WO2016034110A1 - 通过miRNA抑制埃博拉病毒的方法 - Google Patents

通过miRNA抑制埃博拉病毒的方法 Download PDF

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WO2016034110A1
WO2016034110A1 PCT/CN2015/088802 CN2015088802W WO2016034110A1 WO 2016034110 A1 WO2016034110 A1 WO 2016034110A1 CN 2015088802 W CN2015088802 W CN 2015088802W WO 2016034110 A1 WO2016034110 A1 WO 2016034110A1
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ebola virus
mir
protein
ebola
virus
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French (fr)
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张辰宇
梁宏伟
周桢
曾科
陈熹
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江苏命码生物科技有限公司
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Priority to US15/507,984 priority Critical patent/US10155947B2/en
Priority to EP15837490.0A priority patent/EP3189856B1/en
Priority to CN201580046895.5A priority patent/CN106659805B/zh
Publication of WO2016034110A1 publication Critical patent/WO2016034110A1/zh

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    • C12N2760/14111—Ebolavirus, e.g. Zaire ebolavirus
    • C12N2760/14121—Viruses as such, e.g. new isolates, mutants or their genomic sequences

Definitions

  • the invention relates to the field of bioinformatics and public health.
  • the invention relates to a method of inhibiting Ebola virus by miRNA.
  • the invention also provides a method and application for regulating Ebola virus protein gene by microRNA.
  • Ebola virus disease is a serious, acute viral disease with typical features and signs including: onset, fever, extreme weakness, muscle pain, headache and sore throat. Vomiting, diarrhea, rash, kidney and liver damage, and, in some cases, internal and external bleeding can occur. Laboratory results included a decrease in white blood cell and platelet counts and an increase in liver enzymes. Human blood and secretions are contagious when they contain a virus. The incubation period can last from 2 days to 21 days.
  • the disease affects humans and non-human primates (monkeys, gorillas and chimpanzees).
  • the virus is transmitted to humans through wild animals (if possible natural hosts such as bats) and spreads through the interpersonal spread.
  • the interpersonal transmission includes: direct contact with blood, secretions, organs or other body fluids of the infected person (through broken skin or mucous membranes), and indirect contact with an environment contaminated by such body fluids.
  • the mortality rate of EVD is as high as 90%, and patients with severe conditions need severe supportive care.
  • the people at higher risk of infection are: health workers, family members or other people who are in close contact with the infected person, mourners who have direct contact with the body of the deceased during the funeral, and are found in rainforest areas and forests. The dead animals are exposed to hunters and so on.
  • Ebola virus infections have been confirmed only through laboratory tests. Patient samples are at risk of extreme biohazard; testing is only possible under the highest level of bioprotective conditions (Grade 4 Biosafety Laboratories). When large-scale epidemics break out, health care workers and laboratory workers in other regions around the world often face enormous risks.
  • the Ebola virus is mainly transmitted through the patient's blood, saliva, sweat and secretions. Laboratory tests for common lymphopenia, severe thrombocytopenia and elevated transaminases (AST>ALT), and sometimes elevated blood amylase. Diagnosis ELISA can be used to detect specific IgG antibodies (IgM antibodies suggest infection); ELISA for detection of antigens in blood, serum or tissue homogenates; detection of viral antigens in hepatocytes by monoclonal antibodies with IFA; or by cell culture or guinea pigs Inoculate the virus. Viruses can sometimes be observed in liver sections using an electron microscope. Detection of antibodies with IFA often leads to misjudgments, especially when conducting serological investigations of previous infections.
  • a research system is urgently needed to study and analyze the pathogenic and replication mechanism of Ebola virus from the perspective of genomics, to further understand the cause and mechanism of Ebola virus, and to design Eb based on the research results. Specific detection methods and treatment methods for pulling viruses.
  • the diagnosis is mainly carried out by detecting the specific IgM and IgG antibodies of Ebola virus, and the antibody in the blood of the patient can appear only after a few days of onset, and there is a window period problem, and the virus in the window period
  • the replication has begun, the patient is highly contagious, but the antibody has not yet been fully produced, so it is easy to cause false negative problems.
  • One of the objects of the present invention is to provide a medicament which is effective for inhibiting Ebola virus replication or treating Ebola virus.
  • Another object of the invention is a method for modulating an Ebola virus protein gene using plant-derived or synthetic microRNAs.
  • Another object of the present invention is to provide a method and reagent that can be accurately used for early detection of Ebola virus.
  • a microRNA miRNA miR-2911 for the preparation of (a) a medicament for the treatment of Ebola virus; (b) a medicament for regulating the expression of an Ebola virus protein gene; / or (c) drugs that inhibit the growth of Ebola virus.
  • the medicament is for inhibiting replication of the Ebola virus.
  • the medicament is for inhibiting endothelial cell damage or death caused by the GP protein of Ebola virus.
  • the Ebola virus protein is selected from the group consisting of GP, VP40, or a combination thereof.
  • the Ebola virus comprises: Benedict Ebola virus (BDBV), Zaire Ebola virus (EBOV) and Sudan Ebola virus (SUDV).
  • BDBV Benedict Ebola virus
  • EBOV Zaire Ebola virus
  • SUDV Sudan Ebola virus
  • the Ebola virus comprises: Reston Ebola virus (RESTV), and Tay Forest Ebola virus (TAFV).
  • RESTV Reston Ebola virus
  • TAFV Tay Forest Ebola virus
  • the MiR-2911 comprises: a synthetic MiR-2911, a plant MiR-2911, a MiR-2911 precursor and/or a mature form; and/or
  • the plant is selected from the group consisting of honeysuckle, indigo, turfgrass, horse blue, populus, cowpea, cotton, Chinese cabbage, potato or a combination thereof.
  • the plant is selected from the group consisting of honeysuckle, indigo, turfgrass, horse blue, populus euphratica or combinations thereof.
  • the plant is a honeysuckle.
  • the Ebola virus protein comprises GP, VP40 or a combination thereof.
  • the inhibition is by binding to the CDS region of the GP protein and the CDS region or the 3' UTR region of VP40.
  • compositions for inhibiting Ebola virus replication and/or treating Ebola virus infection comprising (a) a pharmaceutically acceptable carrier or food science An acceptable carrier; and (b) an active ingredient, which comprises miR2911.
  • a method for non-therapeutic inhibition of Ebola virus replication or inhibition of Ebola virus protein gene expression in vitro comprising the steps of: miR2911 and Ebola virus or angstrom Bola virus-infected cells are exposed.
  • the cells comprise endothelial cells.
  • the cell is a mammalian cell.
  • a method of preventing or treating Ebola virus disease comprising the step of administering MiR-2911 or a MiR-2911-containing extract or composition to a subject in need thereof.
  • a plant extract containing an effective amount of MiR-2911, or a composition formed by mixing the MiR-2911 with a pharmaceutically or food acceptable carrier is applied to a subject in need thereof.
  • the plant is a medicinal plant, a fruit or vegetable plant, or an ornamental plant.
  • the plant is selected from the group consisting of honeysuckle, indigo, turfgrass, horse blue, populus, cowpea, cotton, Chinese cabbage, potato or a combination thereof.
  • the plant is selected from the group consisting of honeysuckle, indigo, turfgrass, horse blue, populus euphratica or combinations thereof.
  • the plant is a honeysuckle.
  • the administration method comprises: oral, respiratory, injection, transdermal, mucosal or intracavitary administration;
  • the mode of administration comprises injecting a plasmid.
  • a piconucleotide miR-2911 for the preparation of an inhibitor of GP protein of Ebola virus; or for the preparation of a preparation or composition, or The composition is used to inhibit endothelial cell damage or death caused by the GP protein of Ebola virus.
  • Figure 1 shows a schematic representation of the regulation of the GP gene by MiR-2911.
  • Figure 2 shows a schematic representation of the MiR-2911 regulation of the VP40 gene.
  • Fig. 3 is a view showing the structure of a part of the plasmid of the present invention, wherein Fig. 3A is a plasmid map of luciferase; and Fig. 3B is a plasmid map of a ⁇ -galactosidase reporter plasmid.
  • FIG. 4 shows the regulation of Ebola virus-associated genes by MiR-2911.
  • Figure 5 shows the schematic of Oligo DNA synthesis. Among them, N1 represents the first base, N2 represents the second base, and so on.
  • Figure 6 shows a schematic of the 970 nm filamentous Ebola virus.
  • Figure 7 is a schematic representation of the Ebola genome.
  • Figure 8 shows a schematic diagram of adenovirus expressing eGFP-encoded GP gene encoded by Ebola.
  • Figure 9 shows the MIR2911 overexpression plasmid map.
  • FIG 10 shows the expression level of MIR2911 in HUVEC cells after transfection of MIR2911.
  • Figure 11 shows the expression level of eGFP after transfection of MIR2911.
  • Figure 12 shows the results of Q-PCR measurement of GP mRNA.
  • Figure 13 shows the results of western blotting of GP protein levels.
  • Figure 14 shows trypan blue staining to observe the survival rate of GP protein on HUVEC cells.
  • Figure 15 shows the effect of cytotoxicity assay kits on the integrity of endothelial cell membranes detected by GP protein.
  • Figure 16 shows the mortality of mice during the experiment.
  • Figure 17 shows the expression levels of AST, ALT and TB.
  • Figure 18 shows the levels of TGF ⁇ , IL-6 in serum.
  • Figure 19 shows the results of HE staining of the liver.
  • Figure 20 shows the results of HE staining of the spleen.
  • the present inventors have identified, for the first time, a small ribonucleic acid which can efficiently bind to a protein gene encoded by Ebola virus and inhibit Ebola virus through extensive screening and experiments.
  • the present inventors identified a small ribonucleic acid, MiR-2911, which binds to the genes of the GP protein and the VP40 protein of Ebola virus by bioinformatics and luciferase assay.
  • the microRNA MiR-2911 can effectively inhibit the replication of the protein gene of Ebola virus.
  • the method provided by the present invention has a significant inhibitory effect on the pathogenicity and viral replication of Ebola virus, and the present invention has been completed on the basis of this.
  • ETD Ebola virus disease
  • Ebola virus disease As used herein, “Ebola virus disease”, “Ebola virus disease”, “EVD” and “Ebola virus disease” are used interchangeably, formerly known as “Ebola Hemorrhagic Fever (EBH) ",” is a serious infectious disease that is often fatal to humans and primates, mainly in remote villages in Central and West Africa near tropical rainforests.
  • Ebola Hemorrhagic Fever Ebola Hemorrhagic Fever
  • Ebola virus is one of the three members of the filamentous virus family (wire-loaded virus), including five different genera: Bundibugyo ebolavirus (Bundibugyo virus, BDBV), Zaire Zaire ebolavirus (Ebola virus, EBOV), Reston ebolavirus (Reston virus, RESTV), Sudan ebolavirus (Sudan virus, SUDV) and Tay forest Tai Forest ebolavirus (Tai Forest virus, TAFV).
  • Bundibugyo virus Boundibugyo virus, BDBV
  • Zaire Zaire ebolavirus Ebola virus, EBOV
  • Reston ebolavirus Reston virus, RESTV
  • Sudan ebolavirus Sud virus, SUDV
  • Tay forest Tai Forest ebolavirus Tai Forest virus, TAFV
  • Benedictine Ebola virus, Zaire Ebola virus and Sudan Ebola virus are associated with a large outbreak of Ebola virus disease in Africa.
  • Ebola virus is transmitted to humans through close contact with the blood, secretions, organs or other body fluids of infected animals.
  • a typical Ebola virus belongs to the family Filofiridae, which is a filamentous, single-stranded negative-strand RNA virus with 18,959 bases and a molecular weight of 4.17 ⁇ 10 6 .
  • the virus particle diameter is about 80nm, the size is 100nm ⁇ (300 ⁇ 1500) nm, the virus with strong infection ability is generally long (665 ⁇ 805) nm, there are branches, U-shaped, 6-shaped or ring, branch The shape is more common.
  • the pure virion consists of a helical ribonucleocapsid complex containing a negative-stranded linear RNA molecule and four virion-structured proteins.
  • the replication mechanism of Ebola virus is as follows: First, the viral RNA-dependent RNA polymerase binds to the leader region sequence of the capsid genome, and then sequentially transcribes the genome by recognizing the revelation and termination signals of the flanking genome. During the synthesis, mRNA is capped and polyadenylated by the L protein. During transcription, the unprocessed primary product transcribed from the GP gene produces a small molecule non-structural glycoprotein, sGP, which is efficiently secreted in infected cells. Subsequent RNA processing allows full-length GP gene expression.
  • Figure 6 shows a schematic representation of a filamentous Ebola virus (about 80 nm in diameter)
  • Figure 7 is a linear negative-strand RNA genome of Ebola virus, 18-19 kb in size, encoding seven proteins.
  • Adsorption The virus first attaches to the host cell receptor through the GP glycoprotein, and then the endocytosis of the host cell is mediated by the GP protein, and enters the cytoplasm of the host cell through the microbubble;
  • replication initiation of replication when nuclear proteins are sufficient to coat the newly synthesized positive and negative genomes
  • Budding The nucleocapsid is in contact with the matrix protein under the plasma membrane, and the endogenous modification is defined by the ESCRT complex of the host plasma membrane (Endosomal sorting complex required for transport (ESCRT)). Membrane proteins mediate the formation of endocytic vesicle budding and multivesicular bodies (MVBs). In addition, in a similar topology, ESCRT is also involved in processes such as cytokinesis, autophagy, and budding of enveloped viruses. ) to carry out budding.
  • ESCRT Endosomal sorting complex required for transport
  • GP protein gene and “GP gene” are used interchangeably and refer to a gene encoding an Ebola GP protein. Among them, “GP” refers to the Ebola virus glycoprotein.
  • the GP gene can express multiple products through translational modification and post-expression modification, which are: secreted glycoprotein (secreted.GP; sGP), glycoprotein (GP), small secreted protein (small sGP, ssGP). ).
  • sGP is a protein that is expressed and secreted by the viral genome and consists of 364 amino acid residues. After expression, modification, and furin cleavage, sGP can constitute a 110 kDa homodimer by disulfide bonds. At present, the function of sGP is not fully understood, and it may be related to the virus evading host humoral immunity and endothelial cell repair.
  • ssGP is another non-viral structural protein obtained by transcriptional modification of the GP gene, which is also known as small sGP. ssGP has the same structure as GP and sGP with 295 amino acid residues, but its current role in the pathogenesis of Ebola virus is unclear.
  • the Ebola virus glycoprotein is a type I transmembrane glycoprotein encoded by the viral GP gene and consists of 676 amino acid residues (REBOV type is 677 amino acid residues). Among them, GP has 295 amino acid residues at the amino terminus identical to sGP, but the difference in the spindle end determines the large difference in its conformation.
  • two subunits of GP1 and GP2 are formed by furin cleavage, and are connected by disulfide bonds to form a heterodimer. Thereafter, the GP protein composed of the GP1 and GP2 subunits forms a trimer having a molecular weight of about 450 kDa on the surface of the virus.
  • GP is a key component of the Ebola virus envelope and plays a key role in the virus's invasion into the host and its toxic effects.
  • the mature Ebola virus glycoprotein contains two subunits, GP1 and GP2.
  • GP1 subunit to virus Entry and toxicity are essential. It contains 469 amino acid residues and can be divided into three subdomains: the base, the head and the glycan cap.
  • the basal part of GP1 acts closely with the GP2 subunit through a disulfide bond to stabilize the conformation of the GP2 protein before fusion.
  • the head of GP1 is located between the connecting base and the glycan cap, which contains a collection of receptors associated with the entry of the virus into the cell.
  • the glycan cap of the GP1 subunit contains a mucin-like domain associated with GP protein toxicity.
  • the GP2 subunit is immobilized on the cell membrane by a transmembrane segment, which not only immobilizes the stable GP2 subunit, but also is responsible for the fusion of the viral cell membrane with the host cell membrane.
  • GP2 is a type I transmembrane protein
  • the fusion portion of GP2 resembles the beta sheet of type II and type III transmembrane proteins.
  • GP is not only involved in the early stages of viral infection, but also participates in virus sprouting. Studies have shown that during Ebola virus infection, GP preferentially binds to endothelial cells, and GP first anchors Ebola virus to target cells through its transmembrane form, and then transmits the components of the virus to monocytes and (or) macrophages, which stimulate these cells to release pro-inflammatory factors IL21 ⁇ , TNF ⁇ , IL26 and chemokines IL28, pro2 ⁇ , and the like. These cytokines act on endothelial cells, destroying the integrity of blood vessels and causing symptoms of hemorrhagic fever. GP is expressed in cells after virus-infected endothelial cells, which can induce cell rounding and shedding, causing cytopathic effects.
  • VP40 protein gene and “VP40 gene” are used interchangeably and refer to a gene encoding the Ebola virus VP40 protein.
  • VP40 is the most abundant class of proteins in filamentous virions, and plays an important role in the budding process of filovirus.
  • VP40 is composed of two structurally similar domains rich in beta sheets, which are joined by a "bridge" of 6 amino acid residues.
  • VP40 is tightly bound to the cell membrane through its C-terminus and is therefore resistant to high salinity.
  • the most prominent feature of VP40 is the ability to undergo oligomerization (oligomerzation).
  • the full-length EBOV VP40 undergoes auto-oligomerization after exposure to lipid bilayers and exposes its N-terminal domain. It can be combined with other VP40 monomers.
  • VP40 dimers are a cyclic structure composed of four antiparallel dimers, and the dimers form a "pocket" in the junction of each other, and can be 5'-UGA-3 with RNA. 'Sequences combine to make their structure more stable.
  • This VP40 octamer may be involved in the formation of nucleocapsids of virions and may also be involved in the regulation of transcription and translation of virion RNA.
  • the structure of the VP40 hexamer is similar to that of the octamer, and is also a cyclic structure, which can also bind to nucleic acids.
  • EBOV VP40 expressed by mammalian cells can be released into the culture medium in a membrane-bound form, wherein the C-terminal domain of VP40 plays an irreplaceable role in the germination of virions. Further studies have shown that a conservative motif, the late domain, in VP40 also plays a very important role in the sprouting process of virions. There are three main forms of late domains: PTAP, PPXY and YXXL.
  • the late domain can play the same role in different parts of VP40: when the late domain of the N-terminal of EBOV VP40 is After insertion into the C-terminus, the release activity of VP40-mediated virus like particles (VLPs) did not change.
  • the late domain and cytokines can also interact to promote their budding process. These cytokines include cellular proteins such as the ubiquitin ligase Need4, Tsg101, and AP-2 protein complexes. Among them, Need4 can bind to PPXY motif, Tsg101 can bind to PTAP motif, and AP-2 protein complex can bind to YXXL motif.
  • Nedd4 is a ubiquitin ligase that regulates the expression of related proteins (such as Pinna channel, EnaC) on the cell surface.
  • the epithelial channel enables the PPXY motif to interact directly with the WW domain of Nedd4 for recognition.
  • Nedd4 can directly ubiquitinate VP40 and ubiquitinate cell surface-associated VP40-related host proteins, which is essential for efficient release of VLPs.
  • Lipid rafts can play a role in the assembly and budding of EBOV.
  • the oligomer of VP40 binds to the microdomains of lipid rafts, and the C-terminal domain of VP40 plays a key role in this binding.
  • EBO V virions it is currently believed that the assembly and budding process of EBO V virions is such that the VP40 monomer first binds to the multivesicular bodies (MVB) through its C-terminus, and this binding changes the conformation of VP40 to auto-oligomerization.
  • MVB multivesicular bodies
  • Nedd4 binds to the PPXY motif of VP40 to ubiquitinate VP40 and adjacent proteins.
  • the ESCRT complex n and the ESCRT complex 111 are combined with the ubiquitinated VP40-MVB complex and then transported together to the plasma membrane.
  • the VP40-MVB complex binds to a viral protein trimer and gradually forms vesicles under the eversion of the ESCRT complex 111-induced membrane.
  • the ESCRT complex also promotes the accumulation of mature virions. , eventually leading to the release of virions.
  • VP40 function mainly includes: matrix protein VP40 plays an important role in the process of virus assembly and budding.
  • the VP40 monomer first binds to the multi-vesicular bodies (MVB) through its C-terminus, and this binding changes the conformation of VP40 to auto-oligomerization.
  • Nedd4 binds to the PPXY motif of VP40 to ubiquitinate VP40 and adjacent proteins. After binding of the Tsg101 to the ESCRT-1 complex, the ESCRT complex and the ESCRT complex are combined with the ubiquitinated VP40-MVB complex and then transported together to the plasma membrane.
  • the VP40-MVB complex binds to a viral protein trimer and gradually forms vesicles under the eversion of the ESCRT complex-induced membrane.
  • the ESCRT complex also promotes the accumulation of mature virions. Lead to the release of virions.
  • microRNA of the invention As used herein, "microRNA of the invention”, “microRNA of the invention”, “MiR-2911 of the invention” and “MiR-2911” are used interchangeably, including but not limited to: synthetic MiR- 2911, Plant MiR-2911, a plasmid obtained by a fermentation method, expresses MiR-2911 produced in vivo and various precursor and/or mature forms of the above substances. It should be understood that the term includes, but is not limited to, for example, pri-MiR-2911, pre-MiR-2911, and MiR-2911 mature bodies, and the like.
  • MiR-2911 is 20 nt in length and has the sequence: GGCCGGGGGACGGGCUGGGA (SEQ ID NO.: 1); its GC content is as high as 85%, which makes it have a wide range of potential sites of action.
  • Naturally derived MiR-2911 is one of many plant microRNAs, which was first discovered in Populus euphratica and subsequently detected in other plants. It is produced differently from the traditional plant microRNA processing and maturation process, but by the plant 26s ribosome. RNA (26s rRNA) expression is produced.
  • MiR-2911 itself is very high. Compared with other plant microRNAs, MiR-2911 can still be used in pharmaceutical products after high temperature cooking, RNase treatment, etc., with high content and strong stability. By using real-time quantitative PCR detection, MiR-2911 is abundantly present in honeysuckle at a concentration of 0.34 pmol/g, which is a potential active ingredient.
  • Plant MiR-2911 is MiR-2911 enriched in the water-soluble and/or fat-soluble extract of the plant.
  • the plant comprises a medicinal plant, a fruit and vegetable plant, an ornamental plant; preferably comprising honeysuckle, indigo, turf, maling, populus, cowpea, cotton, Chinese cabbage or potato;
  • the plant is honeysuckle, indigo, grass daqing, horse blue or Populus euphratica; optimally, the plant is honeysuckle.
  • Modes of administration of the MiR-2911 of the present invention include, but are not limited to, oral, respiratory, injection, transdermal, mucosal or intraluminal administration.
  • the mode of administration of MiR-2911 includes injection of a plasmid (such as a plasmid expressing MiR-2911).
  • the extraction method of the plant microRNA (such as MiR2911) of the present invention mainly adopts a solvent extraction method, that is, a microRNA is extracted from a plant by using a solvent.
  • the solvent comprises water, a hydrophilic solvent, or a combination thereof.
  • the combination includes adding an appropriate amount of a hydrophilic solvent to water or adding an appropriate amount of water to a hydrophilic solvent.
  • an auxiliary agent such as a pH adjuster (such as an acid or a base) may be added to the solvent.
  • the extraction can be carried out at any suitable temperature (e.g., normal temperature to the temperature at which the solvent is refluxed), and preferably, a dipping method, a percolation method, a boiling method, a reflux extraction method, a continuous extraction method, or the like.
  • a suitable temperature e.g., normal temperature to the temperature at which the solvent is refluxed
  • a dipping method e.g., a percolation method, a boiling method, a reflux extraction method, a continuous extraction method, or the like.
  • the plants can be pretreated, for example, by pulverizing the plants or by enzymatic treatment (such as cellulase, hemicellulase, pectinase, xylanase, neutral protease, papain, dextran). Enzyme, as well as Xia synthase), etc.; the extracted mixture may also be post-treated, for example, after the plant is extracted with water, a hydrophilic solvent (such as ethanol) may be added to the extracted mixture to make the mixture aged. precipitation.
  • a hydrophilic solvent such as ethanol
  • the liquid material obtained after the extraction can be used as it is, or can be filtered, concentrated, dried (such as lyophilized), etc. A solid is obtained and then used.
  • the method for extracting the plant microRNA of the present invention is an aqueous extraction method.
  • the method comprises the steps of: taking an appropriate amount of honeysuckle, and after pulverizing, placing the honeysuckle powder in a water bath at a certain temperature (such as room temperature to reflux), heating for several times (such as 1 to 5 times), each holding for a period of time (such as 0.1 ⁇ ) 10 hours), collect the liquid and set aside.
  • a certain temperature such as room temperature to reflux
  • heating for several times such as 1 to 5 times
  • a period of time such as 0.1 ⁇ 10 hours
  • honeysuckle after pulverization, at a certain temperature (such as room temperature ⁇ reflux), the honeysuckle powder is placed in a water bath, heated several times (such as 1 to 5 times), each holding for a period of time (such as 0.1 ⁇ 10 hours), after the extract is concentrated to a certain volume, an appropriate amount of ethanol is added to precipitate most of the mucus, filtered, and the filtrate is collected for use.
  • a certain temperature such as room temperature ⁇ reflux
  • the honeysuckle powder is placed in a water bath, heated several times (such as 1 to 5 times), each holding for a period of time (such as 0.1 ⁇ 10 hours)
  • a period of time such as 0.1 ⁇ 10 hours
  • the test method used may be a conventional method in the art, such as (but not limited to): Solexa sequencing technology, Real-time PCR, RT-PCR, microarray chip, in situ hybridization, Northern Blotting, constant temperature rolling circle amplification, based on total Detection of microRNAs of conjugated polymers, etc.
  • composition of the present invention may comprise: (a) a pharmaceutically acceptable carrier or a food acceptable carrier; and (b) an active ingredient (ie The miRNA of the invention which inhibits Ebola virus).
  • the composition consists of or consists essentially of components (a) and (b).
  • component (b) is present in an amount of from 0.01 to 99% by weight, based on the total weight of the composition, preferably from 0.1 to 90% by weight, based on the microRNA.
  • the method of preparing the composition comprises the steps of: mixing the miRNA of the invention or a plant extract comprising the miRNA of the invention with a pharmaceutically or food acceptable carrier to form the composition.
  • the pharmaceutical composition of the present invention comprises a safe and effective amount of the active ingredient (e.g., miR2911) and a pharmaceutically acceptable excipient or carrier.
  • safe and effective amount it is meant that the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical compositions contain from 1 to 2000 mg of active ingredient per dose, more preferably from 10 to 200 mg of active ingredient per dose. Or it may contain 0.01 to 100 micromoles of active ingredient/agent, preferably 0.1 to 10 micromoles per dose; preferably, the "one dose" is an oral solution.
  • “Pharmaceutically acceptable carrier” means: one or more compatible solid or liquid fillers or gel materials which are suitable for human use and which must be of sufficient purity and of sufficiently low toxicity. By “compatibility” it is meant herein that the components of the composition are capable of intermingling with the compounds of the invention and with each other without significantly reducing the efficacy of the compound.
  • pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid).
  • magnesium stearate magnesium stearate
  • calcium sulfate vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifier (such as ), a wetting agent (such as sodium lauryl sulfate), a coloring agent, a flavoring agent, a stabilizer, an antioxidant, a preservative, a pyrogen-free water, and the like.
  • compositions of the invention include oral, respiratory, injection, transdermal, mucosal or intraventricular administration.
  • the dosage form of the composition of the present invention comprises: a tablet, a capsule, a powder, a pill, a granule, a syrup, a solution, a suspension, an emulsion, a suspension, a spray, an aerosol, a powder, a volatile liquid , injection, powder injection, topical solution, lotion, pouring agent, expectorant, paste, eye drops, nasal drops, ophthalmic ointment, gargle, sublingual tablet or suppository.
  • the present invention provides the use of a microRNA molecule MIR2911 or an extract containing MIR2911 for the preparation of a medicament for the treatment of Ebola.
  • the extract (unconcentrated or concentrated) contains from 0.01 to 100 nM (preferably from 0.1 to 20 nM) of MIR2911.
  • microribonucleic acid of the present invention is effective for inhibiting the replication of the Ebola virus GP and/or VP40 protein genes.
  • the microribonucleic acid of the present invention can inhibit the pathogenicity and replication of Ebola virus and help to reduce the infection rate.
  • microribonucleic acid or the food or the drug containing the active ingredient of the invention has a certain therapeutic or palliative effect on Ebola virus infection.
  • microribonucleic acid of the present invention is highly targeted.
  • Example 1 MiR-2911 reduces the expression of protein genes encoded by Ebola virus
  • the Ebola virus is extremely dangerous, and its live virus research must be carried out in the Biosafety Level 4 laboratory and is subject to strict control worldwide.
  • the pseudovirus can't be replicated in the body, only the host cell can be infected once, and it can be used to replace the entry mechanism of living cells.
  • transgenic mice of the Ebola virus-encoded protein gene artificially synthesized NC (negative control of microRNA), MiR-2911, MiR-156a, MiR-168a, MiR-162a and the like were fed separately.
  • NC negative control of microRNA
  • the transgenic mouse is induced to express the protein encoded by Ebola virus.
  • MiR-2911 GGCCGGGGGACGGGCUGGGA (SEQ ID NO.: 1)
  • MiR-156a UGACAGAAGAGAGUGAGCAC (SEQ ID NO.: 2)
  • MiR-168a UCGCUUGGUGCAGGUCGGGAA (SEQ ID NO.: 3)
  • MiR-162a UGGAGGCAGCGGUUCAUCGAUC (SEQ ID NO.: 4)
  • Ebola virus-encoding protein gene was significantly decreased in the serum and major organs (liver, spleen, lung) of mice fed MiR-2911, while other microRNAs, Ebola There was no change in the expression level of the encoded protein gene.
  • MiR-2911 can efficiently bind to the protein gene encoded by Ebola virus and effectively inhibit the transcription and replication of the protein gene of Ebola virus.
  • This example uses bioinformatics and luciferase assays to verify that MiR-2911 regulates the GP and VP40 protein genes encoded by Ebola virus.
  • MiR-2911 regulates the gene GP encoded by Ebola virus
  • MiR-2911 regulation of GP gene is shown in Figure 1.
  • MiR-2911 and Ebola virus-encoded gene GP have a binding site in the CDS region (coding region) of GP gene (GGTACCACCACCACCGGGAAGCTCCCCCGGCCCAAGCTT, SEQ ID NO.: 5), and its Gibbs free energy (mfe) reaches -35.4 Kcal/mol, mfe indicates the lowest folding free energy of the candidate target gene binding to MiR-2911. The larger the absolute value of mfe, the higher the matching degree of candidate target gene with MiR-2911 sequence.
  • the seed sequence of MiR-2911 and the binding site of the GP gene CDS region are completely complementary, the largest loop is only 5 bases, and only 4 bases of MiR-2911 are not complementary to the binding sites of the GP gene CDS region. Based on this, MiR-2911 can bind to the GP gene, thereby further verifying that MiR-2911 can inhibit the expression of the GP gene through this binding site.
  • MiR-2911 regulates Ebola virus-encoded gene VP40
  • FIG. 2 A schematic diagram of the MiR-2911 regulation of the VP40 gene is shown in Figure 2.
  • the first binding site is located in the CDS region (coding region) of the VP40 gene (GGTACCATTCCTGCCACTCCCCGGCCAAAGCTT, SEQ ID NO.: 6), and its Gibbs free energy (mfe) reaches -37.2 kcal/mol, the seed sequence of MiR-2911 and
  • the binding site of the VP40 gene CDS region is completely complementary, the largest loop is only 3 bases, and only 4 bases of MiR-2911 are not complementary to the binding site of the VP40 gene CDS region; the second binding site is located at the VP40 gene.
  • 3'UTR region non-coding region (non-coding region) (GGTACCACAATCAACCCCGGCAAAGCTT, SEQ ID NO.: 7), whose Gibbs free energy (mfe) reaches -24.0 kcal/mol, the seed sequence of MiR-2911 and the 3'UTR region of VP40 gene
  • the binding sites are fully complementary, the largest loop is only 4 bases, and only 9 bases of MiR-2911 are not complementary to the binding sites of the VP40 gene CDS region. Based on this, MIR2911 can bind to the VP40 gene, thereby further confirming that MiR-2911 inhibits the expression of the VP40 gene through this binding site.
  • the Ebola virus fragment which can be bound by MiR-2911 (40 bp upstream and downstream of the binding site) predicted by bioinformatics was synthesized, and then the product was inserted into a luciferase reporter gene p-MIR-report (Ambion).
  • the pMIR-REPORT miRNA expression reporter vector system was used to verify whether MIR2911 can regulate Ebola-encoded genes.
  • the plasmid map of the pMIR-REPORT miRNA expression reporter vector system is shown in Figure 3.
  • CDS pGL3-GP
  • the sequence of the pGL3-VP40 (3'UTR) vector is shown in SEQ ID NO.: 10, wherein the VP40 (3' UTR) sequence is located at positions 7-34.
  • the sequence of the pGL3-VP40 (CDS) vector is shown in SEQ ID NO.: 11, wherein the VP40 (CDS) sequence is located at positions 7-40.
  • the synthesized complementary oligo DNA was dissolved into 100 ⁇ M with ddH 2 O, and 5 ⁇ l of each of the complementary single strands were mixed, and the system was annealed according to Table 2.
  • the oligo mixture was heated at 95 ° C for 5 minutes and then left at room temperature for 20 minutes to form double-stranded DNA.
  • the enzyme was digested with KpnI and MluI. After the digestion is completed, the digested product is recovered using a DNA recovery kit.
  • the oligo DNA recovered by digestion and the empty pgl3 plasmid were ligated with T4 DNA ligase at room temperature.
  • 10 ⁇ l of the ligation product was transformed into 100 ⁇ l of competent E. coli cells DH5 ⁇ , LB plates (containing 50 ⁇ g/ml kanamycin), and then incubated at 37 °C.
  • the pMIR-REPORT miRNA expression reporter vector system consists of an experimental firefly luciferase reporter vector (Fig. 3A) and the associated beta-galactosidase reporter control plasmid (Fig. 3B). By inserting predicted miRNA target sequences at the multiple cloning site, the pMIR-REPORT luciferase reporter miRNA expression reporter vector can be used to perform accurate, quantitative, assessment of intracellular miRNA function.
  • the pMIR-REPORT luciferase vector contains a firefly luciferase reporter gene under the control of a CMV promoter and terminator.
  • a non-coding region at the 3' end of the luciferase gene comprises a multiple cloning site for insertion of a predicted miRNA binding target sequence or other nucleotide sequence.
  • Luciferase reports expression is regulated by insertion of the predicted miRNA target sequence clone into the pMIR-REPORT vector. This mimics the mode of action of the miRNA target sequence.
  • the pMIR-REPORT ⁇ -gal plasmid is a beta-galactosidase reporter plasmid designed for standardized exploration of cell transfection protocols. The ⁇ -galactosidase expressed by the control plasmid can be used to normalize the diversity of cell expression levels due to differences in cell viability and transfection efficiency.
  • the luciferase recombinant plasmid was first transferred into the 293T cells together with the ⁇ -galactosidase reporter plasmid (the ⁇ -galactosidase reporter plasmid was used to determine the transfection efficiency).
  • the ⁇ -galactosidase reporter plasmid was used to determine the transfection efficiency.
  • Transfected into 293T cells with an equal amount of miRNA precursor or synthetic negative control microRNA so that after 24 hours, luciferase activity can be detected by the luciferase activity assay kit (Promega). Reflects the regulation of miRNAs on Ebola related genes.
  • MIR2911 has two genes for Ebola virus, a total of three sites [GP: MIR2911 and its binding site are located in the CDS region of the GP gene, GP (CDS); VP40: MIR2911 and it has Two binding sites, the first binding site is located in the CDS region of the VP40 gene, VP40 (CDS), the second binding site is located in the 3'UTR region of the VP40 gene, and VP40 (3'UTR) can be combined. And the inhibition efficiency is 60%.
  • MiR-2911 can bind to the GP and VP40 genes encoded by Ebola virus, can inhibit the invasion and replication of Ebola virus, and thus can be used for the treatment of Ebola virus infection.
  • MiR-2911 can still bind to two important genes encoded by Ebola virus, GP and VP40. It is further verified by luciferase assay that MIR2911 can indeed bind to the protein gene encoded by Ebola virus. .
  • Synthetic MiR-2911 is preferably synthesized by Oligo DNA/RNA artificial chemical synthesis method using ⁇ -acetonitrile phosphoramidite chemical synthesis from the 3' ⁇ 5' direction, usually the first base at the 3' end. Combined on the Glass (Poleglass) (Controlled Pore Glass, CPG). The detailed process of synthesis is shown in Figure 5, which is briefly described as follows:
  • a specific method includes the steps of:
  • Two universal primers A and B were synthesized according to the template plasmid sequence of MiR-2911, and four specific oligonucleotide primer sequences (I, II, III, IV) were designed according to the MiR-2911 sequence;
  • the plasmid containing MiR-2911 was used as a template, and PCR amplification was carried out by using A and IV, III and II, I and B as primer combinations respectively.
  • the PCR reaction conditions were: 95 ° C, 2 minutes for 1 cycle ⁇ 95 ° C, 30 Second, 55 ° C, 30 seconds, 72 ° C, 40 seconds for 24 cycles ⁇ 72 ° C, 7 minutes; respectively, product 1, product 2, product 3;
  • a variety of plants including medicinal plants, fruit and vegetable plants, ornamental plants are rich in MiR-2911; such as honeysuckle, Indigo, grass daqing, horse blue, populus, cowpea, cotton, Chinese cabbage or potato.
  • the extraction method of plant MiR-2911 mainly adopts the solvent extraction method, that is, the MiR-2911 is extracted from the plant by using a solvent.
  • the solvent comprises water, a hydrophilic solvent, or a combination thereof.
  • the combination includes adding an appropriate amount of a hydrophilic solvent to water or adding an appropriate amount of water to a hydrophilic solvent.
  • an auxiliary agent such as a pH adjuster (such as an acid or a base) may be added to the solvent.
  • the extraction can be carried out at any suitable temperature (e.g., normal temperature to the temperature at which the solvent is refluxed), and preferably, a dipping method, a percolation method, a boiling method, a reflux extraction method, a continuous extraction method, or the like.
  • a suitable temperature e.g., normal temperature to the temperature at which the solvent is refluxed
  • a dipping method e.g., a percolation method, a boiling method, a reflux extraction method, a continuous extraction method, or the like.
  • the plants can be pretreated, for example, by pulverizing the plants or by enzymatic treatment (such as cellulase, hemicellulase, pectinase, xylanase, neutral protease, papain, dextran). Enzyme, as well as Xia synthase), etc.; the extracted mixture may also be post-treated, for example, after the plant is extracted with water, a hydrophilic solvent (such as ethanol) may be added to the extracted mixture to make the mixture aged. precipitation.
  • a hydrophilic solvent such as ethanol
  • the liquid material obtained after the extraction can be used as it is, or can be processed by filtration, concentration, drying (for example, lyophilization) to obtain a solid matter, and then used.
  • the method for extracting the plant microRNA of the present invention is an aqueous extraction method.
  • the MiR-2911 was prepared and extracted from the honeysuckle plant as follows.
  • the plant material for preparing MiR-2911 is not limited to honeysuckle, and the preparation and extraction methods are applicable to medicinal plants, fruit and vegetable plants, and ornamental plants.
  • Honeysuckle contains a naturally occurring broad-spectrum antiviral drug MiR-2911.
  • Honeysuckle MiR-2911 was extracted by water extraction. Appropriate amount (50 g) of dried honeysuckle medicinal herbs, heated in a 100 ml water bath of 500 ml (the ratio of honeysuckle mass to water: 1:10) for 0.5 hours, and the extract was concentrated under reduced pressure at 60 ° C to 1/10 of the original volume. . Concentrated and unconcentrated honeysuckle aqueous extracts were collected and honeysuckle MiR-2911 was used for subsequent experiments.
  • the precursor of MiR-2911 was constructed into a plasmid by an artificially designed method, and the plasmid was transformed into Escherichia coli.
  • the fermentation product was recovered by fermentation, and the plasmid was extracted and further purified for subsequent experiments.
  • Example 4 inhibits Ebola virus-encoding protein gene
  • Synthetic MiR-2911, plant MiR-2911, and the plasmid obtained by the fermentation method are expressed in vivo to produce MiR-2911 by oral, respiratory, injection, transdermal, mucosal or intracavitary administration to Ebola.
  • the virus has an inhibitory effect.
  • the Ebola virus is extremely dangerous, and its live virus research must be carried out in a biosafety level 4 laboratory and is subject to strict control worldwide.
  • a pseudovirus cannot replicate in the body and can only infect the host cell at one time. It can be used to replace the entry mechanism of live virus research cells. Firstly, the transgenic mice were induced to express the protein gene encoded by Ebola virus, and then the physiological indexes such as body weight and mortality were observed.
  • mice After the mice were sacrificed, the mRNA expression of the encoded protein gene was detected by Real-time PCR; The expression level of GFP is used to reflect the expression of Ebola virus-encoded protein; the main organs (heart, liver, spleen, lung, kidney) and mouse cardiovascular system, immune system are observed by freezing or paraffin section and flow cytometry. Various pathological changes.
  • the artificially synthesized NC (MiR-2911 control) and MiR-2911 of the transgenic mice were fed separately, and then the physiological indexes such as body weight and mortality were observed.
  • the Ebola was detected by Real-time PCR.
  • mRNA expression of viral-encoded protein gene Western blotting was used to detect the expression of Ebola virus-encoded protein by detecting the expression level of GFP; the main organs of the mouse (heart, liver, spleen, lung) were observed by freezing or paraffin section and flow cytometry. , kidney) and mouse cardiovascular system, various pathological changes of the immune system.
  • the expression level of the Ebola virus-encoding gene mRNA was detected by Real-time, and the specific procedure was as described in Example 1.
  • the expression level of the protein encoded by Ebola virus was detected by conventional Western blotting.
  • the method comprises the steps of extracting protein, SDS-PAGE, transmembrane, immune reaction, chemiluminescence, gel image analysis and the like.
  • the film is scanned or photographed, and the molecular weight and net optical density values of the target tape are analyzed by a gel image processing system.
  • mice fed with synthetic NC The results showed that compared with the transgenic mice fed with synthetic NC, the expression level of Ebola-encoded protein in the transgenic mice fed with synthetic MiR-2911 was significantly decreased, and the main organs of the mice (heart, liver, spleen, lung) The kidneys and the cardiovascular system of mice have significantly improved the immune system, and the symptoms of mice have been significantly reduced.
  • the transgenic mice were injected with a blank control plasmid and MiR-2911 overexpression plasmid in the tail vein, and then observed the body weight, mortality and other physiological indicators; after the mice were sacrificed, the Ebola virus code was detected by Real-time PCR. Gene mRNA expression; western blotting was used to detect the expression of Ebola virus by detecting the expression level of GFP; the main organs of the mouse (heart, liver, spleen, lung, kidney) were observed by freezing or paraffin section and flow cytometry. And the pathological changes of the immune system of the mouse cardiovascular system.
  • Real-time is used to detect the expression level of Ebola coding gene mRNA, and the specific operation steps are as in Example 1. Said.
  • the expression level of the protein encoded by Ebola virus was detected by western blotting.
  • mice injected with the blank control plasmid in the tail vein showed that compared with the transgenic mice injected with the blank control plasmid in the tail vein, the expression level of the Ebola virus-encoded protein in the transgenic mice injected with the MiR-2911 overexpression plasmid in the tail vein was significantly decreased, and the main organs of the mouse (heart) The liver system of the mice, the liver, the spleen, the lungs and the kidneys, and the immune system of the mice were significantly improved, and the symptoms of the mice were significantly alleviated.
  • Transgenic mice were fed with MiR-2911-free plants (rice) and MIR2911-rich plants (Honeysuckle), and observed physiological indexes such as body weight and mortality. After killing mice, they were detected by Real-time PCR. mRNA expression of Ebola-encoded protein gene; western blotting was used to detect the expression of Ebola virus-encoded protein by detecting the expression level of GFP; the main organs of the mouse (heart, liver, etc.) were observed by freezing or paraffin sectioning and flow cytometry. Spleen, lung, kidney) and mouse cardiovascular system, various pathological changes of the immune system.
  • Ebola encoded protein gene mRNA The expression level of Ebola encoded protein gene mRNA was detected by Real-time.
  • the expression level of the protein encoded by Ebola virus was detected by western blotting.
  • Ice or paraffin sections and flow cytometry were used to observe the pathological changes of the immune system in the main organs of mice and the cardiovascular system of mice.
  • mice fed MiR-2911-rich plants were significantly decreased compared with transgenic mice fed MiR-2911-free plants, and the main organs of the mice (heart, Liver, spleen, lung, kidney) and the cardiovascular system of mice, the immune system was significantly improved, and the symptoms of mice were significantly reduced.
  • the present invention demonstrates that MiR-2911 is capable of inhibiting the expression of the Ebola encoded protein gene under a variety of modes of administration.
  • HUVEC cells were infected with adenovirus expressing GP gene encoded by Ebola virus coupled with eGFP. After 12 h, MIR2911 precursor mimic or MIR2911 overexpression plasmid was transfected into HUVEC cells infected with adenovirus, and 48 h later in fluorescence microscope The expression of green fluorescent protein was observed, and then RNA and protein were collected. The mRNA and protein levels of GP gene in HUVEC cells were detected by Q-PCR and western blotting, respectively.
  • FIG 10 shows the expression level of MIR2911 in HUVEC cells after transfection of MIR2911.
  • the MIR2911 precursor mimic or the MIR2911 overexpression plasmid was transfected compared to the transfected random control nucleic acid or the empty pcDNA6.2 plasmid, and the level of MIR2911 was significantly increased in HUVEC cells.
  • Figure 11 shows the expression level of eGFP after transfection of MIR2911.
  • the MIR2911 precursor mimetic or the MIR2911 overexpression plasmid can significantly inhibit the expression of the adenoviral green fluorescent protein of the GP gene encoded by the Ebola-conjugated eGFP, but has no effect on the green fluorescent protein expressed by the empty adenovirus.
  • Figure 12 shows the results of Q-PCR measurement of GP mRNA.
  • ncRNA infected with eGFP-conjugated Ebola-encoded GP gene adenovirus for 12 h and then transfected with random control nucleic acid
  • MIR 2911 infected with eGFP-conjugated Ebola-encoded GP gene adenovirus for 12 h MIR2911 precursor mimic
  • Control Plasmid infected with eGFP transfected with EPG-encoded GP gene for 12h and then transfected with empty pcDNA6.2 plasmid
  • MIR2911Plasmid infected with eGFP-expressing Ebola virus The adenovirus encoding the GP gene was transfected with the pcDNA6.2 plasmid expressing MIR2911 12 h later.
  • MIR2911 precursor mimic MIR 2911
  • MIR2911 Plasmid MIR2911 overexpression plasmid
  • Figure 13 shows the results of western blotting of GP protein levels.
  • ncRNA infected with eGFP-conjugated Ebola-encoded GP gene adenovirus for 12 h and then transfected with random control nucleic acid
  • MIR 2911 infected with eGFP-conjugated Ebola-encoded GP gene adenovirus for 12 h MIR2911 precursor mimic
  • Control Plasmid infected with eGFP transfected with EPG-encoded GP gene for 12h and then transfected with empty pcDNA6.2 plasmid
  • MIR2911Plasmid infected with eGFP-expressing Ebola virus The adenovirus encoding the GP gene was transfected with the pcDNA6.2 plasmid expressing MIR2911 12 h later.
  • the MIR2911 precursor mimic or the MIR2911 overexpression plasmid was transfected, and the protein level of GP was also decreased.
  • GP protein can induce a large number of non-programmed death of endothelial cells, which leads to a series of symptoms such as internal bleeding caused by incomplete blood vessels in Ebola patients.
  • tryptophan staining was used to observe the cell death of GP protein in HUVEC cells at 12h, 24h, and 36h, and the CytoTox-GloTM cytotoxicity assay (purified from promega) was used. Detection of the effect of GP protein on endothelial cell membrane integrity
  • viable cell rate (%) total number of viable cells / (total number of viable cells + total number of dead cells) ⁇ 100%
  • Figure 14 shows trypan blue staining to observe the survival rate of GP protein on HUVEC cells.
  • Mock stands for: cells that have not been treated, are negative controls; AD: empty adenovirus; AD (GP): an adenovirus that conjugates eGFP to express the GP gene encoded by Ebola virus.
  • ncRNA infected with eGFP-conjugated Ebola-encoded GP gene adenovirus for 12 h and then transfected with random control nucleic acid;
  • MIR 2911 infected with eGFP-conjugated Ebola-encoded GP gene adenovirus for 12 h MIR2911 precursor mimic; Control Plasmid: infected with eGFP transfected with EPG-encoded GP gene for 12h and then transfected with empty pcDNA6.2 plasmid; MIR2911Plasmid: infected with eGFP-expressing Ebola virus The adenovirus encoding the GP gene was transfected with the pcDNA6.2 plasmid expressing MIR
  • the cell viability of transfected MIR2911 and MIR2911plasmid was significantly reduced compared to ncRNA and control plasmid.
  • GP protein can promote cell death, while MIR2911 and MIR2911 overexpression plasmids can inhibit the promotion of cell death by GP protein.
  • Figure 15 shows the effect of cytotoxicity assay kits on the integrity of endothelial cell membranes detected by GP protein.
  • the meaning of each legend is the same as that of FIG.
  • the endothelial cell membrane integrity of transfected MIR2911 and MIR2911plasmid was significantly improved compared to ncRNA and control plasmid.
  • the results showed that GP protein can destroy the integrity of endothelial cell membrane, while MIR2911 and MIR2911 overexpression plasmid can inhibit the destruction of endothelial cells by GP protein.
  • Example 12 In vivo verification of the preventive effect of honeysuckle soup on Ebola virus and the therapeutic effect of MIR2911-expressing plasmid on Ebola virus
  • the experimental method is as follows:
  • mice In the control group, 1:5 mice were raised normally and were not treated;
  • mice In the control group, 2:5 mice were normally reared, and on the third day, empty adenovirus (AD) was injected through the tail vein at a dose of 10 8 IU for 3 consecutive days. The mice were sacrificed on the eighth day.
  • AD adenovirus
  • mice were normally reared, and on the third day, GP-derived adenovirus [AD(GP)] was injected through the tail vein at a dose of 10 8 IU for 3 consecutive days. The mice were sacrificed on the eighth day.
  • AD(GP) GP-derived adenovirus
  • mice were given honeysuckle decoction on the first day (3 ml per mouse per day), and on the third day, eGFP was fused to the EGP-expressing GP gene-encoded adenovirus by tail vein ( Adenovirus expressing GP) was injected at a dose of 10 8 IU for 3 consecutive days. The mice were sacrificed on the eighth day.
  • mice were given honeysuckle decoction on the first day (3 ml per mouse per day), and adenovirus expressing EPG-encoded GP gene was coupled by tail vein injection through the tail vein for three days. The injection dose was 10 8 IU for 3 consecutive days. After the last injection of adenovirus on day 5, MIR2911 plasmid was injected through the tail vein at a dose of 5 mg/kg body weight for 3 consecutive days. Mice were sacrificed on day 8.
  • Figure 16 shows the mortality during the experiment.
  • the empty adenovirus had no effect on the survival rate of mice, and the adenovirus expressing GP protein could kill the mice strongly, and the mortality rate was 40%.
  • the mice were given honeysuckle decoction for prophylactic treatment, the mice died.
  • the rate was significantly reduced to 90%, and mice given the MIR2911 overexpression plasmid in the tail vein were all cured.
  • honeysuckle decoction can significantly prevent Ebola virus infection, and significantly reduce the mortality and MIR2911 plasmid has a therapeutic effect on Ebola virus.
  • Figure 17 shows the expression levels of AST, ALT, TB.
  • Adenovirus expressing GP caused damage to the liver of mice.
  • AST, ALT, and TB increased significantly in adenovirus mice injected with GP in the tail vein.
  • MIR2911 expression plasmid was given or honeysuckle soup was given, AST, ALT, TB has dropped significantly.
  • FIG. 18 shows the levels of TGF ⁇ , IL-6 in serum.
  • TGF ⁇ , IL-6 was significantly increased in mice injected with adenovirus expressing GP in the tail vein, however, when MIR2911 expression plasmid was administered or treated with honeysuckle soup, TGF ⁇ and IL-6 were significantly decreased.
  • Figure 19 shows the results of HE staining of the liver. Significant damage after injection of GP adenovirus MIR2911 expression plasmid was significantly relieved when treated with honeysuckle soup.
  • Figure 20 shows the results of HE staining of the spleen. Significant damage occurred after injection of GP adenovirus, and significant relief was obtained when MIR2911 expression plasmid was administered or treated with honeysuckle soup.
  • the MIR2911 expression plasmid or honeysuckle soup has a significant inhibitory effect on GP adenovirus, the preventive effect of honeysuckle soup on Ebola virus, and the therapeutic effect of MIR2911 plasmid on Ebola virus.

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Abstract

提供了微小核糖核酸MiR-2911在调控埃博拉病毒中的方法和应用。具体地，提供了一种分离的微小核糖核酸(microRNA)MiR-2911调控埃博拉病毒蛋白基因的方法及应用。

Description

通过miRNA抑制埃博拉病毒的方法 技术领域
本发明涉及生物信息学和公共卫生领域。具体地，本发明涉及通过miRNA抑制埃博拉病毒的方法。本发明还提供了一种通过微小核糖核酸(microRNA)调控埃博拉病毒蛋白基因的方法及应用。
背景技术
埃博拉病毒病是一种严重、急性的病毒性疾病，其典型特征和体征包括：起病急，有发热、极度虚弱、肌肉疼痛、头痛和咽喉痛。随后会出现呕吐、腹泻、皮疹、肾脏和肝脏功能受损，某些情况下会有内出血和外出血。化验结果包括白血细胞和血小板计数降低，而肝酶升高。人的血液和分泌物中含有病毒时就会具有传染性。潜伏期可持续2天至21天。
该病会影响人类和非人类灵长目动物(猴子、大猩猩和黑猩猩)。其病毒通过野生动物(如果蝠等可能的自然宿主)传到人，并且通过人际间传播在人群中蔓延。其中，所述的人际传播包括：与感染者的血液、分泌物、器官或其它体液直接接触(通过破损皮肤或粘膜)，和间接接触受到这类体液污染的环境。
依据世卫组织截止2014年4月的报道，EVD的死亡率高达90％，病情严重的患者需要获得重症支持治疗。
出现疫情时，感染风险较高的人员主要为：卫生工作者、与感染者存在密切接触的家庭成员或其他人、在葬礼期间与死者尸体发生直接接触的哀悼者、在雨林地区与森林中发现的死亡动物发生接触的猎人等。
目前为止，埃博拉病毒病感染只有通过实验室检验才可获得确认。病人样本具有极端生物危害风险；只有在最高级别的生物防护条件下(4级生物安全实验室)才可进行检测。而当大型疫情区域性爆发时，世界范围内其它地域的卫生保健工作人员和实验室工作人员也往往面临着巨大的风险。
埃博拉病毒主要是通过病人的血液、唾液、汗水和分泌物等途径传播。实验室检查常见淋巴细胞减少，血小板严重减少和转氨酶升高(AST>ALT)，有时血淀粉酶也增高。诊断可用ELISA检测特异性IgG抗体(出现IgM抗体提示感染)；用ELISA检测血液、血清或组织匀浆中的抗原；用IFA通过单克隆抗体检测肝细胞中的病毒抗原；或者通过细胞培养或豚鼠接种分离病毒。用电子显微镜有时可在肝切片中观察到病毒。用IFA检测抗体常导致误判，特别是在进行既往感染的血清学调查时。
目前对于埃博拉病毒的研究较少，全世界范围内对该病毒都没有较好的预防、检测和治疗办法。就治疗而言，截止目前，无论对人还是对动物都无可用的已获 证实学科的特异性治疗方法或者疫苗。
目前急需一套研究体系，能从基因组学的角度对埃博拉病毒的致病和复制机理进行研究和分析，进一步了解埃博拉病毒的致病原因及机理，并根据研究结果设计针对埃博拉病毒的特异性检测方法和治疗方法。
就检测而言，在现有技术中，主要通过检测埃博拉病毒的特异性IgM和IgG抗体进行诊断，病人血液中的抗体在发病几天后才能出现，存在窗口期问题，在窗口期病毒已经开始复制，病人具有很强的传染性，但抗体还未完全产生，因此很容易产生假阴性问题。
综上所述，本领域迫切需要研发可有效抑制埃博拉病毒复制或治疗埃博拉病毒的药物，还迫切需要开发能用于准确地检测埃博拉病毒的方法。
发明内容
本发明的目的之一是提供一种可有效抑制埃博拉病毒复制或治疗埃博拉病毒的药物。
本发明另一目的是利用植物来源或人工合成的微小核糖核酸调控埃博拉病毒蛋白基因的方法。
本发明的另一目的是提供一种准确地可用于早期检测埃博拉病毒的方法和试剂。
在本发明的第一方面，提供了一种微小核糖核酸miR-2911的用途，用于制备(a)治疗埃博拉病毒的药物；(b)调控埃博拉病毒蛋白基因表达的药物；和/或(c)抑制埃博拉病毒生长的药物。在另一优选例中，所述的药物用于抑制埃博拉病毒的复制。
在另一优选例中，所述的药物用于抑制埃博拉病毒的GP蛋白所导致的内皮细胞受损或死亡。
在另一优选例中，所述的埃博拉病毒蛋白选自下组：GP，VP40、或其组合。
在另一优选例中，所述的埃博拉病毒包括：本迪布焦埃博拉病毒(BDBV)、扎伊尔埃博拉病毒(EBOV)和苏丹埃博拉病毒(SUDV)。
在另一优选例中，所述的埃博拉病毒包括：雷斯顿埃博拉病毒(RESTV)、和塔伊森林埃博拉病毒(TAFV)。
在另一优选例中，所述的MiR-2911包括：人工合成的MiR-2911、植物MiR-2911、MiR-2911前体和/或成熟体形式；和/或
含有MiR-2911的植株、植株部分、或提取物。
在另一优选例中，所述的植物选自下组：金银花、菘蓝、草大青、马蓝、胡杨、豇豆、棉花、大白菜、马铃薯或其组合。
更佳地，所述植物选自下组：金银花、菘蓝、草大青、马蓝、胡杨或其组合。
最佳地，所述植物为金银花。
在另一优选例中，所述的埃博拉病毒蛋白包括GP、VP40或其组合。
在另一优选例中，所述抑制是通过结合于GP蛋白的CDS区和VP40的CDS区或3’UTR区。
在本发明的第二方面，提供了一种用于抑制埃博拉病毒复制和/或治疗埃博拉病毒感染的组合物，所述组合物含有(a)药学上可接受的载体或食品学上可接受的载体；以及(b)活性成分，所述活性成分包括miR2911。
在本发明的第三方面，提供了一种体外的非治疗性地抑制抑制埃博拉病毒复制或抑制埃博拉病毒蛋白基因表达的方法，包括步骤：将miR2911与埃博拉病毒或受埃博拉病毒感染的细胞进行接触。
在另一优选例中，所述的细胞包括内皮细胞。
在另一优选例中，所述的细胞为哺乳动物的细胞。
在本发明的第四方面，提供了一种预防或治疗埃博拉病毒病的方法，包括以下步骤：给需要的对象施用MiR-2911或含MiR-2911的提取物或组合物。
在另一优选例中，对需要的对象施用含有有效量MiR-2911的植物提取物、或将所述的MiR-2911与药学上或食品学上可接受的载体混合从而形成的组合物。
在另一优选例中，所述的植物是药用植物、果蔬植物、观赏植物。
在另一优选例中，所述的植物选自下组：金银花、菘蓝、草大青、马蓝、胡杨、豇豆、棉花、大白菜、马铃薯或其组合。
更佳地，所述植物选自下组：金银花、菘蓝、草大青、马蓝、胡杨或其组合。
最佳地，所述植物为金银花。
在另一优选例中，所述给药方式包括：口服、呼吸道、注射、透皮、粘膜或腔道给药；
在另一优选例中，所述给药方式包括注射质粒。
在本发明的第五方面，提供了一种微小核糖核酸miR-2911的用途，被用于制备埃博拉病毒的GP蛋白的抑制剂；或用于制备一制剂或组合物，所述制剂或组合物用于抑制埃博拉病毒的GP蛋白所导致的内皮细胞受损或死亡。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了MiR-2911调控GP基因的示意图。
图2显示了MiR-2911调控VP40基因的示意图。
图3显示了本发明部分质粒的结构示意图，其中，图3A是荧光素酶的质粒图谱；图3B是β半乳糖苷酶报告质粒的质粒图谱。
图4显示了MiR-2911对埃博拉病毒相关基因的调控情况。
图5显示了Oligo DNA合成原理图。其中，N1代表第一个碱基，N2代表第二个碱基，依此类推。
图6显示了970纳米丝状埃博拉病毒示意图。
图7是埃博拉基因组示意图。
图8显示了偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒示意图。
图9显示了MIR2911过表达质粒图谱。
图10显示了转染MIR2911后HUVEC细胞中MIR2911的表达水平。
图11显示了转染MIR2911后eGFP的表达水平。
图12显示了GP mRNA的Q-PCR测定结果。
图13显示了GP蛋白水平的western blotting结果。
图14显示了台盼蓝染色观察GP蛋白对HUVEC细胞存活率。
图15显示了细胞毒性检测试剂盒检测GP蛋白对内皮细胞膜完整性的影响。
图16显示了实验过程中的小鼠死亡率。
图17显示了AST、ALT和TB的表达水平。
图18显示了血清中TGFα，IL-6的水平。
图19显示了肝脏的HE染色结果。
图20显示了脾脏的HE染色结果。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选和实验，首次鉴定出可与埃博拉病毒编码的蛋白基因有效结合并抑制埃博拉病毒的微小核糖核酸。具体地，本发明人利用生物信息学及荧光素酶检测方法，鉴定出一种可与埃博拉病毒的GP蛋白和VP40蛋白的基因结合的微小核糖核酸，即MiR-2911。实验证实，微小核糖核酸MiR-2911可有效抑制埃博拉病毒所述蛋白基因的复制。进一步的实验还验证，本发明提供的方法对埃博拉病毒致病性和病毒复制具有明显的抑制作用，在此基础上完成了本发明。
基于上述发现，本发明人提供了以下技术方案：
(a)一种筛选与埃博拉病毒编码蛋白基因结合的微小核糖核酸的方法。
(b)一种抑制埃博拉病毒蛋白基因的复制的方法；
(c)一种对埃博拉病毒感染有治疗作用的微小核糖核酸及其在埃博拉病毒感染治疗中的用途；
(d)提供了MiR-2911制成的食物、药物治疗和/或抑制埃博拉病毒的用途。
术语
埃博拉病毒病(EVD)
如本文所用，“埃博拉病毒病”、“伊波拉病毒病”、“EVD”和“埃博拉病毒Disease”可互换使用，旧称“埃博拉病毒性出血热(Ebola Hemorrhagic Fever，EBHF)”，是一种严重且对于人类和灵长类动物往往致命的传染性疾病，主要发生在中非和西非靠近热带雨林的边远村庄。
埃博拉病毒
如本文所用，“埃博拉”、“伊波拉”、“埃博拉病毒”、“伊波拉病毒”、“埃博拉病毒”和“EBV”可互换使用。
埃博拉病毒属是丝状病毒科(线装病毒)的三位成员之一，包括5个不同的属种：本迪布焦埃博拉病毒(Bundibugyo ebolavirus,Bundibugyo virus,BDBV)、扎伊尔埃博拉病毒(Zaire ebolavirus,Ebola virus,EBOV)、雷斯顿埃博拉病毒(Reston ebolavirus,Reston virus,RESTV)、苏丹埃博拉病毒(Sudan ebolavirus,Sudan virus,SUDV)和塔伊森林埃博拉病毒(Tai Forest ebolavirus,Tai Forest virus,TAFV)。其中，本迪布焦埃博拉病毒、扎伊尔埃博拉病毒和苏丹埃博拉病毒与非洲埃博拉病毒病大型疫情相关。
研究认为，埃博拉病毒通过密切接触到感染动物的血液、分泌物、器官或其它体液而传到人类。
典型的埃博拉病毒(EBV)属丝状病毒科(Filoviridae)，呈长丝状体，单股负链RNA病毒，有18,959个碱基，分子量为4.17×106。外有包膜，病毒颗粒直径大约80nm，大小100nm×(300～1500)nm，感染能力较强的病毒一般长(665～805)nm左右，有分支形、U形、6形或环形，分支形较常见。有囊膜，表面有(8～10)nm长的纤突。纯病毒粒子由一个螺旋形核糖核壳复合体构成，含负链线性RNA分子和4个毒粒结构蛋白。
研究表明，埃博拉病毒的复制机制如下：首先，病毒RNA依赖性的RNA聚合酶与衣壳基因组的前导区序列结合，然后通过识别侧翼基因组的启示和终止信号，按顺序依次转录基因组。在合成的过程中，通过L蛋白对mRNA进行加帽和聚腺苷酸化。在转录过程中，GP基因转录出的未加工的初级产物会产生一个小分子非结构性糖蛋白，即sGP，sGP在感染细胞中是高效分泌的。随后的RNA加工过程允许全长的GP基因表达。
图6显示了丝状埃博拉病毒的示意图(直径约80纳米)，图7是埃博拉病毒线性负链RNA基因组，大小18-19kb，编码7种蛋白质。
通常认为，埃博拉病毒的感染涉及以下过程：
(a)吸附：病毒首先通过GP糖蛋白附着到宿主细胞受体，随后由GP蛋白介导宿主细胞的内吞作用，通过微泡进入宿主细胞细胞质内；
(b)融合：病毒外膜与微泡膜融合，核壳体被释放到细胞质中；
(c)后随转录：病毒通过细胞质内的聚合酶对自身的mRNA进行加帽和聚腺苷酸化；
(d)复制：在核蛋白足够用于包被新合成的正反基因组时开始启动复制；
(5)出芽：核壳体与质膜下的基质蛋白接触，通过宿主质膜的ESCRT复合体(内吞体分选转运复合体(Endosomal sorting complex required for transport，ESCRT)主要识别泛素化修饰的膜蛋白，介导内吞小泡出芽和多泡体(Multivesicular bodies，MVBs)的形成。此外，以类似的拓扑方式，ESCRT也参与胞质分裂、自体吞噬、以及包膜病毒的出芽等过程)进行出芽。
目前，埃博拉病毒的一些信息参考以下公共数据库：数据库链接核苷酸数据库：NCBI蛋白质数据库：UniProtKB。
GP蛋白及其基因
如本发明所用，“GP蛋白基因”和“GP基因”可以互换使用，是指编码埃博拉病毒GP蛋白的基因。其中，“GP”是指埃博拉病毒糖蛋白(glycolprotein)。
GP基因通过翻译修饰及表达后修饰的方法，可表达出多个产物，分别为:分泌型糖蛋白(secreted.GP；sGP)、糖蛋白(glycoprotein，GP)、小分泌蛋白(small sGP，ssGP)。
sGP是由病毒基因组表达并分泌出细胞的蛋白，其由364个氨基酸残基组成。在经过表达、修饰并经过弗林蛋白酶(furin)剪切后，sGP可通过二硫键组成110kDa的同源二聚体。目前sGP的功能尚未完全明了，其可能与病毒逃避宿主体液免疫及内皮细胞修复相关。ssGP是GP基因通过转录修饰得到的另一种非病毒结构蛋白，其又被称为小sGP。ssGP与GP及sGP在结构上有295个氨基酸残基相同，但其目前在埃博拉病毒发病过程中所扮演的角色尚不清楚。
埃博拉病毒糖蛋白(glycoprotein,GP)是病毒GP基因所编码I型跨膜糖蛋白，由676个氨基酸残基组成(REBOV型为677个氨基酸残基)。其中，GP在氨基端有295个氨基酸残基与sGP相同，但其梭基端的差异决定了其构象上巨大的差别。GP蛋白表达后通过弗林蛋白酶剪切形成GP1与GP2两个亚单位，其间通过二硫键相连，形成异源二聚体。之后，GP1与GP2亚基组成的GP蛋白在病毒表面形成分子量约为450kDa的三聚体。
研究表明，GP是埃博拉病毒包膜的关键组成部分，在病毒侵入宿主及发挥毒性作用中起关键作用。
成熟的埃博拉病毒糖蛋白含有GP1与GP2两个亚基。其中，GP1亚基对病毒 的进入及毒性至关重要。其含有469个氨基酸残基，又可被分为三个亚结构域(subdomain):基底部、头部与聚糖帽(glycan cap)。其中，GP1的基底部通过二硫键与GP2亚基紧密作用，稳定GP2蛋白在融合前的构象。GP1的头部位于连接基底部与聚糖帽之间，其含有与病毒进入细胞相关的受体集合区域。此外，GP1亚基的聚糖帽中含有与GP蛋白毒性相关的粘蛋白样结构域(mucin-like domain)。
GP2亚基通过跨膜段固定于细胞膜上，其不但固定稳定GP2亚基，并且负责病毒细胞膜与宿主细胞膜的融合。虽然GP2是I型跨膜蛋白，但GP2的融合部分却类似II型、III型跨膜蛋白的β折叠(βsheet)。
GP不仅与病毒感染早期阶段有关，并且参与病毒出芽。研究表明，在埃博拉病毒的感染过程中,GP优先结合于内皮细胞,GP首先通过其跨膜形式将埃博拉病毒锚定于靶细胞,然后将病毒的组分传递给单核细胞和(或)巨噬细胞，这可刺激这些细胞释放前炎症因子IL21β、TNFα、IL26和趋化因子IL28、pro2α等。这些细胞因子再作用于内皮细胞，破坏血管的完整性，导致出血热症状。GP在病毒感染内皮细胞后在细胞内表达，可诱导细胞变圆和脱落，引起细胞病变。
VP40蛋白及其基因
如本发明所用，“VP40蛋白基因”和“VP40基因”可以互换使用，是指编码埃博拉病毒VP40蛋白的基因
VP40是丝状病毒毒粒中含量最丰富的一类蛋白，在丝状病毒的出芽过程中起着十分重要的作用。VP40是由两个富含β折叠的结构相似的结构域组成，而这两个结构域由6个氨基酸残基构成的“桥段”连接。VP40可以通过其C端与细胞膜紧密结合，因此具有抵抗高盐度的特点。相对于其他病毒蛋白来说，VP40最突出的特是能够发生寡聚化作用(oligomerzation)全长的EBOV VP40在与脂双层合后会发生自体寡聚化，并暴露出其N端结构域而可与其他VP40单体结合。科学家已经分离出了EBOV VP40的六聚体和八聚体，并发现无论是VP40六聚体还是八聚体，其结构元件都是VP40二聚体。研究显示，EBOV VP40八聚体是一个环状结构，由四个反平行的二聚体组成，而二聚体在彼此连接的地方形成“口袋”状，能与RNA的5’-U-G-A-3’序列结合，从而使自身的结构更加稳定。这种VP40八聚体可能与毒粒的核衣壳形成有关，还可能参与对毒粒RNA转录和翻译的调控过程。VP40六聚体的结构与八聚体的相似，也是环状结构，同样能与核酸结合。利用哺乳动物细胞表达的EBOV VP40能以与膜结合的形式释放到培养基中，其中VP40的C端结构域在毒粒出芽过程中起着不可替代的作用。进一步的研究表明，VP40中一种保守的模体—晚期结构域(late domain)，在毒粒的出芽过程中也起着非常重要的作用。晚期结构域主要有三种形式:PTAP，PPXY和YXXL。另外，晚期结构域可以在VP40的不同部位发挥相同的作用:当把EBOV VP40 N端的晚期结构域去 除而插人C端后，由VP40介导的病毒样颗粒(virus like particles，VLPs)的释放活性不会发生改变。晚期结构域与细胞因子还可发生相互作用促进其出芽过程。这些细胞因子包括泛素连接酶Need4，Tsg101以及AP-2蛋白复合物等细胞蛋白。其中Need4能与PPXY模体结合，Tsg101能与PTAP模体结合，而AP-2蛋白复合物则能与YXXL模体结合。
人的Need4是由4个富含脯氨酸的WW结构域组成，而其第三个WW结构域对于与VP40结合是必需的。Timmins等发现，只有VP40的寡聚体才能与Nedd4发生强烈的相互作用，这提示，这种相互作用可能是在VP40与细胞膜结合后发生的。VP40也可以与Tsg101结合，但与Nedd4不同的是，Tsg101与VP40的单体和寡聚体均能结合，从而导致V LPs释放量的增加。Nedd4是一种能够调控相关蛋白(如上皮纳通道，EnaC)在细胞表面表达的泛素连接酶，上皮纳通道能够使PPXY模体直接与Nedd4的WW结构域作用，从而进行识别。Nedd4既能直接泛素化VP40，又能泛素化细胞表面与VP40相关的宿主蛋白，这对于VLPs的高效释放是至关重要的。脂筏(lipid rafts)在EBOV的组装和出芽过程中能起作用。VP40的寡聚体能与脂筏的微结构域(microdomains)结合，而VP40的C端结构域在这种结合中起着关键作用。目前认为EBO V病毒颗粒的组装和出芽过程是这样的:VP40单体首先通过其C端与多囊泡体(multivesicular bodies，MVB)结合，这种结合使VP40的构像发生变化从而自体寡聚化。Nedd4与VP40的PPXY模体结合后能够泛素化VP40和邻近的蛋白。Tsg101与ESCRT-1复合物结合后，再协同ESCRT复合物n和ESCRT复合物111与泛素化了的VP40-MVB复合物结合，然后一起被运送到质膜。在质膜上，VP40-MVB复合物与一种病毒蛋白三聚体结合，并在ESCRT复合物111诱导膜的外翻作用下逐渐形成小泡，ESCRT复合物班还能促进成熟毒粒的聚集，最终导致毒粒的释放。
目前的研究表明，VP40功能主要包括：基质蛋白VP40在病毒的装配和出芽过程中起着重要的作用。VP40单体首先通过其C端与多囊泡体(mult ivesicular bodies，MVB)结合，这种结合使VP40的构像发生变化从而自体寡聚化。Nedd4与VP40的PPXY模体结合后能够泛素化VP40和邻近的蛋白。Tsg101与ESCRT-1复合物结合后，再协同ESCRT复合物和ESCRT复合物与泛素化了的VP40-MVB复合物结合，然后一起被运送到质膜。在质膜上，VP40-MVB复合物与一种病毒蛋白三聚体结合，并在ESCRT复合物诱导膜的外翻作用下逐渐形成小泡，ESCRT复合物还能促进成熟毒粒的聚集，最终导致毒粒的释放。
MiR-2911
如本文所用，“本发明的微小RNA”、“本发明的微小核糖核酸”、“本发明的MiR-2911”和“MiR-2911”可互换使用，包括但不限于：人工合成的MiR-2911、 植物MiR-2911、通过发酵方法所得到的质粒在生物体内表达产生的MiR-2911以及上述物质的各类前体和/或成熟体形式。应理解，该术语包括(但并不限于)：例如pri-MiR-2911、pre-MiR-2911和MiR-2911成熟体等。
MiR-2911长度为20nt，序列为：GGCCGGGGGACGGGCUGGGA(SEQ ID NO.:1)；其GC含量高达85％，使其存在着广泛的潜在的作用位点。
天然来源的MiR-2911为众多植物microRNA的一种，最早在胡杨中被发现，之后陆续在其他植物中被检测出，其产生不同于传统的植物microRNA加工成熟过程，而是由植物26s核糖体RNA(26s rRNA)表达产生。
MiR-2911自身的稳定性非常的高。相较于其他的植物microRNA，MiR-2911经过高温的蒸煮，RNA酶等处理之后，依然能够存在较高的含量，有很强的稳定性，能够被广泛用于医药产品中。通过使用实时定量PCR的检测，MiR-2911大量存在于金银花中，浓度达到0.34pmol/g，是其潜在的有效成分。
植物MiR-2911是所述植物的水溶性和/或脂溶性的提取物中富含的MiR-2911。
在另一优选例中，所述的植物包括药用植物、果蔬植物、观赏植物；较佳地包括金银花、菘蓝、草大青、马蓝、胡杨、豇豆、棉花、大白菜或马铃薯；更佳地，所述植物为金银花、菘蓝、草大青、马蓝或胡杨；最佳地，所述植物为金银花。
本发明的MiR-2911的给药方式包括但不限于：口服、呼吸道、注射、透皮、粘膜或腔道给药。
在另一优选例中，MiR-2911的给药方式包括注射质粒(如表达MiR-2911的质粒)。
提取方法(植物提取物的制法)
本发明所述的植物microRNA(如MiR2911)的提取方法主要采用溶剂提取法，即采用溶剂从植物中提取其microRNA。其中，所述的溶剂包括水、亲水性溶剂、或其组合。所述组合包括：在水中添加适量的亲水性溶剂或在亲水性溶剂中添加适量的水。应理解，溶剂中还可添加适量的辅助试剂，如pH调节剂(如酸或碱)等。
提取可以在任何适宜的温度(如常温～溶剂回流的温度)下进行，优选采用浸渍法、渗漉法、煎煮法、回流提取法、连续提取法等。
在提取过程中，可对植物进行预处理，例如将植物粉碎或进行酶处理(如纤维素酶、半纤维素酶、果胶酶、木聚糖酶、中性蛋白酶、木瓜蛋白酶、葡聚糖酶、以及夏合酶)等；也可对提取的混合物进行后处理，如将植物用水进行提取后，可在提取后的混合物中加入亲水性溶剂(如乙醇等)，使得混合物经陈化沉淀。
提取后得到的液体物可直接使用，也可进行过滤、浓缩、干燥(如冻干)等处理后 制得固体物，然后再使用。
优选地，本发明所述的植物microRNA的提取方法为水提法。
例如包括步骤：取适量金银花，粉碎后，在一定温度(如室温～回流)下，将金银花粉末置于水浴中，加热若干次(如1～5次)，每次保温一段时间(如0.1～10小时)，收集液体，备用。
或包括步骤：取适量金银花，粉碎后，在一定温度(如室温～回流)下，将金银花粉末置于水浴中，加热若干次(如1～5次)，每次保温一段时间(如0.1～10小时)，将提取液浓缩至一定体积后，加入适量乙醇，沉淀出大部分的粘液质，过滤，收集滤液，备用。
对植物进行提取后，收集植物提取物，检测提取物中植物microRNA的种类及其含量。所用的测试方法可以是本领域常规方法，例如(但不限于)：Solexa测序技术，Real-time PCR、RT-PCR、微阵列芯片、原位杂交、Northern Blotting、恒温滚环扩增、基于共轭聚合物的microRNA检测等。
组合物
本发明所述组合物(包括药物组合物、食品组合物或保健品组合物)可包含：(a)药学上可接受的载体或食品学上可接受的载体；以及(b)活性成分(即可抑制埃博拉病毒的本发明miRNA)。
优选地，所述的组合物由或基本上由组分(a)和(b)构成。
在另一优选例中，组分(b)的含量为组合物总重量0.01-99wt％，较佳地0.1-90wt％(按microRNA计)。
所述组合物的制备方法包括步骤：将所述的本发明miRNA或含所述本发明miRNA的植物提取物与药学上或食品学上可接受的载体混合，从而形成所述的组合物。
现以药物组合物为例，对组合物作进一步说明：本发明的药物组合物包含安全有效量范围内的活性成分(如miR2911)及药理上可以接受的赋形剂或载体。其中“安全有效量”指的是：活性成分的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg活性成分/剂，更佳地，含有10-200mg活性成分/剂。或者含有0.01～100微摩尔活性成分/剂，较佳地为0.1～10微摩尔/剂；较佳地，所述的“一剂”为一口服液。
“药学上可以接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和，而不明显降低化合物的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘 露醇、山梨醇等)、乳化剂(如
Figure PCTCN2015088802-appb-000001
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明组合物的给药方式包括：口服、呼吸道、注射、透皮、粘膜或腔道给药。
本发明组合物的剂型包括：片剂、胶囊剂、粉剂、丸剂、颗粒剂、糖浆剂、溶液、混悬液、乳剂、混悬剂、喷雾剂、气雾剂、粉雾剂、挥发性液体、注射液、粉针剂、外用溶液剂、洗剂、浇淋剂、搽剂、糊剂、滴眼剂、滴鼻剂、眼用软膏剂、含漱剂、舌下片剂或栓剂。
优选地，本发明提供了一种microRNA分子MIR2911或含MIR2911的提取物的用途，用于制备治疗埃博拉病的药物。较佳地，所述的提取物(未浓缩或浓缩)中含0.01-100nM(较佳地0.1-20nM)的MIR2911。
发明的主要优点包括：
1)首次鉴别出一种可与博拉病毒编码的GP、VP40蛋白基因结合的微小核糖核酸。
2)本发明的微小核糖核酸可有效抑制埃博拉病毒GP和/或VP40蛋白基因的复制。
3)本发明的微小核糖核酸可抑制埃博拉病毒的致病性和复制，有助于降低感染率。
4)本发明微小核糖核酸或含所述活性成分的食物、药物对埃博拉病毒感染有一定治疗或缓解作用。
5)本发明微小核糖核酸的靶向性强。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1.MiR-2911降低埃博拉病毒编码的蛋白基因的表达
1.1 动物模型
埃博拉病毒的极具危险性，其活病毒研究必须在生物安全四级实验室中进行，且在全球受到及其严格的控制。假病毒不能在体内进行复制，只能一次性感染宿主细胞，可以很好的用来代替活病毒研究细胞的进入机理等。利用假病毒构建埃博拉病毒编码蛋白基因的转基因小鼠。
1.2 实验方法
对于所述埃博拉病毒编码蛋白基因的转基因小鼠，分别喂食人工合成的NC(microRNA的阴性对照物)、MiR-2911、MiR-156a、MiR-168a、MiR-162a等众多不同的miRNA，并诱导转基因小鼠表达埃博拉病毒编码的蛋白。
然后，利用Real-time PCR检测小鼠血清及主要脏器(肝，脾，肺)中埃博拉编码蛋白基因mRNA表达情况。
其中，部分经测试的miRNA序列如下：
MiR-2911:GGCCGGGGGACGGGCUGGGA(SEQ ID NO.:1)
MiR-156a:UGACAGAAGAGAGUGAGCAC(SEQ ID NO.:2)
MiR-168a:UCGCUUGGUGCAGGUCGGGAA(SEQ ID NO.:3)
MiR-162a:UGGAGGCAGCGGUUCAUCGAUC(SEQ ID NO.:4)
Real-time PCR的检测方法，具体操作步骤如下：
(1)根据埃博拉病毒编码基因设计引物；
(2)提取样本中总RNA，通过RNA逆转录反应得到cDNA样品；
(3)加入TaqMan探针或者荧光染料进行PCR反应；
(4)检测样本中埃博拉编码蛋白基因的量的变化。
与喂食人工合成的NC相比，喂食MiR-2911的小鼠血清及主要脏器(肝，脾，肺)中埃博拉病毒编码蛋白基因表达水平均显著下降，而其他的microRNA，埃博拉编码蛋白基因的表达水平没有变化。
这表明，MiR-2911可有效结合于埃博拉病毒编码的蛋白基因，并有效抑制埃博拉病毒所述蛋白基因的转录和复制。
实施例2.MiR-2911调控埃博拉病毒编码的蛋白基因
本实施例利用生物信息学和荧光素酶检测方法验证MiR-2911调控埃博拉病毒编码的GP、VP40蛋白基因
2.1 MiR-2911调控埃博拉病毒编码的基因GP
MiR-2911调控GP基因示意图如图1所示。MiR-2911与埃博拉病毒编码的基因GP在GP基因的CDS区(编码区)(GGTACCACCACCGGGAAGCTCCCCCGGCCCAAGCTT，SEQ ID NO.:5)有一个结合位点，其吉布斯自由能(mfe)达到-35.4kcal/mol，mfe表示候选靶基因与MiR-2911结合的最低折叠自由能，mfe绝对值越大，候选靶基因与MiR-2911序列匹配度越高。
MiR-2911的种子序列和GP基因CDS区的结合位点完全互补，最大的loop只有5个碱基，并且MiR-2911只有4个碱基不和GP基因CDS区的结合位点互补， 基于此，MiR-2911可以和GP基因结合，从而进一步验证，MiR-2911可通过这个结合位点抑制GP基因的表达。
2.2 MiR-2911调控埃博拉病毒编码的基因VP40
MiR-2911调控VP40基因示意图如图2所示。MiR-2911与埃博拉病毒编码的基因VP40一共有两个结合位点。第一个结合位点位于VP40基因的CDS区(编码区)(GGTACCATTCCTGCCACTCCCCGGCCAAAGCTT，SEQ ID NO.:6)，其吉布斯自由能(mfe)达到-37.2kcal/mol，MiR-2911的种子序列和VP40基因CDS区的结合位点完全互补，最大的loop只有3个碱基，并且MiR-2911只有4个碱基不和VP40基因CDS区的结合位点互补；第二个结合位点位于VP40基因的3’UTR区(非编码区)(GGTACCACAATCAACCCCGGCAAAGCTT，SEQ ID NO.:7)，其吉布斯自由能(mfe)达到-24.0kcal/mol，MiR-2911的种子序列和VP40基因3’UTR区的结合位点完全互补，最大的loop只有4个碱基，并且MiR-2911只有9个碱基不和VP40基因CDS区的结合位点互补。基于此，MIR2911可以和VP40基因结合，从而进一步证实，MiR-2911通过这个结合位点抑制VP40基因的表达。
2.3.利用荧光素酶检测方法验证MiR-2911调控埃博拉病毒编码的蛋白基因
2.3.1 基本信息
人工合成通过生物信息学预测的可以被MiR-2911结合的埃博拉病毒片段(结合位点上下游各延生40bp)，然后此产物被嵌入一个荧光素酶的报告基因p-MIR-report(Ambion)的3’-UTR端，利用pMIR-REPORT miRNA表达报告基因载体系统验证MIR2911是否可以调控埃博拉病毒编码的基因。pMIR-REPORT miRNA表达报告基因载体系统质粒图谱如图3所示。
在图3中pGL3-Basic载体的全长为4818bp(SEQ ID NO.:8)，部分元件的信息如下：
表1
启动子 (无)
增强子 (无)
多克隆区 1-58
荧光素酶基因(luc+) 88-1740
GLprimer2结合位点 89-111
SV40 late poly(A)信号 1772-1993
RVprimer4结合位点 2061-2080
ColE1-源性质粒复制起点 2318
beta-内酰胺酶基因(Ampr) 3080-3940
f1起点 4072-4527
Synthetic(upstream)poly(A)信号 4658-4811
RVprimer3结合位点 4760-4779
pGL3-GP(CDS)载体的序列如SEQ ID NO.:9所示，其中GP(CDS)序列位于第7-42位。
pGL3-VP40(3’UTR)载体的序列如SEQ ID NO.:10所示，其中VP40(3’UTR)序列位于第7-34位。
pGL3-VP40(CDS)载体的序列如SEQ ID NO.:11所示，其中VP40(CDS)序列位于第7-40位。
2.3.2 载体构建过程：
(a)Oligo DNA的设计与合成
根据已知GP(CDS),VP40(CDS),VP40(3’UTR)序列设计并合成2对互补oligo DNA，对应oligo DNA序列见表2：
表2 oligo DNA序列
Figure PCTCN2015088802-appb-000002
(b)荧光素酶质粒载体的构建与验证
将合成好的互补oligo DNA用ddH2O溶解成100μM，互补单链各取5μl两两混合，按表2给出体系进行退火。oligo混合物在95℃加热5分钟，然后放置室温20分钟，形成双链DNA。
按照对合成的oligo DNA，以及空载pgl3质粒，用KpnI和MluI进行酶切， 酶切完成后利用DNA回收试剂盒回收酶切产物。
将酶切后回收的oligo DNA，以及空载pgl3质粒，在室温下用T4DNA连接酶进行连接反应。
取10μl连接产物转化100μl感受态大肠杆菌细胞DH5α，涂LB平板(含50μg/ml卡那霉素)后，37℃孵育。
每个转化平板分别挑取3个克隆，摇菌抽提质粒后进行测序，以验证重组克隆中插入片段序列是否与设计的oligo DNA序列一致。
pMIR-REPORT miRNA表达报告基因载体系统由一个实验萤火虫荧光素酶报告载体(图3A)和相关的β-半乳糖苷酶报告对照质粒(图3B)组成。通过在多克隆位点插入预测的miRNA靶序列，pMIR-REPORT荧光素酶报告miRNA表达报告载体可以被用来进行准确的，定量的，评估细胞内miRNA的功能。pMIR-REPORT荧光素酶载体包含一个CMV启动子和终止子控制下的萤火虫荧光素酶报告基因。在荧光素酶基因的3’端非编码区包含一个多克隆位点，用于插入的预测miRNA的结合靶序列或其他核苷酸序列。通过将预测的miRNA靶序列克隆插入到pMIR-REPORT载体，荧光素酶报告表达就收到调节。这模仿的miRNA靶序列的作用方式。pMIR-REPORTβ-gal质粒是一个β-半乳糖苷酶报告质粒，其被设计用于细胞转染操作流程的标准化探索。该对照质粒表达的β-半乳糖苷酶，可以用来标准化由于细胞活力和转染效率差异导致的细胞表达水平的多样性。
测序结果表明，显示质粒构建是正确的。
在进行荧光素酶活性检验时，先将荧光素酶的重组质粒与β半乳糖苷酶报告质粒共同转入293T细胞中(β半乳糖苷酶报告质粒是用来确定转染效率的)，同时转染入293T细胞的还有等量的miRNA的前体或者人工合成的阴性control microRNA，这样24小时后，就可以用荧光素酶活性检验试剂盒(Promega)检测荧光素酶活性，通过它来反映miRNA对埃博拉病毒相关基因的调控作用。
具体结果
如图4所示，MIR2911对埃博拉病毒的2个基因，共3个位点【GP:MIR2911与它的结合位点位于GP基因的CDS区，GP(CDS)；VP40：MIR2911与它有两个结合位点，第一个结合位点位于VP40基因的CDS区，VP40(CDS)，第二个结合位点位于VP40基因的3’UTR区，VP40(3’UTR)】都可以结合，并且抑制效率都在60％。
从图4可以得出，MiR-2911可以和埃博拉病毒编码的GP、VP40基因结合，可以抑制埃博拉病毒的侵入和复制，因此可用于对埃博拉病毒感染的治疗。
在极其严格的筛选条件下，MiR-2911依然可以和埃博拉病毒编码的两个重要基因GP和VP40结合，通过荧光素酶实验进一步验证，MIR2911确实可以和埃博拉病毒编码的蛋白基因结合。
实施例3.MiR-2911的制备
3.1.人工合成的MiR-2911的制备及提取方法
人工合成的MiR-2911优选采用oligo DNA/RNA人工化学合成方法采用β-乙腈亚磷酰胺化学合成Oligo DNA/RNA，合成时从3’→5’方向进行，通常3’端的第一个碱基结合在Glass担体(Controlled Pore Glass，CPG)上。合成的详细过程见图5，现简要说明如下：
(a).脱掉附加在CPG担体上的第一个碱基5’-OH基团上的保护基(DMTr)，准备附加下一个新的碱基；
(b).活化新的碱基单体(Phosphoramidite)，准备与第一个碱基进行反应；
(c).第二个碱基与第一个碱基发生偶联反应；
(d).将没有反应的第一个碱基的5’-OH加帽封死(Capping)，使其不再进一步参与反应；
(e).将核苷亚磷酸酯氧化成更稳定的核苷磷酸酯(即将三价磷氧化成五价磷)。
(f).重复进行1～5的循环，直至合成完所需的Oligo DNA/RNA序列。
(g).合成结束后，将Oligo DNA/RNA分子从CPG上切下，再进行进一步的纯化。
3.2 MiR-2911的其他合成方法
一种具体方法包括步骤：
(a)设计合成MiR-2911的引物：
根据MiR-2911的模板质粒序列合成两条通用引物A、B，根据MiR-2911序列设计4条特异的寡核苷酸引物序列(I、II、III、IV)；
(b)第一轮PCR扩增：
以包含MiR-2911的质粒作为模板，分别以A与IV、III与II、I与B作为引物组合进行PCR扩增，PCR反应条件是：95℃、2分钟进行1个循环→95℃、30秒，55℃、30秒，72℃、40秒进行24个循环→72℃、7分钟；分别得到产物1、产物2、产物3；
(c)第二轮PCR扩增：以第一轮PCR扩增得到的产物1、产物2、产物3作为模板，以A与B作为引物进行PCR扩增，PCR反应条件是：95℃、2分钟进行1个循环→95℃、30秒，55℃、30秒，72℃、1分305秒进行24个循环→72℃、7分钟，PCR产物琼脂糖凝胶回收，得到合成的MiR-2911；
(d)将合成的MiR-2911甲基化，形成稳定的甲基化产物MiR-2911。
3.3 植物MiR-2911的制备及提取方法
多种植物包括药用植物、果蔬植物、观赏植物中富含MiR-2911；比如金银花、 菘蓝、草大青、马蓝、胡杨、豇豆、棉花、大白菜或马铃薯。
植物MiR-2911的提取方法主要采用溶剂提取法，即采用溶剂从植物中提取其MiR-2911。其中，所述的溶剂包括水、亲水性溶剂、或其组合。所述组合包括：在水中添加适量的亲水性溶剂或在亲水性溶剂中添加适量的水。应理解，溶剂中还可添加适量的辅助试剂，如pH调节剂(如酸或碱)等。
提取可以在任何适宜的温度(如常温～溶剂回流的温度)下进行，优选采用浸渍法、渗漉法、煎煮法、回流提取法、连续提取法等。
在提取过程中，可对植物进行预处理，例如将植物粉碎或进行酶处理(如纤维素酶、半纤维素酶、果胶酶、木聚糖酶、中性蛋白酶、木瓜蛋白酶、葡聚糖酶、以及夏合酶)等；也可对提取的混合物进行后处理，如将植物用水进行提取后，可在提取后的混合物中加入亲水性溶剂(如乙醇等)，使得混合物经陈化沉淀。
提取后得到的液体物可直接使用，也可进行过滤、浓缩、干燥(如冻干)等处理后制得固体物，然后再使用。
优选地，本发明所述的植物microRNA的提取方法为水提法。
以下以金银花植物为原料，制备和提取MiR-2911。但是制备MiR-2911的植物原料不局限于金银花，所述制备和提取方法适用于药用植物、果蔬植物、观赏植物。
金银花中含有一种天然存在的广谱抗病毒药物MiR-2911。
>peu-miR-2911 GGCCGGGGGACGGGCUGGGA(SEQ ID NO.:1)
利用水提法提取金银花MiR-2911。取适量(50克)干燥金银花药材，在500ml(金银花质量与水体积比为1：10)水的100℃水浴下加热0.5小时，提取液在60℃下减压浓缩至原体积的1/10。收集浓缩及未浓缩金银花水提液，金银花MiR-2911用于后续实验。
3.4 通过发酵方法所得到的质粒在生物体内表达
通过人工设计的方法将MiR-2911的前体构建到质粒中，将质粒转化进大肠杆菌中，通过发酵的方法，回收发酵产物，提取质粒并进一步纯化，用于后续实验。
实施例4 MiR-2911对埃博拉病毒编码蛋白基因有抑制作用
人工合成的MiR-2911、植物MiR-2911、通过发酵方法所得到的质粒在生物体内表达产生的MiR-2911，通过口服、呼吸道、注射、透皮、粘膜或腔道给药，对埃博拉病毒有抑制作用。
埃博拉病毒极具危险性，其活病毒研究必须在生物安全四级实验室中进行，且在全球受到及其严格的控制。假病毒不能在体内进行复制，只能一次性感染宿主细胞， 可以很好的用来代替活病毒研究细胞的进入机理等。首先诱导转基因小鼠表达Ebola病毒编码的蛋白基因，然后观察小鼠体重，死亡率等各项生理指标；处死小鼠后，利用Real-time PCR检测编码蛋白基因mRNA表达情况；利用western blotting通过检测GFP的表达水平来反应Ebola病毒编码蛋白表达情况；利用冰冻或者石蜡切片和流式细胞技术技术观察小鼠主要脏器(心，肝，脾，肺，肾)以及小鼠心血管系统，免疫系统的各项病理变化。
结果
(a)MiR-2911改善染病小鼠的症状
(a1)喂食人工合成的MiR-2911改善染病小鼠的症状
首先分别喂食转基因小鼠人工合成的NC(MiR-2911对照物)和MiR-2911，然后观察小鼠体重，死亡率等各项生理指标；处死小鼠后，利用Real-time PCR检测埃博拉病毒编码蛋白基因mRNA表达情况；利用western blotting通过检测GFP的表达水平来反应Ebola病毒编码蛋白表达情况；利用冰冻或者石蜡切片和流式细胞技术观察小鼠主要脏器(心，肝，脾，肺，肾)以及小鼠心血管系统，免疫系统的各项病理变化。
利用Real-time检测埃博拉病毒编码基因mRNA表达水平，具体操作步骤如实施例1所述。
利用常规的Western blotting方法通过检测Ebola病毒编码蛋白表达水平。该方法包括提取蛋白、SDS-PAGE、转膜、免疫反应、化学发光、凝胶图像分析等步骤。
在凝胶图像分析步骤中，将胶片进行扫描或拍照，用凝胶图象处理系统分析目标带的分子量和净光密度值。
此外，利用常规的冰冻或者石蜡切片和流式细胞技术观察小鼠主要脏器以及小鼠心血管系统，免疫系统的各项病理变化。
结果表明，与喂食人工合成NC的转基因小鼠相比，喂食人工合成MiR-2911的转基因小鼠体内埃博拉编码蛋白的表达水平明显下降，小鼠主要脏器(心，肝，脾，肺，肾)以及小鼠心血管系统，免疫系统得到显著改善，小鼠患病症状明显减轻。
上述实验结果表明，喂食人工合成的MiR-2911能够抑制埃博拉编码蛋白的表达。
(a2)静脉注射MiR-2911的过表达质粒改善染病小鼠的症状
分别给转基因小鼠尾静脉注射空白对照质粒和MiR-2911的过表达质粒，然后观察小鼠体重，死亡率等各项生理指标；处死小鼠后，利用Real-time PCR检测埃博拉病毒编码基因mRNA表达情况；利用western blotting通过检测GFP的表达水平来反应Ebola病毒编码蛋白表达情况；利用冰冻或者石蜡切片和流式细胞技术技术观察小鼠主要脏器(心，肝，脾，肺，肾)以及小鼠心血管系统，免疫系统的各项病理变化。
利用Real-time检测埃博拉编码基因mRNA表达水平，具体操作步骤如实施例1 所述。
利用western blotting通过检测Ebola病毒编码蛋白表达水平。
利用冰冻或者石蜡切片和流式细胞技术观察小鼠主要脏器以及小鼠心血管系统，免疫系统的各项病理变化
结果表明，与尾静脉注射空白对照质粒的转基因小鼠相比，尾静脉注射MiR-2911过表达质粒的转基因小鼠体内埃博拉病毒编码蛋白的表达水平明显下降，小鼠主要脏器(心，肝，脾，肺，肾)以及小鼠心血管系统，免疫系统得到显著改善，小鼠患病症状明显减轻。
上述实验结果表明，尾静脉注射MiR-2911过表达质粒能够抑制埃博拉编码蛋白的表达。
(a3)富含MIR2911的植物改善染病小鼠的症状
分别给转基因小鼠喂食不含MiR-2911的植物(大米)和富含MIR2911的植物(金银花)，观察小鼠体重，死亡率等各项生理指标；处死小鼠后，利用Real-time PCR检测埃博拉编码蛋白基因mRNA表达情况；利用western blotting通过检测GFP的表达水平来反应Ebola病毒编码蛋白表达情况；利用冰冻或者石蜡切片和流式细胞技术技术观察小鼠主要脏器(心，肝，脾，肺，肾)以及小鼠心血管系统，免疫系统的各项病理变化。
利用Real-time检测埃博拉编码蛋白基因mRNA表达水平。
利用western blotting通过检测Ebola病毒编码蛋白表达水平。
利用冰冻或者石蜡切片和流式细胞技术观察小鼠主要脏器以及小鼠心血管系统，免疫系统的各项病理变化。
结果发现，与喂食不含MiR-2911植物的转基因小鼠相比，喂食富含MiR-2911植物的转基因小鼠体内埃博拉病毒编码蛋白的表达水平明显下降，小鼠主要脏器(心，肝，脾，肺，肾)以及小鼠心血管系统，免疫系统得到显著改善，小鼠患病症状明显减轻。
上述实验结果表明，喂食富含MiR-2911的植物能够强烈抑制埃博拉编码蛋白的表达。
总之，本发明证实了在多种给药方式下，MiR-2911都能够抑制埃博拉编码蛋白基因的表达。
实施例5 在HUVEC细胞中验证MIR2911对埃博拉病毒编码的GP基因的抑制作用
常规实验方法，构建偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒(图8)和MIR2911过表达质粒(图9)。
利用偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒感染HUVEC细胞，12h后，在感染腺病毒的HUVEC细胞中转染MIR2911前体模拟物或者MIR2911过表达质粒，48h后在荧光显微镜下观测绿色荧光蛋白表达情况，随后收集RNA和蛋白，分别利用Q-PCR技术和western blotting技术检测HUVEC细胞中GP基因mRNA和蛋白质水平的变化。
结果如下：
图10显示了转染MIR2911后HUVEC细胞中MIR2911的表达水平。
与转染随机对照核酸或者空载pcDNA6.2质粒相比，转染MIR2911前体模拟物或者MIR2911过表达质粒，MIR2911的水平在HUVEC细胞中显著上升。
图11显示了转染MIR2911后eGFP的表达水平。
与感染空载腺病毒12h后转染随机对照核酸、和感染偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒12h后转染空载pcDNA6.2质粒的对照组相比，转染MIR2911前体模拟物或者MIR2911过表达质粒能够明显抑制偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒绿色荧光蛋白的表达，而对空载的腺病毒表达的绿色荧光蛋白没有影响。
图12显示了GP mRNA的Q-PCR测定结果。
ncRNA：感染偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒12h后转染随机对照核酸；MIR 2911:感染偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒12h后转染MIR2911前体模拟物；Control Plasmid:感染偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒12h后转染空载pcDNA6.2质粒；MIR2911Plasmid:感染偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒12h后转染过表达MIR2911的pcDNA6.2质粒。
利用Q-PCR测定GP的mRNA水平，结果发现，
与ncRNA和Control Plasmid相比，转染MIR2911前体模拟物(MIR 2911)或者MIR2911过表达质粒(MIR 2911 Plasmid)，GP的mRNA都显著下降。
图13显示了GP蛋白水平的western blotting结果。
ncRNA：感染偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒12h后转染随机对照核酸；MIR 2911:感染偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒12h后转染MIR2911前体模拟物；Control Plasmid:感染偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒12h后转染空载pcDNA6.2质粒；MIR2911Plasmid:感染偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒12h后转染过表达MIR2911的pcDNA6.2质粒。
与ncRNA和Control Plasmid相比，转染MIR2911前体模拟物或者MIR2911过表达质粒，GP的蛋白水平也下降。
实施例11在HUVEC细胞中验证MIR2911对埃博拉病毒编码的GP基因导致的内皮细胞死亡的影响
GP蛋白可以诱导内皮细胞大量非程序性死亡，从而导致埃博拉患者血管不完整而导致内出血等一系列症状。为了观察GP对细胞状态的影响，在12h、24h、36h利用台盼蓝染色观察GP蛋白对HUVEC细胞细胞死亡情况，同时，利用细胞毒性检测试剂盒(CytoTox-GloTM cytotoxicity assay)(购自promega)检测GP蛋白对内皮细胞膜完整性的影响
台盼蓝染色操作步骤如下：
1、4％台盼蓝母液：称取4g台盼蓝，加少量蒸馏水研磨，加双蒸水至100ml，用滤纸过滤，4度保存。使用时。用PBS稀释至0.4％。
2、胰酶消化贴壁细胞，制备单细胞悬液，并作适当稀释。
3、染色：细胞悬液与0.4％台盼蓝溶液以9:1混合混匀。(终浓度0.04％)
4、计数：在三分钟内，分别计数活细胞和死细胞。
5、镜下观察，死细胞被染成明显的蓝色，而活细胞拒染呈无色透明状。
6、统计细胞活力：活细胞率(％)＝活细胞总数/(活细胞总数+死细胞总数)×100％
结果如下：
图14显示了台盼蓝染色观察GP蛋白对HUVEC细胞存活率。
Mock代表：没做任何处理的细胞，是阴性对照；AD:空载腺病毒；AD(GP):偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒。ncRNA：感染偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒12h后转染随机对照核酸；MIR 2911:感染偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒12h后转染MIR2911前体模拟物；Control Plasmid:感染偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒12h后转染空载pcDNA6.2质粒；MIR2911Plasmid:感染偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒12h后转染过表达MIR2911的pcDNA6.2质粒。
与ncRNA和control plasmid相比，转染MIR2911和MIR2911plasmid的细胞存活率显著降低升高。GP蛋白可以促进细胞死亡，而MIR2911和MIR2911过表达质粒可以抑制GP蛋白对细胞死亡的促进作用。
图15显示了细胞毒性检测试剂盒检测GP蛋白对内皮细胞膜完整性的影响。各图例的意义与图14相同。
与ncRNA和control plasmid相比，转染MIR2911和MIR2911plasmid的内皮细胞膜完整性得到显著改善。结果表明，GP蛋白可以破坏内皮细胞膜的完整性，而MIR2911和MIR2911过表达质粒可以抑制GP蛋白对内皮细胞的破坏作用。
实施例12小鼠体内验证金银花汤对埃博拉病毒的预防作用以及表达MIR2911的质粒对埃博拉病毒的治疗作用
实验方法如下：
对照组1：5只小鼠正常饲养，不做处理；
对照组2：5只小鼠正常饲养，在第三天通过尾静脉注射空载腺病毒(AD)，注射剂量为108IU连续注射3天。第八天处死小鼠。
对照组3：5只小鼠正常饲养，在第三天通过尾静脉注射表达GP的腺病毒[AD(GP)]，注射剂量为108IU连续注射3天。第八天处死小鼠。
实验组1：5只小鼠在第一天喝金银花汤剂(每只小鼠每天3ml)，在第三天通过尾静脉注射偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒(表达GP的腺病毒)，注射剂量为108IU连续注射3天。第八天处死小鼠。
实验组2：5只小鼠在第一天喝金银花汤剂(每只小鼠每天3ml)，在饲养三天时通过尾静脉注射偶联eGFP的表达埃博拉病毒编码的GP基因的腺病毒，注射剂量为108IU连续注射3天。在第5天最后一次注射腺病毒后同时通过尾静脉注射MIR2911质粒，注射剂量为5mg/kg体重，连续注射3天。在第8天处死小鼠。
统计各组死亡率，收取组织和血液测定的相关生化指标。ELASA法(Real time PCR)检测小鼠血清中谷丙转氨酶(AST)、门冬酸氨基转移酶(ALT)、总胆红素(TB)的含量。在处死小鼠后，解剖小鼠，观察小鼠肝脏和脾脏的变化。并采用real-time PCR方法检测血清中TGFα，IL-6的水平；对肝脏和脾脏进行HE染色观察。
结果如下：
图16显示了实验过程中的死亡率。空载腺病毒对小鼠生存率没有影响，而注射表达GP蛋白的腺病毒可以强烈致死小鼠，致死率为40％，当给小鼠喝金银花汤剂进行预防性治疗时，小鼠的死亡率显著降到90％，而给予尾静脉注射MIR2911过表达质粒的小鼠，小鼠基本被全部治愈。结果表明，金银花汤剂可以显著预防埃博拉病毒感染，并显著降低了死亡率并且MIR2911质粒对于埃博拉病毒有治疗效果。
图17显示了AST，ALT，TB表达水平。表达GP的腺病毒会对小鼠肝脏造成了损伤，AST，ALT，TB在尾静脉注射GP的腺病毒小鼠中显著上升，然而当给予MIR2911表达质粒或者给予金银花汤治疗时，AST，ALT，TB发生明显下降。
同时，通过对解剖小鼠的观察，发现注射表达GP的腺病毒的小鼠肝脏和脾脏明显肿大，而给予MIR2911表达质粒或者给予金银花汤治疗时得到显著缓解。
图18显示了血清中TGFα，IL-6的水平。TGFα，IL-6在尾静脉注射表达GP的腺病毒的小鼠中显著上升，然而当给予MIR2911表达质粒或者给予金银花汤治疗时，TGFα，IL-6发生明显下降。
图19显示了肝脏的HE染色结果。在注射GP腺病毒后出现明显损伤，而给予 MIR2911表达质粒或者给予金银花汤治疗时得到显著缓解。
图20显示了脾脏的HE染色结果。在注射GP腺病毒后出现明显损伤，而给予MIR2911表达质粒或者给予金银花汤治疗时得到显著缓解。
综上所述，MIR2911表达质粒或金银花汤对GP腺病毒有显著抑制作用，金银花汤对埃博拉病毒的预防作用，表达MIR2911的质粒对埃博拉病毒的治疗作用。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种微小核糖核酸miR-2911的用途，其特征在于，用于制备(a)治疗埃博拉病毒的药物；(b)调控埃博拉病毒蛋白基因表达的药物；和/或(c)抑制埃博拉病毒生长的药物。
  2. 如权利要求1所述的用途，其特征在于，所述的药物用于抑制埃博拉病毒的复制。
  3. 如权利要求1所述的用途，其特征在于，所述的药物用于抑制埃博拉病毒的GP蛋白所导致的内皮细胞受损或死亡。
  4. 如权利要求1所述的用途，其特征在于，所述的MiR-2911包括：人工合成的MiR-2911、植物MiR-2911、MiR-2911前体和/或成熟体形式；和/或
    含有MiR-2911的植株、植株部分、或提取物。
  5. 如权利要求1所述的用途，其特征在于，所述的埃博拉病毒蛋白包括GP、VP40或其组合。
  6. 如权利要求1所述的用途，其特征在于，所述的埃博拉病毒包括：本迪布焦埃博拉病毒(BDBV)、扎伊尔埃博拉病毒(EBOV)和苏丹埃博拉病毒(SUDV)。
  7. 一种用于抑制埃博拉病毒复制和/或治疗埃博拉病毒感染的组合物，其特征在于，所述组合物含有(a)药学上可接受的载体或食品学上可接受的载体；以及(b)活性成分，所述活性成分包括miR2911。
  8. 一种体外的非治疗性地抑制抑制埃博拉病毒复制或抑制埃博拉病毒蛋白基因表达的方法，其特征在于，包括步骤：将miR2911与埃博拉病毒或受埃博拉病毒感染的细胞进行接触。
  9. 一种预防或治疗埃博拉病毒病的方法，其特征在于，包括以下步骤：给需要的对象施用MiR-2911或含MiR-2911的提取物或组合物。
  10. 一种微小核糖核酸miR-2911的用途，其特征在于，用于制备埃博拉病毒的GP蛋白的抑制剂；或用于制备一制剂或组合物，所述制剂或组合物用于抑制埃博拉病毒的GP蛋白所导致的内皮细胞受损或死亡。
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CN112014557A (zh) * 2019-05-28 2020-12-01 中国人民解放军军事科学院军事医学研究院 一种可用于埃博拉病毒病治疗的靶点
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CN114470212B (zh) * 2022-03-22 2022-10-11 中国人民解放军军事科学院军事医学研究院 EIF4B siRNA在制备治疗埃博拉病毒病药物中的应用
CN115844009A (zh) * 2022-09-15 2023-03-28 北京元露健康科技有限公司 含有2911植物元的组合物和含有2911植物元的金银花提取物的制备方法
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102416185A (zh) * 2011-12-12 2012-04-18 武汉大学 miR29在制备抗流感病毒药物中的应用
CN103589721A (zh) * 2012-08-15 2014-02-19 北京命码生科科技有限公司 植物微小核糖核酸的提取、制备及其应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101285100A (zh) * 2008-03-11 2008-10-15 浙江理工大学 一种利用原位合成微流体芯片的检测方法
WO2013066442A2 (en) * 2011-07-29 2013-05-10 Hodge Thomas W Mammalian genes and gene products involved in infection
EP2756102B1 (en) * 2011-09-13 2019-04-24 Commonwealth Scientific and Industrial Research Organisation Detection of viral infection
WO2013123503A1 (en) * 2012-02-17 2013-08-22 The Children's Hospital Of Philadelphia Aav vector compositions and methods for gene transfer to cells, organs and tissues
WO2014026333A1 (zh) * 2012-08-15 2014-02-20 北京命码生科科技有限公司 植物微小核糖核酸的提取、制备及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102416185A (zh) * 2011-12-12 2012-04-18 武汉大学 miR29在制备抗流感病毒药物中的应用
CN103589721A (zh) * 2012-08-15 2014-02-19 北京命码生科科技有限公司 植物微小核糖核酸的提取、制备及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIN ZHANG ET AL.: "Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA", CELL RESEARCH, vol. 22, no. 1, 31 January 2012 (2012-01-31), pages 107 - 126, XP055075505, ISSN: 1001-0602, DOI: doi:10.1038/cr.2011.158 *
See also references of EP3189856A4 *

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