WO2014026627A1 - 植物微小核糖核酸的提取、制备及其应用 - Google Patents

植物微小核糖核酸的提取、制备及其应用 Download PDF

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WO2014026627A1
WO2014026627A1 PCT/CN2013/081583 CN2013081583W WO2014026627A1 WO 2014026627 A1 WO2014026627 A1 WO 2014026627A1 CN 2013081583 W CN2013081583 W CN 2013081583W WO 2014026627 A1 WO2014026627 A1 WO 2014026627A1
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plant
microrna
mir2911
extract
gene
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PCT/CN2013/081583
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English (en)
French (fr)
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张辰宇
张峻峰
曾科
董磊
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北京命码生科科技有限公司
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Priority to US14/422,117 priority Critical patent/US9752148B2/en
Priority to EP13829580.3A priority patent/EP2886652B1/en
Priority to CN201380042918.6A priority patent/CN104640987B/zh
Priority to KR1020157006402A priority patent/KR101724218B1/ko
Priority to JP2015526868A priority patent/JP6231099B2/ja
Publication of WO2014026627A1 publication Critical patent/WO2014026627A1/zh
Priority to HK15109799.7A priority patent/HK1209157A1/zh

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    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/178Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention belongs to the field of biology.
  • the invention relates to methods of extracting plant microRNAs and uses thereof. Background technique
  • a small ribonucleic acid is a non-coding single-stranded small ribonucleic acid molecule of about 19 to 23 nucleotides in length. They are widely found in animal and plant cells and are highly conservative in evolution. MicroRNAs do not completely complement each other by recognizing the 3' untranslated sequence of the target messenger RNA (mR A), thereby inhibiting translation of the corresponding protein. As a powerful regulator of mR A, microRNAs are closely related to physiological activities, including biological activities such as development, tissue differentiation, apoptosis, and energy metabolism. At the same time, microRNAs are closely related to the occurrence and development of many diseases.
  • Patent PCT/CN2010/000677 discloses the regulation of rice roots by MIR164 derived from rice, and proposes to construct a nucleic acid fragment comprising a MIR164 sequence and transfer it to a rice plant to obtain a transgenic rice having a stronger root than that of ordinary rice.
  • Patent PCT/IB2010/055600 discloses that up-regulation of several microRNAs, including MIR156, increases plant tolerance to environmental stress factors, thereby increasing plant biomass, vigor and yield.
  • One of the objects of the present invention is to provide a plant microRNA having a function of regulating a non-plant target gene or a plant extract containing the microRNA, and a preparation method and use thereof.
  • Another object of the invention is to provide a method of identifying plant functional microRNAs.
  • an isolated plant functional microRNA or an extract comprising the plant functional microRNA comprising the plant functional microRNA, the plant functional microRNA being derived from an endogenous microRNA of a plant and present In the water-soluble and/or fat-soluble extract of the plant, and the plant functional microRNA has a function of regulating a non-plant target gene.
  • the plant functional microRNA comprises MIR2911; and the isolated plant functional microRNA or the extract containing the plant functional microRNA, the content of MIR2911 is 70%, based on the total microRNA .
  • the content of MIR2911 is 80%; preferably, 90%; more preferably, 100%.
  • the total number of microRNAs refers to the sum of the number of microRNAs having a length of 18-24 nt.
  • the modulation comprises inhibiting (downregulating) expression of a target gene and promoting (upregulating) expression of a target gene.
  • the non-plant target gene includes a bacterial gene, a viral gene, a Chlamydia gene, a yeast gene, Animal gene.
  • the plant comprises: a medicinal plant, a fruit and vegetable plant, an ornamental plant; more preferably: honeysuckle, indigo, grass daqing, horse blue, populus, cowpea, cotton, Chinese cabbage or potato .
  • the plant is honeysuckle, indigo, turmeric, horse blue or Populus euphratica; more preferably, the plant is honeysuckle.
  • the plant functional micraRNA is a microRNA species enriched in the water-soluble and/or fat-soluble extract of the plant (eg, the top 20 in abundance, preferably the top 10) microR A species).
  • the plant functional microRNA further comprises one or more selected from the group consisting of MIR156h, MIR166f, MIR396a, MIR166a, MIR168a, MIR1440, MIR2910 MIR2915 MIR2916 MIR818d, MIR159e, MIR159c, MIR156j, MIR1432, MIR166k, MIR167b, MIR396c, MIR156e, MIR169k, MIR167c, MIR160d, MIR399a, MIR156d, MIR160e, MIR169n, MIR166K MIR159f, MIR166c, MIR159b, MIR166j, MIR167i, MIR169c, MIR164c, MIR167j, MIR167g, MIR160c, MIR399e, MIR399b, MIR529b , MIR164e, MIR166d, MIR166h, MIR164b, MIR156f, MIR164a, MIR169K M
  • the plant extract comprises a water soluble and/or fat soluble extract of the plant.
  • the plant extract comprises an extract of branches, leaves, roots, flowers, fruits and/or stems of the plant.
  • an isolated plant functional microRNA or an extract comprising the plant functional microRNA according to the first aspect of the invention which (a) is used for the preparation of a regulatory non- a composition of a plant target gene; or (b) a medicament for the preparation of a disease associated with a non-plant target gene.
  • the non-plant target gene includes a bacterial gene, a viral gene, a Chlamydia gene, a yeast gene, and an animal gene.
  • the non-plant target gene is a gene of a pathogen (including bacteria, viruses, chlamydia, etc.).
  • the non-plant target gene-related diseases include: tumors (such as liver cancer, lung cancer); acute and chronic infectious diseases (such as viral influenza, viral hepatitis, AIDS, SA S, etc., such as viral diseases, such as Bacterial diseases such as tuberculosis and bacterial pneumonia, and acute and chronic infectious diseases caused by pathogenic microorganisms; Other acute and chronic diseases (such as respiratory diseases, immune system diseases, blood and hematopoietic diseases, circulatory systems such as cardiovascular and cerebrovascular diseases) Disease, endocrine system metabolic disease, digestive system disease, nervous system disease, urinary system disease, reproductive system disease and motor system disease).
  • tumors such as liver cancer, lung cancer
  • acute and chronic infectious diseases such as viral influenza, viral hepatitis, AIDS, SA S, etc.
  • viral diseases such as Bacterial diseases such as tuberculosis and bacterial pneumonia
  • the plant functional microRNA comprises MIR2911. More preferably, the medicament is for the treatment of viral influenza.
  • composition comprising (a) a pharmaceutically acceptable carrier or a food acceptable carrier and (b) the isolated plant function of the first aspect of the invention A microRNA and/or a plant extract containing the plant functional microRNA.
  • 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 microRNA if.
  • the composition comprises a pharmaceutical composition, a food composition or a health care product composition.
  • the method of preparing the composition comprises the steps of: mixing the plant functional microR A or the plant extract containing the functional micraRNA with a pharmaceutically or food acceptable carrier, Thereby the composition is formed.
  • the plant functional micraR A is derived from the following plants: a medicinal plant, a fruit and vegetable plant, an ornamental plant, and a non-plant target gene selected from the group consisting of: a bacterial gene, a viral gene, a Chlamydia gene, Yeast gene, animal gene.
  • the plant functional micraRNA is derived from honeysuckle, indigo, turfgrass, mala, or populus. More preferably, the plant functional microR A comprises MIR2911.
  • a method for non-therapeutic regulation of non-plant target gene expression in vitro wherein the non-plant target gene comprises a bacterial gene, a viral gene, a Chlamydia gene, a yeast gene, an animal gene, and the method
  • the method comprising the steps of: cultivating the biological material containing the target gene in the presence of the isolated plant functional microRNA of the first aspect of the invention or the extract containing the plant functional microRNA, thereby regulating the non-plant target gene expression.
  • the target gene is a gene of a pathogen (including bacteria, viruses, chlamydia, etc.).
  • the biological material comprises a virus, a cell, a tissue.
  • the plant functional micraRNA is derived from the following plants: medicinal plants, fruit and vegetable plants, ornamental plants.
  • the plant functional micraRNA is derived from honeysuckle, indigo, turfgrass, mala, or populus. More preferably, the plant functional microRNA comprises MIR2911.
  • a method for identifying a plant functional microRNA, wherein the plant functional micraRNA has a function of regulating a non-plant target gene comprising the steps of:
  • the non-plant target gene comprises a gene in a gene database.
  • the non-plant target gene includes a bacterial gene, a viral gene, a Chlamydia gene, a yeast gene, and an animal gene.
  • the non-plant target gene is a pathogen gene.
  • the plant comprises a medicinal plant, a fruit and vegetable plant.
  • the microRNA species in the top 20 are selected in step (3) for comparison and analysis.
  • a plant microRNA species having a ratio Lm/La ⁇ 130% is selected and compared in step (3), wherein Lm is a certain The abundance (or level) of the plant microRNA in the extract, and La is the average abundance (or level) of the total microRNA of the plant.
  • a plant functional microRNA molecule identified by the method of the fifth aspect of the invention.
  • the microRNA molecule comprises MIR2911.
  • a method of preparing a composition comprising the steps of:
  • the plant functional microRNA molecule is mixed with a pharmaceutically or food acceptable carrier to form a composition.
  • the extract (unconcentrated or concentrated) contains 0.01-100 nM (preferably 0.1-20 nM) of MIR2911; or
  • the content of MIR2911 is 70%; preferably, 80%; more preferably, ⁇ 90; optimally, 100%, based on the total microRNA.
  • a method for preventing or treating a disease wherein the disease is a disease associated with a non-plant target gene, the method comprising the step of administering to a subject in need thereof the first aspect of the invention An isolated plant functional microRNA or an extract comprising the plant functional microRNA, or a composition according to the third aspect of the invention, thereby preventing or treating the disease, wherein the plant functional microRNA has regulation The function of non-plant target genes.
  • the subject comprises a mammal (e.g., a human).
  • the non-plant target gene includes a bacterial gene, a viral gene, a Chlamydia gene, a yeast gene, and an animal gene.
  • the non-plant target gene is a pathogen gene.
  • a method for screening a candidate substance for an antiviral active ingredient comprising the steps of:
  • the microRNA species is selected as a candidate substance for the antiviral active ingredient.
  • the method further includes the steps of:
  • the high stability microRNA is selected from the group consisting of:
  • the top 5 most stable, preferably the first 3, more preferably the first microRNA;
  • C Q is the concentration at which the hour is placed
  • C 12 is the concentration when placed for 12 hours.
  • Co is not zero.
  • the non-plant target gene is a viral gene.
  • the miR A ranks 5th in abundance (high to low) in fresh plants. After that, it is preferably the type after the 10th place.
  • the plant functional microRNA molecule is mixed with a pharmaceutically or food acceptable carrier to form a composition.
  • a method of increasing the abundance of MIR2911 comprising the steps of:
  • n is a positive integer of 2 (preferably 5 or 10), and one of the microRNAs is MIR2911 ;
  • the extract is an aqueous extract or an alcohol extract.
  • the aging treatment is carried out at 20-50 ° C for 2 hours to 10 days.
  • the method further comprises using the obtained MIR2911 abundance-enhanced extract for pharmaceutical use, or for storage at a low temperature (e.g., 0 ° C to -4 ° C or lower).
  • the predetermined value is 60%, 70%, 80%, 90%, 95%, or 99%.
  • the abundance is calculated as follows
  • Figure 1 shows the results of Solexa sequencing of plant microRNA in honeysuckle aqueous extract.
  • Figure 2A is a Real-time PCR result of the expression of honeysuckle microRNA in serum at different time points after oral administration of honeysuckle water extract in mice.
  • the detection time points were 0 h (0 hours), 2 h (2 hours), 4 h (4 hours), and 6 h (6 hours).
  • Fig. 2B is a Real-time PCR result of the expression of honeysuckle microRNA in liver and lung at different time points after oral administration of honeysuckle water extract in mice.
  • the detection time points were 0 h (0 hours), 5 h (5 hours), 10 h (10 hours), 15 h (15 hours), 20 h (20 hours), and 25 hours (25 hours).
  • Figure 3 shows the results of sequence analysis of MIR2911 predicted target genes.
  • Mfe indicates the lowest folding free energy of the candidate target gene, and the larger the absolute value of mfe, the higher the matching degree of the candidate target gene with the Peu-MIR2911 sequence.
  • Figure 4 shows the results of luciferase assay for predicting target genes.
  • the predicted genes include the ADV gene, the HPIV gene, the H1N1 gene, and the H5N1 gene.
  • Figure 5A is a diagram showing the content of the viral gene ADV5 in MDCK cells by Real-time PCR.
  • Figure 5B is a diagram showing the content of the viral gene H1N1 in MDCK cells by Real-time PCR.
  • Figure 5C shows the detection of the viral gene H5N1 in MDCK cells by Real-time PCR.
  • Fig. 5A, Fig. 5B and Fig. 5C "uninfected” represents cells not infected with influenza virus; “infected untreated” represents cells infecting influenza virus and not treated with any drug; “MIR2911” represents infecting influenza virus And cells treated with MIR2911; 'WC' stands for cells infected with influenza virus and treated with the nonsense sequence RA of MIR2911; “Duffy” stands for influenza virus infection and Tamiflu (Osmivir, specific influenza virus) Cells treated with a neuraminidase inhibitor).
  • Figure 6 shows Real-time PCR results of MIR2911 in MIR2911-loaded cell microsomes secreted by HEK 293T cells transfected with MIR2911.
  • Figure 7A shows the expression level of influenza virus ADV in HEIR 293T cells treated with MIR2911-loaded microparticles.
  • Figure 7B shows the expression level of influenza virus H1N1 in HEIR 293T cells treated with MIR2911-loaded microparticles.
  • Fig. 7A and Fig. 7B HEK 293T cells treated with cell microparticles not loaded with MIR2911 after infection with influenza virus were used as a negative control.
  • Fig. 8A shows the rapid expression of the cold virus ADV in the lung after the mouse drinking honeysuckle aqueous extract, the honeysuckle aqueous extract + anti-MIR2911 (oral group), the irrigated honeysuckle aqueous extract + anti-MIR291 K irrigation group) Time PCR results.
  • Fig. 8B shows the real-time expression of the cold virus H1N1 in the lung after the mouse drinking honeysuckle aqueous extract, the honeysuckle aqueous extract + anti-MIR2911 (oral group), the lung filling honeysuckle aqueous extract + anti-MIR2911C irrigation lung group) PCR results.
  • Figure 9 shows the real-time PCR results of MIR2911 in human blood after taking the honeysuckle water extract.
  • the detection time points are 0 h (0 hours), 1 h (1 hour), 2 h (2 hours), 3 h (3 hours), 4 hours (4 hours), 5 hours (5 hours), 6 hours (6 hours). hour).
  • Figure 10 shows the results of Solexa sequencing of plant miR As in fresh honeysuckle.
  • Figure 11 shows the results of Solexa sequencing of plant miRNAs in honeysuckle aqueous extracts.
  • Figure 12 shows the Real-time PCR results of MIR2911 or synthetic MIR2911 in honeysuckle extract.
  • Figure 13 shows Real-time PCR results for the expression levels of MIR2911 or MIR167 in serum or water.
  • Figure 14A shows the expression of MIR2911 in serum at different time points after MIR2911 synthesis in mice.
  • the detection time points were 0 h (0 hours), 0.5 h (0.5 hours), 1 h (1 hour), 3 h (3 hours), 6 hours (6 hours), 12 hours (12 hours), and 24 hours (24 hours).
  • Fig. 14B shows the distribution of MIR2911 in organs after oral administration of honeysuckle water in mice.
  • Figure 15 shows the results of sequence analysis of MIR2911 predicted target genes.
  • Mfe indicates the lowest folding free energy of the candidate target gene, and the larger the absolute value of mfe, the higher the matching degree of the candidate target gene with the Peu-MIR2911 sequence.
  • Figure 16 shows the results of luciferase assay for predicting target genes.
  • the predicted genes include the H3N2 gene, the H71N9 gene, the H1N1 gene, and the H5N1 gene.
  • Figure 17 shows the real-time PCR results of MIR2911 in MDCK cells after MIR2911 and AG02 complexes were combined into MDCK cells.
  • Figure 18 shows the virus titer of influenza virus H1N1 in MDCK cells transfected with MIR2911 and AG02 complexes.
  • Figure 19 shows the virus titer of influenza virus H5N1 in MDCK cells transfected with MIR2911 and AG02 complexes.
  • Figure 20 shows the virus titer of influenza virus H7N9 in MDCK cells transfected with MIR2911 and AG02 complexes.
  • Figure 21 shows lung virus titers at different time points after drinking MIR2911 in mice infected with H1N1 virus. Changes in EID50.
  • Figure 22 shows changes in lung virus titer EID50 at different time points after drinking the honeysuckle soup +anti-MIR2911 in mice infected with H1N1 virus.
  • Figure 23 shows changes in lung virus titer EID50 at different time points after drinking MIR2911 in mice infected with H1N1 virus.
  • Figure 24 shows changes in lung virus titer EID50 at different time points after drinking the honeysuckle soup +anti-MIR2911 in mice infected with H1N1 virus.
  • Figure 25 shows the real-time PCR results of MIR2911 in fresh honeysuckle, dried honeysuckle, and mouse feeders.
  • an isolated plant endogenous microR A or a plant extract containing the microR A stably present in a plant extract can be used to regulate the body of the animal.
  • the expression of the endogenous microR A target gene is used to regulate the physiological and pathological activities of the animal. Therefore, it can be used to guide the preparation of drugs or functional foods and the like.
  • the inventors have completed the present invention.
  • microRNA species such as MIR2911 have an unusually high stability (far higher than general microRNA) and can be stably existed for a long period of time, and thus are particularly suitable as an active ingredient for use in medicines or other products.
  • osa means rice; “peu” means Populus euphratica.
  • the plant functional microRNA of the present invention is an endogenous microRNA of the plant which is stably present in the water-soluble and/or fat-soluble extract of the plant.
  • the plant functional microRNA is a microRNA species enriched in a water-soluble and/or fat-soluble extract of the plant (e.g., the top 20 abundance, more preferably the top 10 microRNA species).
  • the microRNA of the present invention includes various open forms such as pri-microR A, pre-microRNA, and microRNA mature bodies.
  • plant-functional microRNAs include, but are not limited to, one or more microRNAs selected from Table 1, particularly one or more selected from the group consisting of MIR156h, MIR166f, MIR396a, MIR166a, MIR168a, MIR1440, MIR2910 MIR291 MIR2915 MIR2916.
  • the plant comprises a medicinal plant, a fruit and vegetable plant, an ornamental plant; preferably comprises honeysuckle, indigo, grass daqing, horse blue, populus, cowpea, cotton, Chinese cabbage or potato;
  • the plant is honeysuckle, indigo, turfgrass, horse blue or Populus euphratica; optimally, the plant is honeysuckle.
  • Extracts containing the plant functional microRNA of the present invention include water-soluble and/or fat-soluble extracts of plants, such as extracts of branches, leaves, roots, flowers, fruits and/or stems of plants. Extraction method (manufacturing method of plant extract)
  • the method for extracting plant microRNA according to the present invention mainly adopts a solvent extraction method in which a micraR A is extracted from a plant 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 (e.g., acid or base) may be added to the solvent.
  • the extraction can be carried out at any suitable temperature (such as normal temperature ⁇ solvent reflux temperature), preferably by dipping method, percolation method, Decoction method, reflux extraction method, continuous extraction method, and the like.
  • suitable temperature such as normal temperature ⁇ solvent reflux temperature
  • the plants can be pretreated, for example by pulverizing plants or enzymatically treating them (eg cellulase, hemicellulase, pectinase, xylanase, neutral protease, papain, dextran) Enzyme, and Xia synthase), etc.; the extracted mixture may also be post-treated.
  • a hydrophilic solvent such as ethanol
  • ethanol may be added to the extracted mixture to make the mixture aged. precipitation.
  • the liquid obtained after the extraction can be used as it is, or it can be subjected to filtration, concentration, drying (e.g., lyophilization) to obtain a solid, which is then used.
  • the method for extracting the plant micraRNA of the present invention is an aqueous extraction method.
  • 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.
  • the Solexa sequencing technology method preferably includes the steps of:
  • the adaptor primer is ligated to the 3' and 5' ends of the small RNA molecule
  • Purified DNA was used directly for cluster generation and sequenced using the Illumina Genome Analyzer.
  • the Real-time PCR method preferably includes the steps of:
  • the invention provides a method for identifying a plant functional microRNA, wherein the plant functional microRNA has the function of regulating a non-plant target gene, comprising the steps of:
  • the non-plant target gene comprises a gene in a gene database.
  • the non-plant target gene comprises a bacterial gene, a viral gene, a Chlamydia gene, a yeast gene, an animal gene; or the non-plant target gene is a pathogen gene.
  • the microRNA species in the top 20 are selected in step (3) for comparison and analysis.
  • a plant microRNA species having a ratio Lm/La ⁇ 130% is selected and compared in step (3), wherein Lm is a certain The abundance (or level) of the plant microRNA in the extract, and La is the average abundance (or level) of the total microRNA of the plant.
  • the plant functional microRNA molecule identified by the method includes MIR2911. Use
  • the plant functional microRNA of the present invention or a plant extract comprising the plant functional microRNA has the following applications:
  • the active ingredient of the composition of the invention is the plant functional microRNA or a plant extract comprising the plant functional microRNA.
  • the plant functional microRNA e.g., MIR2911, etc.
  • the regulation comprises inhibiting (down-regulating) expression of a target gene and promoting (upregulating) expression of a target gene.
  • the non-plant target gene comprises a bacterial gene, a viral gene, a Chlamydia gene, a yeast gene, an animal gene; or the non-plant target gene is a gene of a pathogen (including bacteria, virus, chlamydia, etc.) .
  • the non-plant target gene-related diseases include: tumors (such as liver cancer, lung cancer); acute and chronic infectious diseases (such as viral influenza, viral hepatitis, AIDS, SA S, etc., such as viral diseases, such as Bacterial diseases such as tuberculosis and bacterial pneumonia, and acute and chronic infectious diseases caused by pathogenic microorganisms; Other acute and chronic diseases (such as respiratory diseases, immune system diseases, blood and hematopoietic diseases, circulatory systems such as cardiovascular and cerebrovascular diseases) Disease, endocrine system metabolic disease, digestive system disease, nervous system disease, urinary system disease, reproductive system disease and motor system disease). combination
  • tumors such as liver cancer, lung cancer
  • acute and chronic infectious diseases such as viral influenza, viral hepatitis, AIDS, SA S, etc.
  • viral diseases such as Bacterial diseases such as tuberculosis and bacterial pneumonia, and acute and chronic infectious diseases caused by pathogenic microorganisms
  • Other acute and chronic diseases such as respiratory diseases, immune system diseases, blood
  • composition of the present invention may comprise: (a) a pharmaceutically acceptable carrier or a food acceptable carrier; and (b) an active ingredient.
  • 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, preferably from 0.1 to 90% by weight based on the total weight of the composition (by microRNA if)
  • the method of preparing the composition comprises the steps of: mixing the plant functional microRNA or the plant extract containing the functional microRNA with a pharmaceutically or food acceptable carrier to form the composition.
  • the pharmaceutical composition of the present invention comprises the active ingredient in a safe and effective amount and a pharmaceutically acceptable excipient or carrier.
  • safe and effective amount is meant: The amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition contains from 1 to 2000 mg of active ingredient / More preferably, it contains 10 to 200 mg of active ingredient per dose. Or containing 0.01 to 100 micromoles of active ingredient per agent, 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 intermixing 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 oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween®), moist Wet agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.
  • vegetable oils such as soybean oil, sesame oil, peanut oil, olive oil, etc.
  • polyols such as propylene glycol, glycerin, mannitol, sorbitol, etc.
  • emulsifiers such as Tween®
  • moist Wet agents such as sodium lauryl sulfate
  • colorants such as sodium lauryl sulfate
  • flavoring agents such as pepperminophen®
  • 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-off agent, tincture, barb cream, plaster, rubber ointment, ointment, plaster, paste, eye drops, nasal drops, ophthalmic ointment Agent, gargle, sublingual tablet or suppository.
  • the present invention provides the use of a microR A molecule MIR2911 or an extract containing MIR2911 for the preparation of a medicament for the treatment of viral influenza.
  • the extract (unconcentrated or concentrated) contains 0.01 to 100 nM (preferably 0.1-20 nM) of MIR2911. 2.
  • the plant functional microRNA present in plants is identified by the method of the present invention, and the skilled person can directly direct the skilled person to artificially synthesize the microRNA, thereby increasing the production of the microRNA.
  • the synthetic plant functional microRNA molecule is combined with a pharmaceutically or food acceptable carrier to form a composition.
  • the method of artificially synthesizing microRNA can be a method familiar to those skilled in the art.
  • the preferred method includes:
  • MIR2911 According to the template plasmid sequence of MIR2911, two universal primers A and B were synthesized, and four specific oligonucleotide primer sequences (1, II, III, IV) were designed according to the MIR2911 sequence;
  • the plasmid containing MIR2911 was used as a template, and PCR amplification was carried out by using A and IV, III and ⁇ , I and B as primer combinations respectively.
  • the PCR reaction conditions were: 95 ° C, 2 minutes for 1 cycle ⁇ 95 ° C, 30 Seconds, 55 °C, 30 seconds, 72 24 cycles of 40 ° ⁇ 72 ° C, 7 minutes; 40, respectively, product 1, product 2, product 3;
  • the first round of PCR amplification of product 1, product 2, product 3 was used as a template, and A and B were used as primers for PCR amplification.
  • the PCR reaction conditions were: 95 ° C, 2 minutes for 1 cycle ⁇ 95 °C 30 seconds, 55 ° C, 30 seconds, 72 ° C, 1 minute 30 seconds for 24 cycles ⁇ 72 ° C, 7 minutes, PCR product agarose gel recovery, to obtain synthetic MIR2911;
  • the non-plant target gene eg, bacterial gene, viral gene, chlamydial gene, yeast gene, animal gene
  • the non-plant target gene is cultured in the presence of the isolated plant functional microRNA or the extract containing the plant functional microRNA of the present invention.
  • Biomaterials including viruses, cells, tissues) to achieve in vitro regulation of expression of the non-plant target gene
  • the target gene is a gene of a pathogen (including bacteria, viruses, chlamydia, etc.).
  • the plant functional microRNA is derived from the following plants: a medicinal plant, a fruit and vegetable plant, an ornamental plant; preferably, from honeysuckle, indigo, grass daqing, horse blue or Populus euphratica; more preferably The plant functional microRNA comprises MIR2911. Fourth, disease prevention or treatment
  • the isolated plant functional microRNA of the present invention or the extract containing the plant functional microRNA, or the composition of the present invention is administered to a subject in need thereof (such as a mammal or a human) to achieve prevention or treatment
  • a subject in need thereof such as a mammal or a human
  • the plant functional microRNA has a function of regulating a non-plant target gene (including a bacterial gene, a viral gene, a Chlamydia gene, a yeast gene, an animal gene).
  • the non-plant target gene is a pathogen gene.
  • the inventors have developed a set of methods for efficient and stable extraction of plant functional microRNAs through a series of studies on plant functional microRNAs and their presence, delivery pathways and functions after entering animals.
  • a method for identifying plant functional microRNAs can be used to guide the selective synthesis of plant functional microRNAs, which is beneficial to accelerate the production of the microRNA; and can also be used to guide the screening of plants rich in plant functional microRNAs, Conducive to the identification of the advantages and disadvantages of herbs.
  • a set of methods to guide the manufacture of functional foods or drugs 4.1 The method utilizes isolated plant functional microRNAs or plant extracts containing plant functional microRNAs or selected functional microRNA-rich plants, available For the manufacture of functional foods or pharmaceutical compositions; 4.2 methods for extracting plant microRNAs and identifying plant functional microRNAs, and performing artificial synthesis of the plant functional microRNAs, and then using synthetic microRNAs for manufacturing functionality Food or medicine.
  • the main advantages of the invention include:
  • the microR A is a regulatable expression of a non-plant target gene and has various uses (such as a standard which can be used as an active ingredient of a Chinese herbal medicine, and is advantageous for guiding the development and manufacture of a functional composition, etc.).
  • a composition comprising an isolated plant functional microRNA and/or a plant extract comprising the plant functional microRNA as an active ingredient.
  • the action mechanism of the active ingredient is clear and the effect is remarkable, which is beneficial to the exploration of the scientific mechanism of traditional Chinese medicine, and has a simple preparation method and low cost, and is suitable for industrial production.
  • honeysuckle microRNA was extracted by water extraction. Take an appropriate amount (50 g) of dried honeysuckle medicinal herbs and heat them in a 100 ° C water bath of 500 ml (the honeysuckle mass to water volume ratio of 1: 10) for 0.5 hours. The extract is concentrated under reduced pressure at 60 ° C. 1/10 of the volume. Concentrated and unconcentrated honeysuckle aqueous extracts were collected for subsequent experiments.
  • the plant microRNA stably present in the aqueous extract of the honeysuckle prepared by the above procedure was detected, and the Solexa was loaded at 10 R A .
  • the concentration of total microRNA in the unconcentrated aqueous extract was determined to be about 1 M, and the total microRNA concentration in the concentrated aqueous extract was about 10 M.
  • honeysuckle microRNA can enter the animal by feeding and is stable.
  • mice were orally administered with an aqueous extract of honeysuckle (concentrated water prepared in Example 1), and the expression levels of honeysuckle microRNA in serum, liver and lung were examined.
  • mice were starved for 12 hours, and then the mice were intragastrically administered with 10 ml/kg of mouse body weight concentrated aqueous extract of honeysuckle, using Real-time PCR at 0 h (0 hours), 2 h (2 hours). , 4 h (4 hours), 6 h (6 hours), etc., to detect the expression level of honeysuckle microRNA MIR2911 in mouse serum, liver and lung.
  • Primer sequences for real-time PCR detection of MIR2911 are:
  • Probe sequence ⁇ 'J TCCCAGCCCGTCCCCCGGCC (SEQ ID NO.: 88).
  • Figure 2A is a Real-time PCR result of the expression of honeysuckle microRNA in serum at different time points after oral administration of honeysuckle water extract in mice. It can be seen from the results that the content of MIR2911 in the serum increased significantly after the mice were administered with the aqueous extract of honeysuckle. After 1.5 hours of oral administration of honeysuckle aqueous extract, the serum MIR2911 content reached the highest value, and the serum MIR2911 content decreased to the initial level after 6 hours.
  • Fig. 2B is a Real-time PCR result of the expression levels of honeysuckle microRNA in liver and lung at different time points after oral administration of honeysuckle water extract in mice. It can be seen from the results that the expression level of MIR2911 in the liver and lung increased after the mice were administered with the aqueous extract of honeysuckle. The expression of MIR2911 in the liver reached the maximum after 6 hours of oral administration of the honeysuckle aqueous extract to the mice. After 12 hours of oral administration of the honeysuckle aqueous extract, the expression of MIR2911 in the lung reached a maximum. The results showed that honeysuckle micraR A can enter the animal through feeding and is stable.
  • Example 3 Honeysuckle mkroRNA can regulate physiological and pathological activities
  • honeysuckle microR A regulates physiological and pathological activities in animals.
  • Bioinformatics was used to predict multiple target genes in the genome of respiratory infectious virus to match the MIR2911 sequence.
  • the specific results are shown in Figure 3.
  • Figure 3 shows the results of sequence analysis of MIR2911 predicted target genes. Mfe indicates the lowest folding free energy of the candidate target gene, and the larger the absolute value of mfe, the higher the matching degree of the candidate target gene with the Peu-MIR2911 sequence. The results indicated that the ADV gene, HPIV1 gene, H1N1 gene and H5N1 gene are potential target genes of honeysuckle microRNA.
  • Figure 4 shows the results of luciferase assay for predicting target genes.
  • NC i.e., an equivalent amount of R A containing the mismatch control sequence of MIR2911
  • the results indicate that most of the predicted target genes bind to mature MIR2911 to form a double-stranded structure. This suggests that MIR2911 can regulate the binding of genes such as ADV gene, HPIV gene, H1N1 gene and H5N1.
  • MDCK cells were cultured in a 24-well cell culture plate.
  • MIR2911 (20 pmol/10 6 cells) was transfected into MDCK cells (MIR2911 group) by commercial transfection reagent lipofectamine2000, and the nonsense sequence RA of MIR2911 was transfected into MDCK cells as control (NC) at the same dose. And at the same time set up a positive drug (Tuffy).
  • the specific steps for detecting the viral gene are as follows: After the cells are digested and collected, the cells are repeatedly washed three times with PBS buffer, and total RA is extracted with a protein denaturation reagent, and then the content of the viral marker PB1 mR A is identified by real-time PCR. The virus content was calculated in comparison with the standard curve.
  • Figure 5A shows the detection of the viral gene ADV by Real-Time PCR.
  • the content in MDCK cells is a Real-Time PCR technique for detecting the content of the viral gene H1N1 in MDCK cells.
  • Figure 5C is a Real-Time PCR technique for detecting the content of the viral gene H5N1 in MDCK cells.
  • MIR2911 significantly inhibited and blocked ADV5, H1N1 and H5N1.
  • Example 4 Honeysuckle mkroRNA enters other cells through intestinal epithelial cells Caco-2 cell microparticles (MVs) and plays a regulatory role in other cells.
  • MVs intestinal epithelial cells
  • MVs Caco-2 cell microparticles
  • honeysuckle microR A can be encapsulated into the animal body through the intestinal epithelial cells Caco-2 cell microparticles, and is transported into other cells by the cell microparticles, affecting the physiological and pathological conditions of other cells, such as suppressing the cold virus.
  • the cells were cultured for 24 or 48 hours and used for Real-time PCR analysis.
  • MIR2911 has been encapsulated by microparticles released by Caco-2 cells: RNA in microparticles was extracted, and the content of MIR2911 was determined by absolute quantitative real-time PCR.
  • microparticles containing MIR2911 were collected and treated with HEK 293T cells to confirm that the honeysuckle microRNA was delivered by cell microparticles into other cells.
  • Real-time PCR was used to detect the expression level of MIR2911 in HEK 293T cells.
  • the specific steps of the Real-time PCR experiment are as described in Example 2.
  • Figure 6 shows the results of Real-time PCR in cell microparticles secreted by HEK 293T cells after treatment with cell microparticles secreted by MIR2911-loaded Caco-2 cells.
  • Cellular microparticles secreted by HEK 293T cells treated with cell microparticles (; control MV) not secreted by MIR2911-loaded Caco-2 cells served as controls.
  • MIR2911 was significantly increased in HEK 293T cells after treatment with MIR2911-containing microparticles as compared with the control.
  • honeysuckle microRNA can be encapsulated into the animal body through Caco-2 secreting cell microparticles and delivered to other cells.
  • Example 4.4 The microparticles obtained in the portion of Example 4.4 were added to the cell culture medium, and the amount of the microparticles was 0.1 pmol of MIR2911/10 6 cells.
  • Virus infection After 24 hours, the virus content was detected by Real-time PCR. Real-time PCR was used to detect the content of influenza virus ADV or H1N1 in HEK 293T cells. The specific steps are as described in Example 3.
  • Figure 7A shows the expression level of influenza virus ADV in HEK 293T cells treated with MIR2911-loaded microparticles.
  • Figure 7B shows the expression level of influenza virus H1N1 in HEK 293T cells treated with microparticles loaded with MIR2911.
  • Fig. 7A and Fig. 7B are HEK 293T cells (blank control) which were left untreated after infection with influenza virus, and HEK 293T cells treated with microparticles (negative control) which were not loaded with MIR2911 as a control.
  • influenza virus ADV and H1N1 were significantly decreased in HEK 293T cells treated with MIR2911-loaded microparticles.
  • the results showed that MIR2911 significantly inhibited influenza virus ADV and H1N1.
  • honeysuckle microRNA can be introduced into an animal by oral or the like, and is delivered by cell microparticles into other cells and functions in the cell, such as suppressing a cold virus. It can be seen that the honeysuckle microRNA can enter the animal tissues and organs through oral administration and regulate the physiological and pathological conditions of the animal.
  • Example 5 Honeysuckle microRNA can significantly inhibit cold virus in animals
  • mice were allowed to drink the unconcentrated honeysuckle water extract (prepared in Example 1). After 3 days, the ADV or H1N1 virus was inoculated, and the unconcentrated honeysuckle aqueous extract was continued for 3 days, and then the mouse lung was detected by real-time PCR. The viral content. The results showed that the honeysuckle soup had a strong inhibitory effect on the proliferation of ADV and H1H1 viruses.
  • honeysuckle microRNA MIR2911 that is used to verify the viral inhibition.
  • the following control group is used:
  • mice were given a honeysuckle soup containing anti-MIR2911 (the amount equivalent to that of MIR2911), including: honeysuckle aqueous extract + anti-MIR2911 (oral group) and honeysuckle aqueous extract + anti-MIR2911 (administered lung group) ; anti-MIR2911 is an antisense nucleic acid of MIR2911, which is fully complementary to MIR2911.
  • mice are irrigated with lungs.
  • the specific steps are:
  • mice were allowed to drink an equal volume of pure water with honeysuckle soup as a blank control group. Then, Real-time PCR was used to detect the levels of ADV or H1N1 in the lungs of each group.
  • Reverse primer sequence 5'-AATGCAACACTCGGTTCACA-3' (SEQ ID NO.: 90);
  • Probe sequence TCAGGC CCC CTCAAAGCCGA (SEQ ID NO.: 91).
  • Real-time PCR detects the primer sequence of the cold virus H1N1 as follows:
  • Probe sequence TCAACAGTGGCGAGTTCCCTAGCA (SEQ ID NO.: 94).
  • Fig. 8A shows the expression of cold virus ADV in the lung after drinking honeysuckle water extract, honeysuckle water extract + anti-MIR2911 (oral group), and lung honeysuckle water extract + anti-MIR2911C irrigation group). Time PCR results.
  • mice fed the same volume of water as a blank control after drinking honeysuckle aqueous extract, ADV expression in the lungs of mice significantly decreased, indicating that honeysuckle microRNA inhibits the expression of cold virus ADV.
  • ADV increased the expression of ADV in the lungs of mice after drinking the honeysuckle aqueous extract +anti-MIR2911, indicating that anti-MIR2911 disrupted the inhibitory effect of MIR2911 on the ADV of the cold virus.
  • Fig. 8B shows the expression of cold virus H1N1 in the lung after drinking honeysuckle water extract, honeysuckle water extract + anti-MIR2911 (oral group), and lung honeysuckle water extract + anti-MIR2911C irrigation group). Time PCR results.
  • honeysuckle microRNA is absorbed by the body through the digestive tract and enters the circulatory system.
  • the dried honeysuckle is boiled for 30 minutes to make 1000 ml of water extract.
  • the content of MIR2911 in the aqueous extract is about 0.4 n mol/L.
  • recruit 20 healthy volunteers each taking 1,000 ml of water extract, at 0 h (0 hours), lh (1 hour), 2 hours (2 hours), 3 hours (3 hours), 4 hours Volunteer blood was collected (4 hours), 5 h (5 hours), 6 h (6 hours), and the content of MIR2911 was detected by Real-time PCR.
  • the specific steps of the Real-time PCR experiment were as described in Example 2.
  • Figure 9 shows the real-time PCR results of MIR2911 in human blood after taking honeysuckle water extract. It can be seen from the results that the content of MIR2911 in human blood is significantly increased after taking the honeysuckle water extract. After 1.5 hours of taking the honeysuckle water extract, the content of MIR2911 in the human blood reached its maximum value, and after 3 hours, the content of MIR2911 in the human blood decreased to the initial level. These results are consistent with the results of the mouse experiments in Example 2 (Fig. 1K). The results showed that honeysuckle microRNA can enter the human body through feeding, is absorbed by the digestive tract, and enters the circulatory system. Example 7 Honeysuckle microRNA can significantly inhibit the cold virus in human body
  • Groups 4-6 are viral influenza patients carrying the H1N1 virus:
  • Group 1 1000 mL of honeysuckle water extract (prepared in Example 6) was taken.
  • Group 2 Take 1000 ml of honeysuckle water extract + anti-MIR2911 (the amount is equivalent to the amount of MIR2911); wherein anti-MIR2911 is an antisense nucleic acid of MIR2911, which is completely complementary to MIR2911 and can specifically degrade MIR2911.
  • Group 3 Take an equal volume of pure water with the water extract.
  • Group 5 Take the honeysuckle aqueous extract lOOOOmL + anti-MIR2911 (the amount is equivalent to the amount of MIR2911); wherein anti-MIR2911 is an antisense nucleic acid of MIR2911, which is completely complementary to MIR2911 and can specifically degrade MIR2911.
  • Group 6 Take an equal volume of pure water with the water extract.
  • the honeysuckle microR A was extracted by water extraction. Take an appropriate amount (100 g) of fresh honeysuckle medicinal herbs and heat them in a 100 ° C water bath of 1000 ml (the honeysuckle mass to water volume ratio of 1: 10) for 0.5 hours. The extract is concentrated under reduced pressure at 60 ° C. 1/10 of the volume. The honeysuckle aqueous extract was collected for subsequent experiments.
  • a variety of plant microRNAs are stably present in fresh honeysuckle, among which MIR167a, MIR166f, MIR166b, MIR164a, MIR168a, MIR156h, MIR172a, MIR162b, MIR159d, MIR827b, MIR396b, MIR2911, and MIR2916 are high.
  • MIR167a, MIR166f, MIR166b, MIR164a, MIR168a, MIR156h, MIR172a, MIR162b, MIR159d, MIR827b, MIR396b, MIR2911, and MIR2916 are high.
  • a variety of plant microRNAs are stably present in the aqueous extract of honeysuckle, including MIR167a, MIR166f, MIR166b, MIR164a, MIR168a, MIR156h, MIR172a, MIR162b, MIR159d, MIR827b, MIR396b, MIR2911, MIR2916, among which MIR2911 is the highest. As shown in Figure 11.
  • honeysuckle micraR A was extracted by water extraction.
  • the specific experimental procedure is as described in Example 8.1. And follow conventional methods Synthetic MIR2911.
  • the concentration of MIR2911 or synthetic MIR2911 in the honeysuckle extract was determined by Real-time PCR. The specific procedure was as described in Example 2.
  • the concentration of MIR2911 and MIR167 in serum and water was detected by Real-time PCR method, and the specific procedure was as described in Example 2.
  • MIR2911 which is synthesized or extracted from honeysuckle, does not degrade in water or serum at various time points, and has high stability; in contrast, MIR167 degrades rapidly. After one hour, the content is greatly reduced. It can be seen that MIR2911 is more stable in water and serum.
  • MIR2911 Three hours after the mice were orally administered with honeysuckle aqueous extract, the fluorescently labeled synthetic MIR2911 was specifically delivered to the lungs of mice. As shown in Figure 14B, the labeled MIR2911 (red) accumulated rapidly in the lungs of mice. Description After entering the blood, MIR2911 can reach the department that needs to function, that is, it can successfully reach the target organ and play its role. Example 9 MIR2911 can regulate physiological and pathological activities
  • MIR2911 targets various influenza viruses and inhibits their replication in virus-infected cells.
  • MIR2911 predicts the sequence analysis results of viral target genes.
  • Figure 15 shows the results of sequence analysis of the MIR2911 predicted target gene. The results showed that the viral genes H1N1, H3N2, H5N1 and H7N9 are potential target genes of honeysuckle microR A.
  • the target gene was verified using a luciferase assay.
  • the specific operation steps are as described in Example 3.2.
  • H1N1, H3N2, H5N1, H7N9 are the potential of honeysuckle MIR2911 At the target gene.
  • plant MIR2911 can bind to the AG02 complex, and AG02 can help transport MIR2911 into cells.
  • MDCK cells were cultured in a 24-well cell culture plate.
  • Example 3.3 The specific operation steps are as described in Example 3.3. The difference is that MIR2911 is transfected into MDCK cells by MIR2911 and AG02 complexes. The transfection time is divided into 12 hours and 24 hours.
  • MIR2911 is transfected into MDCK cells by MIR2911 and AG02 complexes.
  • the transfection time is divided into 12 hours and 24 hours.
  • MIR2911 is transfected into MDCK cells by MIR2911 and AG02 complexes.
  • the transfection time is divided into 12 hours and 24 hours.
  • MDCK cells were infected with the avian influenza virus gene H7N9.
  • Example 10 MIR2911 inhibits replication of mouse IAVs (representing influenza A virus).
  • mice were treated with three different subtypes of influenza virus prior to inoculation. After 3, 5, and 7 days of infection, changes in body weight were recorded daily. The mice were sacrificed and the amount of virus in the lungs was measured.
  • mice inoculated with H1N1 virus had no significant changes in body weight after drinking MIR2911-containing solution or honeysuckle soup. Drinking MIR2911-free solution significantly reduced the body weight of mice. It can be seen that MIR2911 has a significant inhibitory effect on H1N1 virus, maintains the body weight balance of mice, and improves the physiological condition of mice.
  • the virus was serially diluted 10 times and inoculated separately for 1 week;
  • mice were sacrificed 3 days, 5 days, and 7 days after infection, and the blood of the lungs was taken for hemagglutination test;
  • H1N1 is directly administered to the H1N1 virus infection group
  • H1N1+NC is infected with H1N1 virus, and a negative control (nonsense sequence) is added at the same time;
  • H1N1+MIR2911 is infected with H1N1 virus and directly administered to MIR2911).
  • Figure 21 shows: There was no significant difference between the two control groups (H1N1, H1N1+NC) 3 days after infection, indicating that the nonsense sequence did not kill the virus; compared with the control group (H1N1+NC), administration The lung virus content of the group (H1N1+MIR2911) was greatly reduced, and there was a significant difference, indicating that the drug had an extremely obvious killing effect on the lung virus.
  • Figure 22 shows: 3 days and 5 days after infection, compared with the control group (HlNl+LJ soup administered with honeysuckle soup and infected with H1N1), the experimental group (giving the honeysuckle soup +anti-MIR2911 and infected with H1N1)
  • the mouse, H1N1+LJ soup + MIR2911 had a significantly higher lung virus content with significant differences.
  • Description anti-MIR2911 Degraded MIR2911 in honeysuckle soup, the honeysuckle soup lost its antiviral effect, indicating that MIR2911 is active.
  • mice inoculated with H5N1 virus had no significant change in body weight after drinking MIR2911-containing solution or honeysuckle soup. Drinking MIR2911-free solution significantly reduced the body weight of mice. It can be seen that MIR2911 has a significant inhibitory effect on H5N1 virus, maintains the body weight balance of mice, and improves the physiological condition of mice.
  • mice inoculated with H7N9 virus had no significant changes in body weight after drinking MIR2911-containing solution or honeysuckle soup.
  • the body weight of the mice was significantly reduced. It can be seen that MIR2911 has a significant inhibitory effect on H7N9 virus, maintains the body weight balance of mice, and improves the physiological condition of mice.
  • mice infected with H7N9 virus the changes in lung virus titer EID 5Q were determined at different time points after infection. The specific operation steps are as described in Example 10.1.
  • H7N9 was directly administered to the H7N9 virus infection group
  • H7N9+NC is infected with H7N9 virus, and a negative control (nonsense sequence) is added at the same time;
  • H7N9+MIR2911 is infected with H7N9 virus and directly administered to MIR2911).
  • Figure 23 shows that there was no significant difference between the two control groups (H7N9, H7N9+NC) after infection, indicating that the nonsense sequence did not kill the virus; compared with the control group (H7N9+NC), the administration group ( H7N9+MIR2911) The lung virus content was greatly reduced, and there was a significant difference, indicating that the drug had an extremely significant killing effect on the lung virus.
  • Figure 24 shows: After infection, compared with the control group (money-infested with H7N9, H7N9+LJ soup), the experimental group (Hypergillin + anti-MIR2911 and H7N9-infected mice, H7N9) +LJ soup + MIR2911)
  • the lung virus content is significantly higher, with significant differences.
  • Example 11 Comparison of MIR2911 content in fresh honeysuckle, dried honeysuckle and mouse feed
  • the content of MIR2911 in fresh honeysuckle, dried honeysuckle, and mouse was measured by Real-time PCR method. The specific procedure was as described in Example 2.

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Abstract

提供了一种分离的植物功能性microRNA或含该植物功能性microRNA的提取物、其制备方法及其用途。该植物功能性microRNA来源于某一植物的内源性microRNA且存在于该植物的水溶性和/或脂溶性的提取物中,而且具有调控非植物靶基因的功能。

Description

植物微小核糖核酸的提取、 制备及其应用
技术领域
本发明属于生物领域。 具体地, 本发明涉及提取植物 microRNA的方法及其应用。 背景技术
微小核糖核酸(microR A、 miR A或 mi ) 是一类长约 19至 23个核苷酸的非编码单链 小核糖核酸分子。 它们广泛存在于动植物细胞中, 在进化上高度保守。 MicroRNA通过识别靶 信使 RNA (mR A) 的 3' 端非翻译序列, 与之不完全互补, 从而抑制相应蛋白质的翻译。 作 为 mR A强有力的调节因子, microRNA与生理活动密切相关, 涉及生物个体发育、 组织分 化、 细胞调亡及能量代谢等生命活动; 同时, microRNA也与许多疾病的发生和发展存在紧密 联系。
目前, 对植物 microRNA研究都集中在 microRNA对植物本身的调控作用, 包括对植物生 长发育、信号转导和逆境胁迫活动的作用。对研究成果的应用则集中于对植物物种的改良, 如 调节农作物在其食用部分对营养元素的表达。
专利 PCT/CN2010/000677公开了源自水稻的 MIR164对植株根系的调控作用, 提出构建 包括 MIR164序列的核酸片段, 并将其转入水稻植株, 从而获得根系比普通水稻强大的转基因 水稻。 专利 PCT/IB2010/055600公开了上调包括 MIR156的若干 microRNAs可提高植物对环 境胁迫因素的耐受能力, 进而提高植物的生物质、 活力和产量。
目前, 现有的研究仅限于植物 microRNA 对植物本身生理活动的调控作用, 但植物 microRNA对动物的生理活动的调控作用以及对植物 microRNA 的提取还有待研究。 发明内容
本发明的目的之一是提供一种具有调控非植物靶基因的功能的植物 microRNA或含有所 述 microRNA 的植物提提取物及其制法和用途。
本发明的另一目的是提供一种鉴定植物功能性 microRNA的方法。
在本发明第一方面中, 提供了一种分离的植物功能性 microRNA或含所述植物功能性 microRNA的提取物, 所述的植物功能性 microRNA是来源于某一植物的内源性 microRNA且 存在于所述植物的水溶性和 /或脂溶性的提取物中, 而且所述植物功能性 microRNA具有调控 非植物靶基因的功能。
在另一优选例中, 所述植物功能性 microRNA包括 MIR2911; 且所述分离的植物功能性 microRNA 或含所述植物功能性 microRNA 的提取物中, MIR2911 的含量 70 %, 按总 microRNA的数量计。
在另一优选例中, MIR2911的含量 80%; 较佳地, 90%; 更佳地, 100%。
在另一优选例中, 所述的总 microRNA的数量指长度为 18-24nt的 microRNA的数量之 和。
在另一优选例中, 所述的调控包括抑制 (下调)靶基因的表达、 促进 (上调)靶基因的表达。 在另一优选例中,所述的非植物靶基因包括细菌基因、病毒基因、衣原体基因、酵母基因、 动物基因。
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; 更佳地包括: 金银 花、 菘蓝、 草大青、 马蓝、 胡杨、 豇豆、 棉花、 大白菜或马铃薯。 优选地, 所述植物为金银花、 菘蓝、 草大青、 马蓝或胡杨; 更优选地, 所述植物为金银花。
在另一优选例中, 所述的植物功能性 micraRNA是所述植物的水溶性和 /或脂溶性的提取 物中富含的 microRNA种类 (如丰度列前 20位, 更佳地前 10位的 microR A种类)。
在另一优选例中,所述的植物功能性 microRNA还包括选自下组的一种或多种: MIR156h、 MIR166f、 MIR396a、 MIR166a、 MIR168a、 MIR1440、 MIR2910 MIR2915 MIR2916 MIR818d、 MIR159e、 MIR159c、 MIR156j、 MIR1432、 MIR166k、 MIR167b、 MIR396c、 MIR156e、 MIR169k、 MIR167c、 MIR160d、 MIR399a、 MIR156d、 MIR160e、 MIR169n、 MIR166K MIR159f、 MIR166c、 MIR159b、 MIR166j、 MIR167i、 MIR169c、 MIR164c、 MIR167j、 MIR167g、 MIR160c、 MIR399e、 MIR399b、 MIR529b、 MIR164e、 MIR166d、 MIR166h、 MIR164b、 MIR156f、 MIR164a、 MIR169K MIR166m、 MIR164f、 MIR156k、 MIR166g、 MIR166b、 MIR160b、 MIR166e、 MIR159d、 MIR818e、 MIR172a、 MIR156b、 MIR399g、 MIR169b、 MIR399f、 MIR167a、 MIR394、 MIR156a、 MIR166i、 MIR167f、 MIR319a、 MIR156g、 MIR166n、 MIR399c、 MIR160a、 MIR159a.l、 MIR156c、 MIR319b、 MIR169o、 MIR167h、 MIR156i、 MIR167d、 MIR169a、 MIR172d MIR818b、 MIR164d、 MIR167e、 MIR396b、 MIR2914(表 1)。
在另一优选例中, 所述的植物提取物包括植物的水溶性和 /或脂溶性的提取物。
在另一优选例中, 所述的植物提取物包括植物的枝、 叶、 根、 花、 果和 /或茎的提取物。 在本发明第二方面中, 提供了一种如本发明第一方面所述的分离的植物功能性 microRNA 或含所述植物功能性 microRNA的提取物的用途,它 (a)用于制备调控非植物靶基因的组合物; 或 (b)用于制备治疗非植物靶基因相关疾病的药物。
在另一优选例中, 所述非植物靶基因包括细菌基因、 病毒基因、 衣原体基因、 酵母基因、 动物基因。
在另一优选例中, 所述的非植物靶基因是病原体 (包括细菌、 病毒、 衣原体等)的基因。 在另一优选例中, 所述非植物靶基因相关疾病包括: 肿瘤 (如肝癌、 肺癌); 急慢性传染病 (如病毒性流感、病毒性肝炎、 艾滋病、 SA S等的病毒性疾病, 如结核、 细菌性肺炎等的细菌 性疾病, 以及病原微生物导致的急慢性传染病); 其它急慢性疾病 (如呼吸系统疾病、 免疫系统 疾病、血液与造血系统疾病、 如心脑血管疾病的循环系统疾病、 内分泌系统代谢性疾病、 消化 系统疾病、 神经系统疾病、 泌尿系统疾病、 生殖系统疾病和运动系统疾病)。
在另一优选例中, 所述的植物功能性 microRNA包括 MIR2911。更佳地, 所述的药物用于 治疗病毒性感冒。
在本发明第三方面中, 提供了一种组合物, 其包含 (a)药学上可接受的载体或食品学上可 接受的载体以及 (b)本发明第一方面所述的分离的植物功能性 microRNA和 /或含所述植物功 能性 microRNA的植物提取物。
在另一优选例中, 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 0.1-90 %(按 microRNA if) o 在另一优选例中, 所述组合物包括药物组合物、 食品组合物或保健品组合物。 在另一优选例中,所述组合物的制备方法包括步骤: 将所述的植物功能性 microR A或含 所述功能性 micraRNA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。
在另一优选例中, 所述的植物功能性 micraR A来自以下植物: 药用植物、 果蔬植物、 观 赏植物, 并且调控选自下组的非植物靶基因: 细菌基因、 病毒基因、 衣原体基因、 酵母基因、 动物基因。
在另一优选例中,所述的植物功能性 micraRNA来自金银花、菘蓝、草大青、马蓝或胡杨。 更佳地, 植物功能性 microR A包括 MIR2911。
在本发明第四方面中, 提供了一种体外非治疗性调控非植物靶基因表达的方法, 其中, 非 植物靶基因包括细菌基因、病毒基因、衣原体基因、酵母基因、动物基因, 所述方法包括步骤: 在本发明第一方面所述的分离的植物功能性 microRNA或含所述植物功能性 microRNA的提取 物存在下, 培养含所述靶基因的生物材料, 从而调控所述非植物靶基因的表达。
在另一优选例中, 所述的靶基因是病原体 (包括细菌、 病毒、 衣原体等)的基因。
在另一优选例中, 所述的生物材料包括病毒、 细胞、 组织。
在另一优选例中, 所述的植物功能性 micraRNA来自以下植物: 药用植物、 果蔬植物、 观 赏植物。
在另一优选例中,所述的植物功能性 micraRNA来自金银花、菘蓝、草大青、马蓝或胡杨。 更佳地, 植物功能性 microRNA包括 MIR2911。
在本发明第五方面中,提供了一种植物功能性 microRNA的鉴定方法, 其中所述植物功能 性 micraRNA具有调控非植物靶基因的功能, 所述方法包括步骤:
(1)提供某一植物的提取物;
(2)检测所述提取物中植物内源性 micraRNA的种类或水平;
(3)将被检测到的植物 microRNA的序列与非植物靶基因进行比对和分析, 从而鉴定出具 有调控非植物靶基因的功能的植物功能性 microR A。
在另一优选例中, 所述的非植物靶基因包括基因数据库中的基因。
在另一优选例中, 所述非植物靶基因包括细菌基因、 病毒基因、 衣原体基因、 酵母基因、 动物基因。
在另一优选例中, 所述的非植物靶基因是病原体基因。
在另一优选例中, 所述的植物包括药用植物, 果蔬植物。
在另一优选例中, 在步骤 (3)中挑选提取物中丰度列前 20位 (更佳地前 10位)的 microRNA 种类进行比对和分析。
在另一优选例中, 在步骤 (3)中挑选比值 Lm/La≥130% (较佳地≥150%; 更佳地≥200%)的植 物 microRNA种类进行对比和分析, 其中 Lm为某一植物 microRNA在提取物中的丰度 (或水 平), La为所述植物总 microRNA的平均丰度 (或水平)。
在本发明第六方面中, 提供了一种由本发明第五方面所述的方法所鉴定出的植物功能性 microRNA分子。
在另一优选例中, 所述 microRNA分子包括 MIR2911。 在本发明第七方面中, 提供了一种制备组合物的方法, 包括步骤:
人工合成本发明第六方面所述的植物功能性 microRNA分子; 以及
将所述的植物功能性 microRNA分子与药学上或食品学上可接受的载体混合,从而形成组 合物。
在本发明第八方面中, 提供了一种 microRNA分子 MIR2911或含 MIR2911的提取物的用 途, 用于制备治疗病毒性感冒的药物。
在另一优选例中, 所述的提取物 (未浓縮或浓縮)中含 0.01-lOOnM (较佳地 0.1-20nM)的 MIR2911 ; 或
所述的提取物 (未浓縮或浓縮)中, MIR2911 的含量 70% ; 较佳地, 80%; 更佳地, ^90 ; 最佳地, 100%, 按总 microRNA的数量计。
在本发明第九方面中,提供了一种预防或治疗疾病的方法,其中所述疾病是与非植物靶基 因相关的疾病,所述方法包括步骤:给需要的对象施用本发明第一方面所述分离的植物功能性 microRNA或含所述植物功能性 microRNA的提取物、 或本发明第三方面所述的组合物, 从而 预防或治疗所述的疾病, 其中所述的植物功能性 microRNA具有调控非植物靶基因的功能。
在另一优选例中, 所述的对象包括哺乳动物 (如人)。
在另一优选例中,所述的非植物靶基因包括细菌基因、病毒基因、衣原体基因、酵母基因、 动物基因。
在另一优选例中, 所述的非植物靶基因是病原体基因。
在本发明第十方面, 提供了一种筛选抗病毒活性成分候选物质的方法, 包括步骤:
(a) 提供分离的植物功能性 microRNA或含所述植物功能性 microRNA的提取物;
(b) 测定所述植物功能性 microRNA中各 microRNA的稳定性, 从而挑选出高稳定性的 microRNA禾中^ I;
(c) 将上一步骤挑选出的所述高稳定性的 microRNA, 与非植物靶基因进行比对, 从而确 定所述的高稳定性的 microRNA是否匹配于或结合于非植物靶基因;
其中, 如果比对结果表明, 所述的高稳定性的 microRNA可匹配于或结合于非植物靶基 因, 则选出该 microRNA种类作为抗病毒活性成分候选物质。
在另一优选例中, 所述方法还包括步骤:
(d) 在体外试验或动物试验中, 验证抗病毒活性成分候选物质是否具有抗病毒活性。 在另一优选例中, 所述的高稳定性的 microRNA选自下组:
(i) 在所述植物功能性 microRNA或含所述植物功能性 microRNA的提取物中,稳定性最 高的前 5种, 较佳地前 3种, 更佳地前 1种 microRNA;
(ii) 稳定性满足下式的 microRNA
Figure imgf000006_0001
式中, CQ为放置 0小时时的浓度;
C12为放置 12小时时的浓度.
较佳地, Co不为零。
在另一优选例中, 所述的非植物靶基因为病毒基因。
在另一优选例中, 步骤 (a)中, 所述 miR A在新鲜植物中丰度排名 (从高到低)第 5名之 后, 较佳地第 10名之后的种类。
在本发明第十一方面, 提供了一种制备组合物的方法, 包括步骤:
人工合成植物功能性 microR A分子, 其中所述的植物功能性 microR A分子是用第十 方面所述方法筛选出的抗病毒活性成分候选物质; 以及
将所述的植物功能性 microRNA分子与药学上或食品学上可接受的载体混合, 从而形成 组合物。
在本发明第十二方面中, 提供了一种提高 MIR2911丰度的方法, 包括步骤:
(a) 提供含植物功能性 microRNA的提取物, 其中, 所述的植物功能性 microRNA包括 n 种 microRNA, 其中 n 为 2(较佳地 5 或 10)的正整数, 并且其中一种 microRNA 为 MIR2911 ;
(b) 将所述提取物进行陈化处理 (如放置),得到经陈化的提取物, 并且测定所述经陈化的 提取物中 MIR2911的丰度, 并与预定值进行比较, 并且当 MIR2911的丰度 预定值时, 停 止陈化处理, 得到 MIR2911丰度提高的提取物。
在另一优选例中, 所述的提取物为水提取物或醇提取物。
在另一优选例中, 所述的陈化处理是在 20-50°C, 放置 2小时一 10天。
在另一优选例中,所述的方法还包括将得到的 MIR2911丰度提高的提取物用于制药、或 置于低温 (如 0°C至 -4°C或更低温度)保藏。
在另一优选例中, 所述的预定值为 60%、 70%、 80%、 90%、 95%、 或 99%。
在另一优选例中, 所述的丰度按下式计算
丰度 = S1/∑SiX 100%
式中, Si为 MIR2911的数量 (;或浓度), i= l到 n, Si为第 i种 microRNA的数量 (;或浓度), 其中 microRNA是长度为 18-26nt的 RNA。
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显示植物 microRNA在金银花水提液中 Solexa测序结果。
图 2A是小鼠灌胃金银花水提液后, 金银花 microRNA在不同时间点血清中表达量的 Real-time PCR结果。 检测时间点为 0 h(0小时)、 2 h(2小时)、 4 h(4小时)、 6 h(6小时)。
图 2B是小鼠灌胃金银花水提液后, 金银花 microRNA在不同时间点肝脏和肺中表达量的 Real-time PCR结果。 检测时间点为 0 h(0小时)、 5 h(5小时)、 10 h(10小时)、 15 h(15小时)、 20 h(20小时)、 25 h(25小时)。
图 3显示 MIR2911预测靶基因的序列分析结果。 mfe表示候选靶基因的最低折叠自由能, mfe绝对值越大, 候选靶基因与 Peu-MIR2911序列匹配度越高。
图 4显示荧光素酶检测预测靶基因的结果。 预测基因包括 ADV基因、 HPIV基因、 H1N1 基因、 H5N1基因。
图 5A是利用 Real-time PCR技术检测病毒基因 ADV5在 MDCK细胞中的含量。 图 5B是利用 Real-time PCR技术检测病毒基因 H1N1在 MDCK细胞中的含量。
图 5C是利用 Real-time PCR技术检测病毒基因 H5N1在 MDCK细胞中的含量。
图 5A、 图 5B和图 5C中, "未感染"代表未侵染流感病毒的细胞; "感染未处理 "代表侵染 流感病毒且未用任何药物处理的细胞; "MIR2911"代表侵染流感病毒且用 MIR2911 处理的细 胞; 'WC"代表侵染流感病毒且用 MIR2911 的无义序列 R A处理的细胞; "达菲 "代表侵染流 感病毒且用达菲 (奥塞米韦, 特异性流感病毒神经氨酸酶抑制药)处理的细胞。
图 6显示 MIR2911在转染 MIR2911的 HEK 293T细胞分泌的载有 MIR2911的细胞微粒 子中的 Real-time PCR结果。以未转染 MIR2911的 HEK 293T细胞分泌的细胞微粒子作为对照 图 7A是流感病毒 ADV在载有 MIR2911的细胞微粒子处理的 HEK 293T细胞中的表达量。 图 7B是流感病毒 H1N1在载有 MIR2911的细胞微粒子处理的 HEK 293T细胞中的表达量。 图 7A和图 7B中, 以感染流感病毒后不作处理作为空白对照和未载有 MIR2911的细胞微 粒子处理的 HEK 293T细胞作为阴性对照。
图 8A显示小鼠饮用金银花水提液、 金银花水提液 +anti-MIR2911(口服组)、 灌肺金银花水 提液 +anti-MIR291 K灌肺组)后, 感冒病毒 ADV在肺表达的 Real-time PCR结果。
图 8B显示小鼠饮用金银花水提液、 金银花水提液 +anti-MIR2911(口服组)、 灌肺金银花水 提液 +anti-MIR2911C灌肺组)后, 感冒病毒 H1N1在肺表达的 Real-time PCR结果。
图 9显示服用金银花水提液后, 人血液中 MIR2911的 Real-time PCR结果。
检测时间点为 0 h(0小时)、 1 h(l小时)、 2 h(2小时)、 3 h(3小时)、 4 h(4小时)、 5 h(5小时)、 6 h(6小时)。
图 10显示植物 miR As在新鲜金银花中 Solexa测序结果。
图 11显示植物 miRNAs在金银花水提液中 Solexa测序结果。
图 12显示金银花提取液中 MIR2911或合成的 MIR2911的 Real-time PCR结果。
图 13显示 MIR2911或 MIR167在血清或水中表达量的 Real-time PCR结果。
图 14A 显示小鼠灌胃合成的 MIR2911 后, MIR2911 在不同时间点血清中表达量的
Real-time PCR结果。检测时间点为 0 h(0小时)、 0.5 h(0.5小时)、 1 h(l小时)、 3 h(3小时)、 6h(6 小时)、 12h(12小时) 、 24h(24小时)。
图 14B显示小鼠灌胃金银花水提掖后, MIR2911在器官中的分布。
图 15显示 MIR2911预测靶基因的序列分析结果。 mfe表示候选靶基因的最低折叠自由能, mfe绝对值越大, 候选靶基因与 Peu-MIR2911序列匹配度越高。
图 16显示荧光素酶检测预测靶基因的结果。预测基因包括 H3N2基因、 H71N9基因、 H1N1 基因、 H5N1基因。
图 17显示 MIR2911与 AG02复合物结合转入 MDCK细胞后, MIR2911在 MDCK细胞 中的 Real-time PCR结果。
图 18显示流感病毒 H1N1在 MIR2911与 AG02复合物转染的 MDCK细胞中的病毒滴度。 图 19显示流感病毒 H5N1在 MIR2911与 AG02复合物转染的 MDCK细胞中的病毒滴度。 图 20显示流感病毒 H7N9在 MIR2911与 AG02复合物转染的 MDCK细胞中的病毒滴度。 图 21显示感染 H1N1病毒的小鼠, 饮用 MIR2911溶液后, 在不同时间点肺部病毒滴度 EID50的变化。
图 22显示感染 H1N1病毒的小鼠, 饮用金银花汤药 +anti-MIR2911溶液后, 在不同时间 点肺部病毒滴度 EID50的变化。
图 23显示感染 H1N1病毒的小鼠, 饮用 MIR2911溶液后, 在不同时间点肺部病毒滴度 EID50的变化。
图 24显示感染 H1N1病毒的小鼠, 饮用金银花汤药 +anti-MIR2911溶液后, 在不同时间 点肺部病毒滴度 EID50的变化。
图 25显示新鲜金银花、 干金银花、 小鼠伺料中 MIR2911的 Real-time PCR结果。 具体实施方式
本发明人通过长期而深入的研究,意外地发现一种稳定存在于植物提取物中的可分离的植 物内源性 microR A或含有所述 microR A的植物提取物, 可用于调控动物体内所述内源性 microR A靶基因的表达, 进而用于调控动物的生理病理活动。 因此, 可用于指导制备药物或 功能性食物等。 在此基础上, 发明人完成了本发明。
本发明人还发现, 诸如 MIR2911等 microRNA种类具有异乎寻常的高稳定性 (远高于一般 microRNA), 可以长期稳定存在, 因此特别适合作为活性成分而应用于药物或其他产品。
如本文所用, "osa"表示水稻; "peu"表示胡杨。
分离的植物功能性 microRNA
本发明所述的植物功能性 microRNA为所述植物内源性的 microRNA, 可稳定存在于该植 物的水溶性和 /或脂溶性的提取物中。 优选为所述的植物功能性 microRNA是所述植物的水溶 性和 /或脂溶性的提取物中富含的 microRNA种类 (如丰度列前 20 位, 更佳地前 10 位的 microRNA种类)。此夕卜,本发明的 microRNA包括各种开式,例如 pri-microR A、 pre-microRNA 以及 microRNA成熟体。
所述植物功能性 microRNA的例子包括 (但并不限于)选自表 1的一种或多种 microRNA, 尤其是选自下组的一种或多种: MIR156h、 MIR166f、 MIR396a、 MIR166a、 MIR168a、 MIR1440、 MIR2910 MIR291 MIR2915 MIR2916。
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。
本发明所述含所述植物功能性 microRNA的提取物包括植物的水溶性和 /或脂溶性的提取 物, 例如植物的枝、 叶、 根、 花、 果和 /或茎的提取物。 提取方法 (植物提取物的制法)
本发明所述的植物 microRNA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 micraR A。 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、 浓縮、 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 micraRNA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), 将提取液浓縮至一定 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。 检测
对植物进行提取后, 收集植物提取物, 检测提取物中植物 microR A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。
所述 Solexa测序技术方法, 优选包括步骤:
收集金银花组织或汤药样本;
通过 Trizol试剂提取样本总 R A;
进行 PAGE电泳回收 17-27nt RNA分子;
将接头引物(adaptor primer) 酶联在小 RNA分子的 3'与 5 '端;
纯化的 DNA直接用于集群生成, 利用 Illumina Genome Analyzer进行测序分析。
Real-time PCR方法, 优选包括步骤:
提取样本中总 R A, 通过 R A逆转录反应得到 cDNA样品;
用植物 microRNA设计引物;
加入 TaqMan探针或者荧光染料进行 PCR反应;
检测样本中植物 microRNA的量的变化。 鉴定
本发明提供了一种植物功能性 microRNA鉴定方法,其中所述植物功能性 microRNA具有 调控非植物靶基因的功能, 包括步骤:
(1)提供某一植物 (包括药用植物, 果蔬植物)的提取物;
(2)检测所述提取物中植物内源性 microRNA的种类或水平;
(3)将被检测到的植物 microRNA的序列与非植物靶基因进行比对和分析, 从而鉴定出具 有调控非植物靶基因的功能的植物功能性 microR A。
在另一优选例中, 所述的非植物靶基因包括基因数据库中的基因。优选地, 所述非植物靶 基因包括细菌基因、 病毒基因、 衣原体基因、 酵母基因、 动物基因; 或所述的非植物靶基因是 病原体基因。
在另一优选例中, 在步骤 (3)中挑选提取物中丰度列前 20位 (更佳地前 10位)的 microRNA 种类进行比对和分析。
在另一优选例中, 在步骤 (3)中挑选比值 Lm/La≥130% (较佳地≥150%; 更佳地≥200%)的植 物 microRNA种类进行对比和分析, 其中 Lm为某一植物 microRNA在提取物中的丰度 (或水 平), La为所述植物总 microRNA的平均丰度 (或水平)。
由所述方法鉴定出的植物功能性 microRNA分子包括 MIR2911。 用途
本发明所述植物功能性 microRNA或包含所述植物功能性 microRNA的植物提取物具有如 下多个应用:
一、 指导功能性组合物的开发或制造
本发明所述组合物的活性成分为所述植物功能性 microRNA 或包含所述植物功能性 microRNA的植物提取物。 其中, 所述的植物功能性 microRNA (如 MIR2911等)具有调控非植 物靶基因的功能。 可用于 (a)制备调控非植物靶基因的组合物; 或 (b)制备治疗非植物靶基因 相关疾病的药物。 其中, 所述的调控包括抑制 (下调)靶基因的表达、 促进 (上调)靶基因的表达。
在另一优选例中, 所述非植物靶基因包括细菌基因、 病毒基因、 衣原体基因、 酵母基因、 动物基因; 或所述的非植物靶基因是病原体 (包括细菌、 病毒、 衣原体等)的基因。
在另一优选例中, 所述非植物靶基因相关疾病包括: 肿瘤 (如肝癌、 肺癌); 急慢性传染病 (如病毒性流感、病毒性肝炎、 艾滋病、 SA S等的病毒性疾病, 如结核、 细菌性肺炎等的细菌 性疾病, 以及病原微生物导致的急慢性传染病); 其它急慢性疾病 (如呼吸系统疾病、 免疫系统 疾病、血液与造血系统疾病、 如心脑血管疾病的循环系统疾病、 内分泌系统代谢性疾病、 消化 系统疾病、 神经系统疾病、 泌尿系统疾病、 生殖系统疾病和运动系统疾病)。 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。
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 0.1-90 %(按 microRNA if)
所述组合物的制备方法包括步骤: 将所述的植物功能性 microRNA 或含所述功能性 microRNA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 l-2000mg 活性成分 / 剂, 更佳地, 含有 10-200mg活性成分 /剂。 或者含有 0.01〜100微摩尔活性成分 /剂, 较佳地为
0.1〜10微摩尔 /剂; 较佳地, 所述的"一剂"为一口服液。
"药学上可以接受的载体"指的是: 一种或多种相容性固体或液体填料或凝胶物质, 它们适 合于人使用, 而且必须有足够的纯度和足够低的毒性。 "相容性"在此指的是组合物中各组份能 和本发明的化合物以及它们之间相互掺和,而不明显降低化合物的药效。药学上可以接受的载 体部分例子有纤维素及其衍生物 (如羧甲基纤维素钠、 乙基纤维素钠、纤维素乙酸酯等)、 明胶、 滑石、 固体润滑剂 (如硬脂酸、 硬脂酸镁)、 硫酸钙、 植物油 (如豆油、 芝麻油、 花生油、 橄榄油 等)、 多元醇 (如丙二醇、 甘油、 甘露醇、 山梨醇等)、 乳化剂 (如吐温 ®)、 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、 巴布膏剂、 膏药、 橡胶膏剂、 软膏剂、 硬膏剂、 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 microR A分子 MIR2911或含 MIR2911的提取物的用途, 用 于制备治疗病毒性感冒的药物。 较佳地, 所述的提取物 (未浓縮或浓縮)中含 0.01-100 nM (较佳 地 0.1-20 nM)的 MIR2911。 二、 指导人工合成植物功能性 microRNA
通过本发明所述的方法鉴定出植物中 (尤其是植物提取中)存在的植物功能性 microRNA, 可直接指导技术人员对所述 microRNA进行人工合成, 从而提高所述的 microRNA的生产。并 将合成的植物功能性 microRNA分子与药学上或食品学上可接受的载体混合, 从而形成组合 物。
人工合成 microRNA的方法可以本领域技术人员所熟悉的方法。例如,人工合成 MIR2911, 优选方法包括:
1. 化学合成 MIR2911的方法, 具体操作步骤包括:
(1)制备四种脱氧核苷 (脱氧腺嘌呤核苷 dA, 脱氧鸟嘌呤核苷 dG, 脱氧胞嘧啶核苷 dC, 脱氧胸腺嘧啶核苷 dT)的甲氧基亚磷酞二异丙胺单体;
(2)经四唑活化后, 起始保护脱氧核苷与载体相连接;
(3)利用固相亚磷酞胺法, 在载体上逐个延长核苷酸链, 合成 MIR2911 ;
2. 其他 MIR2911的合成方法, 具体操作步骤包括:
(1)设计合成 MIR2911的引物:
根据 MIR2911的模板质粒序列合成两条通用引物 A、 B , 根据 MIR2911序列设计 4条 特异的寡核苷酸引物序列 (1、 II、 III、 IV);
(2)第一轮 PCR扩增:
以包含 MIR2911的质粒作为模板, 分别以 A与 IV、 III与 Π、 I与 B作为引物组合进行 PCR扩增, PCR反应条件是 : 95 °C、 2分钟进行 1个循环→95 °C、 30秒, 55 °C、 30秒, 72 °C、 40秒进行 24个循环→72°C、 7分钟; 分别得到产物 1、 产物 2、 产物 3 ;
(3)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 °C、 2分钟进行 1个循环→95 °C、 30秒, 55 °C、 30秒, 72 °C、 1分 30秒进行 24个循环→72°C、7分钟, PCR产物琼脂糖凝胶回收,得到合成的 MIR2911;
(4)将合成的 MIR2911甲基化, 形成稳定的甲基化产物 MIR2911。 三、 体外非治疗性调控非植物靶基因表达
在本发明所述的分离的植物功能性 microRNA或含所述植物功能性 microRNA的提取物存 在下, 培养含非植物靶基因 (如细菌基因、 病毒基因、 衣原体基因、 酵母基因、 动物基因)的生 物材料 (包括病毒、 细胞、 组织), 从而实现体外调控所述非植物靶基因的表达
在另一优选例中, 所述的靶基因是病原体 (包括细菌、 病毒、 衣原体等)的基因。
在另一优选例中, 所述的植物功能性 microRNA来自以下植物: 药用植物、 果蔬植物、 观 赏植物;较佳地,来自金银花、菘蓝、草大青、马蓝或胡杨;更佳地,所述植物功能性 microRNA 包括 MIR2911。 四、 疾病预防或治疗方法
给需要的对象 (如哺乳动物或人))施用本发明所述分离的植物功能性 microRNA或含所述 植物功能性 microRNA的提取物、或本发明所述的组合物, 从而实现预防或治疗与非植物靶基 因相关的疾病, 其中所述的植物功能性 microRNA具有调控非植物靶基因 (包括细菌基因、 病 毒基因、 衣原体基因、 酵母基因、 动物基因)的功能。
在另一优选例中, 所述的非植物靶基因是病原体基因。 发明人通过对植物功能性 microRNA及其进入动物体内后的存在形式、递送途径和功能等 —系列研究, 1. 形成了一套高效、 稳定地提取植物功能性 microRNA的方法,提供了一种包含 植物功能性 microRNA的植物提取物。2. 发现了通过多种途径 (如摄食或静脉注射等),植物 (如 金银花等)功能性 microRNA可进入动物体并在血液中富集, 并调节非植物靶基因, 进而参与 动物体 (如人)的生理病理活动。 3. 形成了一套鉴定植物功能性 microRNA的方法, 可用于指导 选择性合成植物功能性 microRNA的方法, 有利于加快该 microRNA的生产; 还可用于指导筛 选富含植物功能性 microRNA的药材, 有利于鉴别药材的优劣。 4. 形成了一套指导制造功能 性食物或药物的方法: 4.1 所述方法利用分离的植物功能性 microRNA 或包含植物功能性 microRNA的植物提取物或筛选出的富含功能性 microRNA的植物, 可用于制造功能性食物或 药物等组合物; 4.2所述方法通过提取植物 microRNA并鉴定出植物功能性 microRNA, 并进 行所述植物功能性 microRNA的人工合成,然后将人工合成的 microRNA用于制造功能性食物 或药物。 本发明主要优点包括:
1. 提供了一种分离的植物功能性 microRNA和 /或含所述植物功能性 microRNA的植物提 取物及其用途。 所述 microR A为可调控非植物靶基因的表达, 具有多种用途 (如可作为中草 药有效成分的标准, 有利于指导功能性组合物的开发和制造等)。
2. 提供了一种以分离的植物功能性 microRNA和 /或含所述植物功能性 microRNA的植物 提取物为活性成分的组合物。所述活性成分的作用机理明确, 效果显著, 有利于对中医药的科 学机制的探索, 且制法简单、 成本低廉, 适合工业化生产。
3. 提供了一种鉴定植物功能性 microRNA的方法。 下面结合具体实施, 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,否则百分比和份数按重量计算。 实施例 1使用 Solexa测序技术检测稳定存在于金银花水提液中的植物 microRNA 首先, 利用水提法提取金银花 microRNA。 取适量 (50克)干燥金银花药材, 在 500 ml (;金 银花质量与水体积比为 1 : 10)水的 100°C水浴下加热 0.5小时, 提取液在 60°C下减压浓縮至原 体积的 1/10。 收集浓縮及未浓縮金银花水提液, 用于后续实验。
然后, 使用 Solexa测序技术检测稳定存在于经上述步骤制备出的金银花水提液中的植物 microRNA, Solexa上样量为 10 R A。经测定, 未浓縮水提液中总 microRNA的浓度约为 1 M, 浓縮水提液中总 microRNA浓度约为 10 M。
检测结果显示, 金银花水提液中稳定存在多种植物 microRNA, 能被检测到的包括表 1中 所列出的 85种 microRNA, 其序列详见表 1。 其中 MIR156h、 MIR166f、 MIR396a、 MIR166a、 MIR168a、 MIR1440、 MIR2910、 MIR291 MIR2915、 MIR2916含量较高, MIR2911的含量 最高。具体结果见图 1。表 1中其他 75种 miRNA在提取物中检测出拷贝数均较低 (都在 2-1000 之间)。
表 1. 稳定存在于金银花中且可检测的 microR As
microRNA名称 对应序列
SEQ ID NO.: 1 MIR156h uugacagaagauagagagcac
SEQ ID NO. :2 MIR166f ucggaccaggcuucauucccc
SEQ ID NO. :3 MIR396a uuccacagcuuucuugaacug
SEQ ID NO. :4 MIR166a ucggaccaggcuucauucccc
SEQ ID NO. :5 MIR168a ucgcuuggugcaggucgggaa
SEQ ID NO. :6 MIR1440 ugcucaaauaccacucuccu
SEQ ID NO. :7 MIR2910 uaguugguggagcgauuuguc
SEQ ID NO. :8 MIR2911 ggccgggggacgggcuggga
SEQ ID NO. :9 MIR2915 cccgucuagcucaguuggua
SEQ ID NO.: 10 MIR2916 uggggacucgaagacgaucauau
SEQ ID NO.: 11 MIR818d aaucccuuauauuaugggacgg
SEQ ID NO.: 12 MIR159e auugguuugaagggagcucca
SEQ ID NO.: 13 MIR159c auuggauugaagggagcucca
SEQ ID NO.: 14 MIR156j ugacagaagagagagagcac
SEQ ID NO.: 15 MIR1432 cucaggagagaugacaccgac
SEQ ID NO.: 16 MIR166k ucggaccaggcuucauucccc SEQ ID NO.: 17 MIR167b ugaagcugccagcaugaucua
SEQ ID NO.: 18 MIR396c uuccacagcuuucuugaacuu
SEQ ID NO.: 19 MIR156e ugacagaagagagugagcaca
SEQ ID NO. :20 MIR169k uagccaaggaugacuugccug
SEQ ID NO.:21 MIR167c uaagcugccagcaugaucuug
SEQ ID NO. :22 MIR160d ugccuggcucccuguaugcca
SEQ ID NO. :23 MIR399a ugccaaaggagaauugcccug
SEQ ID NO. :24 MIR156d ugacagaagagagugagcac
SEQ ID NO. :25 MIR160e ugccuggcucccuguaugcca
SEQ ID NO. :26 MIR169n ugagccaaggaugacuugccg
SEQ ID NO. :27 MIR1661 ucggaccaggcuucaaucccu
SEQ ID NO. :28 MIR159f uuuggauugaagggagcucug
SEQ ID NO. :29 MIR166c ucggaccaggcuucauucccc
SEQ ID NO. :30 MIR159b uuuggauugaagggagcucug
SEQ ID NO. :31 MIR166J ucggaucaggcuucauuccuc
SEQ ID NO. :32 MIR167i ugaagcugccagcaugaucug
SEQ ID NO. :33 MIR169c cagccaaggaugacuugccgg
SEQ ID NO. :34 MIR164c uggagaagcagggcacgugca
SEQ ID NO. :35 MIR167J ugaagcugccagcaugaucug
SEQ ID NO. :36 MIR167g ugaagcugccagcaugaucug
SEQ ID NO. :37 MIR160c ugccuggcucccuguaugcca
SEQ ID NO.:38 MIR399e ugccaaaggagauuugcccgg
SEQ ID NO. :39 MIR399b ugccaaaggagauuugcccgg
SEQ ID NO. :40 MIR529b agaagagagagaguacagcuu
SEQ ID NO.:41 MIR164e uggagaagcaggacacgugag
SEQ ID NO. :42 MIR166d ucggaccaggcuucauucccc
SEQ ID NO. :43 MIR166h ucggaccaggcuucauucccc
SEQ ID NO. :44 MIR164b uggagaagcagggcacgugca
SEQ ID NO. :45 MIR156f ugacagaagagagugagcac
SEQ ID NO. :46 MIR164a uggagaagcagggcacgugca
SEQ ID NO. :47 MIR1691 uagccaaggaugacuugccug
SEQ ID NO. :48 MIR166m ucggaccaggcuucauucccu
SEQ ID NO. :49 MIR164f uggagaagcagggcacgugcu
SEQ ID NO. :50 MIR156k ugacagaagagagggagcac
SEQ ID NO. :51 MIR166g ucggaccaggcuucauucccc
SEQ ID NO. :52 MIR166b ucggaccaggcuucauucccc
SEQ ID NO. :53 MIR160b ugccuggcucccuguaugcca
SEQ ID NO. :54 MIR166e ucggaccaggcuucauucccc
SEQ ID NO. :55 MIR159d cuuggauugaagggagcuccu
SEQ ID NO. :56 MIR818e aaucccuuauauuaugggacgg
SEQ ID NO. :57 MIR172a agaaucuugaugaugcugcau
SEQ ID NO.:58 MIR156b ugacagaagagagugagcac
SEQ ID NO. :59 MIR399g ugccaaaggagauuugcccag
SEQ ID NO. :60 MIR169b cagccaaggaugacuugccgg
SEQ ID NO.:61 MIR399f ugccaaaggagauuugcccag
SEQ ID NO. :62 MIR167a ugaagcugccagcaugaucua
SEQ ID NO. :63 MIR394 uuggcauucuguccaccucc
SEQ ID NO. :64 MIR156a ugacagaagagagugagcac
SEQ ID NO. :65 MIR166i ucggaucaggcuucauuccuc SEQ ID NO. :66 MIR167f ugaagcugccagcaugaucug
SEQ ID NO. :67 MIR319a agcugccgaaucauccauuca
SEQ ID NO. :68 MIR156g ugacagaagagagugagcac
SEQ ID NO. :69 MIR166n ucggaccaggcuucauucccc
SEQ ID NO. :70 MIR399c ugccaaaggagaauugcccug
SEQ ID NO.:71 MIR160a ugccuggcucccuguaugcca
SEQ ID NO. :72 MIR159a.l uuuggauugaagggagcucug
SEQ ID NO. :73 MIR156c ugacagaagagagugagcac
SEQ ID NO. :74 MIR319b uuggacugaagggugcuccc
SEQ ID NO. :75 MIR169o uagccaagaaugacuugccua
SEQ ID NO. :76 MIR167h ugaagcugccagcaugaucug
SEQ ID NO. :77 MIR156i ugacagaagagagugagcac
SEQ ID NO. :78 MIR167d ugaagcugccagcaugaucug
SEQ ID NO. :79 MIR169a cagccaaggaugacuugccga
SEQ ID NO. :80 MIR172d agaaucuugaugaugcugcau
SEQ ID NO. :81 MIR818b aaucccuuauauuaugggacgg
SEQ ID NO. :82 MIR164d uggagaagcagggcacgugcu
SEQ ID NO. :83 MIR167e ugaagcugccagcaugaucug
SEQ ID NO. :84 MIR396b uuccacagcuuucuugaacug
SEQ ID NO. :85 MIR2914 uaugguggugacgggugacggag 实施例 2利用 Real-time PCR方法检测通过摄食进入动物体内且稳定存在的金银花 microRNA
本实施例证实金银花 microRNA可通过摄食进入动物体内, 且稳定存在。
向小鼠灌胃金银花水提液 (实施例 1制备的浓縮水提掖), 检测金银花 microRNA在血清、 肝脏和肺中的表达水平。
具体步骤为: 首先将小鼠饥饿 12小时, 然后向小鼠灌胃 10 ml/kg小鼠体重的金银花浓縮 水提液, 采用 Real-time PCR在 0 h(0小时)、 2 h(2小时)、 4 h(4小时)、 6 h(6小时)等, 检测金 银花 microRNA MIR2911在小鼠血清、 肝脏和肺中的表达水平。
用于 Real-time PCR检测 MIR2911的引物序列为:
正向弓 I物: ACACTCCAGCTGGGGGCCGGGGGACGGG (SEQ ID NO.: 86);
NO.: 87);
探针序歹 'J: TCCCAGCCCGTCCCCCGGCC (SEQ ID NO.: 88)。
具体结果见图 2A和图 2B。
图 2A是小鼠灌胃金银花水提液后, 在不同时间点, 金银花 microRNA在血清中表达量的 Real-time PCR结果。 从结果可以看出, 小鼠灌胃金银花水提液后, 血清中 MIR2911的含量显 著增加。 灌胃金银花水提液 1.5 小时后, 血清中 MIR2911 的含量达到最高值, 6小时后血清 中 MIR2911的含量下降到最初水平。
图 2B是小鼠灌胃金银花水提液后, 在不同时间点, 金银花 microRNA在肝脏和肺中表达 量的 Real-time PCR结果。 从结果可以看出, 小鼠灌胃金银花水提液后, MIR2911在肝脏和肺 中的表达水平增加。 向小鼠灌胃金银花水提液 6小时后, MIR2911在肝脏中的表达达到最大 值; 灌胃金银花水提液 12小时后, MIR2911在肺中的表达达到最大值。 结果表明, 金银花 micraR A可通过摄食进入动物体内, 且稳定存在。 实施例 3金银花 mkroRNA可调控生理病理活动
本实施例证实金银花 microR A可在动物体内调节生理病理活动。
3.1 利用生物信息学预测金银花 microRNA的靶基因
采用生物信息学预测呼吸道传染病病毒的基因组中多个靶基因与 MIR2911序列匹配。 具 体结果见图 3。 图 3是 MIR2911预测靶基因的序列分析结果。 mfe表示候选靶基因的最低折叠 自由能, mfe绝对值越大, 候选靶基因与 Peu-MIR2911序列匹配度越高。 结果表明, ADV基 因、 HPIV1基因、 H1N1基因、 H5N1基因是金银花 microRNA的潜在靶基因。
3.2利用荧光素酶检测方法验证靶基因。 具体步骤如下:
(1) 构建一个将预测的 MIR2911靶基因序列特定片段插入到荧光素酶表达质粒的 3'-UTR 区;
(2)筛选阳性克隆, 测序; 扩增克隆并提纯质粒备用;
(3)扩增预测的靶基因质粒, 提纯备用; 同时准备相应的空载质粒对照, 提纯备用;
(4)培养相关细胞, 并接种于 24孔板中, 生长 10-24小时;
(5)将要检测的载有预测靶基因的荧光素酶表达质粒与 MIR2911共转染细胞;
(6)加入荧光素酶底物, 荧光素酶与底物反应, 产生荧光素, 通过检测荧光的强度可以测 定荧光素酶的活性;
(7)与空载对照 (NC)比较, 从而判断预测靶点是否能被 MIR2911抑制。
具体结果见图 4。 图 4是荧光素酶检测预测靶基因的结果。 以 NC (即等量的含有 MIR2911 的错配对照序列的 R A)作为对照。 结果表明, 大多数预测的靶基因可与成熟 MIR2911结合, 形成双链结构。 这提示, MIR2911可对 ADV基因、 HPIV基因、 H1N1基因、 H5N1等基因通 过结合而起到调节作用。
3.3 检测马丁达比狗肾(MDCK)细胞中 MIR2911对病毒基因 ADV、H1N1、H5N1的影响。 具体步骤如下:
(1)在 24孔细胞培养板中培养 MDCK细胞。
(2)将 MIR2911(20 pmol/106细胞)通过商用转染试剂 lipofectamine2000转染进 MDCK细 胞 (MIR2911组), 同时将 MIR2911的无义序列 R A按照相同剂量转染至 MDCK细胞作为对 照 (NC), 并同时设立阳性药物 (达菲)。
(3)使用 H1N1, H5N1和腺病毒 ADV按照感染复数 (MOI) 为 0.001感染上述细胞。
(4)病毒感染 24小时后, 通过 real-time PCR检测细胞中病毒基因 ADV、 H1N1、 H5N1的 含量, 通过与对照组比较, 判断 MIR2911是否对病毒复制具有抑制作用。
检测病毒基因的具体步骤为: 将细胞消化收集后, 用 PBS缓冲液冲反复洗 3次, 用蛋白 变性试剂提取总 R A, 然后通过 real-time PCR方法鉴定其中病毒标志 PBl mR A的含量, 并 与标准曲线对照, 计算病毒的含量。
具体结果见图 5A、 图 5B和图 5C。 图 5A是 Real-Time PCR技术检测病毒基因 ADV在 MDCK细胞中的含量。 图 5B是 Real-Time PCR技术检测病毒基因 H1N1在 MDCK细胞中的 含量。 图 5C是 Real-Time PCR技术检测病毒基因 H5N1在 MDCK细胞中的含量。 结果表明, MIR2911对 ADV5、 H1N1和 H5N1有显著的抑制和阻断作用。 实施例 4金银花 mkroRNA通过肠道上皮细胞 Caco-2细胞微粒子 (MVs)进入其他细胞, 并在其他细胞内起调控作用
本实施例证实金银花 microR A可通过肠道上皮细胞 Caco-2细胞微粒子包裹进入动物体, 并被细胞微粒子递送进入其他细胞, 对其他细胞的生理病理状况产生影响, 如抑制感冒病毒。
4.1 将金银花 microRNA转入肠道上皮细胞 Caco-2, 具体步骤为:
(1)将肠道上皮细胞 Caco-2, 接种于 12孔板或 10毫米培养皿, 过夜;
(2)第二天使用脂质体 2000转染;
(3) MIR2911转染 Caco-2细胞;
(4)转染后, 细胞培养 24或 48小时后, 用于 Real-time PCR分析。
4.2采用差速离心法分离肠道上皮细胞 Caco-2释放的细胞微粒子:
(1)先将培养肠道上皮细胞 Caco-2于 300 g离心 5分钟, 取上清;
(2)将上清于 1200 g离心 20分钟, 取上清;
(3)将上清于 10000 g离心 30分钟, 取上清;
4)将上清于 110000 g离心 2小时, 全部操作在 4°C中, 在 FBS-free介质中收集沉淀, 得 到总细胞微粒子。
4.3 检测 Caco-2细胞释放的细胞微粒子中 MIR2911水平,
证实 MIR2911 已被 Caco-2细胞释放的细胞微粒子包裹: 提取微粒子中的 RNA, 通过绝 对定量 real-time PCR技术确定其中的 MIR2911的含量。
4.4 收集载有 MIR2911的细胞微粒子, 用其处理 HEK 293T细胞, 证实金银花 microRNA 由细胞微粒子递送进入其他细胞。
用载有 MIR2911 的细胞微粒子处理 HEK 293T细胞后, 采用 Real-time PCR实验检测 MIR2911在 HEK 293T细胞中的表达水平。 Real-time PCR实验具体步骤如实施例 2所述。
具体结果见图 6。 图 6是经载有 MIR2911的 Caco-2细胞分泌的细胞微粒子处理后, HEK 293T细胞分泌的细胞微粒子中的 Real-time PCR结果。 以未经载有 MIR2911的 Caco-2细胞分 泌的细胞微粒子 (;对照 MV)处理后的 HEK 293T细胞分泌的细胞微粒子作为对照。 从结果可以 看出, 与对照相比, MIR2911在用载有 MIR2911细胞微粒子处理后 HEK 293T细胞中表达显 著增加。
结果表明, 金银花 microRNA可通过 Caco-2分泌细胞微粒子包裹进入动物体, 并递送至 其他细胞。
4.5 金银花 microRNA由细胞微粒子递送进入其他细胞后, 对细胞生理 /病理状况的影响: 用载有 MIR2911的细胞微粒子 (制法同本实施例第 4.2部分的差速离心法) 处理被流感病 毒 ADV或 H1N1感染的 HEK 293T细胞。 具体步骤为: (1)在 24孔板中培养 HEK 293T细胞。
(2) 将本实施例 4.4 部分获得的微粒子加入细胞培养基, 微粒子用量为 0.1 pmol MIR2911/106 细胞。
(3)步骤 (2)处理 6小时后, 按照 MOI = 0.001感染腺病毒 ADV或 H1N1感冒病毒。
(4)病毒感染 24小时后, 通过 Real-time PCR检测病毒的含量。 采用 Real-time PCR方法 检测 HEK 293T细胞中流感病毒 ADV或 H1N1的含量。 具体步骤如实施例 3所述。
具体结果见图 7。图 7A是流感病毒 ADV在载有 MIR2911的细胞微粒子处理的 HEK 293T 细胞中的表达量。图 7B是流感病毒 H1N1在载有 MIR2911的细胞微粒子处理的 HEK 293T细 胞中的表达量。图 7A和图 7B以感染流感病毒后不作处理的 HEK 293T细胞 (空白对照)和以未 载有 MIR2911的细胞微粒子 (阴性对照)处理的 HEK 293T细胞作为对照。
从结果可以看出,在载有 MIR2911的细胞微粒子处理的 HEK 293T细胞中,流感病毒 ADV 和 H1N1表达显著降低。 结果表明, MIR2911对流感病毒 ADV和 H1N1有显著抑制作用。
本实施例证实了金银花 microRNA可通过口服等途径进入动物体,被细胞微粒子包裹递送 进入其他细胞, 并在细胞中发挥作用, 如抑制感冒病毒。 可见, 金银花 microRNA通过可通过 口服等途径进入动物组织、 器官, 并调节动物生理病理状况。 实施例 5金银花 microRNA可在动物体内显著抑制感冒病毒
本实施例证实抑制感冒病毒的有效成分为金银花 microRNA, 而非其他。
使小鼠随意饮用未浓縮金银花水提液 (实施例 1制备) 3天后, 接种 ADV或 H1N1病毒, 继续服用未浓縮金银花水提液 3天, 然后利用 real-time PCR检测小鼠肺部的病毒含量。 结果 表明, 金银花汤药对 ADV和 H1H1病毒的增殖有强烈的抑制作用。
为验证发挥病毒抑制作用的正是金银花 microRNA MIR2911, 采用如下对照组:
1. 使小鼠饮用含有 anti-MIR2911(用量与 MIR2911 的用量相当)的金银花汤药, 包括: 金 银花水提液 +anti-MIR2911(口服组)和金银花水提液 +anti-MIR2911(灌肺组);其中 anti-MIR2911 是 MIR2911的反义核酸, 与 MIR2911完全互补。
小鼠灌肺, 具体步骤为:
(1)将小鼠麻醉并固定, 置于超净工作台;
(2)颈部去毛并消毒;
(3)无菌条件下暴露气管, 插导管并固定;
(4)用 1次性注射器抽取金银花水提液 +anti-MIR2911灌肺, 反复抽吸。
2. 使小鼠饮用与金银花汤药等体积的纯水, 作为空白对照组。 然后, 使用 Real-time PCR 方法检测各组小鼠肺部感冒病毒 ADV或 H1N1的含量。
Real-time PCR检测感冒病毒 ADV的引物序列如下:
正向引物序列: 5'-CAAAGACTTCTCATCGGTTGC-3, (SEQ ID NO. :89);
反向引物序列: 5'-AATGCAACACTCGGTTCACA-3' (SEQ ID NO.: 90);
探针序列: TCAGGC CCC CTCAAAGCCGA (SEQ ID NO.: 91)。
Real-time PCR检测感冒病毒 H1N1的引物序列如下:
正向引物序列: 5 '-CCCAAAGTGAGGGATCAAGA-3 ' (SEQ ID NO.: 92); 反向引物序列: 5'-CCCTTGGGTGTCTGACAAGT-3' (SEQ ID NO.: 93);
探针序列: TCAACAGTGGCGAGTTCCCTAGCA (SEQ ID NO.: 94)。
具体结果见图 8A和图 8B。
图 8A是向小鼠饮用金银花水提液、金银花水提液 +anti-MIR2911 (口服组)、灌肺金银花水 提液 +anti-MIR2911C灌肺组)后, 感冒病毒 ADV在肺表达的 Real-time PCR结果。
从结果可以看出: 以喂食相同体积的水的小鼠作为空白对照,饮用金银花水提液后, ADV 在小鼠肺部表达显著降低, 表明金银花 microRNA抑制感冒病毒 ADV的表达。 与饮用金银花 水提液相比, 小鼠饮用金银花水提液 +anti-MIR2911 后, ADV在小鼠肺部表达增加, 说明 anti-MIR2911破坏 MIR2911对感冒病毒 ADV的抑制作用。结果表明,金银花 microRNA有直 接抗病毒作用。
图 8B是向小鼠饮用金银花水提液、金银花水提液 +anti-MIR2911 (口服组)、灌肺金银花水 提液 +anti-MIR2911C灌肺组)后, 感冒病毒 H1N1在肺表达的 Real-time PCR结果。
从结果可以看出:与饮用纯水的空白对照组小鼠相比,饮用金银花水提液的小鼠肺部 H1N1 表达显著降低, 表明金银花 microRNA抑制感冒病毒 H1N1 的表达。 而饮用金银花水提液 +anti-MIR2911后, 小鼠肺部 H1N1表达增加。 由于 anti-MIR2911破坏 MIR2911 , 结果表明, 金银花 microRNA有直接抗病毒作用。 实施例 6金银花 microRNA通过消化道进入人体
本实施例证实金银花 microRNA通过消化道被人体吸收, 进入循环系统。
首先将干金银花水煮 30 min, 制成 1000 ml的水提液, MIR2911在水提液中的含量约为 0.4 n mol/L。然后招募 20名健康志愿者, 每人服用制成的水提液 1000 ml, 在 0 h (0小时)、 l h (1小时)、 2 h (2小时)、 3 h (3小时)、 4 h (4小时)、 5 h (5小时)、 6 h (6小时)收集志愿者的血 液, 利用 Real-time PCR检测其中 MIR2911的含量, Real-time PCR实验具体步骤如实施例 2 所述。
具体结果见图 9。图 9是服用金银花水提液后,人血液中 MIR2911的 Real-time PCR结果。 从结果可以看出, 服用金银花水提液后, 人血液中 MIR2911的含量显著增加。 服用金银花水 提液 1.5 小时后, 人血液中 MIR2911的含量达到最大值, 3小时后人血液中 MIR2911的含量 下降到最初水平。 这些结果与实施例 2 中小鼠实验结果一致 (图 1K)。 结果表明, 金银花 microRNA可通过摄食进入人体, 被消化道吸收, 进入循环系统。 实施例 7金银花 microRNA可显著抑制人体内的感冒病毒
本实施例证实抑制人体感冒病毒的有效成分为金银花 microRNA, 而非其他。
首先招募 15名携带 ADV病毒的病毒性感冒患者, 15名携带 H1N1病毒的病毒性感冒患 者, 然后将上述病毒性感冒患者分成 6组, 每组 5人, 组 1-3为携带 ADV病毒的病毒性感冒 患者, 组 4-6为携带 H1N1病毒的病毒性感冒患者:
组 1 : 服用金银花水提液 (实施例 6制备) 1000mL。
组 2: 服用金银花水提液 lOOOmL + anti-MIR2911(用量与 MIR2911 的用量相当); 其中 anti-MIR2911是 MIR2911的反义核酸, 与 MIR2911完全互补, 可特异性降解 MIR2911。 组 3: 服用与水提液等体积纯水。
组 4: 服用金银花水提液 (实施例 6制备) 1000mL。
组 5: 服用金银花水提液 lOOOmL + anti-MIR2911(用量与 MIR2911 的用量相当); 其中 anti-MIR2911是 MIR2911的反义核酸, 与 MIR2911完全互补, 可特异性降解 MIR2911。
组 6: 服用与水提液等体积纯水。
服用 6天后, 采用 Real-time PCR检测人血液中感冒病毒 ADV或 H1N1的含量。 具体步 骤如实施例 5所述。
结果表明:
1. 以服用水的感冒患者 (组 3, 组 6)作为对照, 服用金银花水提液后, ADV或 H1N1在人 血液中表达显著降低, 表明金银花 microR A抑制人体感冒病毒 ADV或 H1N1的表达。
2. 以服用金银花水提液的感冒患者 (组 1, 组 4M乍为对照, 感冒患者服用金银花水提液 +anti-MIR2911后, ADV或 H1N1在人血液中表达增加。 可见 anti-MIR2911破坏 MIR2911对 感冒病毒 ADV或 H1N1的抑制作用。 结果表明, 金银花 microRNA有直接抗病毒作用。 实施例 8 MIR2911的稳定性研究以及 MIR2911在人和小鼠中的吸收和组织分布情况
8.1 新鲜金银花和金银花水提液中存在的植物 miRNAs
实验方法:
首先,利用水提法提取金银花 microR A。取适量 (100克)新鲜金银花药材,在 1000 ml (;金 银花质量与水体积比为 1 : 10)水的 100°C水浴下加热 0.5小时, 提取液在 60°C下减压浓縮至原 体积的 1/10。 收集金银花水提液, 用于后续实验。
取适量 (1克)新鲜金银花药材, 液氮条件下研磨, 通过 Trizol法提取总 RNA, 用于后续实 验。
然后, 使用 Solexa测序技术检测稳定存在于经上述步骤制备出的新鲜金银花和金银花水 提液中的植物 microRNA, Solexa上样量为 10 μ g RNA。
实验结果:
a. 植物 miRNAs在新鲜金银花中 Solexa测序结果;
新鲜金银花中稳定存在多种植物 microRNA, 其中 MIR167a、 MIR166f、 MIR166b、 MIR164a、 MIR168a、 MIR156h、 MIR172a、 MIR162b、 MIR159d、 MIR827b、 MIR396b、 MIR2911、 MIR2916 含量较高。 如图 10所示。 b. 植物 miRNAs在金银花水提液中 Solexa测序结果;
金银花水提液中稳定存在多种植物 microRNA, 包括 MIR167a、 MIR166f、 MIR166b、 MIR164a、 MIR168a、 MIR156h、 MIR172a、 MIR162b、 MIR159d、 MIR827b、 MIR396b、 MIR2911、 MIR2916 , 其中 MIR2911含量最高。 如图 11所示。
8.2 提取的或合成的 MIR2911的稳定性分析
实验方法:
利用水提法提取金银花 micraR A。 具体实验步骤如实施例 8.1所述。 并按照常规方法 人工合成的 MIR2911。
利用 Real-time PCR方法检测金银花提取液中 MIR2911或合成的 MIR2911的浓度,具体 操作步骤如实施例 2所述。
利用 Real-time PCR方法检测血清、 水中 MIR2911和 MIR167的浓度, 具体操作步骤如 实施例 2所述。
实验结果:
由图 12可知, 与 MIR2911突变体 (有一个点突变)相比, 不管是合成的还是从金银花提 取的 MIR2911 , 都未发生降解, 稳定性高。 MIR2911 突变体在一小时后浓度就急速降低,说 明降解速度较快。 可见, 金银花提取的 MIR2911或合成的 MIR2911稳定性较高。 有作为药 物发挥作用的基础。
由图 13可知, 与 MIR167相比, 不管是合成的还是从金银花提取的 MIR2911 , 各个时 间点上, 在水、 血清中都未发生降解, 稳定性高; 相比之下 MIR167降解速度快, 从一个小 时后含量就大幅降低。 可见, MIR2911在水、 血清中较稳定。
8.3 小鼠灌胃合成的 MIR2911后, MIR2911在小鼠血清中动力学研究结果。
小鼠灌胃 2 nmol合成的 MIR2911后, 血清中 MIR2911含量显著增加。 如图 14A所示。 灌胃 0.5小时后, 血清中 MIR2911含量达到最高值, 24小时后血清中 MIR2911含量下降到 最初水平。
结果说明合成的 MIR2911可以进入动物体内,通过正常的代谢途径,符合药物代谢动力 学规律, 可以作为潜在治疗药物。
8.4 小鼠灌胃金银花水提掖后, MIR2911在器官中的分布。
小鼠灌胃金银花水提液 3小时后, 荧光标记的合成 MIR2911特异地传输到小鼠肺部。 如图 14B所示, 标记的 MIR2911(红色的)在小鼠肺部快速积累。 说明 MIR2911进入血液后, 可以达到需要发挥作用的部门, 即可以顺利达到靶器官, 发挥作用。 实施例 9 MIR2911可调控生理病理活动
本实施例证实 MIR2911在感染病毒的细胞中, 靶向各种流感病毒、 抑制其复制。
9.1 MIR2911预测病毒靶基因的序列分析结果。
利用生物信息学预测病毒基因 H1N1、 H3N2、 H5N1、 H7N9与 MIR2911相匹配。 具体 结果见图 15。 图 15是 MIR2911预测靶基因的序列分析结果。 结果表明, 病毒基因 H1N1、 H3N2、 H5N1、 H7N9是金银花 microR A的潜在靶基因。
9.2 利用荧光素酶报告基因, 验证预测的 MIR2911病毒靶基因组序列。
实验方法:
利用荧光素酶检测方法验证靶基因。 具体操作步骤如实施例 3.2所述。
实验结果:
结果如图 16所示。 经验证分析, H1N1、 H3N2、 H5N1、 H7N9是金银花 MIR2911的潜 在靶基因。
9.3 在 MDCK细胞中, 植物 MIR2911可以与 AG02复合物结合, AG02可以帮助运 输 MIR2911进入细胞。
实验方法: 具体步骤如下:
(1)将植物 MIR2911与 AG02复合物结合;
(2)在 24孔细胞培养板中培养 MDCK细胞。
(3)使用 MIR2911与 AG02复合物转入上述细胞。
(4) 24小时后, 通过 Real-time PCR检测细胞中 MIR2911的表达水平。
实验结果: 结果如图 17所示。 研究发现, MIR2911可以与 AG02复合物结合, AG02 可以帮助运输 MIR2911进入细胞。
9.4 MIR2911在 MDCK细胞中对抗甲型 H1N1流感病毒基因 (Anti-HINI)的影响。 实验方法:
具体操作步骤如实施例 3.3所述。不同的是, MIR2911通过 MIR2911与 AG02复合物转 染进入 MDCK细胞中。 转染时间分为 12小时、 24小时。 实验结果:
结果如图 18所示。研究发现,在不同作用时间下 (12小时或 24小时给药时间), MIR2911 对 H1N1病毒有显著抑制作用。
9.5 MIR2911在 MDCK细胞中对抗 H5N1禽流感病毒基因 (Anti-H5N1)的影响。 实验方法:
具体操作步骤如实施例 3.3所述。不同的是, MIR2911通过 MIR2911与 AG02复合物转 染进入 MDCK细胞中。 转染时间分为 12小时、 24小时。
实验结果:
结果如图 19所示。研究发现,在不同作用时间下 (12小时或 24小时给药时间), MIR2911 对 H5N1病毒有显著抑制作用。
9.6 MIR2911在 MDCK细胞中对抗 H7N9禽流感病毒基因 (Anti-H7N)的影响。
实验方法:
具体操作步骤如实施例 3.3所述。不同的是, MIR2911通过 MIR2911与 AG02复合物转 染进入 MDCK细胞中。 转染时间分为 12小时、 24小时。 使用禽流感病毒基因 H7N9感染 MDCK细胞。
实验结果:
结果如图 20所示。研究发现,在不同作用时间下 (12小时或 24小时给药时间), MIR2911 对 H7N9病毒有显著抑制作用。 实施例 10 MIR2911抑制小鼠 IAVs (表示甲型流感病毒)的复制。
小鼠在接种前用三种不同的亚型流感病毒处理。 感染病毒 3、 5、 7天后, 每天记录体重 变化。 处死小鼠, 测量肺部的病毒量。
10.1 H1N1病毒
1. 研究 MIR2911对接种 H1N1病毒小鼠的体重的影响
研究发现, 接种 H1N1病毒的小鼠, 饮用含 MIR2911的溶液或金银花汤药后, 体重无显 著变化; 饮用不含 MIR2911的溶液, 小鼠体重显著降低。 可见, MIR2911对 H1N1病毒有显 著抑制作用, 维持小鼠体重平衡, 改善小鼠生理状况。
2. 感染 H1N1病毒的小鼠, 在不同时间点确定感染后, 肺部病毒滴度 EID5Q的变化 实验方法:
具体操作步骤如下:
G)将病毒按 10倍系列稀释, 分别接种生长 1周的小鼠;
(2)每个稀释度接种 6只小鼠为一组, 置 37-38°C培养;
3)分别在感染后 3天、 5天、 7天后, 处死小鼠, 取肺部血液作血球凝集试验;
(4)检测血凝活性, 利用 Reed-Muench法计算肺部 EID50
实验结果如图 21和图 22所示:
H1N1为直接给予 H1N1病毒感染组;
H1N1+NC为感染 H1N1病毒, 同时加入阴性对照 (无义序列);
H1N1+MIR2911为感染 H1N1病毒, 同时直接给予 MIR2911)。
图 21显示: 感染后 3天, 两个对照组 (H1N1, H1N1+NC)之间没有明显差异, 说明无义 序列没有杀灭病毒的作用; 与对照组 (H1N1+NC)相比, 给药组 (H1N1+MIR2911)肺部病毒含 量大大降低, 有显著性差异, 说明药物对肺部的病毒有极其明显的杀灭作用。
图 22显示: 在感染后 3天和 5天, 与对照组 (给予金银花汤药并感染了 H1N1的小鼠, HlNl+LJ soup)相比,实验组 (给予金银花汤药 +anti-MIR2911并感染了 H1N1的小鼠, H1N1+LJ soup + MIR2911)肺部病毒含量大大高出,有显著性差异。说明 anti-MIR2911把金银花汤药中 MIR2911降解后, 金银花汤药失去了抗病毒作用, 说明起作用的是 MIR2911。
10.2 H5N1病毒
1. 研究 MIR2911对接种 H5N1病毒小鼠的体重的影响
研究发现, 接种 H5N1病毒的小鼠, 饮用含 MIR2911的溶液或金银花汤药后, 体重无显 著变化; 饮用不含 MIR2911的溶液, 小鼠体重显著降低。 可见, MIR2911对 H5N1病毒有显 著抑制作用, 维持小鼠体重平衡, 改善小鼠体内生理状况。
2. 感染 H5N1病毒的小鼠, 在不同时间点确定感染后, 肺部病毒滴度 EID5Q的变化 具体操作步骤如实施例 10.1所述。 实验结果表明药物对肺部的病毒有杀灭作用。
10.3 H7N9病毒
1. 研究 MIR2911对接种 H7N9病毒小鼠的体重的影响 研究发现, 接种 H7N9病毒的小鼠, 在饮用含 MIR2911的溶液或金银花汤药后, 其体重 无显著变化; 在饮用不含 MIR2911 的溶液实验组中, 小鼠体重显著降低。 可见, MIR2911 对 H7N9病毒有显著抑制作用, 维持小鼠体重平衡, 改善小鼠体内生理状况。
2. 感染 H7N9病毒的小鼠, 在不同时间点确定感染后, 肺部病毒滴度 EID5Q的变化。 具体操作步骤如实施例 10.1所述。
实验结果如图 23和图 24所示。
H7N9为直接给予 H7N9病毒感染组;
H7N9+NC为感染 H7N9病毒, 同时加入阴性对照 (无义序列);
H7N9+MIR2911为感染 H7N9病毒, 同时直接给予 MIR2911)。
图 23显示, 感染后, 两个对照组 (H7N9, H7N9+NC)之间没有明显差异, 说明无义序列 没有杀灭病毒的作用; 与对照组 (H7N9+NC)相比, 给药组 (H7N9+MIR2911)肺部病毒含量大 大降低, 有显著性差异, 说明药物对肺部的病毒有极其明显的杀灭作用。
图 24显示:在感染后,与对照组 (给予金银花汤药并感染了 H7N9的小鼠, H7N9+LJ soup) 相比, 实验组 (给予金银花汤药 +anti-MIR2911 并感染了 H7N9 的小鼠, H7N9+LJ soup + MIR2911)肺部病毒含量大大高出, 有显著性差异。 说明 anti-MIR2911 把金银花汤药中 MIR2911降解后, 金银花汤药失去了抗病毒作用, 说明起作用的是 MIR2911。 实施例 11 对新鲜金银花、 干金银花、 小鼠饲料中 MIR2911的含量比较
检测方法:
利用 Real-time PCR方法检测新鲜金银花、 干金银花、 小鼠伺料中 MIR2911的含量, 具 体操作步骤如实施例 2所述。
实验结果:
结果如图 25所示, 可见, 在上述实验中, 喂养小鼠的伺料中基本不含有 MIR2911,对实 验结果基本没有影响。 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

权 利 要 求
1. 一种分离的植物功能性 microRNA或含所述植物功能性 microRNA的提取物,其特征 在于, 所述的植物功能性 microRNA是来源于某一植物的内源性 microRNA且存在于所述植 物的水溶性和 /或脂溶性的提取物中,而且所述植物功能性 microRNA具有调控非植物靶基因 的功能。
2. 如权利要求 1所述的分离的植物功能性 microRNA或含所述植物功能性 microRNA的 提取物, 其特征在于, 所述植物功能性 microRNA包括 MIR2911;
且所述分离的植物功能性 microRNA 或含所述植物功能性 microRNA 的提取物中, MIR2911的含量 70%, 按总 microRNA的数量计。
3. 如权利要求 1所述的分离的植物功能性 microRNA或含所述植物功能性 microRNA的 提取物, 其特征在于, 所述的非植物靶基因包括细菌基因、 病毒基因、 衣原体基因、 酵母基 因、 动物基因。
4. 如权利要求 1所述的分离的植物功能性 microRNA或含所述植物功能性 microRNA的 提取物, 其特征在于, 所述的植物包括: 药用植物、 果蔬植物、 观赏植物。
5. 如权利要求 1 或 2 所述的分离的植物功能性 microRNA 或含所述植物功能性 microRNA的提取物, 其特征在于, 所述的植物提取物包括植物的水溶性和 /或脂溶性的提取 物。
6. 一种权利要求 1 或 2 所述的分离的植物功能性 microRNA 或含所述植物功能性 microRNA的提取物的用途,其特征在于, (a)用于制备调控非植物靶基因的组合物;或 (b) 用 于制备治疗非植物靶基因相关疾病的药物。
7. 一种组合物, 其特征在于, 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) 权利要求 1 或 2 所述的分离的植物功能性 microRNA 和 /或含所述植物功能性 microRNA的植物提取物。
8. 一种体外非治疗性调控非植物靶基因表达的方法,其中,非植物靶基因包括细菌基因、 病毒基因、 衣原体基因、 酵母基因、 动物基因, 其特征在于, 包括步骤: 在权利要求 1或 2 所述的分离的植物功能性 microRNA或含所述植物功能性 microRNA的提取物存在下, 培养 含所述靶基因的生物材料, 从而调控所述非植物靶基因的表达。
9. 一种植物功能性 microRNA的鉴定方法,其中所述植物功能性 microRNA具有调控非 植物靶基因的功能, 其特征在于, 包括步骤:
(1) 提供某一植物的提取物;
(2) 检测所述提取物中植物内源性 microRNA的种类或水平; (3) 将被检测到的植物 microRNA的序列与非植物靶基因进行比对和分析, 从而鉴定出 具有调控非植物靶基因的功能的植物功能性 miCr0R A。
10.—种 microRNA分子 MIR2911或含 MIR2911的提取物的用途, 其特征在于, 用于制 备治疗病毒性感冒的药物。
11. 如权利要求 10 所述的用途, 其特征在于, 所述的提取物 (未浓縮或浓縮)中含
0.01-lOOnM (较佳地 0.1-20nM)的 MIR2911 ; 或
所述的提取物 (未浓縮或浓縮)中, MIR2911 的含量 70% ; 较佳地, 80%; 更佳地, ^90 ; 最佳地, 100%, 按总 microRNA的数量计。
12. 一种预防或治疗疾病的方法, 其中所述疾病是与非植物靶基因相关的疾病, 其特征 在于, 包括步骤: 给需要的对象施用权利要求 1或 2所述分离的植物功能性 microRNA或含 所述植物功能性 microRNA的提取物、 或权利要求 7所述的组合物, 从而预防或治疗所述的 疾病, 其中所述的植物功能性 microRNA具有调控非植物靶基因的功能。
13. 一种筛选抗病毒活性成分候选物质的方法, 其特征在于, 包括步骤:
(a) 提供分离的植物功能性 microRNA或含所述植物功能性 microRNA的提取物;
(b) 测定所述植物功能性 microRNA中各 microRNA的稳定性, 从而挑选出高稳定性的 microRNA禾中^ I;
(c) 将上一步骤挑选出的所述高稳定性的 microRNA, 与非植物靶基因进行比对, 从而确 定所述的高稳定性的 microRNA是否匹配于或结合于非植物靶基因;
其中, 如果比对结果表明, 所述的高稳定性的 microRNA可匹配于或结合于非植物靶基 因, 则选出该 microRNA种类作为抗病毒活性成分候选物质。
14. 一种制备组合物的方法, 其特征在于, 包括步骤:
人工合成植物功能性 microRNA分子, 其中所述的植物功能性 microRNA分子是用权利 要求 13所述方法筛选出的抗病毒活性成分候选物质; 以及
将所述的植物功能性 microRNA分子与药学上或食品学上可接受的载体混合, 从而形成 组合物。
15. 一种提高 MIR2911丰度的方法, 其特征在于, 包括步骤:
(a) 提供含植物功能性 microRNA的提取物, 其中, 所述的植物功能性 microRNA包括 n 种 microRNA, 其中 n 为 2(较佳地 5 或 10)的正整数, 并且其中一种 microRNA 为 MIR2911 ;
(b) 将所述提取物进行陈化处理 (如放置),得到经陈化的提取物, 并且测定所述经陈化的 提取物中 MIR2911的丰度, 并与预定值进行比较, 并且当 MIR2911的丰度 预定值时, 停 止陈化处理, 得到 MIR2911丰度提高的提取物。
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CN104640987B (zh) 2018-04-24
JP2015526080A (ja) 2015-09-10
EP2886652B1 (en) 2020-08-12
CN103589721A (zh) 2014-02-19
CN104640987A (zh) 2015-05-20
JP6231099B2 (ja) 2017-11-15
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