WO2019141263A1 - 一种广谱抗肠道病毒的多肽及其应用 - Google Patents

一种广谱抗肠道病毒的多肽及其应用 Download PDF

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WO2019141263A1
WO2019141263A1 PCT/CN2019/072455 CN2019072455W WO2019141263A1 WO 2019141263 A1 WO2019141263 A1 WO 2019141263A1 CN 2019072455 W CN2019072455 W CN 2019072455W WO 2019141263 A1 WO2019141263 A1 WO 2019141263A1
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polypeptide
sequence
virus
cells
amino acid
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PCT/CN2019/072455
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English (en)
French (fr)
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周溪
陆路
方媛
姜世勃
刘泽众
秦成峰
邱洋
穆敬芳
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中国科学院武汉病毒研究所
复旦大学
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Priority to CN201980005805.6A priority Critical patent/CN112261946A/zh
Priority to JP2020560535A priority patent/JP7252575B2/ja
Priority to US16/963,196 priority patent/US11999806B2/en
Priority to AU2019208788A priority patent/AU2019208788B2/en
Publication of WO2019141263A1 publication Critical patent/WO2019141263A1/zh

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    • 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
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • 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
    • C12N2770/32011—Picornaviridae
    • C12N2770/32022—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
    • C12N2770/32011—Picornaviridae
    • C12N2770/32034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of biomedicine, in particular to a broad spectrum anti-enteric virus polypeptide and application thereof.
  • Enterovirus is a class of sense single-stranded RNA viruses belonging to the genus Picornaviridae enterovirus, including human enterovirus (EV) and coxsackie A (Coxsackie A). Virus, CVA), Coxsackie B virus (CVB), Echovirus, Rhinovirus, Poliovirus, and the like. Enterovirus infections are widely distributed throughout the world, with complex clinical manifestations ranging from mild hypothermia, fatigue, respiratory diseases, to herpetic angina, hand, foot and mouth disease, and severe aseptic meningitis, myocarditis, encephalitis, Polio and so on. There is currently no symptomatic drug for effective treatment or protection against enterovirus infection.
  • Herpetic angina is mainly caused by coxsackievirus group A (CVA2), CVA4, CVA6, CVA9, CVA16, CVA22, and group B type 1 (CVB1), CVB2, CVB3, CVB4, CVB5.
  • CVA2 coxsackievirus group A
  • CVA4 CVA6, CVA9, CVA16, CVA22
  • group B type 1 CVB1
  • CVB2 group B type 1
  • the heat stroke is about 2 to 4 days.
  • Older children can complain of sore throat and can affect swallowing. Infants and young children are characterized by hooliganism, refusal to eat, and irritability.
  • Sometimes with headache, abdominal pain or myalgia the 25% of children under 5 years of age can be accompanied by vomiting.
  • Typical symptoms appear in the pharynx. It is characterized by pharyngeal congestion. There are several (1 to 2, as many as 10) small (1 to 2 mm in diameter) gray-white herpes in the oral mucosa within 2 days of onset, surrounded by redness. After 2 to 3 days, the redness increased and the herpes ruptured to form a yellow ulcer. This mucosal rash is more common in the anterior column of the tonsils, but also in the soft palate, uvula, and tonsils, but does not involve the gums and buccal mucosa. The course of the disease is usually 4 to 6 days, occasionally extended to 2 weeks.
  • Hand, foot and mouth disease is mainly caused by enterovirus 71 (EV71), CVA6, CVA8, CVA10, CVA16, CVB3 and CVB5.
  • EV71 enterovirus 71
  • the common clinical manifestations of hand, foot and mouth disease are acute fever, mouth pain, anorexia, scattered herpes or ulcers in the oral mucosa, and are located in the tongue, buccal mucosa, and hard, etc., and can also affect soft palate, gums, tonsils, and pharynx.
  • the number of rashes is as small as a few dozen. No signs left after disappearing, no pigmentation. Some children with hand, foot and mouth disease have herpes angina as the first symptom, and then a red rash can appear in the palm, sole, buttocks and other parts. When the course of disease develops rapidly, a small number of children can develop from severe hand, foot and mouth disease to severe aseptic meningitis and encephalitis. It is characterized by fever, headache, nausea, vomiting, meningeal irritation, body temperature fluctuations, most cases of low fever, can also be as high as 40 ° C or more, often bimodal fever in the course of the disease. Other symptoms such as sore throat, muscle soreness, rash, photophobia, diarrhea, swollen lymph nodes, etc., mild cases such as mild paralysis.
  • Myocarditis is mainly caused by CVB1-61 and Echovirus.
  • the clinical manifestations of patients with viral myocarditis depend on the extent and location of the disease, and mild cases can be asymptomatic. In severe cases, heart failure, cardiogenic shock, and sudden death can occur. Patients often have a history of upper respiratory tract or intestinal infection 1 to 3 weeks before onset, which is characterized by fever, body aches, sore throat, burnout, nausea, vomiting, diarrhea, etc., followed by palpitations, chest tightness, chest pain or pain in the precordial area. Dizziness, difficulty breathing, edema, and even Adams-Stokes syndrome; very few patients develop heart failure or cardiogenic shock.
  • the enterovirus is a sense single-stranded RNA virus with a genome size of approximately 7.5 kb and a large ORF encoding a polyprotein.
  • the polyprotein is further hydrolyzed into four structural proteins (VP1-VP4) and seven non-structural proteins (2A-2C and 3A-3D).
  • the 3A protein is a very conserved non-structural protein in enteroviruses (including EV71, CVA, and CVB, etc.), which exists as a homodimer and localizes to the cell's inner membrane, replicating the virus and regulating the host's natural immunity. Play an important role.
  • RNA interference RNA interference
  • dsRNA virus-derived double-stranded RNA
  • AGO Argonaute
  • RISC RNA-induced silencing complexes
  • the present invention provides polypeptides and uses thereof.
  • the present invention provides the following technical solutions:
  • the present invention provides the use of Enterovirus RNA suppressing protein (ERSP) as a target for the preparation of a medicament for preventing and/or treating a viral disease.
  • ESP Enterovirus RNA suppressing protein
  • the invention also provides for the use of a polypeptide for the preparation of an inhibitor that targets ERSP; the function of the ERSP is inhibited by the polypeptide, and the viral nucleic acid is cleaved by Dicer to produce a vsiRNA.
  • the invention also provides the use of a polypeptide for the preparation of a medicament for the prevention and treatment of viral diseases.
  • the ERSP is enterovirus non-structural protein 3A.
  • the enterovirus is a Piporaviridae enterovirus genus, including Enterovirus (EV) and Coxsackie A virus (Coxsackie A virus). , CVA), Coxsackie B virus (CVB), Echovirus, Rhinovirus, Poliovirus, and the like.
  • EV Enterovirus
  • COxsackie A virus Coxsackie A virus
  • CVA Coxsackie B virus
  • Echovirus Rhinovirus
  • Poliovirus Poliovirus
  • the disease is hand, foot and mouth disease, myocarditis, herpetic angina, aseptic meningitis, encephalitis, viral influenza, and the like caused by the virus.
  • the amino acid sequence of the polypeptide comprises CR, CK and/or DLL.
  • polypeptide has the following sequence:
  • X1 is selected from the group consisting of isoleucine (I);
  • X2 selects serine (S) or alanine (A);
  • X3 is selected from alanine (A) or lysine (K) or glutamine (Q) or arginine (R) or serine (S) or cysteine (C);
  • X4 is selected from serine (S) or alanine (A);
  • X5 is selected from the group consisting of glutamic acid (E) or glutamine (Q);
  • X6 is selected from arginine (R) or lysine (K);
  • Amino acid as used herein includes a natural amino acid or a non-natural amino acid. Amino acid types well known to those skilled in the art are within the scope of the present invention.
  • the sequence as described in I is as set forth in any one of SEQ ID Nos. 1 to 14, but does not include the sequence of the transmembrane peptide and the sequence of the linker peptide.
  • the invention also provides polypeptides capable of inhibiting the activity of ERSP.
  • the amino acid sequence of the polypeptide comprises CR, CK and/or DLL.
  • the amino acid sequence of the polypeptide comprises YCR and/or YCK.
  • polypeptide has the following sequence:
  • X1 is selected from the group consisting of isoleucine (I);
  • X2 selects serine (S) or alanine (A);
  • X3 is selected from alanine (A) or lysine (K) or glutamine (Q) or arginine (R) or serine (S) or cysteine (C);
  • X4 is selected from serine (S) or alanine (A);
  • X5 is selected from the group consisting of glutamic acid (E) or glutamine (Q);
  • X6 is selected from arginine (R) or lysine (K);
  • the polypeptide is as described in any one of SEQ ID Nos. 1 to 14 as described in I, but does not include the sequence of the transmembrane peptide and the sequence of the linker peptide.
  • the present invention also provides a nucleic acid encoding a nucleotide sequence of the polypeptide.
  • the invention also provides recombinant vectors, including the nucleic acids described.
  • the invention also provides host cells, including the recombinant vectors.
  • the invention also provides medicaments, including the polypeptides and pharmaceutically acceptable excipients.
  • the invention also provides a vaccine comprising the polypeptide and a pharmaceutically acceptable excipient.
  • the invention also provides a method of treating an enterovirus-infected disease, taking and/or injecting the medicament.
  • the injection is intramuscular, intraperitoneal or intravenous.
  • the present invention also provides a method of preventing an enterovirus-infected disease by inoculating the vaccine.
  • prevention refers to various means or measures for preventing the occurrence or development of a disease, including medical, physical or chemical means, to prevent and reduce diseases before a disease which is not recognized by clinical standards. The occurrence or development of a symptom.
  • treatment refers to the prevention, suppression, alleviation, amelioration, mitigation, cessation, delay or reversal of the progression or progression of a disease in order to prevent and reduce the occurrence or progression of the disease.
  • Various indicators of disease, disorder, or pathological state at the time of administration and/or administration include alleviating or reducing symptoms or complications, or curing or eliminating a disease, disorder, or condition.
  • drug means a single compound, a composition formed by a plurality of compounds, or a composition or a preparation having a single compound as a main active ingredient, which can be used for preventing or treating a certain disease, It also refers to a composition or formulation from which a plurality of compounds are the active ingredients.
  • “Drug” should be understood to refer not only to the products approved and approved for production by the administrative agencies established by the laws of a country, but also to the inclusion of a single compound as an active ingredient in order to obtain approval and approval of production. Various forms of matter. "Formation” is understood to mean obtained by chemical synthesis, biotransformation or purchase.
  • the present invention also provides an inhibitor of enterovirus type 71, wherein the inhibitor is polypeptide P2, and the amino acid sequence of P2 is represented by SEQ ID NO.
  • the present invention also provides a recombinant prepared by the inhibitor, which is 3A-TAT-EP, 3A-EP-DRI or 3A-EP-PEG4-PA, and the amino acid of the 3A-TAT-EP
  • the amino acid sequence of the 3A-EP-PEG4-PA represented by SEQ ID NO. 4 and the non-natural amino acid sequence of 3A-EP-DRI shown in SEQ ID NO. 3 is represented by SEQ ID NO.
  • the invention also provides the use of said inhibitor or said engineered body for the preparation of an enterovirus inhibitor.
  • RNA interference is an antiviral immune mechanism.
  • double-stranded RNA generated by viral RNA replication is recognized by the host Dicer protein and cleaved into small interfering RNA (siRNA).
  • siRNA small interfering RNA
  • vsiRNAs virus-derived small interfering RNAs
  • the non-structural protein 3A of EV71 can bind to viral double-stranded RNA to prevent Dicer from cleavage and inhibit the production of small interfering RNA derived from vsiRNA virus, thereby achieving the purpose of escape RNA interference against anti-virus immunity.
  • the polypeptides provided by the invention have an efficient, broad spectrum antiviral activity. This provides a new strategy for the prevention and control of EV71 virus, CVA16 virus, CVA6 virus, CVB3 virus and CVB5 virus, and also accelerates the polypeptides against human enterovirus 71, CVA16, CVB3 and CVB5 viruses.
  • the development of molecular drugs provides a new theoretical basis.
  • Figure 1 shows the results of detecting the ability of polypeptide P2 to cross the cell membrane into the cytosol by fluorescent labeling
  • Figure 2 shows the cytotoxicity results of the polypeptide P2 as determined by CCK-8;
  • Figure 3 shows the results of antiviral efficacy of polypeptide P2 in RD cells
  • FIG. 4 shows that polypeptide P2 has an inhibitory effect on viruses in various cells
  • Figure 5 shows the antiviral effect of the polypeptide modified with the polypeptide P2
  • Figure 6 shows the antiviral effects of polypeptides 3A-EP-DRI and 3A-TAT-EP;
  • Figure 7 shows the results of detecting the antiviral activity of polypeptide P1 against EV71 in mice
  • Figure 8 shows the results of detecting the antiviral activity of the polypeptide P1 against CVA16 in mice
  • Figure 9 shows the results of anti-EV71 effect detection of polypeptide CR in RD cells
  • Figure 10 shows the results of antiviral activity of polypeptide CR against EV71 in mice
  • Figure 11 shows the results of antiviral activity of polypeptide CR against CVA16 in mice
  • Figure 12 shows the results of body weight measurement of toxicity evaluation of polypeptide ER-DRI in mice
  • Figure 13 shows the HE staining results of toxicity evaluation of polypeptide ER-DRI in mice
  • Figure 14 shows the results of transmembrane efficiency detection of polypeptide P1 in RD cells
  • Figure 15 shows the results of cytotoxicity experiments of polypeptide P1
  • Figure 16 shows the results of cytotoxicity experiments of the polypeptide 3A-TAT-EP
  • Figure 17 shows the results of cytotoxicity experiments of polypeptides 3A-EP-DRI and 3A-EP-PEG4-PA;
  • Figure 18 shows the results of detection of antiviral effects of the polypeptides EP-PA, EP-CHOL, 3A-EP-DRI and 3A-EP-PEG4-PA;
  • Figure 19 shows the results of toxicity tests of ER-DRI and ER in RD cells
  • Figure 20 shows the results of anti-EV71 virus detection of ER-DRI and ER in RD cells.
  • Figure 21 shows the results of detecting the antiviral activity of the polypeptide ER-DRI against EV71 in mice
  • Figure 22 shows the results of detection of anti-CVA16 virus effects of polypeptides P2, ER, ER-DRI, R8 and TAT;
  • Figure 23 shows the results of toxicity tests of polypeptides BP8, BP10 and BP15 in RD cells
  • Figure 24 shows the results of toxicity tests of polypeptides BP8, BP10 and BP15 in Vero cells
  • Figure 25 shows the results of anti-CVB5 effect detection of polypeptides BP8, BP10 and BP15 in RD cells
  • Figure 26 shows the results of anti-CVB3 effect detection of polypeptide BP8 in Vero cells
  • Figure 27 shows the results of anti-CVB5 effect detection of polypeptide BP8 in mice
  • Figure 28 shows the results of anti-CVA6 effect detection of polypeptide ER-DRI in Vero cells.
  • the invention discloses a polypeptide and an application thereof, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • the EV713A protein contains 86 amino acid residues. 3A has the ability to dimerize, and its dimerization plays a key role in the proper functioning of 3A.
  • the applicant designed the polypeptide P2 (SEQ ID NO. 2) with the EP polypeptide as the core sequence in order to allow the inhibitor to penetrate the cell membrane and enter the cell to inhibit the virus. It is capable of competitively interacting with ⁇ 1, thereby preventing 3A dimerization, inhibiting the anti-innate immunity of 3A, and finally achieving anti-viral purposes.
  • the modified bodies are: 3A-TAT-EP, 3A-EP-DRI and 3A-EP-PEG4-PA; the amino acid sequence of the 3A-TAT-EP is shown in SEQ ID NO. 3, 3A-EP The amino acid sequence of the -DRI is represented by SEQ ID NO. 4, and the amino acid sequence of 3A-EP-PEG4-PA is represented by SEQ ID NO.
  • an inhibitor of enterovirus type 71 comprising preparing an enterovirus 71 type inhibitor using the P2 and P2 recombinants provided by the present invention, or using the P2 and P2 recombinants provided by the present invention and other effective Together with the ingredients, an inhibitor of enterovirus type 71 was prepared.
  • the protection of the present invention also includes an inhibitor which inhibits the activity of EV71 after replacing the different transmembrane sequences, or performing polypeptide modification, or designing and modifying non-natural amino acids according to the P2 polypeptide.
  • Enterovirus 3A is a highly potent ERSP that binds to viral dsRNA to prevent Dicer from cleavage, inhibits the production of viral-derived vsiRNA, and resists host RNAi antiviral immunity.
  • polypeptide and the derivative thereof according to the invention are capable of inhibiting the antiviral immunity of enterovirus 3A, and are an emerging therapeutic drug for EV71, which is important for targeting new targets and fighting against viral resistance.
  • the present invention has the following advantages:
  • the P2 series of polypeptides have potent antiviral activity. This will provide a new strategy for the prevention and control of enterovirus, and also provide a new theoretical basis for accelerating the development of anti-human enterovirus polypeptide small molecule drugs. And the clear anti-viral mechanism of the P2 series of peptides can ensure the safety of its application and the clarity of the optimization route, which is convenient for further development in the future.
  • polypeptides involved in the present invention are shown in Table 1:
  • RRRRRRRR (R8) and YGRKKRRQRRR (TAT) are polypeptides in the polypeptide sequence
  • GSG is a linker peptide
  • the amino acid sequence of each polypeptide is removed from the sequence of the transmembrane peptide and the linker peptide as a core sequence.
  • the polypeptides in various embodiments of the invention were provided with a negative control demonstrating that the core sequences of the polypeptides provided herein have corresponding antiviral effects.
  • polypeptides and the materials and reagents used in the application of the present invention are commercially available.
  • MEM medium (Thermo), serum (Gibco) was purchased from Infineon, Inc., immunofluorescent plate (NEST) was purchased from the promoter company, PBS, DAPI, and paraformaldehyde was purchased from Diyue Innovation Co., Ltd.
  • the polypeptide P2 was synthesized by Nanjing Jinsirui Co., Ltd., and its sequence is shown in SEQ ID NO.
  • the experiment was divided into two groups. In order to avoid the influence of the addition of EV71 virus on the entry of the polypeptide into the cells, one group of experiments added EV71 virus and then added the polypeptide P2, and the other group added the virus without adding the virus, and each group was made a negative control.
  • the immunofluorescence steps are as follows:
  • the transmembrane efficiency in RD cells was examined using a fluorescently labeled (FITC) polypeptide.
  • FITC fluorescently labeled
  • CCK-8 Reagent (MCE) was purchased from a startup subsidiary.
  • Peptide P2 not only inhibits the virus in the process of antiviral, but also ensures that it is not toxic to cells. Therefore, this indicator was tested by a cytotoxicity test, and the cells which were not treated were used as a control group.
  • a 96-well cell plate was plated with RD cells at 100 ⁇ l per well.
  • the MEM medium containing 10% serum is replaced with MEM medium containing 2% serum, and a certain concentration gradient of polypeptide P2 is added to make the final P2 concentration in the well.
  • concentration gradient of polypeptide P2 was added to make the final P2 concentration in the well. They were 0.01 ⁇ M, 0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M, and 100 ⁇ M, respectively.
  • the results are shown in Fig. 2 and Table 2.
  • the cell viability of the untreated cells was 100%. There was no significant difference between the cell viability and the control group (untreated cells) after adding 100 ⁇ M of the polypeptide, which proved that the polypeptide P2 used in this study was 100 ⁇ M. It is not toxic to cells.
  • RNA extraction kit (Omega), 24-well plate, 100mm dish, 50ml syringe were purchased from Diyue Innovation Co., Ltd., 0.22 ⁇ m filter (Millipore) was purchased from the company, and the One step qRT-PCR kit (Takara) was purchased. Since the company, the water used in the extraction of RNA and qRT-PCR was DEPC water, and the whole experiment was carried out in the RNase Free environment.
  • One step qRT-PCR was performed by taking 100 ⁇ l of virus-extracted RNA and previously extracted known titer of viral RNA, and the virus titer was measured.
  • polypeptides were added at a final concentration of 0.01 ⁇ M, 0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M, and 50 ⁇ M, respectively.
  • RD cells supplemented with the R8 transmembrane peptide (sequence: RRRRRRRR) were used as a negative control.
  • the results are shown in Fig. 3 and Table 3.
  • the amount of viral RNA in the experimental group added with R8 by EV71 virus was 100%.
  • the amount of virus decreased with the increase of the polypeptide concentration, and the polypeptide concentration was 50 ⁇ M.
  • the amount of virus decreased to about 4%, which proved that the polypeptide P2 has a good antiviral effect.
  • the virus titer was detected by qRT-PCR method, and the IC 50 value of P2 was determined to be 6.372 ⁇ M; in combination with FIG. 2, the polypeptide P2 was at 100 ⁇ M. There is no cytotoxicity inside, which proves that the peptide has a good antiviral effect under the premise of safe medication.
  • RD cells were used to detect the antiviral effect of polypeptide P2.
  • a 24-well plate was plated with 293T cells, Vero cells, and Huh7.5 cells.
  • polypeptide P2 was added to different cells at a final concentration of 0.01 ⁇ M, 0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M, and 50 ⁇ M, respectively.
  • polypeptide P2 can exert significant antiviral effects in both RD cells, 293T cells, Vero cells and huh7.5 cells.
  • the IC 50 values measured for 293T cells, Vero cells, and H ⁇ h7.5 cells were 9.677 ⁇ M, 1.958 ⁇ M, and 1.842 ⁇ M, respectively.
  • Example 5 Effect of the modified polypeptide of polypeptide P2 on the antiviral effect
  • Polypeptides 3A-TAT-EP shown in SEQ ID NO. 3
  • 3A-EP-DRI shown in SEQ ID NO. 4
  • 3A-EP-PEG4-PA shown in SEQ ID NO. 5
  • a 24-well plate was plated with 293T cells.
  • polypeptides P2, 3A-TAT-EP, 3A-EP-DRI and 3A-EP-PEG-PA were added at a final concentration of 0.01 ⁇ M, 0.1 ⁇ M, 1 ⁇ M, and 10 ⁇ M, respectively.
  • RD cells with good growth conditions were plated in 96-well plates at 1 ⁇ 10 4 per well, and cultured for 24 hours at 37 ° C and 5% CO 2 .
  • the diluted virus was added to the above wells, 100 ⁇ L per well, and the drug-free virus-free wells and the drug-free virus-free wells were set as controls, respectively, and the final concentration of the virus was 0.1 MOI.
  • the mixture was transferred to a 96-well plate in which cells were plated, and cultured at 37 ° C, 5% CO 2 for 24 hours, and then the polypeptide was inhibited against the virus by a CCK8 kit.
  • polypeptide inhibition rate (drug well-viral pore) ⁇ 100% / (no drug pore-viral pore).
  • polypeptide P2 modified polypeptide 3A-TAT-EP shown in SEQ ID NO. 3
  • 3A-EP-DRI shown in SEQ ID NO. 4
  • 3A-EP-PEG4- PA shown in SEQ ID NO. 5
  • the IC 50 of 3A-EP-PEG4-PA was determined by qRT-PCR to be 3.25 ⁇ M.
  • FIG. 6 as determined by viral polypeptide CCK8 method showed inhibitory activity, 3A-TAT-EP IC 50 of 4.36 ⁇ M, and 3A-EP-DRI IC 50 of 3.56 ⁇ M.
  • MEM medium (Thermo), serum (Gibco) was purchased from Infineon, Inc., immunofluorescent plate (NEST) was purchased from the promoter company, PBS, DAPI, and paraformaldehyde was purchased from Diyue Innovation Co., Ltd.
  • the polypeptide P1 was synthesized by Nanjing Kingsray Co., Ltd., and its sequence is shown in SEQ ID NO.
  • the experiment was divided into two groups. In order to avoid the influence of the addition of EV71 virus on the entry of the polypeptide into the cells, one group of experiments added EV71 virus and then added the polypeptide P1, and the other group added the virus without adding the virus, and each group was made a negative control.
  • the immunofluorescence steps are as follows:
  • the transmembrane efficiency in RD cells was examined using a fluorescently labeled (FITC) polypeptide.
  • FITC fluorescently labeled
  • Two sets of experiments were set up. The first group was untreated control, R8 was added, P1 was added, and P2 was added. The second group was infected with EV71, infected with EV71 and added with R8, infected with EV71, and added with P1, infected with EV71 and added with P2.
  • CCK-8 Reagent (MCE) was purchased from a startup subsidiary.
  • Peptide P1 not only inhibits the virus in the process of antiviral, but also ensures that it is not toxic to cells. Therefore, this indicator was tested by a cytotoxicity test, and the cells which were not treated were used as a control group.
  • a 96-well cell plate was plated with RD cells at 100 ⁇ l per well.
  • the MEM medium containing 10% serum is replaced with the MEM medium containing 2% serum, and a certain concentration gradient of the polypeptide P1 is added to make the final P1 concentration in the well.
  • concentration gradient of the polypeptide P1 was added to make the final P1 concentration in the well. They were 0.01 ⁇ M, 0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M, and 100 ⁇ M, respectively.
  • the results are shown in Fig. 15 and Table 4.
  • the cell viability of the untreated cells was 100%. After adding 100 ⁇ M of the polypeptide, the cell viability was not significantly different from that of the control group, which proved that the polypeptide P1 used in the study did not have cells within 100 ⁇ M. toxicity.
  • the cytotoxicity of the polypeptide P1 was detected by RD cells.
  • the concentration gradient peptide P1 was added at a concentration of 0 ⁇ M (control group), 0.01 ⁇ M, 0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M, 100 ⁇ M, respectively.
  • Example 8 Determination of cytotoxicity of polypeptide 3A-TAT-EP
  • CCK-8 Reagent (MCE) was purchased from a startup subsidiary.
  • the polypeptide 3A-TAT-EP not only inhibits the virus but also has no toxicity to the cells during the antiviral process. Therefore, this indicator was tested by a cytotoxicity test, and the cells which were not treated were used as a control group.
  • a 96-well cell plate was plated with RD cells at 100 ⁇ l per well.
  • the MEM medium containing 10% serum is replaced with the MEM medium containing 2% serum, and a certain concentration of the polypeptide EP is added to make the final EP concentration in the well.
  • concentration of the polypeptide EP was added to make the final EP concentration in the well. They were 0.01 ⁇ M, 0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M, and 100 ⁇ M, respectively.
  • the results are shown in Fig. 16 and Table 5.
  • the cell viability of the untreated cells was 100%, and there was no significant difference between the cell viability and the control group (untreated cells) after adding 100 ⁇ M of the polypeptide, which proved that the polypeptide used in the study was 3A-TAT.
  • -EP is not toxic to cells within 100 ⁇ M.
  • the cytotoxicity of the polypeptide 3A-TAT-EP was detected by RD cells.
  • the concentration gradient peptide 3A-TAT-EP was added to the 80% confluent RD cells at concentrations of 0.01 ⁇ M, 0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M, and 100 ⁇ M, respectively. Three concentration experiments were set up for each concentration gradient. After 24 hours, the cells were taken and the cell viability was measured using a CCK-8 kit.
  • Example 9 Determination of cytotoxicity of polypeptides 3A-EP-DRI and 3A-EP-PEG4-PA
  • CCK-8 Reagent (MCE) was purchased from a startup subsidiary.
  • the peptides 3A-EP-DRI and 3A-EP-PEG4-PA not only inhibit the virus but also ensure no toxicity to the cells during the antiviral process. Therefore, this indicator was tested by a cytotoxicity test, and the cells which were not treated were used as a control group.
  • a 96-well cell plate was plated with RD cells at 100 ⁇ l per well.
  • the results are shown in Fig. 17 and Table 6.
  • the cell viability of the untreated cells was 100%, and the cell viability was substantially the same as that of the control group (untreated cells) after adding 100 ⁇ M of the polypeptide, which proved that the polypeptide used in the study was 3A-EP-DRI. And 3A-EP-PEG4-PA is not toxic to cells within 50 ⁇ M.
  • the cytotoxicity of the peptides 3A-EP-DRI and 3A-EP-PEG4-PA was detected by RD cells.
  • the concentration gradient peptides EP-DRI and EP-PEG4-PA were added to the RD cells with 80% confluency, respectively. 0.01 ⁇ M, 0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M, 20 ⁇ M, 30 ⁇ M, 40 ⁇ M, 50 ⁇ M. After 24 hours, the cells were taken and the cell viability was measured using a CCK-8 kit. The results showed that the cell viability was consistent with the control group after adding 50 ⁇ M polypeptide, and it was confirmed that the polypeptides 3A-EP-DRI and 3A-EP-PEG4-PA were not toxic to cells within 50 ⁇ M.
  • RNA extraction kit (Omega), 24-well plate, 100mm dish, 50ml syringe were purchased from Diyue Innovation Co., Ltd., 0.22 ⁇ m filter (Millipore) was purchased from the company, and the One step qRT-PCR kit (Takara) was purchased. Since the company, the water used in the extraction of RNA and qRT-PCR was DEPC water, and the whole experiment was carried out in the RNase Free environment.
  • One step qRT-PCR was performed by taking 100 ⁇ l of virus-extracted RNA and previously extracted known titer of viral RNA, and the virus titer was measured.
  • concentration gradient polypeptides EP-PA, EP-CHOL, 3A-EP-DRI and 3A-EP-PEG4-PA were added, respectively, and all polypeptide concentrations were set to 0.01 ⁇ M, 0.1 ⁇ M, 1 ⁇ M, and 10 ⁇ M.
  • the total RNA was extracted, and the expression level of viral genomic RNA was detected by real-time PCR, so that only the sample without the added polypeptide was used as the control group. The results showed that all of the peptides had anti-EV71 activity, and increasing the polypeptide concentration significantly inhibited the expression level of viral RNA.
  • the antiviral effects of the polypeptides P2, ER and ER-DRI were detected by RD cells, and the transmembrane peptides R8 and TAT were used as controls.
  • the inhibitory activity of the polypeptide against the virus was determined using a CCK-8 kit 24 hours after infection.
  • Polypeptide inhibition rate (drug well-viral well) x 100% / (no drug well-viral well).
  • the results showed that the peptides P2, ER and ER-DRI all significantly inhibited CVA16, while the transmembrane peptides R8 and TAT did not contribute to the virus.
  • RRRRRRRRAISDLLAS is a commercial synthesis. Newly born 2 day old ICR suckling rats.
  • one group was intraperitoneally injected with 10 mg/kg of P1 polypeptide as the treatment group, and the other group was injected with the same amount of PBS as the control group.
  • mice were euthanized, and their hind limb muscles were taken and triturated with Trizol to extract total RNA from the tissues.
  • Example 12 Detection of polypeptide CR inhibiting EV71 activity in RD cells
  • the IC 50 of CR for EV71 was 1.7 ⁇ M.
  • Example 13 Antiviral activity of polypeptide CR against EV71 and CVA16 in mice
  • Peptide CR YGRKKRRQRRRGSGCR is a commercial synthesis. Newly born 2 day old ICR suckling rats.
  • mice were euthanized, and their hind limb muscles were taken and triturated with Trizol to extract total RNA from the tissues.
  • the polypeptide ER-DRI is a commercial synthesis of 12 10-day-old suckling mice.
  • mice were randomly divided into two groups, 6 in each group. One group was intraperitoneally injected with 20 mg/kg ER-DRI once a day for 3 consecutive days. Another group was injected with an equal amount of PBS as a control.
  • mice The body weight of the mice was recorded daily for a total of 15 days.
  • mice were euthanized, and the brain, liver, lungs, and kidneys were dissected and separated for HE staining.
  • CCK-8 Reagent (MCE) was purchased from a startup subsidiary.
  • the polypeptide ER and ER-DRI not only inhibit the virus but also ensure no toxicity to the cells during the antiviral process. Therefore, this indicator was tested by a cytotoxicity test, and the cells which were not treated were used as a control group.
  • a 96-well cell plate was plated with RD cells at 100 ⁇ l per well.
  • the cell viability of the untreated cells was 100%
  • the half cytotoxicity (CC 50 ) of TAT-ER-DRI was 117 ⁇ M
  • the half CC 50 of the TAT-ER was calculated to be 290 ⁇ M. It was proved that the polypeptide ER used in this study is a very low toxicity drug compared with the half-cell cytotoxicity (290 ⁇ M, 117 ⁇ M) of ER-DRI, which is half-inhibitory activity (1.26 ⁇ M, 0.64 ⁇ M).
  • Example 16 ER and ER-DRI inhibit EV71 activity detection on RD cells
  • RNA extraction kit (Omega), 24-well plate, 100mm dish, 50ml syringe were purchased from Diyue Innovation Co., Ltd., 0.22 ⁇ m filter (Millipore) was purchased from Hida Co., Ltd., One step q-pcr kit (Takara) was purchased. Since the company, the water used in the extraction of RNA and qRT-PCR was DEPC water, and the whole experiment was carried out in the RNase Free environment.
  • the polypeptide ER-DRI is a commercial synthesis of 10 2 day old suckling mice.
  • mice After the fifth day, the suckling mice were euthanized, their lungs were taken, and the total RNA in the tissues was extracted after grinding with Trizol.
  • ER-DRI significantly reduced the EV71 viral load in the mouse lung.
  • Example 18 Toxicity of polypeptides BP8, BP10 and BP15 in RD cells
  • CCK-8 Reagent (MCE) was purchased from a startup subsidiary.
  • the peptides BP8, BP10 and BP15 not only inhibit the virus but also ensure no toxicity to the cells during the antiviral process. Therefore, this indicator was tested by a cytotoxicity test, and the cells which were not treated were used as a control group.
  • a 96-well cell plate was plated with RD cells at 100 ⁇ l per well.
  • the MEM medium containing 10% serum is replaced with MEM medium containing 2% serum, and a certain concentration gradient of polypeptide polypeptides BP8, BP10 and BP15 is added to make the pores
  • the final concentrations in the medium were 3.0625 ⁇ M, 6.125 M, 12.5 ⁇ M, 25 ⁇ M, 50 ⁇ M, 100 ⁇ M, and 200 ⁇ M, respectively.
  • the results are shown in Fig. 23 and Tables 27, 28 and 29.
  • the cell viability of the untreated cells was 100%.
  • the cell viability of BP8 and BP10 after adding 200 ⁇ M polypeptide was basically the same as that of the control group (untreated cells), and BP15 was added with 100 ⁇ M polypeptide.
  • the post-cell viability was basically consistent with the control group (untreated cells), and the viability decreased to 20% after the addition of 200 ⁇ M polypeptide. It indicated that BP8 and BP10 were not toxic to cells within 200 ⁇ M, and BP15 was not toxic to cells within 100 ⁇ M.
  • CCK-8 Reagent (MCE) was purchased from a startup subsidiary.
  • the peptides BP8, BP10 and BP15 not only inhibit the virus but also ensure no toxicity to the cells during the antiviral process. Therefore, this indicator was tested by a cytotoxicity test, and the cells which were not treated were used as a control group.
  • the MEM medium containing 10% serum is replaced with MEM medium containing 2% serum, and a certain concentration gradient of polypeptide polypeptides BP8, BP10 and BP15 is added to make the pores
  • the final concentrations in the medium were 3.0625 ⁇ M, 6.125 M, 12.5 ⁇ M, 25 ⁇ M, 50 ⁇ M, 100 ⁇ M, and 200 ⁇ M, respectively.
  • the results are shown in Fig. 24 and Tables 30, 31 and 32.
  • the cell viability of the untreated cells was 100%.
  • the cell viability of BP8 and BP10 after adding 200 ⁇ M polypeptide was basically the same as that of the control group (untreated cells), and BP15 was added with 100 ⁇ M polypeptide.
  • the post-cell viability was basically the same as that of the control group (untreated cells), and the viability decreased to 20% after the addition of 200 ⁇ M of the polypeptide. It indicated that BP8 and BP10 were not toxic to cells within 200 ⁇ M, and BP15 was not toxic to cells within 100 ⁇ M. The toxicity of the drug in RD cells and Vero cells is relatively consistent.
  • Polypeptides BP8 (shown as SEQ ID NO. 12), BP10 (shown as SEQ ID NO. 13), and BP15 (shown as SEQ ID NO. 14). The sequences are all commercially synthesized.
  • a 24-well plate was plated with RD cells.
  • polypeptides BP8, BP10 and BP15 were all able to significantly inhibit CVB5.
  • IC50 BP15 was 6.758 ⁇ M.
  • polypeptide BP8 shown in SEQ ID NO. 12
  • sequences described for polypeptide BP8 are all commercially synthesized.
  • a 24-well plate was plated with Vero cells.
  • polypeptide BP8 at a final concentration of 2.5 ⁇ M, 5 ⁇ M, and 10 ⁇ M was added, respectively.
  • polypeptide BP8 was able to inhibit the replication of CVB3.
  • BP8 an IC 50 of 4.125 ⁇ M ..
  • Polypeptide BP8 (shown as SEQ ID NO. 12) is a commercial synthesis. Newly born 2 day old ICR suckling rats.
  • mice After the fifth day, the suckling mice were euthanized, and their hind limb muscles were taken and ground with Rrizol to extract total RNA from the tissues.
  • the virion copy number of the medicated group (PB10) was significantly lower than that of the blank control group (PBS), and the virion decreased by nearly 80 times.
  • sequences described in the polypeptide ER-DEI (SEQ ID No. 11) are all commercially synthesized.
  • a 24-well plate was plated with Vero cells.
  • polypeptide BP8 at a final concentration of 2.5 ⁇ M, 5 ⁇ M, and 10 ⁇ M was added, respectively.
  • the polypeptide ER-DEI was able to inhibit the replication of CVA6.
  • BP8 less than the IC 50 0.625 ⁇ M.

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Abstract

提供有抗病毒活性的系列多肽，为EV71病毒、CVA16病毒、CVA6病毒、CVB3病毒、CVB5病毒等肠道病毒的防控提供一种新的策略，同时也为加快抗EV71病毒、CVA16病毒、CVA6病毒、CVB3病毒、CVB5病毒等肠道病毒的多肽小分子药物的研发提供了新的理论依据。

Description

一种广谱抗肠道病毒的多肽及其应用
本申请要求于2018年01月20日提交中国专利局、申请号为2018100562971、发明名称为“一种肠道病毒71型的抑制剂及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医药领域，特别涉及一种广谱抗肠道病毒的多肽及其应用。
背景技术
肠道病毒(Enterovirus)是一类正义单链RNA病毒，属于小核糖核酸病毒科(Picornaviridae)肠道病毒属，主要包括人肠道病毒(Enterovirus，EV)、柯萨奇病毒A型(Coxsackie A virus，CVA)、柯萨奇病毒B型(Coxsackie B virus，CVB)、埃可病毒(Echovirus)、鼻病毒(Rhinovirus)、脊髓灰质炎病毒(Poliovirus)等。肠道病毒感染广泛分布于世界各地，临床表现复杂多样，从轻微的低热、乏力、呼吸道疾病，到疱疹性咽峡炎、手足口病，以及严重的无菌性脑膜炎、心肌炎、脑炎、脊髓灰质炎等。目前缺乏有效治疗或抵御肠道病毒感染的对症药物。
疱疹性咽峡炎主要由柯萨奇病毒A组2型(CVA2)、CVA4、CVA6、CVA9、CVA16、CVA22型，以及B组1型(CVB1)、CVB2、CVB3、CVB4、CVB5型引起。疱疹性咽峡炎常急剧发热，热多为低度或中等度，偶见高达40℃以上，甚至引起惊厥。热程约为2～4天。年龄较大的患儿可诉咽痛，可影响吞咽。婴幼儿则表现为流涎、拒食、烦躁不安。有时伴头痛、腹痛或肌痛，5岁以下小儿越有25％可伴发呕吐。典型症状出现在咽部。表现为咽部充血，起病2日内口腔黏膜出现数个(少则1～2个，多达10余个)小的(直径1～2mm)灰白色疱疹，周围绕以红晕。2～3日后红晕加剧扩大，疱疹破溃形成黄色溃疡。此种黏膜疹多见于扁桃体前柱，也可位于软腭，悬雍垂，扁桃体上，但不累及齿龈及颊黏膜。病程一般为4～6天，偶有延至2周者。
手足口病主要由肠道病毒71型(EV71)、CVA6、CVA8、CVA10、CVA16、CVB3及CVB5引起。手足口病的普通临床表现为急发热、口痛、厌食、口腔黏膜出现散在疱疹或溃疡，位于舌、颊黏膜及硬额等处为多，也可波及软腭，牙龈、扁桃体和咽部。手、足、臀部、臂部、腿部出现斑丘疹，后转为疱疹，疱疹周围可有炎性红晕，疱内液体较少。手足部较多，掌背面均有。皮疹数少则几个多则几十个。消退后不留痕迹，无色素沉着。部分手足口病患儿以疱疹性咽峡炎为首发症状，随后可在手掌、足底、臀部等部位出现红色皮疹。当病程发展迅速时，少数患儿可由手足口病发展为严重的无菌性脑膜炎、脑炎等。表现为发热、头痛、恶心、呕吐以后出现脑膜刺激症状，体温波动较大，多数病例为低热，亦可高达40℃以上，病程中往往有双峰热型。其他症状如咽痛、肌肉酸痛、皮疹、怕光、腹泻、淋巴结肿大等，少数病例可出现轻度瘫痪等。
心肌炎主要由CVB1-61及Echovirus引起。病毒性心肌炎患者临床表现取决于病 变的广泛程度和部位，轻者可无症状，重者可出现心力衰竭、心源性休克和猝死。患者常在发病前1～3周有上呼吸道或肠道感染史，表现为发热、全身酸痛、咽痛、倦怠、恶心、呕吐、腹泻等症状，然后出现心悸、胸闷、胸痛或心前区隐痛、头晕、呼吸困难、水肿，甚至发生Adams-Stokes综合征；极少数患者出现心力衰竭或心源性休克。
肠道病毒为正义单链RNA病毒，基因组大小约为7.5kb，包含一个大的ORF可编码一个多聚蛋白。多聚蛋白又被进一步水解成4个结构蛋白(VP1-VP4)与7个非结构蛋白(2A-2C与3A-3D)。3A蛋白在肠道病毒中(包括EV71、CVA及CVB等)是非常保守的非结构蛋白，它以同源二聚体的形式存在并定位于细胞内膜，对于病毒的复制以及调节宿主天然免疫起到重要作用。
当病毒感染后，宿主细胞会启动一系列的天然免疫机制来进行防御，其中包括RNA干扰(RNA interference，RNAi)介导的抗病毒免疫。当病毒感染宿主细胞后，由于病毒基因组自身结构或者复制中间体的原因，会产生病毒来源的长双链RNA(double-stranded RNA，dsRNA)。这些dsRNA被宿主Dicer蛋白识别并被切割成病毒衍生的小分子干扰RNA(viral small interfering RNA，vsiRNA)，随后vsiRNA与Argonaute(AGO)蛋白结合并装配形成RNA诱导的沉默复合体(RNA-induced silencing complex，RISC)，最终介导病毒靶基因RNA的降解，从而抑制病毒的复制达到清除病毒的目的。
发明内容
有鉴于此，本发明提供了多肽及其应用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了肠道病毒抑制蛋白(Enterovirus RNA suppressing protein，ERSP)作为靶标在制备预防和/或治疗病毒疾病的药物中的应用。
本发明还提供了多肽在制备靶向ERSP的抑制剂中的应用；所述ERSP的功能被所述多肽抑制，病毒核酸通过Dicer剪切产生vsiRNA。
本发明还提供了多肽在制备预防和治疗病毒疾病的药物中的应用。
在本发明的一些具体实施方案中，所述ERSP为肠道病毒非结构蛋白3A。
在本发明的一些具体实施方案中，所述肠道病毒为小核糖核酸病毒科(Picornaviridae)肠道病毒属，包括人肠道病毒(Enterovirus，EV)、柯萨奇病毒A型(Coxsackie A virus，CVA)、柯萨奇病毒B型(Coxsackie B virus，CVB)、埃可病毒(Echovirus)、鼻病毒(Rhinovirus)、脊髓灰质炎病毒(Poliovirus)等。
在本发明的一些具体实施方案中，所述疾病为所述病毒引起的手足口病、心肌炎、疱疹性咽峡炎、无菌性脑膜炎、脑炎、病毒性感冒等。
在本发明的一些具体实施方案中，所述多肽的氨基酸序列包括CR、CK和/或DLL。
在本发明的一些具体实施方案中，所述多肽具有如下序列：
I、(X1)(X2)DLL、(X2)DLL(X3)、DLL(X3)(X4)、(X5)YC(X6)、C(X6)；
其中：
X1选自异亮氨酸(I)；
X2选丝氨酸(S)或丙氨酸(A)；
X3选自丙氨酸(A)或赖氨酸(K)或谷氨酰胺(Q)或精氨酸(R)或丝氨酸(S)或半胱氨酸(C)；
X4选自丝氨酸(S)或丙氨酸(A)；
X5选自谷氨酸(E)或谷氨酰胺(Q)；
X6选自精氨酸(R)或赖氨酸(K)；
或II、如I所述序列缺失、添加或取代至少1个氨基酸的序列；
或III、如I或II所述的氨基酸序列具有至少50％同源性且抑制ERSP活性的序列；
或IV、如I或II或III所述序列的互补序列。
本发明所述的“氨基酸”包括天然氨基酸或非天然氨基酸。本领域技术人员公知的氨基酸类型均在本发明的保护范围之内。
在本发明的一些具体实施方案中，如I所述的序列如SEQ ID No.1～14任一项所示，但不包括穿膜肽的序列和连接肽的序列。
本发明还提供了多肽，能够抑制ERSP的活性。
在本发明的一些具体实施方案中，所述多肽的氨基酸序列包括CR、CK和/或DLL。
在本发明的一些具体实施方案中，所述多肽的氨基酸序列包括YCR和/或YCK。
在本发明的一些具体实施方案中，所述多肽具有如下序列：
I、(X1)(X2)DLL、(X2)DLL(X3)、DLL(X3)(X4)、(X5)YC(X6)、C(X6)；
其中：
X1选自异亮氨酸(I)；
X2选丝氨酸(S)或丙氨酸(A)；
X3选自丙氨酸(A)或赖氨酸(K)或谷氨酰胺(Q)或精氨酸(R)或丝氨酸(S)或半胱氨酸(C)；
X4选自丝氨酸(S)或丙氨酸(A)；
X5选自谷氨酸(E)或谷氨酰胺(Q)；
X6选自精氨酸(R)或赖氨酸(K)；
或II、如I所述序列缺失、添加或取代至少1个氨基酸的序列；
或III、如I或II所述的氨基酸序列具有至少50％同源性且抑制ERSP活性的序列；
或IV、如I或II或III所述序列的互补序列。
在本发明的一些具体实施方案中，所述多肽如I所述序列如SEQ ID No.1～14任一项所示，但不包括穿膜肽的序列和连接肽的序列。
在上述研究的基础上，本发明还提供了核酸，编码所述多肽的核苷酸序列。
本发明还提供了重组载体，包括所述的核酸。
在此基础上，本发明还提供了宿主细胞，包括所述的重组载体。
本发明还提供了药物，包括所述多肽及药学上可接受的辅料。
本发明还提供了疫苗，包括所述多肽及药学上可接受的辅料。
本发明还提供了治疗肠道病毒感染疾病的方法，服用和/或注射所述的药物。所述注射为肌肉注射、腹腔注射或静脉注射。
本发明还提供了预防肠道病毒感染疾病的方法，接种所述的疫苗。
在本发明中，术语“预防”是指在未被临床标准认定的疾病前，各种用于防止疾病发生或发展的手段或措施，包括医学、物理或化学的方法，以阻止和降低疾病各种症状的发生或发展。
在本发明中，术语“治疗”是指为了阻止和降低疾病的发生或发展，使疾病病程的发展或加重得以抑制、遏制、减轻、改善、减缓、停止、延迟或反转，所描述的保持和/或用药时的疾病的、紊乱的或病理学状态的各种指标包括减轻或减少症状或并发症，或治愈或消除疾病、紊乱或状况。
在本发明中，术语“药物”是指可以用于预防或治疗某种疾病的单一化合物、多种化合物形成的组合物，或指以单一化合物为主要活性成分的组合物或制剂(formulation)，还指由多种化合物为活性成分的组合物或制剂。“药物”应理解为不仅指根据一国之法律规定，通过其设立的行政机构审批并准予生产的产品，还指在为了获得通过审批和准予生产的过程中，所形成的含单一化合物为活性成分的各类物质形态。“形成”应理解为通过化学合成、生物转化或购买等途径获得。
本发明还提供了一种肠道病毒71型的抑制剂，所述的抑制剂为多肽P2，所述的P2的氨基酸序列为SEQ ID NO.2所示。
本发明还提供了所述抑制剂制备的改造体，所述的改造体为3A-TAT-EP、3A-EP-DRI或3A-EP-PEG4-PA，所述的3A-TAT-EP的氨基酸序列为SEQ ID NO.3所示、3A-EP-DRI的非天然氨基酸序列为SEQ ID NO.4所示、3A-EP-PEG4-PA的氨基酸序列为SEQ ID NO.5所示。
此外，本发明还提供了所述的抑制剂或所述的改造体在制备肠道病毒抑制剂中的应用。
RNA干扰是一种抗病毒免疫机制。在RNA干扰抗病毒过程中，病毒RNA复制产生的双链RNA被宿主Dicer蛋白识别并切割成小干扰RNA(siRNA)。这些病毒衍生的小干扰RNA(vsiRNA)被转移并组装成RNA诱导沉默复合体，介导同源病毒RNA的降解，从而达到抗病毒的目的。例如EV71的非结构蛋白3A能够与病毒双链RNA结合来阻止Dicer对其剪切，抑制vsiRNA病毒来源小干扰RNA的产生，从而达到逃逸RNA干扰抗病毒免疫的目的。
本发明提供的多肽具有高效、广谱的抗病毒活性。这为EV71病毒、CVA16病毒、CVA6病毒、CVB3病毒、CVB5病毒的防控提供了一种新的策略，同时也为加快抗人肠道病毒71型、CVA16病毒、CVB3病毒、CVB5病毒的多肽小分子药物的研发提供了新的理论依据。
附图说明
图1示通过荧光标记检测多肽P2能够穿过细胞膜进入胞浆的结果；
图2示通过CCK-8测定多肽P2的细胞毒性结果；
图3示多肽P2在RD细胞中抗病毒药效结果；
图4示多肽P2在多种细胞中均对病毒有抑制作用；
图5示对多肽P2进行改造后的多肽的抗病毒效果；
图6示多肽3A-EP-DRI和3A-TAT-EP的抗病毒效果；
图7示多肽P1在小鼠体内对EV71的抗病毒活性检测结果；
图8示多肽P1在小鼠体内对CVA16的抗病毒活性检测结果；
图9示多肽CR在RD细胞中的抗EV71效果检测结果；
图10示多肽CR在小鼠体内对EV71的抗病毒活性结果；
图11示多肽CR在小鼠体内对CVA16的抗病毒活性结果；
图12示多肽ER-DRI在小鼠体内的毒性评价的体重测定结果；
图13示多肽ER-DRI在小鼠体内的毒性评价的HE染色结果；
图14示多肽P1在RD细胞中的穿膜效率检测结果；
图15示多肽P1的细胞毒性实验结果；
图16示多肽3A-TAT-EP的细胞毒性实验结果；
图17示多肽3A-EP-DRI及3A-EP-PEG4-PA的细胞毒性实验结果；
图18示多肽EP-PA、EP-CHOL、3A-EP-DRI及3A-EP-PEG4-PA的抗病毒效果检测结果；
图19示多肽ER-DRI及ER在RD细胞中的毒性实验结果；
图20示多肽ER-DRI及ER在RD细胞中的抗EV71病毒效果检测结果
图21示多肽ER-DRI在小鼠体内对EV71的抗病毒活性检测结果；
图22示多肽P2、ER、ER-DRI、R8及TAT的抗CVA16病毒效果检测结果；
图23示多肽BP8、BP10及BP15在RD细胞中的毒性实验结果；
图24示多肽BP8、BP10及BP15在Vero细胞中的毒性实验结果；
图25示多肽BP8、BP10及BP15在RD细胞中的抗CVB5效果检测结果；
图26示多肽BP8在Vero细胞中的抗CVB3效果检测结果；
图27示多肽BP8在小鼠体内抗CVB5效果检测结果；
图28示多肽ER-DRI在Vero细胞中的抗CVA6效果检测结果。
具体实施方式
本发明公开了多肽及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
EV713A蛋白含有86个氨基酸残基。3A具有二聚化的能力，其二聚化对3A正确行使功能起到关键作用。申请人依据3A二聚化结构域两个螺旋结构组成和序列特点，设计了一段EP多肽序列(SEQ ID NO.1所示)，该序列可以与EV71非结构蛋白3A结合，干扰3A蛋白二聚体的形成过程，从而抑制病毒的复制及感染。
根据上述思路，申请人为了能让该抑制剂能穿透细胞膜进入到细胞从而起到抑制病毒的作用，申请人以EP多肽为核心序列，设计了多肽P2(SEQ ID NO.2所示)，其能够与α1竞争性相互作用，从而阻止3A二聚化，起到抑制3A抗天然免疫的作用，最终达到抗病毒的目的。
申请人还在P2的基础上，对其结构进行改造，构建了一系列的改造体，经试验证实，经改造后的P2，提高了抑制病毒的能力，对人肠道病毒的防控和治疗方面具有重大的意义。
所述的改造体为：3A-TAT-EP、3A-EP-DRI和3A-EP-PEG4-PA；所述的3A-TAT-EP的氨基酸序列为SEQ ID NO.3所示，3A-EP-DRI的氨基酸序列为SEQ ID NO.4所示，3A-EP-PEG4-PA的氨基酸序列为SEQ ID NO.5所示。
一种肠道病毒71型的抑制剂的应用，包括利用本发明提供的P2以及P2改造体制备成肠道病毒71型的抑制剂，或是利用本发明提供的P2以及P2改造体与其他有效成分一起，制备成肠道病毒71型的抑制剂。
本发明的保护内容还包括，依据P2多肽，对其替换不同的穿膜序列、或进行多肽修饰、或进行非天然氨基酸的设计和改造后具有抑制EV71的活性的抑制剂。
肠道病毒3A是一种高效ERSP，能够与病毒dsRNA结合来阻止Dicer对其剪切，抑制病毒来源vsiRNA的产生，抵御宿主RNAi抗病毒免疫。
本发明涉及的多肽及其衍生物能够抑制肠道病毒3A的抗病毒免疫能力，是一种新兴的EV71治疗药物，其针对新的靶点，对抗病毒耐药等具有重要意义。
与现有技术相比，本发明具有以下优点：
P2系列多肽有高效的抗病毒活性。这将为肠道病毒的防控提供了一种新的策略，同时也为加快抗人肠道病毒多肽小分子药物的研发提供了新的理论依据。并且P2系列多肽清晰的抗病毒机制，可以保证其应用的安全性和优化途径的明确性，便于以后的进一步开发。
本发明涉及到的多肽如表1：
表1
Figure PCTCN2019072455-appb-000001
Figure PCTCN2019072455-appb-000002
在本发明中，多肽序列中RRRRRRRR(R8)、YGRKKRRQRRR(TAT)为穿膜肽，GSG为连接肽，各多肽的氨基酸序列去掉所述穿膜肽和连接肽的序列为核心序列。本发明各个实施例中的多肽设置了阴性对照，证明本发明提供的多肽的核心序列具有 相应的抗病毒功效。
本发明提供的多肽及其应用中所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1：多肽P2穿膜效率检测
1.材料：
MEM培养基(Thermo)，血清(Gibco)购自英潍捷基公司，免疫荧光平皿(NEST)购自启动子公司，PBS，DAPI，多聚甲醛购自迪跃创新公司。
多肽P2由南京金斯瑞公司合成，其序列为SEQ ID NO.2所示。
2.实验过程
实验分为两组，为了避免加入EV71病毒对多肽进入细胞产生的影响，一组实验加入EV71病毒后加多肽P2，另一组不加病毒只加多肽，且每组做好阴性对照。
免疫荧光步骤如下：
(1)在免疫荧光专用皿中铺1ml RD细胞，待其长到30％融合度时收样。
(2)吸出培养基，用1ml 0.01mol/L，pH7.4的PBS洗去残余培养基，洗三次，每次5min。
(3)配制4％多聚甲醛溶液，将4g多聚甲醛溶解于100ml PBS。将配好的4％多聚甲醛加1ml到每个皿中，反应5min，目的是将细胞固定。
(4)吸出4％多聚甲醛，再加入1ml 0.01mol/L，pH7.4的PBS洗去残余的多聚甲醛，洗三次，每次5min。
(5)将1mg/ml的DAPI溶液用PBS稀释到100ng/ml，加入皿中，反应15min。
(6)吸出反应液，再次加入1ml 0.01mol/L，pH7.4的PBS洗去残余反应液，洗三次，每次5min。
(7)将平皿放置于荧光显微镜下观察。
利用带有荧光标记(FITC)的多肽，检测其在RD细胞中的穿膜效率。分别设置两组实验，第一组为未处理对照组、分别加入R8及P2，第二组为感染EV71组、感染EV71后，再分别加入R8、P2。两组实验同时进行，病毒MOI＝0.1，加入多肽浓度为1μM。加入多肽12小时以后收样，固定细胞并进行免疫荧光实验，DAPI溶液染细胞核。结果显示，在感染或未感染的条件下，多肽均能进入到细胞，具有很好的穿膜能力。
如图1，在未加病毒及加病毒的细胞中，均能观察到多肽入胞，证明多肽P2有很好的穿膜能力。
实施例2：多肽P2的细胞毒性测定
1.实验材料
CCK-8试剂(MCE)购自启动子公司。
2.实验过程
多肽P2在抗病毒的过程中不但要能抑制病毒，还要保证对细胞没有毒性。因此通过细胞毒性测试来检测这一指标，以未做任何处理的细胞为对照组。
步骤如下：
(1)用RD细胞铺96孔细胞板，每孔100μl。
(2)待其长到70％-80％融合度时将含有10％血清的MEM培养基换成含2％血清的MEM培养基，加入一定浓度梯度的多肽P2，使得孔中最终的P2浓度分别为0.01μM、0.1μM、1μM、10μM、100μM。
(3)加入多肽24h后收样，每孔中加入10μl活细胞检测剂CCK-8，混匀。
(4)37度放置2h。
(5)用酶标仪检测OD450的吸光度值。
结果如图2、表2所示，以未处理细胞的细胞活力为100％，加入100μM多肽后细胞活力与对照组(未处理细胞)无显著性差异，证明本研究用到的多肽P2在100μM以内对细胞都没有毒性。
表2
Figure PCTCN2019072455-appb-000003
实施例3：多肽抗病毒效率的测定
1.材料
Total RNA提取试剂盒(Omega)、24孔板、100mm皿、50ml注射器购自迪跃创新公司，0.22μm的滤膜(Millipore)购自飞羿公司，One step qRT-PCR试剂盒(Takara)购自友名公司，提取RNA及qRT-PCR过程中使用的水为DEPC水，整个实验在RNase Free环境中进行。
2.病毒的扩增：
(1)用RD细胞铺5个100mm皿；
(2)待其长到70％-80％融合度时，将含有10％血清的MEM培养基换成含2％血清的MEM培养基，加入1μl 10^7PFU/ml的EV71病毒、CVA16病毒、CVB3病毒、CVB5病毒；
(3)2天后，观察细胞是否发生CPE现象，当病变现象明显时收样；
(4)将100mm皿中的上清吸出到15ml离心管中，500g，5min离心；
(5)再取上清于新的15ml离心管中，并用50ml注射器和0.22μm的滤膜过滤除菌；
(6)取100μl病毒提取RNA与之前提取的已知滴度的病毒RNA进行one step qRT-PCR，测病毒滴度。
3.在RD细胞中测定多肽抗病毒效率：
(1)分别用不同细胞铺24孔板。
(2)待其长到70％-80％融合度时，将含有10％血清的MEM培养基换成含2％血清的MEM培养基(每孔2％血清的MEM培养基的加入量是0.5ml)，每孔加入5μl 10^6PFU/mL EV71病毒。
(3)1h后，分别加入终浓度为0.01μM、0.1μM、1μM、10μM、50μM的不同的多肽。
(4)病毒感染24h后收样，用total RNA提取试剂盒提取RNA。
(5)弃去上清，向孔中加入350μl TRK裂解液，放到摇床上5min。
(6)再向孔中加入350μl 70％乙醇(DEPC)，放到摇床上5min。
(7)取出孔中溶液转移至RNA提取柱子中，12000g离心1min。
(8)将回收管中的溶液重新上一次柱子，12000g离心1min。
(9)加入RNA washing buffer1，12000g离心30s。
(10)加入RNA washing buffer2，12000g离心1min。
(11)重复步骤(10)。
(12)空离柱子12000g，2min，以完全去除残余RNA wasing buffer。
(13)加50μl DEPC水，12000g离心2min。
(14)取2μl RNA样品，用one step qRT-PCR试剂盒进行荧光定量实验。
以添加了R8穿膜肽(序列为：RRRRRRRR)的RD细胞，作为阴性对照。
结果如图3、表3所示，以EV71病毒感染加入R8的实验组病毒RNA的量为100％，加入多肽P2后，病毒量随着多肽浓度的增大而降低，在多肽浓度为50μM时，病毒量降到约4％，证明多肽P2有很好的抗病毒效果，用qRT-PCR的方法检测病毒滴度，测得P2的IC 50值是6.372μM；结合图2，多肽P2在100μM内都没有细胞毒性，证明多肽在用药安全的前提下起到了很好的抗病毒效果。
表3
Figure PCTCN2019072455-appb-000004
利用RD细胞检测多肽P2的抗病毒效果，在80％融合度的RD细胞中加入EV71(MOI＝0.1)，感染1小时后，分别加入指定浓度梯度多肽P2，浓度分别为0.01μM、 0.1μM、1μM、10μM、50μM。24小时后收样，提取细胞总RNA，利用qRT-PCR检测病毒基因组RNA表达水平，以只感染加入穿膜肽R8的样品为对照组。结果显示，随着多肽浓度的增加，病毒RNA表达水平显著降低，证明多肽P2具有明显的抗EV71活性。
实施例4：多肽P2在多种细胞中抗病毒效率的测定
1.材料
HEK293T，Vero，Huh7.5细胞
2.多肽P2在多种细胞中抗病毒效率的测定
(1)用293T细胞、Vero细胞和Huh7.5细胞铺24孔板。
(2)待其长到70％-80％融合度时，将含有10％血清的MEM培养基换成含2％血清的MEM培养基(每孔2％血清的MEM培养基的加入量是0.5ml)，每孔加入5μl 10^6PFU/ml EV71病毒。
(3)1h后，分别在不同细胞中加入终浓度为0.01μM、0.1μM、1μM、10μM、50μM的多肽P2。
(4)病毒感染24h后收样用total RNA提取试剂盒提取RNA。
(5)弃去上清，向孔中加入350μl TRK裂解液，放到摇床上5min。
(6)再向孔中加入350μl 70％乙醇(DEPC)，放到摇床上5min。
(7)取出孔中溶液转移至RNA提取柱子中，12000g离心1min。
(8)将回收管中的溶液重新上一次柱子，12000g离心1min。
(9)加入RNA washing buffer1，12000g离心30s。
(10)加入RNA washing buffer2，12000g离心1min。
(11)重复步骤(10)。
(12)空离柱子12000g，2min，以完全去除残余RNA wasing buffer。
(13)加50μl DEPC水，12000g离心2min。
(14)取2μl RNA样品，用one step qRT-PCR试剂盒进行荧光定量实验。
在图4中可以看到无论是在RD细胞，293T细胞，Vero细胞还是huh7.5细胞中，多肽P2都可以起到明显的抗病毒效果。
293T细胞、Vero细胞、Hμh7.5细胞测得的IC 50值分别是9.677μM，1.958μM，1.842μM。
实施例5：多肽P2的改造体多肽对抗病毒效果的影响
1.材料
多肽3A-TAT-EP(SEQ ID NO.3所示)、3A-EP-DRI(SEQ ID NO.4所示)和3A-EP-PEG4-PA(SEQ ID NO.5所示)。所述序列均为商业合成。
2.多肽P2的改造体对抗病毒效果的影响
(1)用293T细胞铺24孔板。
(2)待其长到70％-80％融合度时，将含有10％血清的MEM培养基换成含2％血清的MEM培养基(每孔2％血清的MEM培养基的加入量是0.5ml)，每孔加入5μl 10^6PFU/ml EV71病毒。
(3)1h后，分别加入终浓度为0.01μM、0.1μM、1μM、10μM的多肽P2、3A-TAT-EP、3A-EP-DRI和3A-EP-PEG-PA。
(4)病毒感染24h后收样用total RNA提取试剂盒提取RNA。
(5)弃去上清，向孔中加入350μl TRK裂解液，放到摇床上5min。
(6)再向孔中加入350μl 70％乙醇(DEPC)，放到摇床上5min。
(7)取出孔中溶液转移至RNA提取柱子中，12000g离心1min。
(8)将回收管中的溶液重新上一次柱子，12000g离心1min。
(9)加入RNA washing buffer1，12000g离心30s。
(10)加入RNA washing buffer2，12000g离心1min。
(11)重复步骤(10)。
(12)空离柱子12000g，2min，以完全去除残余RNA wasing buffer。
(13)加50μl DEPC水，12000g离心2min。
(14)取2μl RNA样品，用one step qRT-PCR试剂盒进行荧光定量实验。
3.CCK8法检测3A-TAT-EP与3A-EP-DRI在RD细胞中的病毒抑制活性
(1)首先将生长状态良好的RD细胞铺96孔板，每孔1×10 4个，37摄氏度、5％CO 2条件下培养24h。
(2)使用含2％FBS的MEM 2倍比梯度稀释多肽药物(3A-TAT-EP和3A-EP-DRI)，稀释浓度为40μM、20μM、10μM、5μM、2.5μM、1.25μM、0.625μM、0.313μM，每孔100μl，加入一个新的96孔板中，每个浓度设置3个复孔。
(3)将稀释好的病毒加入上述孔中，每孔100μL，设置无药物无病毒孔和无药物加病毒孔分别作为对照，病毒终浓度为0.1MOI。
(4)将混合物转入已铺细胞的96孔板中，37摄氏度、5％CO 2条件下培养24h后，利用CCK8试剂盒测定多肽对病毒的抑制活性。
(5)计算不同浓度多肽对病毒感染的抑制率，计算公式为：多肽抑制率＝(药物孔-病毒孔)×100％/(无药物孔-病毒孔)。
结果如图5所示，对多肽P2改造后的多肽3A-TAT-EP(SEQ ID NO.3所示)、3A-EP-DRI(SEQ ID NO.4所示)和3A-EP-PEG4-PA(SEQ ID NO.5所示)，也都可以作为EV71抑制剂的作用，且能起到提高病毒抑制的效果。qRT-PCR测得3A-EP-PEG4-PA的IC 50为3.25μM。
在图6中，通过CCK8法测定多肽的病毒抑制活性表明，3A-TAT-EP的IC 50为4.36μM，而3A-EP-DRI的IC 50为3.56μM。
实施例6：多肽P1穿膜效率检测
1.材料：
MEM培养基(Thermo)，血清(Gibco)购自英潍捷基公司，免疫荧光平皿(NEST)购自启动子公司，PBS，DAPI，多聚甲醛购自迪跃创新公司。
多肽P1由南京金斯瑞公司合成，其序列为SEQ ID NO.6所示。
2.实验过程
实验分为两组，为了避免加入EV71病毒对多肽进入细胞产生的影响，一组实验加入EV71病毒后加多肽P1，另一组不加病毒只加多肽，且每组做好阴性对照。
免疫荧光步骤如下：
(1)在免疫荧光专用皿中铺1ml RD细胞，待其长到30％融合度时收样。
(2)吸出培养基，用1ml 0.01mol/L，pH7.4的PBS洗去残余培养基，洗三次，每次5min。
(3)配制4％多聚甲醛溶液，将4g多聚甲醛溶解于100ml PBS。将配好的4％多聚甲醛加1ml到每个皿中，反应5min，目的是将细胞固定。
(4)吸出4％多聚甲醛，再加入1ml 0.01mol/L，pH7.4的PBS洗去残余的多聚甲醛，洗三次，每次5min。
(5)将1mg/ml的DAPI溶液用PBS稀释到100ng/ml，加入皿中，反应15min。
(6)吸出反应液，再次加入1ml 0.01mol/L，pH7.4的PBS洗去残余反应液，洗三次，每次5min。
(7)将平皿放置于荧光显微镜下观察。
利用带有荧光标记(FITC)的多肽，检测其在RD细胞中的穿膜效率。分别设置两组实验，第一组为未处理对照、加入R8、加入P1及加入P2，第二组为感染EV71、感染EV71并加入R8、感染EV71并加入P1、感染EV71并加入P2。两组实验同时进行，病毒MOI＝0.1，加入多肽浓度为1μM。加入多肽12小时以后收样，固定细胞并进行免疫荧光实验，DAPI溶液染细胞核。结果显示，在感染或未感染的条件下，多肽均能进入到细胞，具有很好的穿膜能力。
如图14，在未加病毒及加病毒的细胞中，均能观察到多肽入胞，证明多肽P1有很好的穿膜能力。
实施例7：多肽P1的细胞毒性测定
1.实验材料
CCK-8试剂(MCE)购自启动子公司。
2.实验过程
多肽P1在抗病毒的过程中不但要能抑制病毒，还要保证对细胞没有毒性。因此通过细胞毒性测试来检测这一指标，以未做任何处理的细胞为对照组。
步骤如下：
(1)用RD细胞铺96孔细胞板，每孔100μl。
(2)待其长到70％-80％融合度时将含有10％血清的MEM培养基换成含2％血清的MEM培养基，加入一定浓度梯度的多肽P1，使得孔中最终的P1浓度分别为 0.01μM、0.1μM、1μM、10μM、100μM。
(3)加入多肽24h后收样，每孔中加入10μl活细胞检测剂CCK-8，混匀。
(4)37度放置2h。
(5)用酶标仪检测OD 450的吸光度值。
结果如图15及表4所示，以未处理细胞的细胞活力为100％，加入100μM多肽后细胞活力与对照组无显著性差异，证明本研究用到的多肽P1在100μM以内对细胞都没有毒性。
表4
Figure PCTCN2019072455-appb-000005
利用RD细胞检测多肽P1的细胞毒性，在80％融合度的RD细胞中，加入浓度梯度多肽P1，浓度分别为0μM(对照组)、0.01μM、0.1μM、1μM、10μM、100μM，每个浓度梯度设置3组平行实验。24小时后收样，利用CCK-8试剂盒检测细胞活性。结果显示，加入100μM多肽P1后细胞存活率与对照组无显著性差异，证明多肽P1在100μM以内对细胞没有毒性。
实施例8：多肽3A-TAT-EP的细胞毒性测定
1.实验材料
CCK-8试剂(MCE)购自启动子公司。
2.实验过程
多肽3A-TAT-EP在抗病毒的过程中不但要能抑制病毒，还要保证对细胞没有毒性。因此通过细胞毒性测试来检测这一指标，以未做任何处理的细胞为对照组。
步骤如下：
(1)用RD细胞铺96孔细胞板，每孔100μl。
(2)待其长到70％-80％融合度时将含有10％血清的MEM培养基换成含2％血清的MEM培养基，加入一定浓度梯度的多肽EP，使得孔中最终的EP浓度分别为0.01μM、0.1μM、1μM、10μM、100μM。
(3)加入多肽24h后收样，每孔中加入10μl活细胞检测剂CCK-8，混匀。
(4)37度放置2h。
(5)用酶标仪检测OD 450的吸光度值。
结果如图16及表5所示，以未处理细胞的细胞活力为100％，加入100μM多肽 后细胞活力与对照组(未处理细胞)无显著性差异，证明本研究用到的多肽3A-TAT-EP在100μM以内对细胞都没有毒性。
表5
Figure PCTCN2019072455-appb-000006
利用RD细胞检测多肽3A-TAT-EP的细胞毒性，在80％融合度的RD细胞中，加入浓度梯度多肽3A-TAT-EP，浓度分别为0.01μM、0.1μM、1μM、10μM、100μM，每个浓度梯度设置3组平行实验。24小时后收样，利用CCK-8试剂盒检测细胞活性。结果显示，以未处理细胞的细胞活性为100％，加入100μM多肽3A-TAT-EP后细胞存活率与未处理对照组基本一致，证明多肽3A-TAT-EP在100μM以内对细胞没有毒性。
实施例9：多肽3A-EP-DRI及3A-EP-PEG4-PA的细胞毒性测定
1.实验材料
CCK-8试剂(MCE)购自启动子公司。
2.实验过程
多肽3A-EP-DRI及3A-EP-PEG4-PA在抗病毒的过程中不但要能抑制病毒，还要保证对细胞没有毒性。因此通过细胞毒性测试来检测这一指标，以未做任何处理的细胞为对照组。
步骤如下：
(1)用RD细胞铺96孔细胞板，每孔100μl。
(2)待其长到70％-80％融合度时将含有10％血清的MEM培养基换成含2％血清的MEM培养基，加入一定浓度梯度的多肽3A-EP-DRI及3A-EP-PEG4-PA，使得孔中最终的3A-EP-DRI及3A-EP-PEG4-PA浓度分别为0.01μM、0.1μM、1μM、10μM、20μM、30μM、40μM、50μM。
(3)加入多肽24h后收样，每孔中加入10μl活细胞检测剂CCK-8，混匀。
(4)37度放置2h。
(5)用酶标仪检测OD 450的吸光度值。
结果如图17及表6所示，以未处理细胞的细胞活力为100％，加入100μM多肽后细胞活力与对照组(未处理细胞)基本一致，证明本研究用到的多肽3A-EP-DRI及3A-EP-PEG4-PA在50μM以内对细胞都没有毒性。
表6
Figure PCTCN2019072455-appb-000007
利用RD细胞检测多肽3A-EP-DRI及3A-EP-PEG4-PA的细胞毒性，在80％融合度的RD细胞中，分别加入浓度梯度多肽EP-DRI及EP-PEG4-PA，浓度分别为0.01μM、0.1μM、1μM、10μM、20μM、30μM、40μM、50μM。24小时后收样，利用CCK-8试剂盒检测细胞活性。结果显示，加入50μM多肽后细胞存活率与对照组基本一致，证明多肽3A-EP-DRI及3A-EP-PEG4-PA在50μM以内对细胞没有毒性。
实施例10：多肽抗病毒效率的测定
1.材料
Total RNA提取试剂盒(Omega)、24孔板、100mm皿、50ml注射器购自迪跃创新公司，0.22μm的滤膜(Millipore)购自飞羿公司，One step qRT-PCR试剂盒(Takara)购自友名公司，提取RNA及qRT-PCR过程中使用的水为DEPC水，整个实验在RNase Free环境中进行。
2.病毒的扩增：
(1)用RD细胞铺5个100mm皿；
(2)待其长到70％-80％融合度时，将含有10％血清的MEM培养基换成含2％血清的MEM培养基，加入1μl 10^7PFU/ml的EV71病毒；
(3)2天后，观察细胞是否发生CPE现象，当病变现象明显时收样；
(4)将100mm皿中的上清吸出到15ml离心管中，500g，5min离心；
(5)再取上清于新的15ml离心管中，并用50ml注射器和0.22μm的滤膜过滤除菌；
(6)取100μl病毒提取RNA与之前提取的已知滴度的病毒RNA进行one step qRT-PCR，测病毒滴度。
3.多种细胞中测定多肽抗病毒效率：
(1)分别用不同细胞铺24孔板。
(2)待其长到70％-80％融合度时，将含有10％血清的MEM培养基换成含2％ 血清的MEM培养基(每孔2％血清的MEM培养基的加入量是0.5ml)，每孔加入5μl 10^6PFU/mL EV71病毒。
(3)1h后，分别加入终浓度为0.01μM、0.1μM、1μM、10μM的不同多肽。未加多肽组为对照。
(4)病毒感染24h后收样，用total RNA提取试剂盒提取RNA。
(5)弃去上清，向孔中加入350μl TRK裂解液，放到摇床上5min。
(6)再向孔中加入350μl 70％乙醇(DEPC)，放到摇床上5min。
(7)取出孔中溶液转移至RNA提取柱子中，12000g离心1min。
(8)将回收管中的溶液重新上一次柱子，12000g离心1min。
(9)加入RNA washing buffer1，12000g离心30s。
(10)加入RNA washing buffer2，12000g离心1min。
(11)重复步骤(10)。
(12)空离柱子12000g，2min，以完全去除残余RNA wasing buffer。
(13)加50μl DEPC水，12000g离心2min。
(14)取2μl RNA样品，用one step qRT-PCR试剂盒进行荧光定量实验。
多肽EP-PA、EP-CHOL、3A-EP-DRI及3A-EP-PEG4-PA的抗病毒效果的抗EV71效果检测结果如图18、表7～表10所示。
表7
Figure PCTCN2019072455-appb-000008
表8
Figure PCTCN2019072455-appb-000009
表9
Figure PCTCN2019072455-appb-000010
表10
Figure PCTCN2019072455-appb-000011
利用RD细胞检测多肽EP-PA、EP-CHOL、3A-EP-DRI及3A-EP-PEG4-PA的抗病毒效果，在80％融合度的RD细胞中加入EV71(MOI＝0.1)，感染1小时后，分别加入浓度梯度多肽EP-PA、EP-CHOL、3A-EP-DRI及3A-EP-PEG4-PA，所有多肽浓度均设置为0.01μM、0.1μM、1μM、10μM。24小时后收样，提取细胞总RNA，利用荧光定量PCR检测病毒基因组RNA表达水平，以只感染未加入多肽的样品为对照组。结果显示，所有的多肽均有抗EV71活性，提高多肽浓度能显著抑制病毒RNA表达水平。
多肽P2、ER、ER-DRI的抗CVA16病毒效果的检测结果见图22、表11～15。
表11
Figure PCTCN2019072455-appb-000012
表12
Figure PCTCN2019072455-appb-000013
表13
Figure PCTCN2019072455-appb-000014
表14
Figure PCTCN2019072455-appb-000015
表15
Figure PCTCN2019072455-appb-000016
利用RD细胞检测多肽P2、ER、ER-DRI的抗病毒效果，穿膜肽R8及TAT作为对照。在80％融合度的RD细胞中加入EV71(MOI＝0.1)，感染1小时后，分别加入指定浓度梯度多肽P2、ER、ER-DRI、R8及TAT，所有多肽浓度均设置为0.15625μM、0.3125μM、0.625μM、1.25μM、2.5μM、5μM。感染24小时后利用CCK-8试剂盒测定多肽对病毒的抑制活性。多肽抑制率＝(药物孔-病毒孔)×100％/(无药物孔-病毒孔)。结果显示，多肽P2、ER、ER-DRI都能够显著抑制CVA16，而穿膜肽R8及TAT对病毒不起作用。P2的IC 50为1.533μM，ER的IC 50为3.211μM，ER-DRI的IC 50为0.856μM。
实施例11：多肽P1在小鼠体内对EV71和CVA16的抗病毒活性检测
1.材料
P1：RRRRRRRRAISDLLAS为商业合成。新出生2日龄的ICR乳鼠。
2.多肽P1在小鼠体内的抗病毒活性
(1)8只2日龄的ICR乳鼠随机分为两组，每组4只。对这8只乳鼠通过腹腔注射的途径进行剂量为10 8PFU/ml EV71的攻毒。
(2)攻毒同时，一组进行腹腔注射10mg/kg的P1多肽作为治疗组，另一组注射等量的PBS作为对照组。
(3)多肽与PBS每隔12h进行一次注射，连续注射到攻毒后的第五天。
(4)第五天后，安乐死乳鼠，取其后肢肌肉，利用Trizol研磨后，提取组织中的总RNA。
(5)利用one step qRT-PCR试剂盒进行荧光定量实验。
结果如图7及表16所示，P1可显著降低小鼠体内的EV71病毒载量。
3.P1对CVA16在小鼠中的抗病毒活性检测步骤同上。
结果如图8及表17所示，P1可显著降低小鼠体内的CVA16的病毒载量。
表16
Figure PCTCN2019072455-appb-000017
Figure PCTCN2019072455-appb-000018
表17
Figure PCTCN2019072455-appb-000019
实施例12：多肽CR在RD细胞内抑制EV71活性的检测
(1)将生长状态良好的RD细胞铺96孔板。
(2)待其长到70％-80％融合度时，将含有10％血清的MEM培养基换成含2％血清的MEM培养基，设置无药物无病毒孔和无药物加病毒孔(DMEM)作为对照，病毒终浓度为MOI＝0.1。
(3)1h后，分别加入终浓度为，5μM、2.5μM、1.25μM、0.625μM、0.3125μM、0.15625μM的CR多肽，并且设定未加多肽只加病毒和不加病毒和多肽的对照组。
(4)病毒感染24h后，待只加病毒的对照组病变明显时，每孔中加入10μl活细胞检测剂CCK-8混匀。
(5)37度放置2h。
(6)用酶标仪检测OD 450的吸光度值。计算公式为：多肽抑制率＝(药物孔-病毒孔)×100％/(无药物孔-病毒孔)。
结果如图9和及表18所示，CR对于EV71的IC 50为1.7μM。
表18
Figure PCTCN2019072455-appb-000020
Figure PCTCN2019072455-appb-000021
实施例13：多肽CR在小鼠体内对EV71和CVA16的抗病毒活性
1.材料
多肽CR：YGRKKRRQRRRGSGCR为商业合成。新出生2日龄的ICR乳鼠。
2.多肽CR在小鼠体内的抗病毒活性
(1)8只2日龄的ICR乳鼠随机分为两组，每组4只。对这8只乳鼠通过腹腔注射进行10 8PFU/ml EV71剂量的攻毒。
(2)攻毒同时，一组进行10mg/kg的CR的腹腔注射，另一组注射等量的PBS作为对照。
(3)多肽与PBS每隔12h进行一次注射，连续注射到攻毒后的第五天。
(4)第五天后，安乐死乳鼠，取其后肢肌肉，利用Trizol研磨后，提取组织中的总RNA。
(5)利用one step qRT-PCR试剂盒进行荧光定量实验。
结果如图10及表19所示，CR可显著降低小鼠体内的EV71病毒载量。
3.CR对CVA16在小鼠中的抗病毒活性检查方法同上。
结果如图11及表20所示，CR可显著降低小鼠体内的CVA16的病毒载量。
表19
Figure PCTCN2019072455-appb-000022
表20
Figure PCTCN2019072455-appb-000023
实施例14：ER-DRI在小鼠体内的毒性评价
1.材料
多肽ER-DRI为商业合成，12只10日龄的乳鼠。
2.ER-DRI在小鼠体内的毒性评价
(1)共12只10日龄的乳鼠随机分为两组，每组6只，一组通过腹腔注射20mg/kg的ER-DRI，每天一次，连续注射3天。另外一组注射等量的PBS作为对照。
(2)每天记录小鼠的体重，共记录15天。
(3)小鼠给药四周后，对小鼠进行安乐死，解剖并分离其脑、肝脏、肺脏、肾脏进行HE染色。
结果如图12所示，20mg/kg的多肽注射组与PBS组的体重无明显差异。图13HE染色表明，20mg/kg的多肽注射组和PBS组的脑、肝脏、肺脏、肾脏无明显病理性变化，无显著性差异。
实施例15ER和ER-DRI在RD细胞上的毒性检测
1.实验材料
CCK-8试剂(MCE)购自启动子公司。
2.实验过程
多肽ER和ER-DRI在抗病毒的过程中不但要能抑制病毒，还要保证对细胞没有毒性。因此通过细胞毒性测试来检测这一指标，以未做任何处理的细胞为对照组。
步骤如下：
(1)用RD细胞铺96孔细胞板，每孔100μl。
(2)待其长到70％-80％融合度时将含有10％血清的MEM培养基换成含2％血清的MEM培养基，加入ER和ER-DRI，使得孔中最终浓度分别为0.46μM、2.34μM、4.68μM、9.37μM、18.75μM、37.5μM、75μM、150μM、300μM。
(3)加入多肽24h后收样，每孔中加入10μl活细胞检测剂CCK-8，混匀。
(4)37度放置2h。
(5)用酶标仪检测OD 450的吸光度值。
结果如图19及表21、22所示，以未处理细胞的细胞活力为100％，TAT-ER-DRI的半数细胞毒性(CC 50)为117μM，计算TAT-ER的半数CC 50为290μM。证明本研究用到的多肽ER与ER-DRI的半数细胞毒性(290μM、117μM)相比其半数抑制活性(1.26μM、0.64μM)，，是一个非常低毒性的药物。
表21
Figure PCTCN2019072455-appb-000024
表22
Figure PCTCN2019072455-appb-000025
实施例16ER和ER-DRI在RD细胞上抑制EV71的活性检测
1.材料
Total RNA提取试剂盒(Omega)、24孔板、100mm皿、50ml注射器购自迪跃创新公司，0.22μm的滤膜(Millipore)购自飞羿公司，One step q-pcr试剂盒(Takara)购自友名公司，提取RNA及qRT-PCR过程中使用的水为DEPC水，整个实验在RNase Free环境中进行。
2.细胞中测定多肽抗病毒效率：
(1)将生长状态良好的RD细胞铺96孔板。
(2)待其长到70％-80％融合度时，将含有10％血清的MEM培养基换成含2％血清的MEM培养基，设置无药物无病毒孔和无药物加病毒孔(DMEM)作为对照，病毒终浓度为MOI＝0.1。
(3)1h后，分别加入终浓度为，2.5μM、1.25μM、0.625μM、0.3125μM、0.15625μM的ER-DRI和ER多肽，并且设定未加多肽只加病毒和不加病毒和多肽的对照组。
(4)病毒感染24h后，待只加病毒的对照组病变明显时，每孔中加入10μl活细胞检测剂CCK-8，混匀。
(5)37度放置2h。
(6)用酶标仪检测OD 450的吸光度值。计算公式为：多肽抑制率＝(药物孔-病毒孔)×100％/(无药物孔-病毒孔)。
结果如图20及表23、24、25所示，ER在RD细胞上抑制EV71的IC 50为1.26μM，ER-DRI在RD细胞上抑制EV71的IC 50为0.64μM。
表23
Figure PCTCN2019072455-appb-000026
表24
Figure PCTCN2019072455-appb-000027
表25
Figure PCTCN2019072455-appb-000028
实施例17ER-DRI在小鼠体内的抗EV71活性检测
1.实验材料
多肽ER-DRI为商业合成，10只2日龄的乳鼠。
2.实验过程
(1)10只2日龄的ICR乳鼠随机分为两组，每组5只。对这10只乳鼠通过腹腔注射进行10 8PFU EV71剂量的攻毒。
(2)攻毒同时，一组通过腹腔注射10mg/kg的ER-DRI多肽作为药物治疗组，另一组注射等量的PBS作为对照组。
(3)多肽与PBS每隔12h进行一次注射，连续注射到攻毒后的第五天。
(4)第五天后，安乐死乳鼠，取其肺脏，利用Trizol研磨后，提取组织中的总RNA。
(5)利用one step qRT-PCR试剂盒进行荧光定量实验。
结果如图21及表26所示，ER-DRI可显著降低小鼠肺脏中的EV71病毒载量。
表26
Figure PCTCN2019072455-appb-000029
实施例18多肽BP8、BP10及BP15在RD细胞中的毒性实验
1.实验材料
CCK-8试剂(MCE)购自启动子公司。
2.实验过程
多肽BP8、BP10及BP15在抗病毒的过程中不但要能抑制病毒，还要保证对细胞没有毒性。因此通过细胞毒性测试来检测这一指标，以未做任何处理的细胞为对照组。
步骤如下：
(1)用RD细胞铺96孔细胞板，每孔100μl。
(2)待其长到70％-80％融合度时将含有10％血清的MEM培养基换成含2％血清的MEM培养基，加入一定浓度梯度的多肽多肽BP8、BP10及BP15，使得孔中最终浓度分别为3.0625μM、6.125M、12.5μM、25μM、50μM、100μM、200μM。
(3)加入多肽24h后收样，每孔中加入10μl活细胞检测剂CCK-8，混匀。
(4)37度放置4h。
(5)用酶标仪检测OD450的吸光度值。
结果如图23及表27、28、29所示，以未处理细胞的细胞活力为100％，BP8、BP10加入200μM多肽后细胞活力与对照组(未处理细胞)基本一致，BP15在加入100μM多肽后细胞活力与对照组(未处理细胞)基本一致，加入200μM多肽后活力下降到20％。说明BP8、BP10在200μM以内对细胞没有毒性，BP15在100μM以内对细胞没有毒性。
表27
Figure PCTCN2019072455-appb-000030
表28
Figure PCTCN2019072455-appb-000031
表29
Figure PCTCN2019072455-appb-000032
实施例19多肽BP8、BP10及BP15在Vero细胞中的毒性实验
1.实验材料
CCK-8试剂(MCE)购自启动子公司。
2.实验过程
多肽BP8、BP10及BP15在抗病毒的过程中不但要能抑制病毒，还要保证对细胞没有毒性。因此通过细胞毒性测试来检测这一指标，以未做任何处理的细胞为对照组。
步骤如下：
(1)用Vero细胞铺96孔细胞板，每孔100μl。
(2)待其长到70％-80％融合度时将含有10％血清的MEM培养基换成含2％血清的MEM培养基，加入一定浓度梯度的多肽多肽BP8、BP10及BP15，使得孔中最终浓度分别为3.0625μM、6.125M、12.5μM、25μM、50μM、100μM、200μM。
(3)加入多肽24h后收样，每孔中加入10μl活细胞检测剂CCK-8，混匀。
(4)37度放置2h。
(5)用酶标仪检测OD450的吸光度值。
结果如图24及表30、31、32所示，以未处理细胞的细胞活力为100％，BP8、BP10加入200μM多肽后细胞活力与对照组(未处理细胞)基本一致，BP15在加入100μM多肽后细胞活力与对照组(未处理细胞)基本一致，加入200μM多肽后活活力下降到20％。说明BP8、BP10在200μM以内对细胞没有毒性，BP15在100μM以内对细胞没有毒性。药物在RD细胞及Vero细胞的毒性较为一致。
表30
Figure PCTCN2019072455-appb-000033
Figure PCTCN2019072455-appb-000034
表31
Figure PCTCN2019072455-appb-000035
表32
Figure PCTCN2019072455-appb-000036
实施例20多肽BP8、BP10及BP15在RD细胞中的抗CVB5效果检测
1.材料
多肽BP8(SEQ ID NO.12所示)、BP10(SEQ ID NO.13所示)和BP15(SEQ ID NO.14所示)。所述序列均为商业合成。
2.多肽BP8、BP10及BP15对抗病毒效果的影响
(1)用RD细胞铺24孔板。
(2)待其长到70％-80％融合度时，将含有10％血清的MEM培养基换成含CVB5病毒的2％血清的MEM培养基，每孔0.5ml，病毒的MOI＝0.01，且对照组不含有CVB5病毒。
(3)1h后，分别加入终浓度为0.78μM、1.56μM、3.13μM、6.25μM的多肽BP8、BP10及BP15。
(4)病毒感染24h后收样用total RNA提取试剂盒提取RNA。
(5)弃去上清，向孔中加入350μl TRK裂解液，放到摇床上5min。
(6)再向孔中加入350μl 70％乙醇(DEPC)，放到摇床上5min。
(7)取出孔中溶液转移至RNA提取柱子中，12000g离心1min。
(8)将回收管中的溶液重新上一次柱子，12000g离心1min。
(9)加入RNA washing buffer1，12000g离心30s。
(10)加入RNA washing buffer2，12000g离心1min。
(11)重复步骤(10)。
(12)空离柱子12000g，2min，以完全去除残余RNA wasing buffer。
(13)加50μl DEPC水，12000g离心2min。
(14)取2μl RNA样品，用one step qRT-PCR试剂盒进行荧光定量实验。
结果如图25及表33、34、35所示，多肽BP8、BP10及BP15都能够显著抑制CVB5。BP8的IC 50为1.545μM，BP10的IC 50为1.335μM，BP15的IC50为6.758μM。
表33
Figure PCTCN2019072455-appb-000037
表34
Figure PCTCN2019072455-appb-000038
表35
Figure PCTCN2019072455-appb-000039
实施例21多肽BP8在Vero细胞中的抗CVB3效果检测
1.材料
多肽BP8(SEQ ID NO.12所示)所述序列均为商业合成。
2.多肽BP8对CVB3抗病毒效果的影响
(1)用Vero细胞铺24孔板。
(2)待其长到70％-80％融合度时，将含有10％血清的MEM培养基换成含CVB3病毒的2％血清的MEM培养基，每孔0.5ml，，且对照组不含有CVB3病毒，病毒的MOI＝0.01。
(3)1h后，分别加入终浓度为2.5μM、5μM、10μM的多肽BP8。
(4)病毒感染24h后收样用total RNA提取试剂盒提取RNA。
(5)弃去上清，向孔中加入350μl TRK裂解液，放到摇床上5min。
(6)再向孔中加入350μl 70％乙醇(DEPC)，放到摇床上5min。
(7)取出孔中溶液转移至RNA提取柱子中，12000g离心1min。
(8)将回收管中的溶液重新上一次柱子，12000g离心1min。
(9)加入RNA washing buffer1，12000g离心30s。
(10)加入RNA washing buffer2，12000g离心1min。
(11)重复步骤(10)。
(12)空离柱子12000g，2min，以完全去除残余RNA wasing buffer。
(13)加50μl DEPC水，12000g离心2min。
(14)取2μl RNA样品，用one step qRT-PCR试剂盒进行荧光定量实验。
结果如图26及表36所示，多肽BP8都能够抑制CVB3的复制。BP8的IC 50为4.125μM.。
表36
Figure PCTCN2019072455-appb-000040
实施例22多肽BP8在小鼠体内抗CVB5效果检测
1.材料：多肽BP8(SEQ ID NO.12所示)为商业合成。新出生2日龄的ICR乳鼠。
2.多肽BP8在小鼠体内的抗病毒活性
(1)10只2日龄的ICR乳鼠随机分为两组，每组5只。对这10只乳鼠通过腹腔注射进行10 8PFU CVB5剂量的攻毒。
(2)攻毒同时，一组进行10mg/kg的BP8的腹腔注射，另一组注射等量的PBS作为对照。
(3)多肽与PBS每隔12h进行一次注射，连续注射到攻毒后的第五天。
(4)第五天后，安乐死乳鼠，取其后肢肌肉，利用Rrizol研磨后，提取组织中的总RNA。
(5)利用one step qRT-PCR试剂盒进行荧光定量实验。
结果如图27及表37所示，加药组(PB10)相较于空白对照组(PBS)，病毒粒子的 拷贝数显著下降，病毒粒子下降将近80倍。
表37
Figure PCTCN2019072455-appb-000041
实施例23多肽ER-DRI在Vero细胞中抗CVA6病毒的效果检测
1.材料
多肽ER-DEI(SEQ ID No.11所示)所述序列均为商业合成。
2.多肽ER-DEI对CVA6抗病毒效果的影响
(1)用Vero细胞铺24孔板。
(2)待其长到70％-80％融合度时，将含有10％血清的MEM培养基换成含CVB3病毒的2％血清的MEM培养基，每孔0.5ml，且对照组不含有CVA6病毒，病毒的MOI＝0.01。
(3)1h后，分别加入终浓度为2.5μM、5μM、10μM的多肽BP8。
(4)病毒感染24h后收样用total RNA提取试剂盒提取RNA。
(5)弃去上清，向孔中加入350μl TRK裂解液，放到摇床上5min。
(6)再向孔中加入350μl 70％乙醇(DEPC)，放到摇床上5min。
(7)取出孔中溶液转移至RNA提取柱子中，12000g离心1min。
(8)将回收管中的溶液重新上一次柱子，12000g离心1min。
(9)加入RNA washing buffer1，12000g离心30s。
(10)加入RNA washing buffer2，12000g离心1min。
(11)重复步骤(10)。
(12)空离柱子12000g，2min，以完全去除残余RNA wasing buffer。
(13)加50μl DEPC水，12000g离心2min。
(14)取2μl RNA样品，用one step qRT-PCR试剂盒进行荧光定量实验。
结果如图28及表38所示，多肽ER-DEI都能够抑制CVA6的复制。BP8的IC 50小于0.625μM。
表38
Figure PCTCN2019072455-appb-000042
Figure PCTCN2019072455-appb-000043
以上对本发明所提供的多肽及其应用进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
Figure PCTCN2019072455-appb-000044
Figure PCTCN2019072455-appb-000045
Figure PCTCN2019072455-appb-000046
Figure PCTCN2019072455-appb-000047
Figure PCTCN2019072455-appb-000048

Claims (25)

  1. ERSP作为靶标在制备治疗病毒疾病的药物中的应用。
  2. 多肽在制备ERSP的抑制剂中的应用；所述ERSP被所述多肽抑制，产生vsiRNA通过Dicer剪切病毒。
  3. 多肽在制备治疗病毒疾病的药物中的应用。
  4. 如权利要求1至3任一项所述的应用，其特征在于，所述ERSP为3A。
  5. 如权利要求1至3任一项所述的应用，其特征在于，所述病毒为肠道病毒。
  6. 如权利要求5所述的应用，其特征在于，所述肠道病毒为小核糖核酸病毒科(Picornaviridae)肠道病毒属，包括人肠道病毒(Enterovirus，EV)、柯萨奇病毒A型(Coxsackie A virus，CVA)、柯萨奇病毒B型(Coxsackie B virus，CVB)、埃可病毒(Echovirus)、鼻病毒(Rhinovirus)、脊髓灰质炎病毒(Poliovirus)等。
  7. 如权利要求6所述的应用，其特征在于，所述疾病为所述病毒引起的手足口病、心肌炎、疱疹性咽峡炎、无菌性脑膜炎、脑炎、病毒性感冒等。
  8. 如权利要求2至7任一项所述的应用，其特征在于，所述多肽的氨基酸序列包括CR、CK和/或DLL。
  9. 如权利要求2至8任一项所述的应用，其特征在于，所述多肽具有如下序列：
    I、(X1)(X2)DLL、(X2)DLL(X3)、DLL(X3)(X4)、(X5)YC(X6)、C(X6)；
    其中：
    X1选自异亮氨酸(I)；
    X2选丝氨酸(S)或丙氨酸(A)；
    X3选自丙氨酸(A)或赖氨酸(K)或谷氨酰胺(Q)或精氨酸(R)或丝氨酸(S)或半胱氨酸(C)；
    X4选自丝氨酸(S)或丙氨酸(A)；
    X5选自谷氨酸(E)或谷氨酰胺(Q)；
    X6选自精氨酸(R)或赖氨酸(K)；
    或II、如I所述序列缺失、添加或取代至少1个氨基酸的序列；
    或III、如I或II所述的氨基酸序列具有至少50％同源性且抑制ERSP活性的序列；
    或IV、如I或II或III所述序列的互补序列。
  10. 如权利要求9所述的应用，其特征在于，如I所述的序列如SEQ ID No.1～14任一项所示，但不包括穿膜肽的序列和连接肽的序列。
  11. 多肽，其特征在于，能够抑制ERSP的活性。
  12. 如权利要求11所述的多肽，其特征在于，其氨基酸序列包括CR、CK和/或DLL。
  13. 如权利要求11所述的多肽，其特征在于，其氨基酸序列包括YCR和/或YCK。
  14. 如权利要求11至13任一项所述的多肽，其特征在于，具有如下序列：
    I、(X1)(X2)DLL、(X2)DLL(X3)、DLL(X3)(X4)、(X5)YC(X6)、C(X6)；
    其中：
    X1选自异亮氨酸(I)；
    X2选丝氨酸(S)或丙氨酸(A)；
    X3选自丙氨酸(A)或赖氨酸(K)或谷氨酰胺(Q)或精氨酸(R)或丝氨酸(S)或半胱氨酸(C)；
    X4选自丝氨酸(S)或丙氨酸(A)；
    X5选自谷氨酸(E)或谷氨酰胺(Q)；
    X6选自精氨酸(R)或赖氨酸(K)；
    或II、如I所述序列缺失、添加或取代至少1个氨基酸的序列；
    或III、如I或II所述的氨基酸序列具有至少50％同源性且抑制ERSP活性的序列；
    或IV、如I或II或III所述序列的互补序列。
  15. 如权利要求14所述的多肽，其特征在于，如I所述序列如SEQ ID No.1～14任一项所示，但不包括穿膜肽的序列和连接肽的序列。
  16. 核酸，其特征在于，编码如权利要求11～15任一项所述多肽的核苷酸序列。
  17. 重组载体，其特征在于，包括如权利要求16所述的核酸。
  18. 宿主细胞，其特征在于，包括如权利要求17所述的重组载体。
  19. 药物，其特征在于，包括如权利要求11～15任一项所述多肽及药学上可接受的辅料。
  20. 疫苗，其特征在于，包括如权利要求11～15任一项所述多肽及药学上可接受的辅料。
  21. 治疗肠道病毒感染疾病的方法，其特征在于，服用或注射如权利要求19所述的药物。
  22. 预防肠道病毒感染疾病的方法，其特征在于，接种如权利要求20所述的疫苗。
  23. 一种肠道病毒71型的抑制剂，所述的抑制剂为多肽P2，所述的P2的氨基酸序列为SEQ ID NO.2所示。
  24. 如权利要求23所述抑制剂制备的改造体，所述的改造体为3A-TAT-EP、3A-EP-DRI或3A-EP-PEG4-PA，所述的3A-TAT-EP的氨基酸序列为SEQ ID NO.3所示、3A-EP-DRI的氨基酸序列为SEQ ID NO.4所示、3A-EP-PEG4-PA的氨基酸序列为SEQ ID NO.5所示。
  25. 如权利要求23所述的抑制剂或权利要求24所述的改造体在制备肠道病毒71型抑制剂中的应用。
PCT/CN2019/072455 2018-01-20 2019-01-21 一种广谱抗肠道病毒的多肽及其应用 WO2019141263A1 (zh)

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