CN102406653B - Anti-virus effect, implementation method and purpose of miRNA(ribose nucleic acid) - Google Patents
Anti-virus effect, implementation method and purpose of miRNA(ribose nucleic acid) Download PDFInfo
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Abstract
The invention relates to an anti-virus effect, an implementation method and a purpose of miRNA(ribose nucleic acid). The invention particularly relates to a purpose of tiny RNA molecule miR-155 to the preparation of compositions for preventing and/or treating virus infection, and an application method thereof. The miR-155 has the obviously improved expression in virus infected macrophage. In addition, the miR-155 can inhibit the virus duplication, so an anti-virus effect is realized. The miR-155 can be used for preparing medicine for effectively resisting virus (particularly the RNA virus) infection, so the clinic application prospects are realized.
Description
Technical field
The invention belongs to biotechnology and medical domain, specifically, the present invention relates to a kind of RNA micromolecule---the antivirus action of miR-155, implementation method and purposes.
Background technology
Virus be by one with nucleic acid (RNA (ribonucleic acid) or DNA (deoxyribonucleic acid) DNA) for core, with protein be shell (capsid, capsid) and composition molecule, its nucleic acid can be divided into sub-thread or bifilar, linear or spherical.
Virus is minimum bio-pathogen, visible reluctantly under ordinary optical microscope from 20nm to 300nm not etc.: wherein maximum is pox viroid, and such as vaccinia virus is 300nm × 200nm; Medium sized virus, as influenza virus, diameter is 80 ~ 100nm; Small-sized virus, as epidemic encephalitis B virus, diameter is only 20nm, must just can see under an electron microscope [basic virology, big gram is waited work by force, Chemical Industry Press, the first edition in 2005; Molecular Virology, yellow galaxy of literary talent etc. work, People's Health Publisher, calendar year 2001 the first edition].
Along with the progress of virusology and immunological investigation, the understanding of people to viral disease (Viral diseases) also develops gradually.The most frequently used virus taxis mode is that the character (RNA or DNA) according to nucleic acid is classified to virus, generally virus is divided into DNA viruses and RNA viruses.Common pathogenic DNA viruses mainly contains: human papillomavirus (HPV), hepatitis B virus (HBV), Epstein Barr (Epstein-Barr virus) etc., common pathogenic RNA viruses comprises: [the basic virologies such as epidemic haemorrhagic fever virus, people's acquired immunodeficiency disease (HIV), severe acute respiratory syndrome (SARS) virus and influenza virus, big gram is waited work by force, Chemical Industry Press, the first edition in 2005; Molecular Virology, yellow galaxy of literary talent etc. work, People's Health Publisher, calendar year 2001 the first edition].Viral disease, according to clinical manifestation and spread path, also can be divided into Respirovirus, arbovirus, eruptive virus, the caused infection of enterovirus etc.
Viral disease often causes more serious clinical manifestation, often causes outbreak of epidemic.Except the local inflammation caused because of viral infection in a short time, long-term viral infection can cause the disease that tumor and organ failure etc. are serious, such as: HPV infects the cervical cancer caused, the hepatocarcinoma that HBV infection causes, liver cirrhosis and liver failure, the nasopharyngeal carcinoma that Epstein-Barr virus causes, the immunodeficiency that HIV causes and Kaposi's sarcoma, [the basic virologies such as the respiratory failure that SARS causes and the respiratory dysfunction that influenza virus causes, big gram is waited work by force, Chemical Industry Press, the first edition in 2005; Molecular Virology, yellow galaxy of literary talent etc. work, People's Health Publisher, calendar year 2001 the first edition].
China is viral disease big country occurred frequently, as: China HBV infection patient estimates about have 1.2 hundred million, is the HBV big country that number of the infected is maximum in the world; What can not forget is SARS in 2002 being very popular in China.In addition, HIV and possible influenza virus are broken out the also moment and threaten national healthy.
Each epidemic virus infects the loss that can cause great human health damage and national economy.But resist virus in this area so far and there is no specific drug, clinical treatment emphasis is just being suited the medicine to the illness and expectant treatment, therefore the research of the molecule of the morbidity of viral disease and the research of amynologic mechanism and therapeutic strategy has great scientific meaning and display meaning.
Microrna (micro RNA, be called for short miRNA) is a class short data records, non-coding, have the strand microRNA of adjusting function, is about 18 ~ 24nt.Report the earliest about miRNA is a kind of microRNA of expressing in temporal found in C. Elegans Automatic Screening ((C.elegants)) body of the reports such as Lee in 1993, i.e. the lin4 of scalable elegans development.2000, Reinhart etc. found again the let-7 that different times is expressed.Up till now, included more than 300 kind of mankind's miRNA sequence in public miRNA data base, wherein 2/3rds be confirmed in experiment.
Microrna derives from the initial transcription product of long-chain RNA (Pri-miRNA) that length is about 1000bp, and Pri-miRNA molecule shears the miRNA precursor (Pre-miRNA) with loop-stem structure forming length about 60 ~ 80nt in nucleus through Drosha enzyme.After miRNA precursor is transported to kytoplasm, be further processed into miRNA.
Microrna by playing a significant role to the cutting of target mRNA and Transcription inhibition, participates in the various procedures such as Growth of Cells, histo-differentiation and tumor formation in animal and plant cells.MiRNA have between species height conservative, time continuing property and tissue specificity.Think at present, miRNA all has important regulating action in apoptosis, propagation, differentiation, angiogenesis and tumor formation etc., participate in the Gene regulation of human body about 30%, relevant with the multiple morbidity such as tumor, cardiovascular disease, hepatopathy, immunologic derangement and metabolism disorder.
From being found at first now, in mammal, found more than 700 kind of miRNA, wherein the biological function of most miRNA is unknown.Have been reported, miRNA participates in many-sided regulating and controlling effect in immunne response.But so far, there is not been reported, and miRNA can regulate and control or affect antiviral natural immunity, does not also report that miRNA participates in the regulating and controlling effect to immune cell function.
By transgenic or knock out mice experiment, miR-155 has been proved and has played indispensable effect [Faraoni in humoral immunization and cellular immunization, I. etc., miR-155gene:A typicalmultifunctional microRNA.Biochim Biophys Acta.2009; 1792:497-505; Rodriguez, A. etc., Requirement of bic/microRNA-155for normal immune function.Science.2007; 316:608-611].And for the natural immunity and inflammatory reaction, miR-155 can by multiple stimulation institute abduction delivering, such as TLR agonist (such as TLR2, TLR3, TLR4 and TLR9 part), cytokine (IL-1, TNF-α and IFN-β etc.) stimulation it all can be induced to express, prompting miR-155 may participate in innate immune reaction [O ' Connell, R.M. etc., MicroRNA-155is induced during the macrophageinflammatory response.Proc Natl Acad Sci USA.2007; 104:1604-1609; Tili, E. etc., Modulation of miR-155and miR-125b levels following lipopolysaccharide/TNF-alpha stimulation and their possible roles in regulating the response toendotoxin shock.J Immunol.2007; 179:5082-5089].But the report of antiviral natural immunne response is not is not also regulated and controled at present about miR-155.
In sum, can effectively for the antiviral natural immunizing agent that viral infection plays a role in the urgent need to developing in this area.
Summary of the invention
Main purpose of the present invention is just to disclose the regulating and controlling effect that miR-155 resists viral innate immunity, the invention provides the effective antiviral novelty teabag of miR-155 thus.
In a first aspect of the present invention, provide Microrna miR-155 or its precursor and preparing the application prevented and/or treated in the compositions of viral infection.
In an embodiment of the invention, the sequence of described miR-155 is as shown in SEQ ID NO:1.
In yet another embodiment of the present invention, the precursor of described miR-155 in subject through process change be miR-155.
In yet another embodiment of the present invention, described miR-155 or its precursor are from people, rat, mice, dog, horse, cattle, rabbit or monkey.
In yet another embodiment of the present invention, described virus is RNA viruses or DNA viruses.
In a preference, described virus is RNA viruses.
In yet another embodiment of the present invention, described virus is selected from: human papillomavirus, hepatitis virus, Epstein Barr, epidemic haemorrhagic fever virus, people's acquired immunodeficiency disease, SARS virus, influenza virus, vesicular stomatitis virus or paramyxovirus.
In a preference, described hepatitis virus is hepatitis B virus.
In another preference, described virus is vesicular stomatitis virus or Sendai virus.
In yet another embodiment of the present invention, described compositions is pharmaceutical composition or vaccine combination.
In a second aspect of the present invention, provide a kind of compositions, described compositions comprises: the Microrna miR-155 of (a) effective dose or its precursor; (b) acceptable carrier pharmaceutically or in immunology.
In a preference, the content of component (a) accounts for 0.001 ~ 99.9wt% of composition total weight, preferably 1 ~ 95wt%, more preferably 5 ~ 90wt%.
In an embodiment of the invention, described compositions also comprises the medicine being selected from lower group: other active component of (c) treatment or prophylaxis of viral infections; And/or (d) chemotherapeutics.
In a preference, described component (c) is selected from: anti-influenza A virus surface M2 receptor, AN, anti-monophosphate carnine acidohydrogenase, antiviral DNA polymerase, AntiHIV1 RT activity reverse transcriptase, AntiHIV1 RT activity protease, anti-sialidase or anti-unwindase.
In another preference, described component (d) is selected from: cell mitogen inhibitor, camptothecine, homoharringtonine, procarbazine, asparaginase, cisplatin, carboplatin, mitoxantrone, tamoxifen, cyclophosphamide, mustine hydrochlcride, lomustine, semustine, phosphinothioylidynetrisaziridine, busulfan, formylmerphalan, chlorambucil, fluorouracil, tegafur, excellent fluorine pyridine, carmofur, mercaptopurine, methotrexate, cytosine arabinoside, ancitabine, mercapto guanine, altretamine, hydroxyurea, mitomycin, amycin, epirubicin, bleomycin, training Lay mycin, acrivastine, Trastuzumab, imatinib mesylate, gemcitabine, hycamtin and/or leuprorelin.
In another preference, described cell mitogen inhibitor is selected from: vinblastine, vincristine, vindesine, vinorelbine, Demecolcine, colchicine, Colchiceinamidum, podophyllotoxin, etoposide, teniposide, paclitaxel or docetaxel.
In yet another embodiment of the present invention, the form of described compositions is suitable for: direct naked RNA injection, liposome RNA direct injection, Jin Bao by rna gene rifle blast technique, breeding unsoundness antibacterial carries plastid rna method or replication defective adenoviral carries object RNA method.
In another aspect of this invention, provide a kind of method preventing and/or treating viral infection, described method comprises: the Microrna miR-155 or its precursor that need the object effective dose prevented and/or treated.
In a preference, described Microrna miR-155 or its precursor give object by the following method: directly naked DNA injection method, liposome DNA direct injection, Jin Bao by DNA particle bombardment, breeding unsoundness antibacterial carries plasmid DNA method or replication defective adenoviral carries target DNA method.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1: the expression of miR-155 after viral stimulating expression of macrophage, wherein:
Figure 1A VSV (MOI=1) infects the miR-155 expression of each time point in 48 hours period;
Figure 1B is for infect each time point miR-155 expression in 96 hours period with SeV (MOI=10);
Fig. 1 C infects the miR-155 expression after 36 hours with the VSV of different MOI (0.01-10);
Fig. 1 D infects the miR-155 expression after 36 hours with the SeV of different MOI (1-100).
(result is shown as mean+SD, n=3).
Fig. 2: miR-155 suppresses VSV copying in macrophage, wherein:
Fig. 2 A is that macrophage transfection miR-155 and tiny RNA contrast VSV TCID in rear culture supernatant
50testing result;
Fig. 2 B is that macrophage transfection miR-155 mortifier and mortifier contrast VSVTCID in rear culture supernatant
50testing result;
Fig. 2 C is the qRT-PCR result that after macrophage transfection miR-155 and tiny RNA contrast, in born of the same parents, VSV viral RNA copies;
Fig. 2 D is the qRT-PCR result that after macrophage transfection miR-155 mortifier and mortifier contrast, in born of the same parents, VSV viral RNA copies.
(result is shown as mean+SD, n=3; *, P < 0.05; *, P < 0.01)
Detailed description of the invention
The present inventor finds through long-term and deep research: in the macrophage of viral infection, the expression of Microrna miR-155 can significantly be raised, thus the miR-155 confirming in macrophage is the response gene of viral infection.Inventor further studies discovery on this basis: miR-155 effectively can suppress the propagation of virus, thus plays antiviral effect.Thus, inventor has found the novelty teabag of miR-155 in antiviral, thus completes the present invention on this basis.
As used herein, include " containing ", " having " or " comprising " " comprising ", " primarily of ... form ", " substantially by ... form " and " by ... form "; " primarily of ... form ", " substantially by ... form " and " by ... formation " belong to the subordinate concept of " containing ", " having " or " comprising ".
As used herein, " separation " or " separation and purification " refers to that material is separated from its primal environment (if crude, namely primal environment is natural surroundings).As the polynucleotide under the native state in active somatic cell and polypeptide do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials existed separately, then for separation and purification.
The feature that the present invention mentions, or the feature that embodiment is mentioned can combination in any.All features that this case description discloses can with any composition forms and use, each feature disclosed in description, anyly can provide identical, alternative characteristics that is impartial or similar object replaces.Therefore apart from special instruction, the feature disclosed is only general example that is impartial or similar features.
miR-155 and precursor thereof
As used herein, term " miR-155 " or " Microrna miR-155 " are used interchangeably, and refer to the Microrna comprising sequence or its homologous sequence shown in SEQ ID NO:1.The miR-155 in known various source, such as people, chimpanzee, horse, chicken etc. in this area, these homologous sequences are all included in term of the present invention " miR-155 ".Also comprise in term of the present invention through replacing, lacking or add one or several nucleotide in above-mentioned naturally occurring miR-155 sequence, or through chemical modification biology, and still there is the derivative RNA of antiviral activity.
As used herein, term " miR-155 precursor " refers to being bestowed in the cell of object or can be formed to the miRNA precursor of miR-155 in body.Those of ordinary skill in the art know the ways and means obtaining naturally occurring miR-155 precursor, also described precursor can be built based on molecule and Heredity theory and means, such as [O ' Connell, RM. etc., Sustained expression of microRNA-155inhematopoietic stem cells causes a myeloproliferative disorder.J Exp Med.2008; 205:585-94].
In this area, known miR-155 is by BIC gene code.BIC gene is Noncoding gene, and its initial transcription product, after a series of processing maturation, forms ripe miR-155.MiR-155 precursor only just has corresponding biological function after being processed into ripe miR-155, therefore, those of ordinary skill in the art all can obtain the effect of acquisition required for the present invention by the rational expectation nucleic acid substances (i.e. " miR-155 precursor " of the present invention) of other forms various that utilizes the mature sequence of miR-155 and can produce or be processed into miR-155, namely play antiviral effect, thus all can be used for the compositions preparing treatment or prophylaxis of viral infections.
The present invention also comprises a kind of carrier, and it contains the construction carrying described miRNA or its precursor.Described expression vector is usually also containing promoter, origin of replication and/or marker gene etc.Method well-known to those having ordinary skill in the art can be used for building construction required for the present invention or expression vector.These methods include but not limited to: gene recombination technology, external synthetic technology etc.Described expression vector preferably comprises one or more selected marker, to be provided for the phenotypic character selecting the host cell transformed, as kalamycin, gentamycin, hygromycin, amicillin resistance.
compositions
Present invention also offers a kind of compositions (especially pharmaceutical composition or vaccine combination), described compositions can be used for preventing and/or treating viral infection and the related symptoms that caused by it and disease.Described compositions contains miR-155 of the present invention and the acceptable carrier pharmaceutically or in immunology of effective dose.
The composition of described " pharmaceutically acceptable " is applicable to people and/or animal and without excessive bad side reaction (as toxicity, stimulation and allergy), namely has the material of rational benefit/risk ratio.Described " effective dose " refer to can to people and/or animal produce function or activity and can by people and/or animal the amount that accepts.
Described " in pharmaceutically/immunology acceptable carrier " refers to the carrier being used for the treatment of agent or vaccine administration, comprises various excipient, diluent and adjuvant.This term refers to some medicaments or vaccine carrier like this: they itself are not necessary active component, and do not have undue toxicity after using.Suitable carrier is well known to those of ordinary skill in the art.Discussing fully about pharmaceutically acceptable excipient can be found in Remington ' s Pharmaceutical Sciences (Mack Pub.Co., N.J.1991).
This kind of carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Usual medicine/bacterin preparation should match with administering mode, and such as, compositions of the present invention can be prepared by conventional method with normal saline or the aqueous solution containing glucose and other adjuvant, with obtained injection form.Described compositions should aseptically manufacture.Preparation of the present invention also can be made into slow releasing preparation.
The effective dose of miR-155 of the present invention can change with order of severity of the pattern of administration and disease to be treated etc.The selection of preferred effective dose can be determined (such as passing through clinical trial) according to various factors by those of ordinary skill in the art.Described factor includes but not limited to: pharmacokinetic parameter such as bioavailability, metabolism, the half-life etc. of miR-155; The disease that patient will treat, the body weight of patient, the immune state of patient, the approach etc. of administration.
Gratifying effect can be obtained when miR-155 of the present invention or its precursor give object with suitable dosage.Such as, when miR-155 of the present invention or its precursor, when every day gives animal with the dosage of about 2.5-25nmol/20g (preferably 20nmol/20g) animal (as mice) body weight, gratifying antiviral effect can be obtained.
administering mode
Compositions of the present invention can be solid-state (as granule, tablet, lyophilized powder, suppository, capsule, sublingual lozenge) or liquid (as oral liquid) or other suitable shape.
Route of administration can adopt such as (but being not limited to): (1) direct naked RNA injection; (2) liposome RNA direct injection; (3) Jin Bao is by rna gene rifle blast technique; (4) breeding unsoundness antibacterial carries plastid rna method; (5) replication defective adenoviral carries object RNA method; (6) electric punch method, etc.
In addition, also can containing other active substance for improving and treat disease of viral infection in compositions of the present invention, other described active substance is selected from lower group (including but not limited to): one or more of clinical conventional antiviral drugs (comprising the surperficial M2 receptor of anti-influenza A virus, AN, anti-monophosphate carnine acidohydrogenase, antiviral DNA polymerase, AntiHIV1 RT activity reverse transcriptase, AntiHIV1 RT activity protease, anti-sialidase, anti-unwindase).
MiR-155 of the present invention can also combine with other medicines and treatment means, for the prevention and therapy of disease of viral infection.
advantage of the present invention
Advantage of the present invention is mainly:
A () present invention is disclosed miR-155 and is preventing and/or treating the novelty teabag in viral infection, the research and development for miR-155 and even miRNA utilizes and provides new thinking and approach;
B () miR-155 of the present invention or its precursor can be effective to viral infection resisting, thus be the viral infection preventive that provides the art a kind of novelty and/or therapeutic agent, have certain potential applicability in clinical practice.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as the people such as Sambrook " molecular cloning: lab guide " (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise percentage ratio and number calculate by weight.
Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
embodiment 1: the rise effect that viral infection is expressed miR-155 in macrophage
Vesicular stomatitis virus (VSV, a kind of minus-strand RNA virus) and Sendai virus (SeV, the RNA viruses of paramyxovirus genus) are purchased from China typical culture collection center (CCTCC).
Mice (C57 strain, female, 4-6 week age, purchased from Shanghai western pul-Bi Kai laboratory animal company limited) lumbar injection meat soup (available from Sigma) 1ml/ is only, abdominal cavity is rinsed with PBS after three days, primary peritoneal macrophage [Hou is obtained according to literature procedure, J. etc., MicroRNA-146a feedback inhibitsRIG-I-dependent Type I IFN production in macrophages by targeting TRAF6, IRAK1, and IRAK2.J Immunol.2009; 183:2150-2158].
By 2 × 10
5individual Turnover of Mouse Peritoneal Macrophages spreads into containing 0.5ml complete medium (RPMI 1640 culture medium+10% hyclone, all purchased from PAA company) 24 orifice plates in overnight incubation, next day, grouping was infected in the following manner: (1) VSV (MOI is 1) infect 48 hours; (2) SeV (MOI is 10) infects 96 hours; (3) VSV (MOI is 0.01-10) infects 36 hours; Or (4) SeV (MOI is 1-100) infects 36 hours.
Real Time RT-PCR (qRT-PCR) method is adopted to analyze the expression of miR-155 in infected cell: to use TRIzol (Invitrogen company) extracting total tissue RNA.QRT-PCR uses SYBRRT-PCR test kit (Takara company) and completes on LightCycler (Roche company) real-time PCR.
MiR-155RT primer is: 5 '-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTCGCA CTG GAT ACG ACC CCC TA-3 ' (SEQ ID NO:2).
Quantification PCR primer is:
5 '-CTC GTG GTT AAT GCT AAT TGT GA-3 ' (upstream, SEQ ID NO:3); With
5 '-GTG CAG GGT CCG AGG T-3 ' (downstream, SEQ ID NO:4).
The reverse transcription reaction primer of internal reference U6 small nuclear rna is identical with the downstream primer of quantitative PCR, and sequence is:
5’-AAC GCT TCA CGA ATT TGC GT-3’(SEQ ID NO：5)；
Quantification PCR primer is:
5 '-CTC GCT TCG GCA GCA CA-3 ' (upstream, SEQ ID NO:6); With
5 '-AAC GCT TCA CGA ATT TGC GT-3 ' (downstream, the same SEQ ID NO:5).
The relative quantification of miRNA uses 2
-Δ Δ Ctmethod calculates (U6 is internal reference) [Livak, KJ. etc., Analysisof relative gene expression data using real-time quantitative PCR and the 2-Δ Δ Ctmethod.Methods.2001; 25:402-408].
After virus stimulating expression of macrophage, the expression of miR-155 as shown in Figure 1.
Result shows: the expression of miR-155 can be induced to significantly improve with VSV stimulating expression of macrophage, and infects arrival plateau (Figure 1A) in latter 24 hours at VSV.Another RNA viruses---SeV also can induce the Expression (Figure 1B) of miR-155 in macrophage as VSV.Result also shows, and this two-strain is all dose-dependent (Fig. 1 C and 1D) to the induction of miR-155.
embodiment 2:miR-155 suppresses vesicular stomatitis virus (VSV) copying in macrophage
Following sequence (being synthesized by Shanghai Ji Ma company) is adopted in Turnover of Mouse Peritoneal Macrophages, to make miR-155 high expressed or suppress the expression of miR-155 respectively:
miR-155：UUAAUGCUAAUCGUGAUAGGGGU(SEQ ID NO：1)；
Tiny RNA contrasts: UUCUCCGAACGUGUCACGUTT (SEQ ID NO:7, it is method of modifying conventional in tiny RNA sequent synthesis that 3 ' end adds that TT gives prominence to, its function is not affected, mainly play the effect [Wilda of stable nucleus thuja acid, M. etc., Killing of leukemic cells with a BCR/ABL fusiongene by RNA interference (RNAi) .Oncogene.2002; 21:5176-5124]);
MiR-155 mortifier: 2 '-O-Me-ACCCCUAUCACGAUUAGCAUUAA (SEQ IDNO:8); And
Mortifier contrasts: 2 '-O-Me-CAGUACUUUUGUGUAGUACAA (SEQ ID NO:9).
Prepare Turnover of Mouse Peritoneal Macrophages as described in Example 1, and adopt INTERFERin transfection reagent (Polyplus company) transfection tiny RNA to macrophage [Hou according to bibliographical information, J. etc., MicroRNA-146afeedback inhibits RIG-I-dependent Type I IFN production in macrophages bytargeting TRAF6, IRAK1, and IRAK2.J Immunol.2009; 183:2150-2158], make the final concentration of miRNA transfection be 10nM.
After transfection 48 hours, infect 1 hour with VSV (MOI is for 1), change fresh complete medium (RPMI1640 culture medium+10% hyclone).After 72 hours, get 0.1ml cells and supernatant and carry out 10 times of gradient doubling dilutions, and add monolayer BHK21 cell (purchased from ATCC).After 72h, observe the death condition of BHK21 cell and calculate median tissue culture infective dose (TCID
50).
Adopt VSV viral RNA in body fluid viral DNA/RNA Miniprep Kit (Body Fluid Viral DNA/RNAMiniprep kit, purchased from Axygen company) extracting cell, and adopt the qRT-PCR standard measure described in embodiment 1.
The quantification PCR primer of serotype VSV is:
5 '-ACG GCG TAC TTC CAG ATG G-3 ' (upstream, SEQ ID NO:10); With
5 '-CTC GGT TCA AGA TCC AGG T-3 ' (downstream, SEQ ID NO:11).
The relative quantification of mRNA uses beta-actin (β-actin) as internal reference.Mice beta-actin mRNA quantification PCR primer reference literature report [Liu, X. etc., CaMKII promotes TLR-triggeredproinflammatory cytokine and type I interferon production by directly binding andactivating TAK 1and IRF3in macrophages.Blood.2008; 112:4961-4970].
VSV TCID in the macrophage culture supernatant that transfection miR-155 and mortifier thereof infect VSV
50the result of impact as shown in Figure 2 A and 2B.Result shows: high expressed miR-155 significantly can suppress the propagation (Fig. 2 A) of VSV, and suppress miR-155 to express can the propagation (Fig. 2 B) of promotion VSV of highly significant;
In the macrophage born of the same parents infected VSV after transfection miR-155 and mortifier thereof, the result of VSV rna replicon impact as illustrated in figs. 2 c and 2d.Result shows: high expressed miR-155 can suppress the RNA (Fig. 2 C) of VSV very significantly, and suppresses miR-155 to express the rna replicon (Fig. 2 D) that significantly can increase VSV.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (5)
1. Microrna miR-155 is preparing the application prevented and/or treated in the compositions of picornavirus infection.
2. apply as claimed in claim 1, it is characterized in that, the sequence of described miR-155 is as shown in SEQ IDNO:1.
3. apply as claimed in claim 1, it is characterized in that, described miR-155 is from people, rat, mice, dog, horse, cattle, rabbit or monkey.
4. apply as claimed in claim 1, it is characterized in that, described virus is selected from: epidemic haemorrhagic fever virus, people's acquired immunodeficiency disease, SARS virus, influenza virus, vesicular stomatitis virus or paramyxovirus.
5. apply as claimed in claim 1, it is characterized in that, described compositions is pharmaceutical composition or vaccine combination.
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101389770A (en) * | 2006-01-05 | 2009-03-18 | 俄亥俄州立大学研究基金会 | Microrna-based methods and compositions for the diagnosis, prognosis and treatment of solid cancers |
Non-Patent Citations (1)
| Title |
|---|
| 郑玉姝 等.MicroRNAs:免疫***的新型调节子.《细胞与分子免疫学杂志》.2009,第25卷(第2期),181-183. * |
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