100 ⁇ M of isopropyl-1-thio- ⁇ -D-thiogalactoside (IPTG) at an optical density of 0.6. induce. After induction, cells were grown overnight (approximately 16-18 hours) at 18°C and collected by centrifugation.
  • IPTG isopropyl-1-thio- ⁇ -D-thiogalactoside
  • binding buffer A containing 20 mM HEPES (pH 7.0), 600 mM NaCl, 10% glycerol, 30 mM imidazole, 2 mM ⁇ -ME. ) column (GE Healthcare).
  • binding buffer A containing 20 mM HEPES (pH 7.0), 600 mM NaCl, 10% glycerol, 30 mM imidazole, 2 mM ⁇ -ME.
  • binding buffer A proteins were eluted with elution buffer containing 20 mM HEPES (pH 7.0), 600 mM NaCl, 10% glycerol, 300 mM imidazole and 2 mM ⁇ -ME.
  • the eluted protein was incubated with PreScission protease (PSP) fused to 20 ⁇ g glutathione S-transferase (GST) to remove the His 6 -tag and treated with 20 mM HEPES (pH 7.0), 600 mM NaCl at 4° C. , 10% glycerol, 2 mM ⁇ -ME in binding buffer B overnight. After dialysis, PSP was removed using a GST column. The protein was reloaded onto a second Ni-NTA column equilibrated with Binding Buffer B and eluted with Binding Buffer A.
  • PSP PreScission protease
  • GST glutathione S-transferase
  • BBRF2 17-278 was expressed as previously described and purified as the native BBRF2 protein.
  • BSRF1 34-159 The cDNA of BSRF1 34-159 (BSRF1 ⁇ ) was cloned into the pGEX-6p-1 vector. Recombinant protein was expressed and collected in lysis buffer containing 50 mM HEPES (pH 7.0), 150 mM NaCl, 1 mM PMSF and 2 mM ⁇ -ME like BBRF2 protein.
  • the collected protein was loaded onto a GST column equilibrated with binding buffer containing 20 mM HEPES (pH 7.0), 150 mM NaCl, and 2 mM ⁇ -ME, and mixed with 20 mM HEPES (pH 7.0), 150 mM NaCl, 10 mM glutathione and 2mM ⁇ -ME buffer was used for elution. After treatment with 20 ⁇ g of PSP, the protein was loaded onto a GST column to remove GST-tag and PSP. SEC was performed in the same manner as for BBRF2 protein.
  • BBRF2 17-278 -BSRF1 34-159 (BBRF2 ⁇ -BSRF1 ⁇ ) complexes were prepared by mixing purified BBRF2 ⁇ and BSRF1 ⁇ in a 1:1 molar ratio and incubating overnight at 4°C, followed by a HiLoad 16/60 Superdex 200 column ( GE) The complex was purified by SEC in buffer C. The cDNA of BSRF1 20-218 was cloned into pSKB vector. Recombinant proteins were expressed, harvested and purified as in the BBRF2 construction with less NaCl (300 mM) and no glycerol in all buffers.
  • Crystallization experiments were performed with a mixture of equal volumes of protein (approximately 7 mg.ml -1 ) and stock solution using the hanging drop vapor diffusion method. Crystals of SeMet BBRF2 ⁇ from 0.09M NPS (0.03M NaNO3, 0.03M Na2HPO4 , 0.03M( NH4 ) 2SO4 ), 0.1M 2- (N-morpholine)ethanesulfonic acid (MES) at 4 °C )/imidazole (pH 6.5), 12.5% PEG1000, 12.5% PEG 3350 and 12.5% MPD.
  • 0.09M NPS (0.03M NaNO3, 0.03M Na2HPO4 , 0.03M( NH4 ) 2SO4
  • MES N-morpholine)ethanesulfonic acid
  • Crystals of the BBRF2 ⁇ -BSRF1 ⁇ complex were grown from 0.1 M magnesium acetate, 0.05 M MES (pH 5.6) and 20% MPD after 1:1000 m/m ⁇ -chymotrypsin treatment overnight at 4°C. The crystals were snap-frozen directly in liquid nitrogen.
  • the X-ray diffraction dataset of BBRF2 ⁇ was collected at beamlines BL17U1 and BL19U1 of the Shanghai Synchrotron Radiation Facility (SSRF).
  • a dataset of BBRF2 ⁇ -BSRF1 ⁇ complexes was collected at beamline BL18U1 of SSRF.
  • the dataset is processed using the XDS program group.
  • the initial phase of the BBRF2 ⁇ structure was obtained by the single-wavelength anomalous dispersion (SAD) method and corrected from the diffraction dataset of SeMet-substituted BBRF2 ⁇ crystals using phenix.
  • the BBRF2 ⁇ -BSRF1 ⁇ complex structure was solved by molecular replacement using Phaser with the structure of BBRF2 ⁇ as the search model.
  • *Numbers in parentheses are values from the layer with the highest resolution.
  • CNE2-EBV cells were donated by Professor Zeng Yi (Chinese Academy of Medical Sciences). Cells were cultured in RPMI 1640 medium (GIBCO) containing 10% FBS and penicillin/streptomycin. The cell line does not contain mycoplasma. CNE2-EBV cells were derived from a parental cell line that had been infected with recombinant EBV.
  • BLI detection was performed using an eight-channel OctetRED Biofilm Layer Interferometer System (FortéBio). To determine the interaction between BBRF2 ⁇ and BSRF1 ⁇ or the peptides P1-P5, His 6 -tagged BBRF2 ⁇ (10 ⁇ g ml ⁇ 1 ) was immobilized on the tip of an NTA biosensor (FortéBio), which had been pretreated with 20 mM Reaction buffer equilibration of HEPES (pH 7.0), 600 mM NaCl, 10% glycerol and 1 mM DTT. BSRF1 ⁇ or peptides P1-P5 were diluted to 4-8 different concentrations and analyzed sequentially at each concentration by a tip coated with His6 - tagged BBRF2 ⁇ .
  • FormBio OctetRED Biofilm Layer Interferometer System
  • BSRF1 ⁇ was biotinylated for 30 min at room temperature using the Biotinylation Kit (Genemore), and then BSRF1 ⁇ was immobilized to streptavidin-coated ( SA) on the biosensor tip.
  • P1 peptides were diluted to different concentrations (100 ⁇ M, 50 ⁇ M, 25 ⁇ M, 12.5 ⁇ M, 6.3 ⁇ M and 3.1 ⁇ M) in reaction buffer and mixed with 200 nM of BBRF2 ⁇ , respectively. All experiments were performed at 25°C.
  • Each measurement involved a baseline of 120s (with reaction buffer), followed by a binding phase of 180s (with protein) or (with peptide), and a dissociation phase of 180s (with reaction buffer).
  • Raw data were processed using Octet Data Analysis software 11.0 provided by Fortebio to derive dissociation constants (K D ). Results were plotted using Origin (version 2019, OriginLab).
  • the TAT-peptide was dissolved in RPMI 1640 medium.
  • CNE2-EBV cells human nasopharyngeal carcinoma cells with persistent EBV infection
  • PMA phorbol 12-myristate 13-acetate
  • CNE2-EBV cells pre-transfected with TAT-peptide were treated with 2.5 mM sodium butyrate and 20 ng ml -1 phorbol 12-myristate 13-acetate (PMA) for 12 h to induce EB virions production. After 48 hours, cells were harvested and washed 3 times with PBS for measurement of viral replication, then the remaining medium was filtered through a 0.45 ⁇ m filter and centrifuged at 1,000 g for 10 minutes at 4° C. to remove cellular debris. The copy number of encapsulated viral genomic DNA was determined by qPCR analysis of viral supernatants. Briefly, (QIAGEN) encapsulated viral genomic DNA was extracted from induced cells.
  • the supernatant was digested with DNase I (105U ml -1 ) for 1 hour at 37°C and then mixed with lysis buffer and 0.1 mg ml -1 proteinase K. The addition of proteinase K removes the viral envelope and capsid. The mixture was heated at 56°C for 10 minutes and then at 75°C for 20 minutes to inactivate the enzymes. The samples were diluted 1:10 with RNase-free water and then subjected to qPCR using primers for the BALF5 DNA polymerase gene. EBV-encoded genes were quantified by qPCR using gene-specific primers (Table 3).
  • BSRF1-derived peptides P1-P5
  • P1-P5 BSRF1-derived peptides
  • BLI analysis revealed that only the longest P1 (covering the N-loop and ⁇ A) showed significant binding to BBRF2 ⁇ with a KD of 7.4 ⁇ M (Fig. 2c, Fig. 2b). This means that a single structural element of BSRF1 is not sufficient for a stable binding between BSRF1 ⁇ and BBRF2.
  • the inventors then examined whether P1 could compete with wild-type BSRF1 ⁇ in the BBRF2 ⁇ -BSRF1 ⁇ complex. As shown by BLI analysis, addition of P1 disrupted the binding between BBRF2 ⁇ and immobilized BSRF1 ⁇ in a concentration-dependent manner (Fig. 2d), indicating that P1 can compete with BSRF1 for binding BBRF2 ⁇ .
  • the inventors investigated the potential cellular effects of P1 in CNE2-EBV cells, a human nasopharyngeal carcinoma cell with persistent EBV infection. To facilitate cellular uptake of P1, the inventors fused the TAT sequence and a tetraglycine linker to the N-terminus of P1 (TAT-P1).
  • TAT-P1 When transfected into CNE2-EBV cells, TAT-P1 reduced the viral genome copy number in a concentration-dependent manner. At a concentration of 5 ⁇ M, TAT-P1 was able to reduce EBV genome copy number by 75%, but this effect was not further enhanced at higher concentrations of TAT-P1 (Fig. 2e). These results suggest that disrupting the BBRF2-BSRF1 interaction may be an effective way to control EBV production.
  • the complex structure of the EBV interlayer proteins BBRF2 and BSRF1 was reported, revealing a conserved pattern among the EBV interlayer proteins.
  • the BBRF2-BSRF1 complex plays an important role in EBV assembly and is important for the secondary envelope.
  • the BBRF2-BSRF1 complex can create a tethering-permissive environment that favors the secondary envelope of EBV.

Landscapes

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

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a peptide for inhibiting the Epstein-Barr virus, DNA encoding the peptide, and the use thereof. By means of using the peptide, the replication of the Epstein-Barr virus can be effectively inhibited, such that diseases related to the Epstein-Barr virus can prevented and/or treated.

Description

一种抑制EB病毒的肽和编码该肽的DNA及其应用A kind of peptide for inhibiting EB virus and DNA encoding the peptide and application thereof 技术领域technical field
本发明属于生物医药领域，具体涉及一种抑制爱泼斯坦-巴尔病毒的肽和编码该肽的DNA及其应用。The invention belongs to the field of biomedicine, and in particular relates to a peptide for inhibiting Epstein-Barr virus, DNA encoding the peptide and application thereof.
背景技术Background technique
爱泼斯坦-巴尔病毒(Epstein-Barr virus，EBV或EB病毒)是一种与多种人类恶性肿瘤有关的γ-疱疹病毒，也即一种双链DNA病毒。据统计，全世界约有95％的人口携带该病毒。EBV是一种致癌病毒，其与多种特定的人类肿瘤的发生密切相关，所述人类肿瘤包括鼻咽癌、霍奇金氏淋巴瘤、伯基特淋巴瘤和胃癌等。国际癌症研究署(IARC)2008年发布的世界癌症报告指出，EBV引发了全球癌症的1％，占所有感染性癌症的5.6％。根据IARC对致癌因子的分类标准，EBV被列在第一组致癌因子中。当前，没有有效的临床方法来预防或消除EBV感染。Epstein-Barr virus (Epstein-Barr virus, EBV or Epstein-Barr virus) is a gamma-herpes virus, also known as a double-stranded DNA virus, associated with a variety of human malignancies. According to statistics, about 95% of the world's population carries the virus. EBV is an oncogenic virus that is closely associated with the development of a variety of specific human tumors, including nasopharyngeal carcinoma, Hodgkin's lymphoma, Burkitt's lymphoma, and gastric cancer. According to the World Cancer Report released by the International Agency for Research on Cancer (IARC) in 2008, EBV causes 1% of global cancers and 5.6% of all infectious cancers. According to the IARC classification criteria for carcinogens, EBV is listed in the first group of carcinogens. Currently, there is no effective clinical approach to prevent or eliminate EBV infection.
因此，本领域亟需找到一种能够预防和/或消除EBV感染的方案。Therefore, there is an urgent need in the art to find a solution that can prevent and/or eliminate EBV infection.
发明内容SUMMARY OF THE INVENTION
如上所述，本发明亟需找到一种预防和/或消除EBV感染的方案，由此来治疗和/或预防与EBV感染和与EBV相关的人类疾病。As mentioned above, there is an urgent need in the present invention to find a solution for preventing and/or eliminating EBV infection, thereby treating and/or preventing EBV infection and EBV-related human diseases.
因此，在第一方面，本发明提供了一种肽，所述肽包含SEQ ID NO：1所示的氨基酸序列。Accordingly, in a first aspect, the present invention provides a peptide comprising the amino acid sequence shown in SEQ ID NO:1.
在第二方面，本发明提供了一种核酸序列，其编码本发明第一方面的肽。In a second aspect, the invention provides a nucleic acid sequence encoding the peptide of the first aspect of the invention.
在第三方面，本发明提供了一种表达载体，其包含本发明第二方面的核酸序列。In a third aspect, the present invention provides an expression vector comprising the nucleic acid sequence of the second aspect of the present invention.
在第四方面，本发明提供了本发明第一方面的肽、本发明第二方面的核酸序列、或者本发明第三方面的表达载体在制备用于治疗爱泼斯坦-巴尔病毒相关的疾病的药物中的用途。In a fourth aspect, the present invention provides the peptide of the first aspect of the present invention, the nucleic acid sequence of the second aspect of the present invention, or the expression vector of the third aspect of the present invention in the preparation for the treatment of Epstein-Barr virus-related diseases Use in medicine.
在第五方面，本发明提供了一种抑制爱泼斯坦-巴尔病毒复制的方法，所述方法包括给予本发明第一方面的肽或者本发明第三方面的表达载体。In a fifth aspect, the present invention provides a method for inhibiting Epstein-Barr virus replication, the method comprising administering the peptide of the first aspect of the present invention or the expression vector of the third aspect of the present invention.
在第六方面，本发明提供了一种治疗爱泼斯坦-巴尔病毒相关的疾病的方法，所述方法包括给予患者本发明第一方面的肽或者本发明第三方面的表达载体。In a sixth aspect, the present invention provides a method of treating an Epstein-Barr virus-related disease, the method comprising administering to a patient the peptide of the first aspect of the present invention or the expression vector of the third aspect of the present invention.
在第七方面，本发明提供了一种试剂盒，包括本发明第一方面的肽、本发明第二方面的核酸序列、或本发明第三方面的表达载体。In a seventh aspect, the present invention provides a kit comprising the peptide of the first aspect of the present invention, the nucleic acid sequence of the second aspect of the present invention, or the expression vector of the third aspect of the present invention.
本发明的有益效果为：The beneficial effects of the present invention are:
本发明的肽可以有效地减少病毒基因组拷贝数，因此根据此肽设计小分子抑制剂可潜在缓解和治疗爱泼斯坦-巴尔病毒感染或与爱泼斯坦-巴尔病毒相关的人类疾病。The peptide of the present invention can effectively reduce the copy number of the virus genome, so the design of small molecule inhibitors according to the peptide can potentially alleviate and treat Epstein-Barr virus infection or human diseases associated with Epstein-Barr virus.
附图说明Description of drawings
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对附图作简单的介绍。显而易见地，下面描述中的附图仅仅涉及本发明的一些实施例。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings are briefly introduced below. Obviously, the drawings in the following description relate only to some embodiments of the invention.
图1示出了BBRF2Δ-BSRF1Δ界面的详细信息，其中：图a示意性地显示了BBRF2Δ-BSRF1Δ晶体复合物中BBRF2Δ(绿色)和BSRF1Δ(蓝色)的可识别残基(有色区域)与全长BBRF2和BSRF1的比较；图b示出了异二聚体的两个视图，颜色同图a，BSRF1Δ的N端和C端被标注；图c示出了BSRF1Δ的两个反平行α螺旋之间的疏水相互作用；图d-f分别示出了BBRF2Δ-BSRF1Δ界面处的疏水界面(d)、极性界面(e)、和BSRF1ΔN端环介导的界面(f)，二级结构元件已标注，所涉及的氨基酸残基被着色为其所属分子的颜色；图g示出了BBRF2Δ(左)和BSRF1Δ(右)的表面保守性图：为了清楚起见，结合的BSRF1Δ(左)和BBRF2Δ(右)显示为透明，用黄色勾勒出BBRF2Δ-BSRF1Δ异二聚体的界面轮廓。Figure 1 shows details of the BBRF2Δ-BSRF1Δ interface, where: Panel a schematically shows the recognizable residues (colored regions) of BBRF2Δ (green) and BSRF1Δ (blue) in the BBRF2Δ-BSRF1Δ crystal complex with full Comparison of long BBRF2 and BSRF1; panel b shows two views of the heterodimer in the same colors as panel a, the N- and C-termini of BSRF1Δ are annotated; panel c shows one of the two antiparallel alpha helices of BSRF1Δ The hydrophobic interaction between the two; Figures d-f show the hydrophobic interface (d), the polar interface (e), and the BSRF1Δ N-terminal loop-mediated interface (f) at the BBRF2Δ-BSRF1Δ interface, respectively, with secondary structural elements annotated, The amino acid residues involved are colored in the color of the molecule to which they belong; panel g shows a surface conservation map of BBRF2Δ (left) and BSRF1Δ (right): bound BSRF1Δ (left) and BBRF2Δ (right) for clarity Shown as transparent, the interface of the BBRF2Δ-BSRF1Δ heterodimer is outlined in yellow.
图2示出了BSRF1Δ衍生肽及其与BBRF2Δ的结合，其中：图a示出了五个BSRF1Δ衍生肽(P1-P5)的氨基酸长度的示意图，其中N表示解析结构中的N-环区域；图b示出了对结合10μg ml -1His 6-BBRF2Δ的五种肽的BLI分析，仅P1显示能与His 6-BBRF2Δ结合；图c示出了通过BLI测量的肽1(P1)和BBRF2Δ的结合亲和力：将不同浓度的P1与10μg ml -1His-标签的BBRF2Δ结合；图d示出了P1与BSRF1Δ竞争结合BBRF2Δ：将200nM BBRF2Δ与不同浓度的P1混合，与10μg ml -1 固定的BSRF1Δ结合；图e示出了TAT-P1以浓度依赖的方式减少EBV基因组拷贝数，其中误差棒表示s.d.(n＝3)。 Figure 2 shows BSRF1Δ-derived peptides and their binding to BBRF2Δ, wherein: Panel a shows a schematic representation of the amino acid lengths of five BSRF1Δ-derived peptides (P1-P5), where N represents the N-loop region in the resolved structure; Panel b shows BLI analysis of five peptides bound to 10 μg ml -1 His6 - BBRF2Δ, only P1 was shown to bind His6 - BBRF2Δ; panel c shows peptide 1 (P1) and BBRF2Δ measured by BLI Binding affinity of: different concentrations of P1 were combined with 10 μg ml -1 His-tagged BBRF2Δ; panel d shows that P1 competes with BSRF1Δ for binding to BBRF2Δ: 200 nM BBRF2Δ was mixed with different concentrations of P1, with 10 μg ml -1 immobilized BSRF1Δ binding; panel e shows that TAT-P1 reduces EBV genome copy number in a concentration-dependent manner, where error bars indicate sd (n=3).
具体实施方式Detailed ways
下面对本发明进行清楚、完整的描述。显然，所描述的实施方案仅仅是本发明的一部分实施方案，而不是全部的实施方案。基于本发明中的实施方案，本领域普通技术人员可以获得的所有其他实施方案，并且它们都属于本发明保护的范围。A clear and complete description of the invention follows. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments can be obtained by a person of ordinary skill in the art, and they all belong to the protection scope of the present invention.
BBRF2和BSRF1(或其同系物)之间的相互作用被认为对于爱泼斯坦-巴尔病毒的生命周期至关重要，而靶向BBRF2和BSRF1结合面有可能成为针对EBV的潜在临床治疗策略。根据BBRF2Δ-BSRF1Δ结合面的结构，本发明人设计出如图2a所示的5种BSRF1衍生肽(P1-P5)，其覆盖了在BSRF1的N-环、ɑA或ɑB上的与BBRF2结合的位点。The interaction between BBRF2 and BSRF1 (or its homologs) is believed to be critical for the life cycle of Epstein-Barr virus, and targeting the binding surface of BBRF2 and BSRF1 has the potential to be a potential clinical therapeutic strategy against EBV. According to the structure of the BBRF2Δ-BSRF1Δ binding surface, the inventors designed five BSRF1-derived peptides (P1-P5) as shown in Fig. 2a, which cover the BBRF2-binding peptides on the N-loop, ɑA or ɑB of BSRF1 site.
因此，在第一方面，本发明提供了一种肽，所述肽包含SEQ ID NO：1所示的氨基酸序列。Accordingly, in a first aspect, the present invention provides a peptide comprising the amino acid sequence shown in SEQ ID NO:1.
如本领域技术人员可以理解的那样，可以使目标肽与具有特定功能的其他肽连接在一起，以实现相应的功能。例如，可以使目标肽与细胞穿透肽连接在一起，由此使该目标肽能容易穿透细胞膜进入细胞。As can be understood by those skilled in the art, the target peptide can be linked with other peptides with specific functions to achieve the corresponding functions. For example, the peptide of interest can be linked to a cell penetrating peptide, thereby allowing the peptide of interest to readily penetrate the cell membrane and enter the cell.
因此，在一个实施方案中，所述肽还包含细胞穿透肽如TAT序列和接头肽。Thus, in one embodiment, the peptide further comprises a cell penetrating peptide such as a TAT sequence and a linker peptide.
在第二方面，本发明提供了一种核酸序列，其编码本发明第一方面的肽。In a second aspect, the invention provides a nucleic acid sequence encoding the peptide of the first aspect of the invention.
本领域技术人员在获知所述肽的氨基酸序列的情况下可以知晓其编码核苷酸序列。Those skilled in the art can know the nucleotide sequence encoding the peptide in the case of knowing the amino acid sequence of the peptide.
在一个实施方案中，所述核酸序列为SEQ ID NO：2所示的序列。In one embodiment, the nucleic acid sequence is the sequence shown in SEQ ID NO:2.
SEQ ID NO:2的序列如下所示：The sequence of SEQ ID NO:2 is shown below:
Figure PCTCN2020120203-appb-000001
Figure PCTCN2020120203-appb-000001
可以理解，SEQ ID NO:2所示的核苷酸序列可以编码本发明的肽，但是其简并序列同样也可以编码本发明的肽。It is understood that the nucleotide sequence shown in SEQ ID NO: 2 can encode the peptide of the present invention, but its degenerate sequence can also encode the peptide of the present invention.
在第三方面，本发明提供了一种表达载体，其包含本发明第二方面的核酸序列。In a third aspect, the present invention provides an expression vector comprising the nucleic acid sequence of the second aspect of the present invention.
在本文中，所谓“表达载体”是指在克隆载体基本骨架的基础上增加表达元件(如启动子、RBS、终止子等)，从而使目的基因能够表达的载体。所述表达载体可以为质粒载体、噬菌体载体、病毒载体等，对其没有特别限制，只要能表达目标蛋白即可。Herein, the so-called "expression vector" refers to a vector that adds expression elements (such as promoter, RBS, terminator, etc.) on the basis of the basic skeleton of the cloning vector, so that the target gene can be expressed. The expression vector can be a plasmid vector, a phage vector, a virus vector, etc., which are not particularly limited, as long as the target protein can be expressed.
在第四方面，本发明提供了本发明第一方面的肽、本发明第二方面的核酸序列、或者本发明第三方面的表达载体在制备用于治疗爱泼斯坦-巴尔病毒相关的疾病的药物中的用途。In a fourth aspect, the present invention provides the peptide of the first aspect of the present invention, the nucleic acid sequence of the second aspect of the present invention, or the expression vector of the third aspect of the present invention in the preparation for the treatment of Epstein-Barr virus-related diseases Use in medicine.
在一个实施方案中，所述爱泼斯坦-巴尔病毒相关的疾病选自传染性单核细胞增多症、连锁淋巴细胞增生综合症、病毒性嗜血细胞综合症、口腔毛状黏膜白斑病、病毒性脑膜炎、周围神经炎、病毒性肺炎病毒性心肌炎、鼻咽癌、霍奇金淋巴瘤、伯基特淋巴瘤、胃癌、肝细胞癌、淋巴上皮样肉瘤、唾液腺肿瘤、乳腺癌、胸腺瘤、原发性渗出性淋巴瘤或B/T/NK细胞淋巴瘤。In one embodiment, the Epstein-Barr virus-related disease is selected from the group consisting of infectious mononucleosis, linked lymphoproliferative syndrome, viral hemophagocytic syndrome, oral hairy leukoplakia, viral Meningitis, peripheral neuritis, viral pneumonia, viral myocarditis, nasopharyngeal carcinoma, Hodgkin lymphoma, Burkitt lymphoma, gastric cancer, hepatocellular carcinoma, lymphoepithelioid sarcoma, salivary gland tumor, breast cancer, thymoma, Primary effusion lymphoma or B/T/NK cell lymphoma.
在第五方面，本发明提供了一种抑制爱泼斯坦-巴尔病毒复制的方法，所述方法包括给予本发明第一方面的肽或者本发明第三方面的表达载体。In a fifth aspect, the present invention provides a method for inhibiting Epstein-Barr virus replication, the method comprising administering the peptide of the first aspect of the present invention or the expression vector of the third aspect of the present invention.
可以理解，在此所述的“抑制爱泼斯坦-巴尔病毒复制”可以出于各种目的进行，比如出于治疗目的，又比如出于科研目的，或者出于某个商业目的。It can be understood that the "inhibition of Epstein-Barr virus replication" described herein can be performed for various purposes, such as for therapeutic purposes, for example for scientific research purposes, or for some commercial purpose.
在第六方面，本发明提供了一种治疗爱泼斯坦-巴尔病毒相关的疾病的方法，所述方法包括给予患者本发明第一方面的肽或者本发明第三方面的表达载体。In a sixth aspect, the present invention provides a method of treating an Epstein-Barr virus-related disease, the method comprising administering to a patient the peptide of the first aspect of the present invention or the expression vector of the third aspect of the present invention.
在一个实施方案中，所述爱泼斯坦-巴尔病毒相关的疾病选自传染性单核细胞增多症、连锁淋巴细胞增生综合症、病毒性嗜血细胞综合症、口腔毛状黏膜白斑病、病毒性脑膜炎、周围神经炎、病毒性肺炎病毒性心肌炎、鼻咽癌、霍奇金淋巴瘤、伯基特淋巴瘤、胃癌、肝细胞癌、淋巴上皮样肉瘤、唾液腺肿瘤、乳腺癌、胸腺瘤、原发性渗出性淋巴瘤或B/T/NK细胞淋巴瘤。In one embodiment, the Epstein-Barr virus-related disease is selected from the group consisting of infectious mononucleosis, linked lymphoproliferative syndrome, viral hemophagocytic syndrome, oral hairy leukoplakia, viral Meningitis, peripheral neuritis, viral pneumonia, viral myocarditis, nasopharyngeal carcinoma, Hodgkin lymphoma, Burkitt lymphoma, gastric cancer, hepatocellular carcinoma, lymphoepithelioid sarcoma, salivary gland tumor, breast cancer, thymoma, Primary effusion lymphoma or B/T/NK cell lymphoma.
在第七方面，本发明提供了一种试剂盒，包括本发明第一方面的肽、本发明第二方面的核酸序列、或本发明第三方面的表达载体。In a seventh aspect, the present invention provides a kit comprising the peptide of the first aspect of the present invention, the nucleic acid sequence of the second aspect of the present invention, or the expression vector of the third aspect of the present invention.
本发明的有益效果为：The beneficial effects of the present invention are:
本发明的肽可以有效地减少病毒基因组拷贝数，并因此可以缓解和治疗爱泼斯坦-巴尔病毒感染和与爱泼斯坦-巴尔病毒相关的人类疾病。The peptides of the present invention can effectively reduce the viral genome copy number, and thus can alleviate and treat Epstein-Barr virus infection and Epstein-Barr virus-related human diseases.
提供以下实施例是为了更好地理解本发明。下述实施例中的试验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规化试剂商店购买所得。应注意，上文的发明内容部分以及下文的详细描述仅为具体阐释本发明之目的，无意于以任何方式对本发明进行限制。在不背离本发明的精神和主旨的情况下，本发明的范围由随附的权利要求书确定。The following examples are provided for a better understanding of the present invention. The test methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional chemical reagent stores. It should be noted that the above summary section and the following detailed description are only for the purpose of specifically illustrating the present invention and are not intended to limit the present invention in any way. The scope of the present invention is to be determined by the appended claims without departing from the spirit and spirit of the present invention.
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不旨在于限制本发明。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention.
实施例Example
一、实验部分：1. Experimental part:
蛋白表达与纯化Protein expression and purification
从人疱疹病毒4型M81病毒株基因组中扩增出BBRF2和BSRF1的cDNA。将全长BBRF2和BBRF2 17-278(BBRF2Δ)的cDNA克隆到经改造的pET28载体(pSKB)中，并在大肠杆菌Rosetta(DE3)细胞中表达N端带有6×His-标签然后是PreScission切割位点的融合蛋白。将转化的细菌在37℃在Terrific Broth(TB)培养基中培养，并在光学密度为0.6时添加100μM的异丙基-1-硫代-β-D-硫代半乳糖糖苷(IPTG)进行诱导。诱导后，使细胞于18℃生长过夜(约16小时-18小时)，并离心收集。 The cDNAs of BBRF2 and BSRF1 were amplified from the genome of human herpesvirus 4 strain M81. The cDNAs of full-length BBRF2 and BBRF2 17-278 (BBRF2Δ) were cloned into the engineered pET28 vector (pSKB) and expressed in E. coli Rosetta (DE3) cells with a 6×His-tag at the N-terminus followed by PreScission cleavage site fusion protein. Transformed bacteria were grown in Terrific Broth (TB) medium at 37°C with the addition of 100 μM of isopropyl-1-thio-β-D-thiogalactoside (IPTG) at an optical density of 0.6. induce. After induction, cells were grown overnight (approximately 16-18 hours) at 18°C and collected by centrifugation.
将收集的细胞在冰冷的缓冲液(含有20mM HEPES(pH 7.0)、600mM NaCl、10％甘油、30mM咪唑、1μM DNase I、1mM苯甲磺酰氟(PMSF)和2mM β-巯基乙醇(β-ME))中使用细胞破碎器(JNBIO)裂解，并于4℃以40,000g离心1小时。将上清液过滤并上样到经结合缓冲液A(包含20mM HEPES(pH 7.0)、600mM NaCl、10％甘油、30mM咪唑、2mM β-ME)平衡后的Ni-NTA(第一Ni-NTA)柱(GE Healthcare)上。用结合缓冲液A洗涤后，蛋白用含有20mM HEPES(pH 7.0)、600mM NaCl、10％甘油、300mM咪唑和2mM β-ME的洗脱缓冲液进行洗脱。将洗脱的蛋 白质同与20μg谷胱甘肽S-转移酶(GST)融合的PreScission蛋白酶(PSP)一起孵育以去除His 6-标签，并于4℃用含有20mM HEPES(pH 7.0)、600mM NaCl、10％甘油、2mM β-ME的结合缓冲液B透析过夜。透析后，使用GST柱除去PSP。将蛋白质重新上样到用结合缓冲液B平衡的第二Ni-NTA柱上，并用结合缓冲液A洗脱。随后，将洗脱的蛋白质应用于使用经缓冲液C(含有20mM HEPES(pH 7.0)、600mM NaCl、10％甘油和1mM二硫苏糖醇(DTT))平衡的HiLoad 16/60 Superdex 75柱(GE Healthcare)的尺寸排阻色谱法(SEC)。蛋白质以离散峰(discrete peak)洗脱并收集。BBRF2 17-278的硒代蛋氨酸(SeMet)衍生物如先前所述进行表达，并如天然BBRF2蛋白那样进行纯化。 Collected cells were incubated in ice-cold buffer (containing 20 mM HEPES (pH 7.0), 600 mM NaCl, 10% glycerol, 30 mM imidazole, 1 μM DNase I, 1 mM phenylmethanesulfonyl fluoride (PMSF) and 2 mM β-mercaptoethanol (β- ME)) using a cell disruptor (JNBIO) and centrifuged at 40,000 g for 1 hour at 4°C. The supernatant was filtered and loaded onto Ni-NTA (first Ni-NTA) equilibrated with binding buffer A containing 20 mM HEPES (pH 7.0), 600 mM NaCl, 10% glycerol, 30 mM imidazole, 2 mM β-ME. ) column (GE Healthcare). After washing with binding buffer A, proteins were eluted with elution buffer containing 20 mM HEPES (pH 7.0), 600 mM NaCl, 10% glycerol, 300 mM imidazole and 2 mM β-ME. The eluted protein was incubated with PreScission protease (PSP) fused to 20 μg glutathione S-transferase (GST) to remove the His 6 -tag and treated with 20 mM HEPES (pH 7.0), 600 mM NaCl at 4° C. , 10% glycerol, 2 mM β-ME in binding buffer B overnight. After dialysis, PSP was removed using a GST column. The protein was reloaded onto a second Ni-NTA column equilibrated with Binding Buffer B and eluted with Binding Buffer A. Subsequently, the eluted protein was applied to a HiLoad 16/60 Superdex 75 column ( Size Exclusion Chromatography (SEC) of GE Healthcare). Proteins eluted as discrete peaks and were collected. The selenomethionine (SeMet) derivative of BBRF2 17-278 was expressed as previously described and purified as the native BBRF2 protein.
将BSRF1 34-159(BSRF1Δ)的cDNA克隆到pGEX-6p-1载体中。表达重组蛋白并如BBRF2蛋白一样将该重组蛋白收集于含有50mM HEPES(pH 7.0)、150mM NaCl、1mM PMSF和2mM β-ME的裂解缓冲液中。将收集的蛋白质上样到经含有20mM HEPES(pH 7.0)、150mM NaCl和2mM β-ME的结合缓冲液平衡的GST柱，并用含有20mM HEPES(pH 7.0)、150mM NaCl、10mM谷胱甘肽和2mM β-ME的缓冲液进行洗脱。在用20μg PSP处理后，将蛋白质上样到GST柱以去除GST-标签和PSP。按照与BBRF2蛋白相同的方法进行SEC。通过将纯化的BBRF2Δ和BSRF1Δ以1:1摩尔比混合并于4℃孵育过夜，从而制备BBRF2 17-278-BSRF1 34-159(BBRF2Δ-BSRF1Δ)复合物，然后用HiLoad 16/60 Superdex 200柱(GE)在缓冲液C中通过SEC纯化该复合物。将BSRF1 20-218的cDNA克隆到pSKB载体。如BBRF2构建一样,表达、收集并纯化重组蛋白，其中所有缓冲液均含更少NaCl(300mM)且没有甘油。 The cDNA of BSRF1 34-159 (BSRF1Δ) was cloned into the pGEX-6p-1 vector. Recombinant protein was expressed and collected in lysis buffer containing 50 mM HEPES (pH 7.0), 150 mM NaCl, 1 mM PMSF and 2 mM β-ME like BBRF2 protein. The collected protein was loaded onto a GST column equilibrated with binding buffer containing 20 mM HEPES (pH 7.0), 150 mM NaCl, and 2 mM β-ME, and mixed with 20 mM HEPES (pH 7.0), 150 mM NaCl, 10 mM glutathione and 2mM β-ME buffer was used for elution. After treatment with 20 μg of PSP, the protein was loaded onto a GST column to remove GST-tag and PSP. SEC was performed in the same manner as for BBRF2 protein. BBRF2 17-278 -BSRF1 34-159 (BBRF2Δ-BSRF1Δ) complexes were prepared by mixing purified BBRF2Δ and BSRF1Δ in a 1:1 molar ratio and incubating overnight at 4°C, followed by a HiLoad 16/60 Superdex 200 column ( GE) The complex was purified by SEC in buffer C. The cDNA of BSRF1 20-218 was cloned into pSKB vector. Recombinant proteins were expressed, harvested and purified as in the BBRF2 construction with less NaCl (300 mM) and no glycerol in all buffers.
结晶：crystallization:
使用悬滴蒸汽扩散法，通过等体积蛋白质(约7mg.ml -1)和储液的混合物进行结晶实验。SeMet BBRF2Δ的晶体在4℃从0.09M NPS(0.03M NaNO 3、0.03M Na 2HPO 4、0.03M(NH 4) 2SO 4)、0.1M 2-(N-吗啡啉)乙磺酸(MES)/咪唑(pH 6.5)、12.5％PEG1000、12.5％PEG 3350和12.5％MPD生长而成。BBRF2Δ-BSRF1Δ复合物的晶体是在1:1000m/m α-胰凝乳蛋白酶在4℃处理过夜后，从0.1M乙酸镁、0.05M MES(pH 5.6)和20％MPD中生长的。将晶体直接在液氮中快速冷冻。 Crystallization experiments were performed with a mixture of equal volumes of protein (approximately 7 mg.ml -1 ) and stock solution using the hanging drop vapor diffusion method. Crystals of SeMet BBRF2Δ from 0.09M NPS (0.03M NaNO3, 0.03M Na2HPO4 , 0.03M( NH4 ) 2SO4 ), 0.1M 2- (N-morpholine)ethanesulfonic acid (MES) at 4 °C )/imidazole (pH 6.5), 12.5% PEG1000, 12.5% PEG 3350 and 12.5% MPD. Crystals of the BBRF2Δ-BSRF1Δ complex were grown from 0.1 M magnesium acetate, 0.05 M MES (pH 5.6) and 20% MPD after 1:1000 m/m α-chymotrypsin treatment overnight at 4°C. The crystals were snap-frozen directly in liquid nitrogen.
结构解析：Structural Analysis:
BBRF2Δ的X射线衍射数据集是在上海同步辐射装置(SSRF)的光束线BL17U1和BL19U1处收集的。在SSRF的光束线BL18U1处收集BBRF2Δ-BSRF1Δ复合物的数据集。使用XDS程序组处理数据集。通过单波长反常色散(SAD)方法获得BBRF2Δ结构的初始相，并使用phenix从SeMet取代的BBRF2Δ晶体的衍射数据集中进行修正。BBRF2Δ-BSRF1Δ复合物结构是通过使用Phaser以BBRF2Δ的结构作为搜索模型进行分子置换来解析的。BSRF1Δ的模型是使用COOT手动构建的。Phenix组中的AutoBuild程序用于最小化模型偏差。使用MolProbity进行结构验证。使用PyMOL分子图形系统(版本0.99，
Figure PCTCN2020120203-appb-000002
http：//www.pymol.org/)和CCP4mg生成结构图。表1中列出了X射线数据收集和优化统计信息。 The X-ray diffraction dataset of BBRF2Δ was collected at beamlines BL17U1 and BL19U1 of the Shanghai Synchrotron Radiation Facility (SSRF). A dataset of BBRF2Δ-BSRF1Δ complexes was collected at beamline BL18U1 of SSRF. The dataset is processed using the XDS program group. The initial phase of the BBRF2Δ structure was obtained by the single-wavelength anomalous dispersion (SAD) method and corrected from the diffraction dataset of SeMet-substituted BBRF2Δ crystals using phenix. The BBRF2Δ-BSRF1Δ complex structure was solved by molecular replacement using Phaser with the structure of BBRF2Δ as the search model. The model of BSRF1Δ was manually constructed using COOT. The AutoBuild program in the Phenix group was used to minimize model bias. Structural verification using MolProbity. Using the PyMOL molecular graphics system (version 0.99,
Figure PCTCN2020120203-appb-000002
http://www.pymol.org/) and CCP4mg to generate a structural map. X-ray data collection and optimization statistics are listed in Table 1.
表1：晶体学数据采集和修正Table 1: Crystallographic data collection and correction
Figure PCTCN2020120203-appb-000003
Figure PCTCN2020120203-appb-000003
Figure PCTCN2020120203-appb-000004
Figure PCTCN2020120203-appb-000004
*括号中的数字是来自解析度最高的层的数值。*Numbers in parentheses are values from the layer with the highest resolution.
细胞培养cell culture
CNE2-EBV细胞由曾毅教授(中国医学科学院)赠予。将细胞在含有10％FBS和青霉素/链霉素的RPMI 1640培养基(GIBCO)中培养。细胞系不含支原体。CNE2-EBV细胞源自母细胞系，该母细胞系已被重组EBV感染。CNE2-EBV cells were donated by Professor Zeng Yi (Chinese Academy of Medical Sciences). Cells were cultured in RPMI 1640 medium (GIBCO) containing 10% FBS and penicillin/streptomycin. The cell line does not contain mycoplasma. CNE2-EBV cells were derived from a parental cell line that had been infected with recombinant EBV.
生物膜干涉技术(BLI)Biofilm Interferometry (BLI)
使用八通道OctetRED生物膜层干涉仪系统(FortéBio)进行BLI检测。为了测定BBRF2Δ和BSRF1Δ或肽P1-P5之间的相互作用，将带有His 6-标签的BBRF2Δ(10μg ml -1)固定在NTA生物传感器的尖端(FortéBio)上，该尖端已预先用含有20mM HEPES(pH7.0)、600mM NaCl、10％甘油和1mM DTT的反应缓冲液平衡。将BSRF1Δ或肽P1-P5稀释至4-8个不同的浓度，并通过由带有His 6-标签的BBRF2Δ包被的尖端在每个浓度依次进行分析。 BLI detection was performed using an eight-channel OctetRED Biofilm Layer Interferometer System (FortéBio). To determine the interaction between BBRF2Δ and BSRF1Δ or the peptides P1-P5, His 6 -tagged BBRF2Δ (10 μg ml −1 ) was immobilized on the tip of an NTA biosensor (FortéBio), which had been pretreated with 20 mM Reaction buffer equilibration of HEPES (pH 7.0), 600 mM NaCl, 10% glycerol and 1 mM DTT. BSRF1Δ or peptides P1-P5 were diluted to 4-8 different concentrations and analyzed sequentially at each concentration by a tip coated with His6 - tagged BBRF2Δ.
为了监测BSRF1Δ和P1肽与BBRF2Δ的竞争结合，使用Biotinylation Kit(Genemore)将BSRF1Δ在室温下生物素化30分钟，然后将BSRF1Δ固定到在反应缓冲液中预平衡的链霉亲和素包被(SA)的生物传感器尖端上。将P1肽在反应缓冲液中稀释至不同浓度(100μM、50μM、25μM、12.5μM、6.3μM和3.1μM)，并分别与200nM的BBRF2Δ混合。所有实验均于25℃进行。每次测量都涉及120s基线(使用反应缓冲液)，然后是180s(使用蛋白质)或(使用肽)的结合阶段，以及180s的解离阶段(使用反应缓冲液)。使用由Fortebio提供的Octet Data Analysis 软件11.0处理原始数据，以得出解离常数(K D)。使用Origin(2019版，OriginLab)绘制结果图。 To monitor the competitive binding of BSRF1Δ and P1 peptides to BBRF2Δ, BSRF1Δ was biotinylated for 30 min at room temperature using the Biotinylation Kit (Genemore), and then BSRF1Δ was immobilized to streptavidin-coated ( SA) on the biosensor tip. P1 peptides were diluted to different concentrations (100 μM, 50 μM, 25 μM, 12.5 μM, 6.3 μM and 3.1 μM) in reaction buffer and mixed with 200 nM of BBRF2Δ, respectively. All experiments were performed at 25°C. Each measurement involved a baseline of 120s (with reaction buffer), followed by a binding phase of 180s (with protein) or (with peptide), and a dissociation phase of 180s (with reaction buffer). Raw data were processed using Octet Data Analysis software 11.0 provided by Fortebio to derive dissociation constants (K D ). Results were plotted using Origin (version 2019, OriginLab).
肽的合成Peptide synthesis
委托Sangon Bio(中国上海)合成以下肽。肽的氨基酸序列如下表2所示：Sangon Bio (Shanghai, China) was commissioned to synthesize the following peptides. The amino acid sequence of the peptide is shown in Table 2 below:
表2.肽的氨基酸序列Table 2. Amino acid sequences of peptides
Figure PCTCN2020120203-appb-000005
Figure PCTCN2020120203-appb-000005
肽转染检测Peptide Transfection Assay
将TAT-肽溶解在RPMI 1640培养基中。将不同浓度的肽添加至预接种了CNE2-EBV细胞(具有持续EBV感染的人鼻咽癌细胞)的12孔板中，并于37℃孵育2小时。随后用PBS将细胞洗涤3次，然后转移至补充了10％胎牛血清(FBS)、青霉素/链霉素、丁酸钠和佛波醇12-十四酸酯13-乙酸酯(PMA)的RPMI 1640中。48小时后，测定这些细胞的EBV基因组拷贝数。The TAT-peptide was dissolved in RPMI 1640 medium. Various concentrations of peptides were added to 12-well plates pre-seeded with CNE2-EBV cells (human nasopharyngeal carcinoma cells with persistent EBV infection) and incubated at 37°C for 2 hours. Cells were subsequently washed 3 times with PBS and then transferred to supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin, sodium butyrate, and phorbol 12-myristate 13-acetate (PMA) The RPMI 1640. After 48 hours, the EBV genome copy number of these cells was determined.
EBV基因拷贝数测定EBV gene copy number determination
将预转染有TAT-肽的CNE2-EBV细胞用2.5mM丁酸钠和20ng ml -1佛波醇12-十四酸酯13-乙酸酯(PMA)处理12小时，以诱导EB病毒体的产生。48小时后，收集细胞并用PBS洗涤3次，用于测量病毒复制情况，然后将剩余的培养基通过0.45μm过滤器过滤，并于4℃以1,000g离心10分钟以去除细胞碎片。通过对病毒上清液进行qPCR分析确定包裹的病毒基因组DNA的拷贝数。简言之，(QIAGEN)从 经诱导的细胞中提取包裹的病毒基因组DNA。将所述上清液用DNase I(105U ml -1)于37℃消化1小时，然后与裂解缓冲液和0.1mg ml -1蛋白酶K混合。加入蛋白酶K去除病毒包膜和衣壳。将所述混合物于56℃加热10分钟，然后于75℃加热20分钟使酶失活。所述样品用不含RNase的水1:10稀释，然后使用BALF5 DNA聚合酶基因的引物进行qPCR。使用基因特异性引物(表3)通过qPCR对EBV编码的基因进行定量。 CNE2-EBV cells pre-transfected with TAT-peptide were treated with 2.5 mM sodium butyrate and 20 ng ml -1 phorbol 12-myristate 13-acetate (PMA) for 12 h to induce EB virions production. After 48 hours, cells were harvested and washed 3 times with PBS for measurement of viral replication, then the remaining medium was filtered through a 0.45 μm filter and centrifuged at 1,000 g for 10 minutes at 4° C. to remove cellular debris. The copy number of encapsulated viral genomic DNA was determined by qPCR analysis of viral supernatants. Briefly, (QIAGEN) encapsulated viral genomic DNA was extracted from induced cells. The supernatant was digested with DNase I (105U ml -1 ) for 1 hour at 37°C and then mixed with lysis buffer and 0.1 mg ml -1 proteinase K. The addition of proteinase K removes the viral envelope and capsid. The mixture was heated at 56°C for 10 minutes and then at 75°C for 20 minutes to inactivate the enzymes. The samples were diluted 1:10 with RNase-free water and then subjected to qPCR using primers for the BALF5 DNA polymerase gene. EBV-encoded genes were quantified by qPCR using gene-specific primers (Table 3).
表3.用于qPCR的引物列表Table 3. List of primers used for qPCR
Figure PCTCN2020120203-appb-000006
Figure PCTCN2020120203-appb-000006
二、结果与讨论：2. Results and discussion:
根据已验证的BBRF2Δ-BSRF1Δ结合界面的结构细节，本发明人设计了5种BSRF1衍生肽(P1-P5)，它们覆盖了在BSRF1的N环、αA或αB的BBRF2的结合位点(图2a)。BLI分析显示，只有最长的P1(覆盖N环和αA)显示出与BBRF2Δ的显著结合，K D为7.4μM(图2c，图2b)。这意味着BSRF1的单个结构元件不足以使BSRF1Δ与BBRF2之间形成稳定的结合。然后，本发明人检测了P1是否能够与BBRF2Δ-BSRF1Δ复合物中的野生型BSRF1Δ竞争。如BLI分析所示，添加P1会以浓度依赖方式破坏BBRF2Δ和固化的BSRF1Δ之间的结合(图2d)，表明P1在结合BBRF2Δ方面可以与BSRF1竞争。鉴于这一结果，本发明人研究了P1在CNE2-EBV细胞(一种具有持续EBV感染的人鼻咽癌细胞)中的潜在细胞效应。为了促进细胞对P1的摄取，发明人将TAT序列和一个四甘氨酸接头与P1的N端融合在一起(TAT-P1)。当转染进CNE2-EBV细胞时，TAT-P1以浓度依赖的方式减少病毒基因组拷贝数。在5μM的浓度下，TAT-P1能够将EBV基因组拷贝数降低75％，但是在更高浓度的TAT-P1下这种作用没有进一步提升(图2e)。这些结果表明，破坏BBRF2-BSRF1相互作用可能是控制EBV产生的有效方法。 Based on the verified structural details of the BBRF2Δ-BSRF1Δ binding interface, the inventors designed five BSRF1-derived peptides (P1-P5) that cover the binding sites of BBRF2 at the N-loop of BSRF1, αA or αB (Fig. 2a). ). BLI analysis revealed that only the longest P1 (covering the N-loop and αA) showed significant binding to BBRF2Δ with a KD of 7.4 μM (Fig. 2c, Fig. 2b). This means that a single structural element of BSRF1 is not sufficient for a stable binding between BSRF1Δ and BBRF2. The inventors then examined whether P1 could compete with wild-type BSRF1Δ in the BBRF2Δ-BSRF1Δ complex. As shown by BLI analysis, addition of P1 disrupted the binding between BBRF2Δ and immobilized BSRF1Δ in a concentration-dependent manner (Fig. 2d), indicating that P1 can compete with BSRF1 for binding BBRF2Δ. In view of this result, the inventors investigated the potential cellular effects of P1 in CNE2-EBV cells, a human nasopharyngeal carcinoma cell with persistent EBV infection. To facilitate cellular uptake of P1, the inventors fused the TAT sequence and a tetraglycine linker to the N-terminus of P1 (TAT-P1). When transfected into CNE2-EBV cells, TAT-P1 reduced the viral genome copy number in a concentration-dependent manner. At a concentration of 5 μM, TAT-P1 was able to reduce EBV genome copy number by 75%, but this effect was not further enhanced at higher concentrations of TAT-P1 (Fig. 2e). These results suggest that disrupting the BBRF2-BSRF1 interaction may be an effective way to control EBV production.
在发明人的先前研究中，报道了EBV间层蛋白BBRF2和BSRF1的复合物结构，揭示了EBV间层蛋白之间的保守模式。基于生物化学和细胞实验结果以及先前对其他疱疹病毒中BBRF2/BSRF1同源物的研究表明，BBRF2-BSRF1复合物在EBV组装中具有重要作用，对二次包膜有重要意义。BBRF2-BSRF1复合物可以创造一个有利于EBV二次包膜的允许束缚的环境。另一方面，据报道BBRF2或BSRF1的敲低会降低EBV感染细胞中的病毒基因组拷贝数，BBRF2-BSRF1复合物可能会在EBV的成熟和流出中进行不同的关键过程。最后，BBRF2的独特折叠使其成为抗EBV的潜在特异性药物靶点。鉴于P1肽在低摩尔水平对EBV基因组拷贝数有抑制作用(图2e)，通过优化的BSRF1衍生肽靶向BBRF2-BSRF1复合物，有望成为治疗EBV感染和EBV相关人类疾病的新策略。In a previous study by the inventors, the complex structure of the EBV interlayer proteins BBRF2 and BSRF1 was reported, revealing a conserved pattern among the EBV interlayer proteins. Based on biochemical and cellular experimental results and previous studies of BBRF2/BSRF1 homologs in other herpesviruses, it has been shown that the BBRF2-BSRF1 complex plays an important role in EBV assembly and is important for the secondary envelope. The BBRF2-BSRF1 complex can create a tethering-permissive environment that favors the secondary envelope of EBV. On the other hand, knockdown of BBRF2 or BSRF1 has been reported to reduce viral genome copy number in EBV-infected cells, and the BBRF2-BSRF1 complex may undergo distinct key processes in EBV maturation and efflux. Finally, the unique folding of BBRF2 makes it a potential specific drug target against EBV. Given the inhibitory effect of P1 peptide on EBV genome copy number at low molar levels (Fig. 2e), targeting the BBRF2-BSRF1 complex via optimized BSRF1-derived peptides is expected to be a novel strategy for the treatment of EBV infection and EBV-related human diseases.

Claims (11)

  1. 一种肽，所述肽包含SEQ ID NO：1所示的氨基酸序列。A peptide comprising the amino acid sequence shown in SEQ ID NO:1.
  2. 根据权利要求1所述的肽，其中所述肽还包含细胞穿透肽如TAT序列和接头肽。The peptide of claim 1, wherein the peptide further comprises a cell penetrating peptide such as a TAT sequence and a linker peptide.
  3. 一种核酸序列，其编码权利要求1-2任一项所述的肽。A nucleic acid sequence encoding the peptide of any one of claims 1-2.
  4. 根据权利要求3所述的核酸序列，其中所述核酸序列为SEQ ID NO：2所示的序列。The nucleic acid sequence according to claim 3, wherein the nucleic acid sequence is the sequence shown in SEQ ID NO: 2.
  5. 一种表达载体，其包含权利要求3或4所述的核酸序列。An expression vector comprising the nucleic acid sequence of claim 3 or 4.
  6. 权利要求1-2任一项所述的肽、权利要求3或4所述的核酸序列、或者权利要求5所述的表达载体在制备用于治疗爱泼斯坦-巴尔病毒相关的疾病的药物中的用途。The peptide of any one of claims 1-2, the nucleic acid sequence of claim 3 or 4, or the expression vector of claim 5 in the preparation of a medicament for the treatment of Epstein-Barr virus-related diseases the use of.
  7. 根据权利要求6所述的用途，其中所述爱泼斯坦-巴尔病毒相关的疾病选自传染性单核细胞增多症、连锁淋巴细胞增生综合症、病毒性嗜血细胞综合症、口腔毛状黏膜白斑病、病毒性脑膜炎、周围神经炎、病毒性肺炎病毒性心肌炎、鼻咽癌、霍奇金淋巴瘤、伯基特淋巴瘤、胃癌、肝细胞癌、淋巴上皮样肉瘤、唾液腺肿瘤、乳腺癌、胸腺瘤、原发性渗出性淋巴瘤或B/T/NK细胞淋巴瘤。The use according to claim 6, wherein the Epstein-Barr virus-related disease is selected from the group consisting of infectious mononucleosis, linked lymphoproliferative syndrome, viral hemophagocytic syndrome, oral hairy leukoplakia Disease, viral meningitis, peripheral neuritis, viral pneumonia, viral myocarditis, nasopharyngeal carcinoma, Hodgkin lymphoma, Burkitt lymphoma, gastric cancer, hepatocellular carcinoma, lymphoepithelioid sarcoma, salivary gland tumor, breast cancer , thymoma, primary effusion lymphoma, or B/T/NK cell lymphoma.
  8. 一种抑制爱泼斯坦-巴尔病毒复制的方法，所述方法包括给予权利要求1-2任一项所述的肽或者权利要求5所述的表达载体。A method of inhibiting Epstein-Barr virus replication, the method comprising administering the peptide of any one of claims 1-2 or the expression vector of claim 5.
  9. 一种治疗爱泼斯坦-巴尔病毒相关的疾病的方法，所述方法包括给予患者权利要求1-2任一项所述的肽或者权利要求5所述的表达载体。A method of treating an Epstein-Barr virus-related disease, the method comprising administering to a patient the peptide of any one of claims 1-2 or the expression vector of claim 5.
  10. 根据权利要求9所述的方法，其中所述爱泼斯坦-巴尔病毒相关的疾病选自传染性单核细胞增多症、连锁淋巴细胞增生综合症、病毒性嗜血细胞综合症、口腔毛状黏膜白斑病、病毒性脑膜炎、周围神经炎、病毒性肺炎病毒性心肌炎、鼻咽癌、霍奇金淋巴瘤、伯基特淋巴瘤、胃癌、肝细胞癌、淋巴上皮样肉瘤、唾液腺肿瘤、乳腺癌、胸腺瘤、原发性渗出性淋巴瘤或B/T/NK细胞淋巴瘤。The method of claim 9, wherein the Epstein-Barr virus-related disease is selected from the group consisting of infectious mononucleosis, linked lymphoproliferative syndrome, viral hemophagocytic syndrome, oral hairy leukoplakia disease, viral meningitis, peripheral neuritis, viral pneumonia, viral myocarditis, nasopharyngeal carcinoma, Hodgkin lymphoma, Burkitt lymphoma, gastric cancer, hepatocellular carcinoma, lymphoepithelioid sarcoma, salivary gland tumor, breast cancer , thymoma, primary effusion lymphoma, or B/T/NK cell lymphoma.
  11. 一种试剂盒，包括权利要求1-2任一项所述的肽、权利要求3或4所述的核酸序列、或权利要求5所述的表达载体。A kit comprising the peptide of any one of claims 1-2, the nucleic acid sequence of claim 3 or 4, or the expression vector of claim 5.
PCT/CN2020/120203 2020-10-10 2020-10-10 Peptide for inhibiting eb virus, dna encoding peptide, and use thereof WO2022073234A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/120203 WO2022073234A1 (en) 2020-10-10 2020-10-10 Peptide for inhibiting eb virus, dna encoding peptide, and use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/120203 WO2022073234A1 (en) 2020-10-10 2020-10-10 Peptide for inhibiting eb virus, dna encoding peptide, and use thereof

Publications (1)

Publication Number Publication Date
WO2022073234A1 true WO2022073234A1 (en) 2022-04-14

Family

ID=81125608

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/120203 WO2022073234A1 (en) 2020-10-10 2020-10-10 Peptide for inhibiting eb virus, dna encoding peptide, and use thereof

Country Status (1)

Country Link
WO (1) WO2022073234A1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6518013B1 (en) * 1993-06-07 2003-02-11 Trimeris, Inc. Methods for the inhibition of epstein-barr virus transmission employing anti-viral peptides capable of abrogating viral fusion and transmission
CN103328003A (en) * 2010-08-25 2013-09-25 亥姆霍兹慕尼黑中心德国研究健康与环境有限责任公司 An epstein-barr-virus vaccine
WO2020074708A1 (en) * 2018-10-12 2020-04-16 Deutsches Krebsforschungszentrum Epstein-barr virus-like particles with broadened antigenic spectrum

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6518013B1 (en) * 1993-06-07 2003-02-11 Trimeris, Inc. Methods for the inhibition of epstein-barr virus transmission employing anti-viral peptides capable of abrogating viral fusion and transmission
CN103328003A (en) * 2010-08-25 2013-09-25 亥姆霍兹慕尼黑中心德国研究健康与环境有限责任公司 An epstein-barr-virus vaccine
WO2020074708A1 (en) * 2018-10-12 2020-04-16 Deutsches Krebsforschungszentrum Epstein-barr virus-like particles with broadened antigenic spectrum

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN 1 December 2020 (2020-12-01), ANONYMOUS : "Chain H, Tegument protein UL51 homolog", XP055919071, retrieved from NCBI Database accession no. 6LQO_H *
DATABASE PROTEIN 2 June 2021 (2021-06-02), ANONYMOUS : "RecName:Full=Tegument protein UL51 homolog", XP055919080, retrieved from NCBI Database accession no. P0CK50 *
JOHANNSEN, E. ET AL.: "Proteins of purified Epstein–Barr virus", PNAS, vol. 101, no. 46, 16 November 2004 (2004-11-16), XP002337075, DOI: 10.1073/pnas.0407320101 *
MASUD H. M. ABDULLAH AL, YANAGI YUSUKE, WATANABE TAKAHIRO, SATO YOSHITAKA, KIMURA HIROSHI, MURATA TAKAYUKI: "Epstein-Barr Virus BBRF2 Is Required for Maximum Infectivity", MICROORGANISMS, vol. 7, no. 12, 16 December 2019 (2019-12-16), XP055919083, DOI: 10.3390/microorganisms7120705 *
SERRANO-SOLIS, V. ET AL.: "Prediction of MicroRNAs in the Epstein–Barr Virus Reveals Potential Targets for the Viral Self-Regulation", INDIAN JOURNAL OF MICROBIOL, vol. 59, no. 1, 28 December 2018 (2018-12-28), XP036669367, DOI: 10.1007/s12088-018-0775-4 *
YANAGI YUSUKE, MASUD H., WATANABE TAKAHIRO, SATO YOSHITAKA, GOSHIMA FUMI, KIMURA HIROSHI, MURATA TAKAYUKI: "Initial Characterization of the Epstein–Barr Virus BSRF1 Gene Product", VIRUSES, vol. 11, no. 3, 21 March 2019 (2019-03-21), XP055919082, DOI: 10.3390/v11030285 *

Similar Documents

Publication Publication Date Title
Yang et al. Structural insight into the SARS-CoV-2 nucleocapsid protein C-terminal domain reveals a novel recognition mechanism for viral transcriptional regulatory sequences
Arold et al. The crystal structure of HIV-1 Nef protein bound to the Fyn kinase SH3 domain suggests a role for this complex in altered T cell receptor signaling
CN107022008B (en) Polypeptide for broad-spectrum inhibition of human coronavirus infection and application thereof
CN112794918B (en) Human ACE2 remodelling protein and ACE2-hFc antibody protein aiming at novel coronavirus
CN113943373B (en) Beta coronavirus polymer antigen, preparation method and application thereof
EP4039696A1 (en) Protein receptacle, polynucleotide, vector, expression cassette, cell, method for producing the receptacle, method of identifying pathogens or diagnosing diseases, use of the receptacle and diagnostic kit
Yuan et al. The crystal structure of KSHV ORF57 reveals dimeric active sites important for protein stability and function
Lei et al. Plasticity at the DNA recognition site of the MeCP2 mCG-binding domain
WO2012018907A2 (en) Polypeptides for treating and/or limiting influenza infection
CN111150833B (en) Application of LTX-315 in preparation of product for inhibiting coronavirus
Tewary et al. Structures of minute virus of mice replication initiator protein N-terminal domain: insights into DNA nicking and origin binding
CN110845624B (en) SUMO-CP fusion protein, preparation method thereof and preparation method of polyclonal antibody thereof
CN107586322B (en) Infectious bovine rhinotracheitis virus gD protein epitope polypeptide, inhibitor and monoclonal antibody thereof, and application of infectious bovine rhinotracheitis virus gD protein epitope polypeptide and inhibitor and monoclonal antibody
Fili et al. Coxsackievirus B3 protease 3C: expression, purification, crystallization and preliminary structural insights
WO2022073234A1 (en) Peptide for inhibiting eb virus, dna encoding peptide, and use thereof
Tan et al. MLL1 is regulated by KSHV LANA and is important for virus latency
Zhang et al. An anti-picornaviral strategy based on the crystal structure of foot-and-mouth disease virus 2C protein
CN112125959B (en) Peptide for inhibiting EB virus, DNA for coding peptide and application thereof
Tassone et al. Probing the role of Arg97 in Heat shock protein 90 N-terminal domain from the parasite Leishmania braziliensis through site-directed mutagenesis on the human counterpart
Chacko et al. A new pentameric structure of rotavirus NSP4 revealed by molecular replacement
EP2800576A2 (en) Polypeptides for treating and/or limiting influenza infection
Mehboob et al. Structure of dihydroorotase from Bacillus anthracis at 2.6 Å resolution
CN114989266A (en) African swine fever virus pA104R protein immunosuppression related amino acid site and application thereof
Nasertorabi et al. Molecular basis for regulation of Src by the docking protein p130Cas
CN117362417A (en) Antibody for broad-spectrum neutralization of saber virus and application thereof

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: 20956549

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20956549

Country of ref document: EP

Kind code of ref document: A1