WO2016034111A1 - 埃博拉病毒特异性的miRNA以及通过miRNA抑制埃博拉病毒的方法 - Google Patents

埃博拉病毒特异性的miRNA以及通过miRNA抑制埃博拉病毒的方法 Download PDF

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WO2016034111A1
WO2016034111A1 PCT/CN2015/088803 CN2015088803W WO2016034111A1 WO 2016034111 A1 WO2016034111 A1 WO 2016034111A1 CN 2015088803 W CN2015088803 W CN 2015088803W WO 2016034111 A1 WO2016034111 A1 WO 2016034111A1
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ebola virus
mir
sequence
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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    • C12N2760/00011—Details
    • C12N2760/14011—Filoviridae
    • C12N2760/14111—Ebolavirus, e.g. Zaire ebolavirus
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Definitions

  • the invention relates to the field of bioinformatics and public health.
  • the invention relates to Ebola virus-specific miRNAs and methods of inhibiting Ebola virus by miRNAs.
  • 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.
  • 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 precursor sequence of Ebola virus or a mature sequence thereof wherein
  • microRNA precursor sequence is selected from the group consisting of:
  • microRNA maturation sequence is selected from the group consisting of:
  • the sequence is a nucleotide sequence of RNA, DNA or RNA/DNA hybrid.
  • a microRNA precursor sequence or a microRNA maturation sequence of the Ebola virus of the first aspect of the invention for the preparation of a reagent or kit for detecting Ebola virus.
  • the reagent or kit is used for early diagnosis of Ebola virus.
  • the reagent comprises a primer, a probe, and a chip.
  • the detection is directed to a sample selected from the group consisting of serum and plasma.
  • the detection is directed to a sample selected from the group consisting of Ebola virus-infected organisms (human, orangutan, monkey, etc.), Ebola-infected organisms (human, orangutan, monkey, etc.) Plasma.
  • the inhibitor is an inhibitor that specifically inhibits the sequence of SEQ ID NO.: 35.
  • composition further comprises microRNA miRNA miR-2911.
  • the composition comprises a pharmaceutical composition, a nutraceutical composition.
  • the composition comprises a pharmaceutical composition.
  • the inhibitor is an antisense nucleic acid sequence or a sponge sequence.
  • the inhibitor specifically binds to a nucleotide sequence selected from the group consisting of:
  • a fourth aspect of the invention there is provided the use of the microRNA precursor sequence of Ebola virus of the first aspect of the invention or a mature sequence thereof, which is used for preparing a state of rehabilitation after the Ebola virus infection or Prognostic reagents or kits.
  • microRNA precursor sequence or the mature sequence thereof is as set forth in SEQ ID NO.:35.
  • a pharmaceutical composition for preventing or treating Ebola virus infection or inhibiting Ebola virus characterized in that said composition contains (i) pharmaceutically acceptable The vector; (ii) an inhibitor of the microRNA precursor sequence of the Ebola virus of the first aspect or a mature sequence thereof.
  • the inhibitor is an antisense nucleic acid sequence or a sponge sequence.
  • the pharmaceutical composition further comprises a piconucleotide MiR-2911 or an extract or composition comprising MiR-2911.
  • a method for preventing or treating Ebola virus disease comprising the step of administering to a subject in need thereof a microRNA precursor sequence directed against the Ebola virus of the first aspect of the invention An inhibitor of its mature sequence.
  • the method further comprises: administering to the subject an additional therapeutic agent, preferably MiR-2911 or an extract or composition comprising MiR-2911.
  • an additional therapeutic agent preferably MiR-2911 or an extract or composition comprising MiR-2911.
  • a microRNA miRNA miR-2911 for the preparation of (a) a medicament for treating Ebola virus; a medicament for regulating expression of an Ebola virus protein gene; and/or ( c) A drug that inhibits the growth of Ebola virus.
  • the medicament is for inhibiting replication of the Ebola virus.
  • the Ebola virus protein is selected from the group consisting of GP, VP40.
  • 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.
  • 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.
  • 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 is a schematic diagram of the secondary structure of EBO-pre-miR-1.
  • Figure 9 is a schematic diagram of the secondary structure of EBO-pre-miR-2.
  • Figure 10 is a schematic diagram of the secondary structure of EBO-pre-miR-3.
  • Figure 11 is a schematic diagram of the secondary structure of EBO-pre-miR-4.
  • Figure 12 is a graphical representation of the results of the conservative analysis of EBO-pre-miR-1.
  • Figure 13 is a graphical representation of the results of the conservative analysis of EBO-pre-miR-2.
  • Figure 14 is a graphical representation of the results of the conservative analysis of EBO-pre-miR-3.
  • Figure 15 is a graphical representation of the results of the conservative analysis of EBO-pre-miR-4.
  • Figure 16 shows the location of the Ebola virus encoded pre-miR-VP in the genome and the predicted neck ring secondary structure.
  • Figure 17 shows the results of Northern blot-specific probe analysis of cellular RNA introduced into the pre-miR-VP sequence.
  • Figure 18 shows the results of a mixed serum sample test in patients with Ebola virus infection.
  • Figure 19 shows the sensitivity of the miR-VP-3p assay.
  • Figure 20 shows the results of qRT-PCR analysis of miR-VP-3p in the serum of patients with Ebola virus.
  • Figure 20A shows the respective CT values of miR-VP-3p in the serum of healthy volunteers and EVD + G+ patients;
  • Figure 20B shows the concentration of miR-VP-3p in serum samples of healthy volunteers and EVD + G+ patients;
  • Figure 20C shows the concentration of miR-VP-3p in the serum of Ebola virus exacerbation disease (EVD + G+ (PN) ) and rehabilitation (EVD - G-(PN) ).
  • PN Ebola virus exacerbation disease
  • PN rehabilitation
  • Figure 21 shows the results of qRT-PCR detection of miR-VP-3p.
  • Figure 22 shows the C T values of miR-VP-3p in the serum of Ebola virus exacerbation (EVD + G+ (PN) ) and rehabilitation (EVD - G-(PN) ).
  • Figure 23 shows the results of serum detection of miR-VP-3p in patients with suspected Ebola virus at different times.
  • Figure 23A shows the results of the detection of 6 patients confirmed to be Ebola positive (black spots, denoted as EVD + G-(NP) and EVD + G+(NP) );
  • Figure 23B shows the confirmation as angstrom Appreciable miR-VP-3p levels were detected in the serum of 6 patients who were positive for Bora virus;
  • Figure 23C shows the results of 9 patients who were still negative for Ebola virus (blue dot, recorded as EVD G-(S) ) and EVD - G-(S) ).
  • Figure 23D shows that no miR-VP-3p was detected in vivo in 9 patients still negative for Ebola virus.
  • Figure 24 shows the C T values of miR-VP-3p in the serum of 15 suspected Ebola patients.
  • the present inventors have also conducted extensive and intensive research to identify, for the first time, a small ribonucleic acid which can efficiently bind to an Ebola-encoded protein gene 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.
  • the GP1 subunit is essential for the entry and toxicity of the virus. 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 structural elements that are VP40 dimers, whether VP40 hexamers or octamers.
  • EBOV VP40 octamer is 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 related to the formation of nucleocapsid of virulence, and possibly Participate 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 EBOV VP40 N-terminal is removed and inserted into the C-terminus, VP40-mediated release of virus like particles (VLPs) No change will happen.
  • 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 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 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 Wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, 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, pour, tincture, paste, eye drops, nasal drops, ophthalmic ointment, gargle, sublingual tablets 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.
  • Ebola virus-specific microRNA or “Ebola virus-specific microRNA of the present invention”, “Ebola virus-encoded microRNA” are used interchangeably and refer to the name EBO-pre-miR. - EBO-pre-miR-2, EBO-pre-miR-3 and EBO-pre-miR-4, four Ebola-derived microRNAs. It should be understood that the term includes a precursor or mature form of microRNA.
  • the inventors used three database comprehensive analysis analysis methods to predict and analyze three different types of Ebola virus, and obtained various prediction results.
  • the four pre-microRNA precursors provided by the present invention include one species of Reston Ebola, one species of Sultan Ebola, and two species of Zaire Ebola.
  • the target genes targeted by the four pre-microRNAs and the possible consequences for the target genes are determined.
  • 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.
  • microRNA of the present invention has strong targeting ability and helps to overcome the problem of misjudgment of Ebola antibodies.
  • Ebola virus Based on the specific microRNA of Ebola virus, specific detection of Ebola virus can be performed to achieve the purpose of rapid diagnosis of Ebola virus infection in the early stage after Ebola virus infection, and avoid the early detection of viral infection. The infection caused.
  • microRNA of Ebola virus it is also possible to design a specific microRNA inhibitor, specifically inhibit the microRNA of Ebola virus, and inhibit the replication and infection of the virus, thereby achieving therapeutic purposes.
  • the target gene sequence targeted by the Ebola virus-specific microRNA is consistent with some pathological symptoms of the Ebola virus, which contributes to the further research and prevention of Ebola virus.
  • 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.
  • 2.1MiR-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 be combined with the GP gene to further verify that MiR-2911 can inhibit the expression of the GP gene through this binding site.
  • 2.2MiR-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
  • sequence of the pGL3-VP40 (3'UTR) vector is set forth 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 ddH2O, 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 recombinant oligo DNA and the empty pgl3 plasmid were digested with KpnI and MluI, and after digestion, the digested product was 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 cell DH5 ⁇ , coated with LB plate (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.
  • the 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 euphratica, 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.
  • 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. 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 reversed The expression of the protein encoded by Ebola virus; the observation of various pathological changes of the immune system by observing the main organs (heart, liver, spleen, lung, kidney) and mouse cardiovascular system by freezing or paraffin section and flow cytometry .
  • 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.
  • Ebola coding gene mRNA The expression level of Ebola coding 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 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.
  • ZEBOV Zaire Ebola virus
  • REBOV Reston Ebola virus
  • SEBOV Sudan Ebola virus
  • TEBOV Ivorian Ebola virus
  • Example 6 Four kinds of Ebola virus-specific microRNA precursors
  • HEK-293T cells (Shenzhen Baienwei Biotechnology Co., Ltd.)
  • T4 ligase (TAKARA)
  • the corresponding oligo DNA was designed, BgIII and HindIII restriction sites were added at both ends, and the pSuper plasmid and the designed oligo DNA were doubled by restriction endonucleases BgIII and HindIII.
  • the linearized pSuper plasmid fragment was digested, and the oligo DNA sequence was introduced into the expression vector, and expressed in Hela cells by lipofection. After expression, RNA was extracted for detection.
  • EBO-pre-miR-1 EBO-pre-miR-1
  • EBO-pre-miR-2 EBO-pre-miR-2
  • EBO-pre-miR-3 EBO-pre-miR-4
  • Example 6 The four pre-miRNAs detected in Example 6 were further subjected to structural analysis.
  • Ebola virus genome Download the Ebola virus genome from the NCBI database, including 6 species of Zaire Ebola virus (ZEBOV), 3 species of Reston Ebola virus (REBOV), 2 species of Sudan Ebola virus (SEBOV), Côte d'Irium Two types of Bora virus (TEBOV), four pre-miRNAs that may be encoded by Ebola virus using BLAST software from NCBI database Sequences were analyzed for conservation.
  • Ebo-miR-3-5p Position 33 CACUAGCGGUACCGCAGGUGCU (SEQ ID NO.: 32)
  • Ebo-miR-3-3p Position 62 UUAUCCUUCUUGAAUCCUGAGA (SEQ ID NO.: 33)
  • RNAhydrid RNAhydrid
  • RNFFOLD Fast Folding
  • Comparison of RNA Secondary Structures were used to predict the target genes that may be regulated by eight microRNAs encoded by Ebola virus.
  • miRNAs have at most 10 bases that are not complementary
  • loop has at most 8 bases.
  • the Ebola-encoded microRNA may regulate an RNA-binding protein that recognizes viral RNA, which may cause the Ebola virus to be incapable of being recognized by the human immune system, thereby allowing a large amount of replication in the human body. Further research on the Ebola virus is very beneficial.
  • the method was the same as in Example 6, and the microRNA mature body of the transfected cells was detected.
  • Ebola-encoded microRNAs can be used as molecular markers for early diagnosis of Ebola virus.
  • the method for detecting the microRNA specific to the Ebola virus includes one or more of an RT-PCR method, a Real-time PCR method, a Northern blotting method, an RNase protection assay method, a Solexa sequencing technology method, and a biochip method.
  • isotope signal detection such as phosphor screen scanning test results.
  • the extracted RNA is dissolved in the hybridization buffer and an antisense RNA probe is added for hybridization reaction;
  • the 9.5Solexa sequencing technology method includes the following steps:
  • Enzyme adaptor is ligated to the 3' and 5' ends of the small RNA molecule
  • the 9.6 biochip method includes the following steps:
  • RT-PCR Real-time PCR
  • Northern blotting Northern blotting
  • RNase protection assay Solexa sequencing technology and biochip were used to analyze the changes and changes of Ebola microRNA, and to quickly diagnose Ebola virus early.
  • the purpose of the infection was used to analyze the changes and changes of Ebola microRNA, and to quickly diagnose Ebola virus early. The purpose of the infection.
  • the hairpin structure of the miRNA precursor sequence was analyzed as described in Example 5 for the genomic sequence of Ebola virus (NC002549).
  • Figure 16 shows the position of the Ebola virus-encoded pre-miR-VP in the genome and the predicted neck ring secondary structure. Among them, the neck ring precursor of the mature miRNA from the 3' end is indicated in red. lie in The three hypothetical precursors of the Ebola virus VP40, NP and L genes were successfully predicted, respectively.
  • mature miRNA sequences were analyzed by the Mature Bayes tool and the Byes-SVM-MiRNA analysis tool.
  • miRNA-VP-3p The results revealed three putative mature miRNAs (currently referred to as miR-VP-3p, miR-NP-3p and miR-L-3p) present at the 3' end of the precursor. Because miRNAs are highly conserved across species, the evolutionary conservation of putative Ebola miRNAs is detected by Blast alignment of the precursor sequences with the existing published full length genomic sequences of the 2014 EBOV Ebola virus strain.
  • the pre-miR-VP sequence was cloned into a plasmid and transfected into HEK293A. In the cell. Cellular RNA was analyzed by a specific probe of Northern blot.
  • pre-miR-VP sequence is essential for the production of miR-VP-3p
  • a mutation was introduced into the hairpin structure, and the mutated pre-miR-VP mut sequence was cloned into a plasmid and transfected into HEK293A cells.
  • Cellular RNA was analyzed by a specific probe of Northern blot.
  • RNAs were extracted from serum samples from Ebola virus-infected patients and healthy volunteers, respectively. The content and sensitivity of miR-VP-3p in serum were detected by Northern blot.
  • Treatment was conducted in accordance with a temporary emergency guidance program developed by the World Health Organization on the management of cases of viral hemorrhagic fever.
  • the program has been approved by the Ethics Committee.
  • Each patient participating in the study received written informed consent prior to the start of the study.
  • All clinical serum samples were taken from samples sent to the Ebola Virus Diagnostic Mobile Laboratory.
  • the serum was inactivated by heat treatment at 60 ° C for 1 hour, then separated and stored at -20 ° C until use. Including age, gender, and Epidemiological information, including the date of onset of symptoms, is recorded by questionnaire and sent along with the serum samples.
  • the conventional Northern blot method is specifically described as described in Example 9.
  • the specific probe sequence is: miR-VP-3p probe (5'-GCCCCAAAGTGCTAATGAAGCA-3'SEQ ID NO.: 36), miR-16 control probe (5'-CGCCAATATTTACGTGCTGCTA-3' (SEQ ID NO) .:37)).
  • miR-VP-3p was detected in a mixed serum sample of Ebola virus-infected patients, but not in healthy volunteers.
  • EVD + G-(NP) represents: serum in which the nucleic acid test is negative in patients with negative Ebola virus nucleic acid detection;
  • EVD + G+(NP) serum in which the nucleic acid test is positive in patients with negative Ebola virus nucleic acid detection
  • EVD + G+ stands for: serum from patients with Ebola recovery who have a positive nucleic acid test during the onset of the disease;
  • EVD + G-(PN) stands for: serum in patients with Ebola recovery who have a negative nucleic acid test after treatment and rehabilitation;
  • EVD - G-(PN) stands for Ebola virus-negative patients with negative Ebola virus nucleic acid test, and after isolation observation, patients with non-Ebola virus infection are confirmed.
  • the level of miR-16 was also detected in serum samples of Ebola virus-infected patients and healthy volunteers, and the results showed no difference in the contents of the two groups.
  • Figure 19 shows the sensitivity of the miR-VP-3p assay.
  • the calculated content of miR-VP-3p in the serum of patients infected with Ebola virus is greater than 100 fmol/L.
  • Small RNA fragments in Ebola virus-infected patients were identified by RT-PCR combined with TA cloning and sequencing techniques. Small RNA isolated from the serum of Ebola virus-infected patients was subjected to RT-PCR to amplify the miRNA of Ebola virus, and then the amplified product was ligated to the TA plasmid and sequenced.
  • Sequencing results showed that the sequence of the product in the Ebola patient sample was identical to the predicted miR-VP-3p sequence, while the sequences present in healthy volunteer samples were diverse and different from the miR-VP-3p sequence. .
  • sequencing of Northern blotting and RT-PCR products did not reveal the presence of a putative Ebola miRNA specific sequence in the serum of Ebola virus-infected patients.
  • the quantitative RT-PCR (qRT-PCR) sequencing method based on TaqMan probe technology was used to detect miR-VP- in 27 Ebola virus-infected patients (denoted as EVD + G+ ) and 13 healthy volunteers. 3p.
  • the synthetic single-stranded miR-VP-3p was fractionally diluted and detected by qRT-PCR sequencing to establish a standard curve, A no-template control was used to determine the specificity of the primers.
  • Figure 20 shows the results of qRT-PCR analysis of miR-VP-3p in the serum of patients with Ebola virus.
  • Fig. 20A shows the respective CT values of miR-VP-3p in the serum of healthy volunteers and EVD + G+ patients.
  • the blue line is the lower limit of the dynamic range.
  • the CT value of patients with EVD + G+ was consistently within the linear range of EVD + G+ , but outside the linear range of healthy volunteers (Table 4), indicating that miR-VP-3p was detectable in EVD + G+ patients, but it was healthy Volunteers do not exist in the body.
  • the concentration of miR-VP-3p was calculated from the standard curve.
  • Figure 20B shows the concentration of miR-VP-3p (SEQ ID NO.: 35) in serum samples from healthy volunteers and EVD + G+ patients.
  • miR-VP-3p was not detected in the serum of healthy volunteers.
  • miR-16 UGCAGCACGUAAAUAUUGGCG
  • Figure 20C shows the concentration of miR-VP-3p in the serum of the Ebola virus exacerbation disease (denoted as EVD + G+ (PN) ) and during the rehabilitation phase (denoted as EVD - G- (PN) ).
  • miR-VP-3p showed high levels during the onset of the disease but disappeared after complete recovery.
  • Northern blot analysis confirmed that miR-VP-3p is absent in the serum of the Ebola virus rehabilitation stage. The results indicate that miR-VP-3p is present during the onset of disease in patients with Ebola and disappears during the recovery phase.
  • Figure 22 shows the C T values of miR-VP-3p in the serum of the Ebola virus exacerbation disease (denoted as EVD + G+ (PN) ) and during the rehabilitation phase (denoted as EVD - G-(PN) .
  • a no-template control was used to determine the specificity of the primers.
  • the synthesized miR-VP-3p can be efficiently amplified, and more rounds of amplification cycles are performed without the template control.
  • the blue line indicates no template negative control. Above the range indicated by the blue line, the amplification is invalid; the blue line below indicates the range, and the amplification is effective.
  • the results indicate that miR-VP-3p is present during the onset of disease in patients with Ebola and disappears during the recovery phase.
  • Ebola virus suspected patients are patients who show symptoms of Ebola virus infection when they start participating in the test, but the Ebola genomic RNA results are negative by RT-PCR.
  • Figure 23 shows the concentration of miR-VP-3p in serum at different times in patients with suspected Ebola virus.
  • Figure 23A shows the results of the detection of 6 patients confirmed to be Ebola positive (black spots, denoted as EVD + G-(NP) and EVD + G+(NP) );
  • Figure 23B shows the confirmation as angstrom Appreciable miR-VP-3p levels were detected in the serum of 6 patients who were positive for Bora virus;
  • Figure 23C shows the results of 9 patients who were still negative for Ebola virus (blue dot, recorded as EVD G-(S) ) and EVD - G-(S) ).
  • Figure 23D shows that no miR-VP-3p was detected in vivo in 9 patients still negative for Ebola virus.
  • Figure 24 shows the C T values of miR-VP-3p in the serum of 15 suspected Ebola patients.
  • the blue line indicates no template negative control. Above the range indicated by the blue line, the amplification is invalid; the blue line below indicates the range, and the amplification is effective.
  • Ebola-encoded microRNAs can be used as molecular markers for early diagnosis of Ebola virus. More notably, it can be noted that the Ebola-encoded microRNA may regulate a large number of ATPase (including Ca++transporting, H+transporting, Na+/K+transporting, etc.) ion channel proteins, which is very likely It causes severe disturbance of electrolytes in the human body, which leads to increased permeability of human vascular endothelial cells, rupture of endothelial cells, and thus a large number of internal bleeding, major organ lysis and other symptoms.
  • ATPase including Ca++transporting, H+transporting, Na+/K+transporting, etc.
  • Ebola-encoded microRNAs can be used as a drug target for the treatment of Ebola virus infection, which provides a new approach and method for the treatment of Ebola virus infection.

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Abstract

提供了埃博拉病毒特异性的microRNAs前体序列或其成熟序列及其在制备检测埃博拉病毒的试剂或试剂盒中的用途。还提供了针对该microRNAs前体序列或其成熟序列的抑制剂及其在治疗埃博拉病毒感染中的应用。

Description

埃博拉病毒特异性的miRNA以及通过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，本发明涉及埃博拉病毒特异性的miRNA以及通过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，提供了一种埃博拉病毒的microRNA前体序列或其成熟序列，其中，
所述的microRNA前体序列选自下组：
(a)SEQ ID NO.:28、22、24和27中任一所述的核苷酸序列；
(b)与组(a)中任一核苷酸序列完全互补或基本互补的核苷酸序列；
所述的microRNA成熟序列选自下组：
(i)SEQ ID NO.:35、21、29-34中任一所述的核苷酸序列；
(ii)与组(i)中任一核苷酸序列完全互补或基本互补的核苷酸序列。
在另一优选例中，所述的序列为RNA、DNA或RNA/DNA杂合的核苷酸序列。
在本发明的第二方面，提供了一种本发明第一方面的埃博拉病毒的microRNA前体序列或microRNA成熟序列的用途，用于制备检测埃博拉病毒的试剂或试剂盒。
在另一优选例中，所述的试剂或试剂盒用于早期诊断埃博拉病毒。
在另一优选例中，所述的试剂包括引物、探针、芯片。
在另一优选例中，所述检测是针对选自下组的样品：血清和血浆。
在另一优选例中，所述检测是针对选自下组的样品：埃博拉病毒感染生物(人，猩猩，猴子等)的血清，埃博拉病毒感染生物(人，猩猩，猴子等)的血浆。
在本发明的的第三方面，提供了一种抑制剂的用途，所述抑制剂是针对本发明第一方面的埃博拉病毒的microRNA前体序列或microRNA成熟序列，所述的抑制剂用于制备治疗埃博拉病毒感染的组合物，或用于制备抑制埃博拉病毒生长的组合物。
在另一优选例中，所述的抑制剂是特异性抑制SEQ ID NO.:35序列的抑制剂。
在另一优选例中，所述的组合物中还含有微小核糖核酸miR-2911。
在另一优选例中，所述的组合物包括药物组合物、保健品组合物。
在另一优选例中，所述的组合物包括药物组合物。
在另一优选例中，所述的抑制剂是反义核酸序列或海绵序列。
在另一优选例中，所述的抑制剂特异性结合于选自下组的核苷酸序列：
(I)SEQ ID NO.:28、22、24、和27中任一所述的核苷酸序列；
(II)SEQ ID NO.:35、21、29-34中任一所述的核苷酸序列。
在本发明的第四方面，提供了本发明第一方面所述的埃博拉病毒的microRNA前体序列或其成熟序列的用途，它们被用于制备判断埃博拉病毒感染后的康复状态或预后的试剂或试剂盒。
在另一优选例中，所述的microRNA前体序列或其成熟序列为如SEQ ID NO.:35所示的。
在本发明的第五方面，提供一种可用于预防或治疗埃博拉病毒感染或抑制埃博拉病毒的药物组合物，其特征在于，所述的组合物含有(i)药学上可接受的载体；(ii)针对第一方面所述的埃博拉病毒的microRNA前体序列或其成熟序列的抑制剂。
在另一优选例中，所述的抑制剂是反义核酸序列或海绵序列。
在另一优选例中，所述药物组合物还含有微小核糖核酸MiR-2911或含MiR-2911的提取物或组合物。
在本发明的第六方面，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：给需要的对象施用针对本发明第一方面所述的埃博拉病毒的microRNA前体序列或其成熟序列的抑制剂。
在另一优选例中，所述的方法还进一步包括：给所述对象施用额外的治疗剂，优选MiR-2911或含MiR-2911的提取物或组合物。
在本发明的第七方面，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；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，所述的埃博拉病毒蛋白选自下组：GP，VP40。
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。
在本发明的第八方面，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。
在本发明的第九方面，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。
在本发明的第十方面，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。
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是EBO-pre-miR-1二级结构示意图。
图9是EBO-pre-miR-2二级结构示意图。
图10是EBO-pre-miR-3二级结构示意图。
图11是EBO-pre-miR-4二级结构示意图。
图12是EBO-pre-miR-1保守性分析结果示意图。
图13是EBO-pre-miR-2保守性分析结果示意图。
图14是EBO-pre-miR-3保守性分析结果示意图。
图15是EBO-pre-miR-4保守性分析结果示意图。
图16显示了埃博拉病毒编码的pre-miR-VP在基因组中的位置和预测的颈环二级结构。
图17显示了导入pre-miR-VP序列的细胞RNA的Northern blot特异探针分析结果。
图18显示了埃博拉病毒感染患者的混合血清样本检测结果。
图19显示了miR-VP-3p检测的灵敏度。
图20显示了埃博拉病毒患者血清中miR-VP-3p的qRT-PCR分析结果。其中，图20A显示了miR-VP-3p在健康志愿者和EVD+ G+患者血清中各自的CT值；图20B显示miR-VP-3p在健康志愿者和EVD+ G+患者血清样本中的浓度；图20C显示了miR-VP-3p在埃博拉病毒康复者疾病发作期(EVD+ G+(P-N))和康复阶段(EVD- G-(P-N))血清中的浓度。
图21显示了miR-VP-3p的qRT-PCR检测结果。
图22显示了miR-VP-3p在埃博拉病毒康复者疾病发作期(EVD+ G+(P-N))和康复阶段(EVD- G-(P-N))血清中的CT值。
图23显示了miR-VP-3p在埃博拉病毒疑似患者不同时期血清检测结果。其中图23A显示了被确认为埃博拉病毒阳性的6名患者的检测结果(黑点，记为EVD+ G-(N-P)和EVD+ G+(N-P))；图23B显示了被确认为埃博拉病毒阳性的6名患者血清中检测到了可观的miR-VP-3p水平；图23C显示了仍为埃博拉病毒阴性的9名患者的检测结果(蓝点，记为EVDG-(S)和EVD- G-(S))。图23D显示了仍为埃博拉病毒阴性的9名患者的体内均未检测到miR-VP-3p。
图24显示了miR-VP-3p在15名疑似埃博拉患者血清中的CT值。
具体实施方式
本发明人经过广泛而深入的研究，对埃博拉病毒进行基因组学分析，基于对三种不同分型的埃博拉病毒的研究，成功发现了埃博拉病毒多种microRNA前体及对应的microRNA成熟体，实验证明，这些microRNA前体可在测试中成功产生对应成熟体的miRNA序列。本发明人还根据这些序列，通过设计好的基因组学分析方法研究了这些前体miRNA针对的靶基因，指明了埃博拉病毒可能产生的microRNA针对的靶基因和可能产生的影响，不仅为埃博拉病毒感染的早期诊断提供了一种全新的方法，还为埃博拉病毒的感染提供了一种可能的致病机理，为埃博拉病毒感染的治疗提供了一个潜在的靶点。
本发明人还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 VP40N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
Figure PCTCN2015088803-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。
埃博拉病毒特有的microRNA
如本文所用，术语“埃博拉病毒特有的microRNA”或“本发明的埃博拉病毒特有的microRNA”、“埃博拉病毒编码的microRNA”可互换使用，指名称为EBO-pre-miR-1、EBO-pre-miR-2、EBO-pre-miR-3和EBO-pre-miR-4等4种源自埃博拉病毒的microRNA。应理解，该术语包括前体或成熟形式的microRNA。
本发明人使用三种数据库综合分析的分析方法对埃博拉病毒的三种不同分型进行预测分析，得到多种预测结果。
基于实验验证，证实了三种埃博拉病毒可产生4种microRNA前体，而所述前体可在被感染的细胞内产生所对应的成熟体，从而作用于潜在的靶基因。
本发明所提供的4种pre-microRNA前体中包括雷斯顿埃博拉1种、苏丹埃博拉1种以及扎伊尔埃博拉2种。
基于对埃博拉病毒的microRNA前体及成熟体的序列保守性分析，进而判断四种pre-microRNA所针对的靶基因，以及针对该靶基因可能产生的后果。
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，有助于克服埃博拉抗体误判问题。
6)基于埃博拉病毒特有的microRNA，可进行埃博拉病毒的特异性检测，以达到埃博拉病毒感染后早期快速诊断埃博拉病毒感染目的，避免了病毒感染早期窗口期无法检测所造成的传染。
7)针对埃博拉病毒特有的microRNA，还可以设计特异性的microRNA抑制剂，特异性抑制埃博拉病毒的microRNA，抑制病毒的复制、传染等通路，从而达到治疗目的。
8)埃博拉病毒特有的microRNA所针对的靶基因序列，与埃博拉病毒部分病理学症状相符，这有助于埃博拉病毒的进一步研究与防治进程。
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
2.1MiR-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。
2.2MiR-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
SV40late 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2oligo DNA序列
Figure PCTCN2015088803-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。
3.2MiR-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-2911GGCCGGGGGACGGGCUGGGA(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4MiR-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基于埃博拉病毒的microRNA分析
从NCBI数据库获得扎伊尔埃博拉病毒(ZEBOV)、雷斯顿埃博拉病毒(REBOV)、苏丹埃博拉病毒(SEBOV)、科特迪瓦埃博拉病毒(TEBOV)的基因组，利用Vir-Mir预测扎伊尔埃博拉病毒(ZEBOV)、雷斯顿埃博拉病毒(REBOV)、苏丹埃博拉病毒(SEBOV)、科特迪瓦埃博拉病毒(TEBOV)能够编码的pre-microRNA。
通过分析，预测埃博拉病毒有以下7条序列可能可以编码pre-microRNA：
(1)>gi|22789222:896-978Reston ebolavirus(雷斯顿埃博拉病毒)全基因组
GCAAAUGCAGGGCAAUUUCUCUCAUUUGCGAGUUUGUUUCUUCCCAAACUGGUUGUGGGAGAGAAGGCUUGCUUGGAAAAAGU(SEQ ID NO.:22)
(2)>gi|22789222:1827-1915Reston ebolavirus(雷斯顿埃博拉病毒)全基因组
CUAAUGGUGCAAUUGACCCCGAGGAUGGUGAUUUUGAAAAUUACAAUGGCUAUCAUGAUGAUGAAGUUGGGACGGCAGGUGACUUGGUC(SEQ ID NO.:23)
(3)>gi|55770807:11450-11536Sudan ebolavirus(苏丹埃博拉病毒)全基因组
AUAGAACGAGGAAGAUUAAGAAAAAGUCCAUAAUGCUGGGGAGGCAAUCCUUGCCACCAUAGGACUUUUUCAAUUCCUCUAUUUUAU(SEQ ID NO.:24)
(4)>gi|55770807:c7690-7601Sudan ebolavirus(苏丹埃博拉病毒)全基因组
GUUUCAUUUGCAAGTTGCCTAAGTCCACAGACTAAGGCATTTTGGTTATGCATCAGGCCTTCAGTGTATATGCCTTCCGCACCCGGTCCA(SEQ ID NO.:25)
(5)>gi|55770807:c11622-11536Sudan ebolavirus(苏丹埃博拉病毒)全基因组
AUGCUCUUGUCACUAGGUCACAUUGGUCUAAGACAAUUGGGGAAGACAAUCUUGCAUCAGGAUAUUGUGUAUGUUGGGUAGCCAUCA(SEQ ID NO.:26)
(6)>gi|10313991:14252-14340Ebola virus-Mayinga，Zaire(扎伊尔博拉病毒)，1976strain Mayinga
GCAAACCUCUGGAUUUCGGAACAAUAUCAUUGGCACUAGCGGUACCGCAGGUGCUUGGAGGGUUAUCCUUCUUGAAUCCUGAGAAAUGU(SEQ ID NO.:27)
(7)>gi|10313991:c4799-4710Ebola virus-Mayinga，Zaire(扎伊尔博拉病毒)，1976strain Mayinga
GCUGUAGGUCUUUUGAUCAGCGACACCUAGAGGAAGCCAAAUUGGAAUUUGCUUCAUUAGCACUUUGGGGCCCGAUAUGACAUUCACCAU(SEQ ID NO.:28)
实施例6 4种埃博拉病毒特有的microRNA前体
6.1试剂和材料：
Hela细胞(上海酶联生物科技有限公司)
HEK-293T细胞(深圳市百恩维生物科技有限公司)
无内毒素质粒大提试剂盒(天根生化科技有限公司，北京)
Balb/c小鼠(南京君科生物工程有限公司)
pSuper质粒(Oligogene)
pSicoR，pCMV-VSV-G,pCMV-dR8.91(上海吉然生物科技有限公司)
限制性内切酶和T4连接酶(TAKARA)
6.2方法
针对预测得到的7种pre-microRNA序列，设计对应的oligo DNA，在两端加入BgIⅡ和HindⅢ酶切位点，利用限制性核酸内切酶BgIⅡ和HindⅢ对pSuper质粒和设计好的oligo DNA进行双酶切得到线性化pSuper质粒片段，将oligo DNA序列导入表达载体，通过脂质体转染Hela细胞表达，表达后，提取RNA进行检测。
检测结果，发现其中产生了筛选得到的4种pre-microRNA，另外3种预测不会产生的pre-microRNA未表达。
4种可被检测到pre-microRNA分别命名为：EBO-pre-miR-1、EBO-pre-miR-2、EBO-pre-miR-3和EBO-pre-miR-4
(1)EBO-pre-miR-1
>gi|22789222:896-978Reston ebolavirus(雷斯顿埃博拉病毒)，全基因组
GCAAAUGCAGGGCAAUUUCUCUCAUUUGCGAGUUUGUUUCUUCCCAAACUGGUUGUGGGAGAGAAGGCUUGCUUGGAAAAAGU(SEQ ID NO.:22)
(2)EBO-pre-miR-2
>gi|55770807:11450-11536Sudan ebolavirus(苏丹埃博拉病毒)，全基因组
AUAGAACGAGGAAGAUUAAGAAAAAGUCCAUAAUGCUGGGGAGGCAAUCCUUGCCACCAUAGGACUUUUUCAAUUCCUCUAUUUUAU(SEQ ID NO.:24)
(3)EBO-pre-miR-3
>gi|10313991:14252-14340Ebola virus-Mayinga，Zaire(扎伊尔埃博拉病毒)，1976strain Mayinga
GCAAACCUCUGGAUUUCGGAACAAUAUCAUUGGCACUAGCGGUACCGCAGGUGCUUGGAGGGUUAUCCUUCUUGAAUCCUGAGAAAUGU(SEQ ID NO.:27)
(4)EBO-pre-miR-4
>gi|10313991:c4799-4710Ebola virus-Mayinga，Zaire(扎伊尔埃博拉病毒)，1976strain Mayinga
GCUGUAGGUCUUUUGAUCAGCGACACCUAGAGGAAGCCAAAUUGGAAUUUGCUUCAUUAGCACUUUGGGGCCCGAUAUGACAUUCACCAU(SEQ ID NO.:28)
实施例7埃博拉病毒特有microRNA的结构分析
对于实施例6中所检测到的4种pre-miRNA，进一步对其进行结构分析。
7.1Dicer切割
结果表明，它们可以被Dicer切割，并最终可以产生成熟体的pre-miRNA。
7.2 4条pre-miRNA的二级结构。
分析得出的二级结构如下：
(1)EBO-pre-miR-1
如图8所示。
(2)EBO-pre-miR-2
如图9所示。
(3)EBO-pre-miR-3
如图10所示。
(4)EBO-pre-miR-4
如图11所示。
7.3保守性分析
从NCBI数据库下载埃博拉病毒基因组，其中扎伊尔埃博拉病毒(ZEBOV)6种，雷斯顿埃博拉病毒(REBOV)3种，苏丹埃博拉病毒(SEBOV)2种,科特迪瓦埃博拉病毒(TEBOV)2种，利用NCBI数据库的BLAST软件对埃博拉病毒可能编码的4条pre-miRNA 序列进行保守性分析。
分析结果表明，4条pre-miRNA序列在各个埃博拉病毒亚型中都非常保守，这充分说明了埃博拉病毒各个亚型都可以编码这4条pre-miRNA，并且和可能在埃博拉病毒感染过程中发挥了重要作用。
(1)EBO-pre-miR-1
保守性分析结果见图12。
(2)EBO-pre-miR-2
保守性分析见图13。
(3)EBO-pre-miR-3
保守性分析见图14。
(4)EBO-pre-miR-4
保守性分析结果见图15。
7.4microRNA成熟体分析
通过计算和分析，确定埃博拉病毒编码的4条pre-miRNA可以编码8条microRNA成熟体。结果如下：
(1)EBO-pre-miR-1
Ebo-miR-1-5p Position 11 GCAAUUUCUCUCAUUUGCGAGU(SEQ ID NO.:21)
Ebo-miR-1-3p Position 49 UGGUUGUGGGAGAGAAGGCUUG(SEQ ID NO.:29)
(2)EBO-pre-miR-2
Ebo-miR-2-5p Position 20 AAAAAGUCCAUAAUGCUGGGGA(SEQ ID NO.:30)
Ebo-miR-2-3p Position 52 GCCACCAUAGGACUUUUUCAAU(SEQ ID NO.:31)
(3)EBO-pre-miR-3
Ebo-miR-3-5p Position 33 CACUAGCGGUACCGCAGGUGCU(SEQ ID NO.:32)
Ebo-miR-3-3p Position 62 UUAUCCUUCUUGAAUCCUGAGA(SEQ ID NO.:33)
(4)EBO-pre-miR-4
Figure PCTCN2015088803-appb-000003
实施例8埃博拉病毒特有microRNA的靶基因分析
根据美国国立图书馆(NCBI)的GENEBANK数据库已经公开的人类(human)蛋白编码基因的3'UTR序列，
利用生物信息学软件RNAhydrid、RNFFOLD、Fast Folding and Comparison of RNA Secondary Structures对埃博拉病毒编码的8条microRNA可能调控的靶基因进行预测。利用microRNA靶基因筛选规则：
1)mfe<-20kcal/mo；
2)种子序列完全互补；
3)miRNA至多有10个碱基不互补；
4)loop至多有8个碱基。
结果表明，埃博拉病毒编码的8条microRNA可能调控的代表性靶基因包括表3中所列的基因：
表3
Figure PCTCN2015088803-appb-000004
Figure PCTCN2015088803-appb-000005
通过结果可以看出，埃博拉病毒编码的microRNA可能调控可以识别病毒RNA的RNA结合蛋白,这很有可能导致埃博拉病毒无法被人体免疫系统识别，从而得以在人体内大量复制，此结果对于埃博拉病毒的进一步研究是非常有利的。
实施例8成熟体microRNA的检测
方法同实施例6，对转染细胞的microRNA成熟体进行检测。
结果表明，检测到4种pre-microRNA前体产生的8种成熟体microRNA。
实施例9
基于埃博拉病毒特有的microRNA的诊断和检测方法
基于血清microRNA作为分子标志物的研究，可以利用埃博拉病毒编码的microRNA作为埃博拉病毒早期诊断的分子标志物。
可用于检测埃博拉病毒特有的microRNA的方法包括：RT-PCR方法、Real-time PCR方法、Northern blotting方法、RNase protection assay方法、Solexa sequencing technology方法以及生物芯片方法中的一种或几种。
9.1RT-PCR方法
包括以下步骤：
(1)提取总RNA，通过RNA逆转录反应得到cDNA样品；或者收集受试者的体液样本，以体液作为缓冲液进行逆转录反应来制备cDNA样品；
(2)用微小核糖核酸设计引物进行PCR反应；
(3)进行PCR产物的琼脂糖凝胶电泳；
(4)EB染色后在紫外灯下观察结果。
9.2Real-time PCR方法
包括以下步骤：
(1)提取受试者的体液总RNA，通过RNA逆转录反应得到cDNA样品；或者收集受试者的体液样本，以体液作为缓冲液进行逆转录反应来制备cDNA样品；
(2)用微小核糖核酸设计引物；
(3)加入荧光探针进行PCR反应；
(4)检测并比较体液样本相对于正常体液中微小核糖核酸的量的变化。
9.3Northern blotting方法
包括以下步骤：
1、收集体液样本；
2、通过Trizol试剂提取体液总RNA；
3、进行变性PAGE电泳和膜转移实验；
4、制备同位素标记微小核糖核酸探针；
5、进行膜杂交反应；
6、同位素信号检测，如磷屏扫描检测结果。
9.4RNase protection assay方法
包括如下步骤：
1、进行反义RNA探针的合成，同位素标记与纯化；
2、收集体液样本并提取RNA；
3、将提取后的RNA溶解在杂交缓冲液中并加入反义RNA探针进行杂交反应；
4、加入RNase消化液进行反应；
5、进行电泳与放射自显影；
6、分析结果。
9.5Solexa sequencing technology方法包括如下步骤：
1、收集体液样本；
2、通过Trizol试剂提取体液总RNA；
3、进行PAGE电泳回收RNA分子；
4、将adaptor prime酶联在小RNA分子的3'与5'端；
5、进行RT-PCR反应后并进行测序；
6、数据分析与处理。
9.6生物芯片方法包括如下步骤：
1、将成熟体微小核糖核酸库点阵并制备生物芯片；
2、收集体液样本；
3、提取体液总RNA；
4、通过柱分离来分离微小核糖核酸；
5、利用T4RNA连接酶进行微小核糖核酸荧光标记；
6、与生物芯片进行杂交反应；
7、数据检测与分析。
通过上述的RT-PCR，Real-time PCR，Northern blotting，RNase protection assay，Solexa sequencing technology和生物芯片等方法分析埃博拉病毒微小核糖核酸的变化趋势及变化量，达到早期快速诊断埃博拉病毒感染的目的。
实施例10埃博拉病毒miRNA前体序列的分析
针对埃博拉病毒的基因组序列(NC002549)，用实施例5所述分析miRNA前体序列的发夹结构。
结果如下：图16显示了埃博拉病毒编码的pre-miR-VP在基因组中的位置和预测的颈环二级结构。其中，来自3'端的成熟体miRNA的颈环前体用红色标出。位于 埃博拉病毒VP40、NP和L基因中的3个假设的前体分别被成功地预测出来。
实施例11埃博拉病毒成熟体miRNA序列
针对实施例10中得到的前体，通过MatureBayes工具和Byes-SVM-MiRNA分析工具，分析成熟体miRNA序列。
分析结果揭示，在前体3'端存在的3种假定的成熟体miRNAs(目前称为miR-VP-3p,miR-NP-3p和miR-L-3p)。因为miRNAs跨物种高度保守，通过将前体序列与现有已公开的2014EBOV埃博拉病毒毒株全长基因组序列进行Blast比对，检测假定埃博拉病毒miRNAs的进化保守性。
结果如下：2014EBOV埃博拉病毒毒株中Pre-miR-VP和pre-miR-L在2014EBOV埃博拉病毒毒株中高度保守，而pre-miR-NP不保守。pre-miR-VP和pre-miR-L被保留以进行进一步分析。
实施例12预测埃博拉病毒miRNA的验证试验
为研究预测的埃博拉病毒miRNA是否可以由前体折叠成为典型的发卡结构并被Dicer酶识别进而在细胞环境内产生成熟体miRNA，将pre-miR-VP序列克隆入质粒并转染进入HEK293A细胞中。通过Northern blot的特异探针对细胞RNA进行了分析。
为确定pre-miR-VP序列对于miR-VP-3p的产生是必不可少的，在发夹结构中引入了一个突变，将突变后的pre-miR-VPmut序列克隆入质粒并转染进入HEK293A细胞中。通过Northern blot的特异探针对细胞RNA进行了分析。
结果如图17所示：导入pre-miR-VP序列的细胞中出现两条假定埃博拉病毒miRNA的成熟体(大概20个核苷酸长度的条带为miR-VP-3p)和发卡前体(大概80个核苷酸长度的条带为pre-miR-VP)。而导入突变后的pre-miR-VPmut序列的细胞中没有miR-VP-3p的条带出现，表明成熟体miR-VP-3p是通过特异性切割pre-miR-VP的发夹结构所产生的。结果表明，在细胞环境中，埃博拉病毒包含可以形成miRNA前体发卡结构的片段，并且其可以进而通过加工而产生miRNA。
实施例13血清样本中miR-VP-3p的检测
分别从埃博拉病毒感染患者和健康志愿者的血清样本中提取小分子RNA。利用Northern blot方法检测血清中miR-VP-3p的含量及灵敏度。
本研究实施例中使用了世界卫生组织建立的标准病例定义，对83名接受治疗的埃博拉病毒确认患者进行了回顾性观察研究。其中，实验室对于埃博拉病毒的确认基于实时定量荧光PCR。
治疗依照世界卫生组织制定的关于病毒性出血热病例管理的临时应急指导方案进行。方案已通过伦理委员会批准。每位参与研究的患者在研究开始之前均收到了书面知情同意书。所有临床血清样本均来自于寄往埃博拉病毒诊断流动实验室的样本。血清通过60℃热处理1小时灭活，随后分离并储存于-20℃备用。包括年龄、性别和 症状发作日期在内的流行病学信息通过调查问卷的方式得到记录，并随血清样本一同发送。
常规的Northern blot方法具体操作如实施例9所述。其中特异性的探针序列为：miR-VP-3p探针(5’-GCCCCAAAGTGCTAATGAAGCA-3’SEQ ID NO.:36)，miR-16对照探针(5’-CGCCAATATTTACGTGCTGCTA-3’(SEQ ID NO.:37))。
结果如图18所示，埃博拉病毒感染患者的混合血清样本中可以检测到miR-VP-3p，而健康志愿者中未检测到。
其中，EVD+ G-(N-P)代表：埃博拉病毒核酸检测阴性转阳性的患者核酸检测呈阴性时的血清；
EVD+ G+(N-P)代表：埃博拉病毒核酸检测阴性转阳性的患者核酸检测呈阳性时的血清；
EVD+ G+(P-N)代表：埃博拉病毒康复患者在疾病发作期核酸检测呈阳性时的血清；
EVD+ G-(P-N)代表：埃博拉病毒康复患者在经过治疗康复后核酸检测呈阴性时的血清；
EVD- G-(P-N)代表：埃博拉病毒核酸检测呈阴性的埃博拉病毒感染疑似患者，经过隔离观察最后确认非埃博拉病毒感染的病人。
作为对照，在埃博拉病毒感染患者和健康志愿者的血清样本中也检测了miR-16的水平，结果显示在两个组别中其含量没有差异。
图19显示了miR-VP-3p检测的灵敏度，通过计算得出miR-VP-3p在埃博拉病毒感染患者血清中存在的含量大于100fmol/L。
实施例14埃博拉病毒感染患者体内小RNA片段的鉴定
通过RT-PCR结合TA克隆和测序技术，对埃博拉病毒感染患者体内小RNA片段的进行鉴定。从埃博拉病毒感染患者血清中分离的小RNA经过RT-PCR以扩增埃博拉病毒的miRNA，随后扩增产物被连接到TA质粒并测序。
测序结果显示，埃博拉病毒患者样本中产物的序列与预测的miR-VP-3p序列完全一致，而存在于健康志愿者样本中的序列是多样的，并且与miR-VP-3p的序列不同。然而，对于miR-L-3p，Northern blotting和RT-PCR产物的测序并未在埃博拉病毒感染患者的血清中发现假定埃博拉病毒miRNA的特异序列存在。
结果表明miR-VP-3p确实出现在埃博拉病毒感染患者的血清中。
实施例15患者血清中miR-VP-3p水平和埃博拉病毒感染严重程度的相关性检验
利用基于TaqMan探针技术的定量RT-PCR(qRT-PCR)测序方法分别检测了27名埃博拉病毒感染者(记为EVD+ G+)和13名健康志愿者血清样本中的miR-VP-3p。
为了确定用于检测miR-VP-3p的qRT-PCR测序的动态范围与敏感性，合成的单链miR-VP-3p被分级稀释、并通过qRT-PCR测序进行检测，用于建立标准曲线，一个无模板对照参与检测用于确定引物的特异性。合成的miR-VP-3p可以高效扩增，无模 板对照进行了更多轮的扩增循环(图21)，表明miR-VP-3p动态量化范围的下限为0.1阿摩尔(1attomole＝10-18mole)。
结果如下：
图20显示了埃博拉病毒患者血清中miR-VP-3p的qRT-PCR分析结果。其中，图20A显示了miR-VP-3p在健康志愿者和EVD+ G+患者血清中各自的CT值。蓝线为动态范围的下限。EVD+ G+患者的CT值一直位于EVD+ G+线性范围内，但位于健康志愿者的线性范围外(表4),表明在EVD+ G+患者体内可检测到miR-VP-3p,但其在健康志愿者体内不存在。
表4qRT-PCR检测EVD+ G+患者与健康志愿者血清中miRNA的原始CT值
Figure PCTCN2015088803-appb-000006
根据标准曲线计算了miR-VP-3p的浓度。
图20B显示miR-VP-3p(SEQ ID NO.:35)在健康志愿者和EVD+ G+患者血清样本中的浓度。在健康志愿者的血清中无法检测到miR-VP-3p，相对的，在EVD+ G+患者血清中的水平为205.3飞摩尔(1femtomole＝1x10-15mole)每升。EVD+ G+患者和健康志愿者血清中的miR-16(UAGCAGCACGUAAAUAUUGGCG，SEQ ID NO.:38)水平没有差异。
实施例16miR-VP-3p和基因组RNA存在的持续性检测
收集8对埃博拉病毒康复患者的血清样本并检测了这些样本在疾病发作期(记为EVD+ G+(P-N)；埃博拉病毒康复患者在疾病发作期核酸检测呈阳性)和康复阶段(记为EVD- G-(P-N)；埃博拉病毒核酸检测呈阴性的埃博拉病毒感染疑似患者，经过隔离观察最后确认非埃博拉病毒感染的病人)的miR-VP-3p含量。
结果如下：
图20C显示了miR-VP-3p在埃博拉病毒康复者疾病发作期(记为EVD+ G+(P-N))和康复阶段(记为EVD- G-(P-N))血清中的浓度。miR-VP-3p在疾病发作期显示出高含量水平，但在完全康复后消失。Northern blot分析确认了miR-VP-3p在埃博拉病毒康复者的康复阶段的血清中不存在。结果表明miR-VP-3p在埃博拉病毒患者患病的疾病发作期存在，并在康复阶段消失。
图22显示了miR-VP-3p在埃博拉病毒康复者疾病发作期(记为EVD+ G+(P-N))和康复阶段(记为EVD- G-(P-N))血清中的CT值。一个无模板对照参与检测用于确定引物的特异性。合成的miR-VP-3p可以高效扩增，无模板对照进行了更多轮的扩增循环。蓝线表示无模板阴性对照。蓝线以上表示范围以外，无效扩增；蓝线以下表示范围以内，有效扩增。结果表明miR-VP-3p在埃博拉病毒患者患病的疾病发作期存在，并在康复阶段消失。
实施例17埃博拉病毒疑似患者中埃博拉病毒的miR-VP-3p的qRT-PCR分析
埃博拉病毒疑似患者是指开始参与检测时，表现出了埃博拉病毒感染症状，但通过RT-PCR检测的埃博拉基因组RNA结果为阴性的患者。
对疑似患者进行后续观察，检测其miR-VP-3p含量。
图23显示了miR-VP-3p在埃博拉病毒疑似患者不同时期血清中的浓度。其中图23A显示了被确认为埃博拉病毒阳性的6名患者的检测结果(黑点，记为EVD+ G-(N-P)和EVD+ G+(N-P))；图23B显示了被确认为埃博拉病毒阳性的6名患者血清中检测到了可观的miR-VP-3p水平；图23C显示了仍为埃博拉病毒阴性的9名患者的检测结果(蓝点，记为EVDG-(S)和EVD- G-(S))。图23D显示了仍为埃博拉病毒阴性的9名患者的体内均未检测到miR-VP-3p。
图24显示了miR-VP-3p在15名疑似埃博拉患者血清中的CT值。蓝线表示无模板阴性对照。蓝线以上表示范围以外，无效扩增；蓝线以下表示范围以内，有效扩增。
分析确认，在疑似患者血清中，在埃博拉病毒基因组RNA被检测到之前，miR-VP-3p就已经存在了。由于诊断窗口期的原因，miR-VP-3p的qRT-PCR测序比埃博拉病毒基因组RNA的检测更为灵敏，因此，在埃博拉病毒阳性患者发展为可检测到的病毒血症之前，该小RNA片段可作为早期诊断埃博拉病毒的潜在生物标志物。
讨论
基于microRNA作为分子标志物的研究，可以利用埃博拉病毒编码的microRNA作为埃博拉病毒早期诊断的分子标志物。更值得注意的是，其中还可以注意到埃博拉病毒编码的microRNA可能调控了大量的ATPase(包括Ca++transporting，H+transporting，Na+/K+transporting等)离子通道蛋白，这很有可能导致了人体内电解质的剧烈紊乱，从而导致人体血管内皮细胞通透性增加，内皮细胞破裂，从而出现大量的内出血，主要脏器溶解等症状。
上述预测结果为埃博拉病毒的高致死率以及造成人体大量出血和脏器溶解等症状提供了一种新的解释。结合目前国内外研究，可以利用埃博拉病毒编码的microRNA作为治疗埃博拉病毒感染的药物靶点，这为埃博拉病毒感染的治疗提供了一种新的途径和方法。
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 (12)

  1. 一种埃博拉病毒的microRNA前体序列或其成熟序列，其特征在于，所述的microRNA前体序列选自下组：
    (a)SEQ ID NO.:28、22、24、和27中任一所述的核苷酸序列；
    (b)与组(a)中任一核苷酸序列完全互补或基本互补的核苷酸序列；
    所述的microRNA成熟序列选自下组：
    (i)SEQ ID NO.:35、21、29-34中任一所述的核苷酸序列；
    (ii)与组(i)中任一核苷酸序列完全互补或基本互补的核苷酸序列。
  2. 如权利要求1所述的microRNA前体序列或其成熟序列，其特征在于，所述的序列为RNA、DNA或RNA/DNA杂合的核苷酸序列。
  3. 一种权利要求1所述的埃博拉病毒的microRNA前体序列或其成熟序列的用途，其特征在于，用于制备检测埃博拉病毒的试剂或试剂盒。
  4. 如权利要求3所述的用途，其特征在于，所述的试剂或试剂盒用于早期诊断埃博拉病毒。5.如权利要求3所述的用途，其特征在于，所述的试剂包括引物、探针、芯片。
  5. 如权利要求3所述的用途，其特征在于，所述检测是针对选自下组的样品：血清和血浆。
  6. 一种抑制剂的用途，所述抑制剂是针对权利要求1所述的埃博拉病毒的microRNA前体序列或其成熟序列，其特征在于，所述的抑制剂用于制备治疗埃博拉病毒感染的组合物，或用于制备抑制埃博拉病毒生长的组合物。
  7. 如权利要求7所述的用途，其特征在于，所述的抑制剂是特异性抑制SEQ ID NO.:35序列的抑制剂。
  8. 如权利要求7所述的用途，其特征在于，所述的抑制剂是反义核酸序列或海绵序列；
    较佳地，所述的抑制剂特异性结合于选自下组的核苷酸序列：
    (I)SEQ ID NO.:28、22、24、和27中任一所述的核苷酸序列；
    (II)SEQ ID NO.:35、21、29-34中任一所述的核苷酸序列。
  9. 如权利要求1所述的埃博拉病毒的microRNA前体序列或其成熟序列的用途，其特征在于，用于制备判断埃博拉病毒感染后的康复状态或预后的试剂或试剂盒。
  10. 一种药物组合物，其特征在于，所述的组合物含有(i)药学上可接受的载体；(ii)针对权利要求1所述的埃博拉病毒的microRNA前体序列或其成熟序列的抑制剂。
  11. 如权利要求11所述的药物组合物，其特征在于，还含有微小核糖核酸MiR-2911或含MiR-2911的提取物或组合物。
  12. 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：给需要的对象施用针对权利要求1所述的埃博拉病毒的microRNA前体序列或其成熟序列的抑制剂。
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