CN105602989A - Recombinant vector and application thereof in preparing or screening anti-influenza drugs - Google Patents

Recombinant vector and application thereof in preparing or screening anti-influenza drugs Download PDF

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CN105602989A
CN105602989A CN201610139667.9A CN201610139667A CN105602989A CN 105602989 A CN105602989 A CN 105602989A CN 201610139667 A CN201610139667 A CN 201610139667A CN 105602989 A CN105602989 A CN 105602989A
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cell
influenza
recombinant vector
sirna
gene
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CN105602989B (en
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马志永
魏建超
史子学
邵东华
李蓓蓓
晏文君
朱紫祥
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Shanghai Veterinary Research Institute CAAS
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/08—Phosphoric triester hydrolases (3.1.8)
    • C12Y301/08001—Aryldialkylphosphatase (3.1.8.1), i.e. paraoxonase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5026—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell morphology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Abstract

The invention provides a recombinant vector and application thereof in preparing or screening anti-influenza drugs. The recombinant vector comprises an opening reading frame of a paraoxonase 3 gene, an opening reading frame fragment of the paraoxonase 3 gene obtained after PCR amplification is conducted is subjected to enzyme digestion and connected into the vector, and the recombinant vector is obtained. The recombinant vector and application thereof in preparing or screening the anti-influenza drugs have the advantages that the supplied recombinant vector can be used for treating influenza through genes or conducting nucleic acid immunization for influenza viruses; a supplied cell model can be used for virus research, specially for swine influenza virus pathogenic mechanism research and can also be used for building an anti-influenza drug screening model, specially for an anti-influenza drug cell model.

Description

A kind of recombinant vector and the application in preparation or screening Tamiflu thereof
The application is that application number is 2012100197129, denomination of invention is for " suppressing influenza virus dependency basisThe siRNA of cause and application thereof ", the applying date is the divisional application of the patent application on January 20th, 2012.
Technical field
The invention belongs to biological technical field, particularly suppress caveolin 2 and Surfactant proteinDThe recombinant vector of the siRNA of gene expression, the nucleotide sequence of siRNA, this siRNA of codified withAnd the recombinant vector of codified transcription factor A and paraoxon acid enzyme 3, and their genes separately existThe purposes of preparation and screening Tamiflu aspect.
Background technology
Poisoning intrusion host process is accompanied by host gene and expresses variation, final decision infection cell and virusDestiny separately. After virus infections host, need to utilize the metabolic process of host cell to complete virus selfCopy, genomic expression in this process virus will inevitably modulate host cell, suppresses antiviral baseBecause expressing, strengthen the gene expression useful to virus replication. Therefore, after virus infections host cell, placeIn chief cell, express these genes that change and often have close relationship with virus replication. Learn to do by biologyIn section modulate host cell in these genes one or regulate and control multiple gene expression simultaneously and may there is inhibitionThe effect that influenza virus is copied, have potential prevention and treatment influenza infection using value.
Influenza virus is the segmented sub-thread minus-stranded rna virus of orthomyxoviridae family's Influenza Virus. VirionSon is polymorph while just separation, after going down to posterity in vivo, become spherical, diameter 80-120nm (Palese, etal.,2007). Virion is by the protein of RNA, the 70-75% of 0.8-1%, 20% lipid and 5-8%Carbohydrate composition, ratio is mass ratio herein.
Influenza virus is divided into A, B, C tri-types, and wherein A type (A type) is the most important. Flu-AVirus can cause natural infection and the morbidity of the mankind, pig, horse and various birds. Due to its surface antigen HAEasily make a variation with NA, known HA has 16 hypotypes (H1-H16), and NA has 9 hypotype (N1-N9), the various combination between them, make influenza A virus have many hypotypes (as H1N1, H2N2,H3N2, H5N1 etc.), Major Epidemic H1N1 and H3N2 hypotype in pig body.
Different animals has different influenza viruses, as swine flu, human influenza, equine influenza, generallyThese influenzas only infect this genus animal separately. But influenza virus has the adaptability that host plants. ?Between the individuality of different animal species, also easily propagate, form the new influenza virus that infects many animals.Another feature is that this animal of pig is more special, and pig is to the influenza virus of other animal also susceptible, and this isThe characteristic that other animal does not have. So once pig has infected human influenza, swine flu, bird flu etc. simultaneously,This multiple virus can be recombinated in pig body, possible will be out a kind of can infected pigs, people, fowlInfluenza virus. So the swine flu of narrow sense is not Amphixenosis. Influenza is in interpersonal spread scopeVery limited, relevant with the route of transmission of influenza. Such as Flu-A in 2009, be exactly human influenza, pig streamThe new influenza virus producing after sense, the common infected pigs of bird flu, the people who starts to infect is most substantially contactCross or with pig people in close relations, have subsequently more people's influenza virus infections. Influenza in human development historyThe great outburst of virus brings great harm all to socio-economic development, people's health. For prevention and treatment streamSense, each state has all dropped into a large amount of manpower and materials, constantly studies methods for the treatment of and medicine. In recent years, from placeGene or albumen that the interior searching of chief cell has antiviral functions are popular domains.
In human development history, to bring all to socio-economic development, people's health great in the great outburst of influenza virusHarm. For prevention and treatment influenza, each state has all dropped into a large amount of manpower and materials, constantly studies methods for the treatment ofAnd medicine. In recent years, grind as target spot for gene or the albumen in host cell with antiviral functionsSending out antiviral drugs is a popular domain.
Target spot using endocellular function molecule as antiviral therapy has broad-spectrum antiviral, difficult generation is resistance toThe advantage of medicine strain, becomes the focus approach of researching and developing antiviral therapy medicine gradually. International in recent yearsHigh level paper is repeatedly reported in host cell screening in signal path and has potential antiviral functionsMolecule breaks trough. For example, zinc refer to antiviral protein (Zinc-fingerantiviralprotein,ZAP) can be by degrading special viral RNA and then suppress copying of the multiple virus such as murine leukemia virus,Be proved to be a kind of important antiviral agent (Chenetal. (2008) Proc.Natl.Acad.Sci.USA.105,4352-4357). Utilize zinc finger protein in carrier carries special enzyme blocking t cellCCR5 expresses, and can promote T cell clearance HIV virus (Holtetal. (2010) Nat.Biotechnol.28,839-847). E3 ubiquitin ligase RNF5 is positioned mitochondria, by ubiquitination MITA(also referred to as STING) also causes its degraded and I type interferon expression (Zhong after negative regulation virus infectionsEtal. (2009) Immunity.30,397-407). Tetherin (host cell proteins) is with dimer anchorDetermine on cell membrane that HIV copies at it, blocking virus is from the endochylema film release (Perez-Caballero that sproutsetal.(2009)Cell.139，499-511)。APOBEC3G(ApolipoproteinBmRNA-editingEnzymecatalyticpolypeptide-like3G) can induce HIV genetic mutation, virus can not be answeredSystem (Shirakawaetal. (2008) Nat.Struct.Mol.Biol.15,1184-1191). Cell cycle at presentElement dependent kinase, prostaglandin, heat shock protein as medicine target molecule be widely used in as, HCMV, HIV,The virosis treatments such as HSV, adenovirus and papilloma.
RNAi is a kind of Gene silence of efficient high specificity, and development in recent years is rapid, soonBecome the powerful of functional genome research. Means are in dsRNA molecule transfered cell by experiment,Specifically in degradation of cell with the mRNA of its sequence homology, sealing endogenous gene expression, from reverse something lostThe function of unknown gene in the angle research mankind that pass or other biological genome. Just someone applies this in early daysItem technical point is from the various compositions in fruit bat insulin signaling transduction pathway path. Recently also there is experiment reportEach the approach relating in lipid within endothelial cells equilibrium process studied in road by RNAi. Before this, Zeng LiSuppress the generation of its phenotype by the characteristic of antisense RNA and the complementation of said target mrna sequence, but due to antisenseRNA to the gene inhibitory action of endogenous expression a little less than, tend to produce some transition phenotypes, it is right easily to causeGene function misjudgment, thinking clinically by examination & approval that tool is medicative at present only has a kind of medicineThing Vitravene.
RNAi specificity is higher, acts on rapidlyer, and side reaction is little, in reticent target gene effectively,Regulator control system on cell itself does not also affect. Recently in mankind's body cell successfully to nearly 20Plant gene function and carried out " knocking out ", especially therefore understood mankind's cavity albumen Tsg101 coupleHIV, in the effect of people's proliferation in vivo, has further deepened the research to HIV. Leonid etc. are with spinal cordPoliovirus is model, utilizes RNAi to carry out the interior immunity of born of the same parents of inducing cell, produces antiviral effect,Especially for RNA virus. For the virus of easy sudden change, can design multiple target viral gene conservativeThe dsRNA of sequence, reduces its opposing to dsRNA. Maen etc. also apply the success of RNAi technologyBlocked the core relevant to cell proliferation and differentiation of a kind of unconventionality expression in MCF-7 breast cancer cellThe function of transcription factor gene 21. The application of RNAi technology, can not only promote gene after the mankind greatlyBatch total is drawn the development of (protein science), and high flux ground screening medicine target gene, detects human genome one by oneExpression inhibiting situation carry out the function of clear and definite gene, and it also will be applied to gene therapy, new drug development,The fields such as biomedical research, carry out the unconventionality expression of suppressor, for treating various diseases by RNAi technologyDisease has been opened up new approach.
Due to the fast development of Protocols in Molecular Biology and cytobiology technology in recent years, molecular pharmacologyResearch also deepens continuously, the variation of new drug target, functional protein, gene expression, biologyActive components etc. are constantly found, for drug screening provides a large amount of new target spots, as new acceptor, enzyme etc.These new target spots provide new information and chance for new medicament screen. Cellular and molecular level drug sieve modelingThe automation mechanized operation that is applied as of type is laid a good foundation, and makes drug screening by traditional manual screening formal transformationFor the new technology system by computer-controlled automation Large-scale Screening, form high-throughput drug sieveChoosing.
The advantage of high-flux medicaments sifting: realized the scale of drug screening, utilized to large extentMedical substance resource, has improved the probability of drug discovery, has improved the quality of finding new drug simultaneously; ScreeningExperiment completes in microscreen system, and generally in Gamma Magnitude, (μ g), has saved sample to amount of samplesProduct resource, has established the material base of " medicine sieves more ", has saved experiment material simultaneously, has reduced listMedicine screening cost; High-flux medicaments sifting is the generation that increasingly automated operation has reduced operate miss, fallsLow labour intensity, and improved the efficiency of drug screening and the accuracy of result; There is multidisciplinary reasonThe feature of opinion and technology combination.
Medicaments sifting model is the essential condition of finding new drug. The foundation of new model will drive newtype drugAppearance. The development of molecular biology, cell biology, computer science, particularly human genomeCompleting of plan, for medical research has brought good opportunity, also for setting up new medicaments sifting model,Many-sided superiority conditions such as theory, technology, material are provided. Therefore, we should make full use of eachThe Development Technology of section is set up how new screening model, promotes the discovery of new drug.
DNA vaccination is the new technology that last century, the nineties grew up, and arrives by exogenous gene cloningConstructed dna vaccine plasmid on carrier for expression of eukaryon, DNA vaccination is expelled to animal base through various approachAfter in body, can be absorbed by host cell and absorb, the nucleus transcription that enters host cell becomes mRNA,In endochylema, translate into again protein. Wherein a part of protein is tied with MHCI quasi-molecule after degradedClose, and arrived cell surface by the Receptor recognition of cd8 t cell by submission, and activating cytotoxic T is thinBorn of the same parents' activity; Another part protein also can be secreted away, then is offered by antigen as foreign proteinCellular uptake is degraded into polypeptide in phagolysosome, and is further combined with mhc class ii molecule,There is antigen presenting cell to offer cell surface by the Receptor recognition of Th2 cell, then by Th2 cellThe Cytokine of secretion, in B cell, is produced as main humoral immunity and stimulate with antibody. DNAVaccine not only can be induced generation humoral immunity, and can induce strong and lasting cellular immunity, also can keep awayExempt to extraneous toxin expelling, and DNA vaccination and attenuation and attenuated vaccine different, it does not exist virulence anti-The problem such as strong. Its security and high efficiency are that traditional vaccine does not reach, so, this technology virus,In the caused infectious disease of cytozoicus, parasite and treatment and prevention of tumour, demonstrate huge potentiality.Wolff can report at mouse expression in vivo recombinant plasmid, and foreign protein can be at Mice BodyInterior expression reaches 60 days, and prompting DNA is expressed in the quite a while in vivo, canTo be used as therapeutic effect, and cause research widely.
Gene therapy is for cancer, atherosclerotic, osteoporosis, arthritis, alzheimer'sThe cause of disease is that the movable prevention and treatment of diseases extremely causing of specific gene has prospect very much. This therapy is oneKind nucleic acid is introduced to people's cell and reaches to be used for revising their hereditary capacity the treatment plan of therapeutic purposesSlightly. Conventionally, this nucleic acid is the therapeutic action of can having encoded, the protein of destruction or mark effectDouble chain DNA molecule (dsDNA). This nucleic acid may be also that the target sequence in host cell is combined,By stoping mRNAs or promoter to suppress antisense RNA or strand that specific gene is expressedDNA (ssDNA). So far, exceeded 600 taking plasmid as basic gene therapy, cancer vaccine etc.Example, the genetic immunization of DNA has been simulated pathogen in intracellular gene expression approach, becomesShould be used for treating to merit the ischaemic of lower limb, cardiovascular regeneration, and the utmost point is hopeful to come by nucleic acid vaccineThe difficult and complicated cases of prevention modern medicine, comprise malaria, AIDS, hepatitis B and tuberculosis etc. For geneTreatment, perhaps plasmid can not be integrated into genomic DNA is a shortcoming, but plasmid can be episomalForm is present in nucleus, also can the continuous expression quite a long time.
Summary of the invention
Therefore, the technical problem to be solved in the present invention designs exactly and suppresses to copy with influenza virus in hostThe siRNA sequence of related gene, the structure recombinant vector of this siRNA of can encoding, and contain thisThe application of the recombinant vector of sequence ORFs aspect preparation Tamiflu.
The present invention solves the problems of the technologies described above one of adopted technical scheme: a kind of for caveolinThe siRNA of 2 genes, the target sequence that wherein siRNA of the present invention is corresponding is caveolin 2 genesMRNA, the length of siRNA of the present invention is 16-30bp, and can make the food in one's mouth after its transfectionMRNA or the protein expression of breast zooblast decline more than 60%.
Wherein said mammal comprises mammals such as being selected from pig, dog, mouse, people, in the present inventionGood is pig, and siRNA of the present invention has the effect of good cryptiogene in pig.
SiRNA described in the present invention, length is 16-30bp, is more preferably 18-25bp. Preferably,Described siRNA contains and is selected from the nucleotide sequence shown in SEQIDNO:1, or as SEQ in sequence tableIDNO:1 represents. More preferably, 3 ' of described siRNA end contains 2 T or contains 2 U'sExtended structure.
The present invention solves the problems of the technologies described above two of adopted technical scheme: a kind of described pressing down of encodingThe recombinant vector of the siRNA that caveolin 2 processed is expressed.
According to the present invention, the carrier that described recombinant vector adopts can be the carrier of this area routine, bagDraw together adenovirus vector, as the various adenovirus vectors taking 5 types (Ad5), 2 types (Ad2) as basis,Slow virus carrier, is selected from HIV-1 type carrier, HIV-2 type carrier, SIV type carrier, FIV type carrierDeng. Described siRNA fragment is cloned in vector plasmid, obtains recombinant vector of the present invention.
The present invention solves the problems of the technologies described above three of adopted technical scheme: a kind of composition, whereinContain the 0.001-99.99wt% siRNA for caveolin 2 of the present invention and acceptable yearBody, diluent or excipient. Wherein, described carrier, diluent or excipient, be more preferably pharmacyUpper acceptable carrier, diluent or excipient, comprise the carrier materials such as water-soluble, slightly solubility or enteric solubilityMaterial, or powder, dextrin, Icing Sugar, calcium sulfate two hydrates, calcium monohydrogen phosphate, magnesia, magnesium carbonate,One or more in calcium carbonate, gel aluminum hydroxide powder and active carbon.
The present invention solves the problems of the technologies described above four of adopted technical scheme: described siRNA existsPurposes in preparation or screening Tamiflu. Wherein, described siRNA can be used as unique anti-currentInfluenza Virus active component, also can combine use with other anti-influenza virus medicaments.
SiRNA of the present invention, in the purposes of preparation Tamiflu, more preferably can adopt as followsTechnical scheme: as by the mode administration sucking, not only easy to use, and can increase it in infectionThe drug concentration at position. And early stage viral load is little owing to infecting, sufficient siRNA can more haveEffect ground suppresses copying of virus, thereby plays the effect of prevention and treatment; RNAi drug target choosing in influenzaSelect more, can compound or multiple medication, be difficult for producing resistance strain; Last RNAi and influenza vaccines are notWith, do not need user to there is sound immune system.
The present invention can also more preferably take following technical scheme: as by intravenous injection or intramuscular injectionSiRNA sequence or the modes such as this siRNA recombinant vector of encoding, be able to this siRNA in vivoThereby efficiently, reach lastingly the object of RNA immunity or gene therapy influenza virus.
The present invention solves the problems of the technologies described above five of adopted technical scheme: described siRNA existsSuppress the purposes of influenza virus in copying.
The present invention solves the problems of the technologies described above six of adopted technical scheme: a kind of screening antiviral drugThe method of thing, comprises the following steps:
(1) make the cells contacting of drug candidate and influenza virus infection, described cell is caveolin 2The cell of gene silencing;
(2) test influenza virus titre;
(3) selection makes the drug candidate that influenza virus titre declines.
Wherein, more preferably the reticent cell of the gene expression of caveolin 2 described in step (1) is by makingThe siRNA that host cell contains described gene or contain coding and have a described siRNARecombinant vector make this gene silencing.
Wherein, described cell can adopt 293T (people's renal epithelial cell system), BHK (hamster nephrocyte),VERO (African green monkey kidney cell), IBRS22 (porcine kidney cell) or MDCK (MDCK) etc.Clone, the preferred mdck cell of the present invention.
Wherein, contact with influenza virus by this cell, examine under a microscope thin through after a period of timeThe injured degree of born of the same parents, relatively process with Tamiflu and the cell of check experiment in influenza virusVirus titer, just can know whether this antiviral drug can reduce the infringement of virus to cell.
The method of the invention, more preferably can adopt following scheme:
The cytotoxicity of medicine detects, and mdck cell is cultivated and formed list in 96 porocyte culture platesAfter layer, add the liquid of variable concentrations, continue to cultivate 3 days, use mtt assay detection of drugs to MDCKThe toxicity of cell.
The effect of medicine resisiting influenza virus detects, and mdck cell is cultivated shape in 96 porocyte culture platesBecome after individual layer, with the influenza infection cell of 100TCID50, then add the series under non-toxic concnThe pastille nutrient solution of concentration, continues to cultivate 3 days, determines the inhibition of medicine infected by influenza through CPE methodEffect.
The method of the invention, preferably can also adopt following technical scheme:
Influenza virus RNA polymerase (RNA-dependentRNApolymerase, RdRp) is mainly joinedWith viral genome duplication with transcribe. For copying, RdRp taking virus genome RNA (vRNA) asTemplate catalysis generates the RNA chain (cRNA) complementary with it, and it is synthetic that cRNA can be used as template subsequentlyVRNA is fitted into progeny virus. For transcribing, RdRp on the basis of synthetic cRNA its 3 'End adds poly A tract, and 5 ' end adds cap sequence, and forming ripe mRNA, to carry out protein synthetic.When the synthetic RNA chain of this experiment basis, consume substrate A TP, UTP, CTP and GTP and make ATP in systemConcentration reduces gradually, and in mensuration system, remaining ATP content carries out degree to reflect reaction.
By detecting influenza virus RNA polymerase change level in this cell line, assessment caveolin 2The sensitivity of reticent cell to Tamiflu.
Wherein said virus titer detection method, MTT method, CPE method, the methods such as cell cultivation,Be this area normal experiment method.
The present invention solves the problems of the technologies described above seven of adopted technical scheme: a kind of for surface-activeThe siRNA of protein D gene, the target sequence that described siRNA is corresponding is Surfactant proteinD geneMRNA, the length of described siRNA is 16-30bp, and can make the mammal after its transfection thinBorn of the same parents' mRNA or expressing quantity decline more than 60%.
Wherein said mammal comprises mammals such as being selected from pig, dog, mouse, people, in the present inventionGood is pig, and siRNA of the present invention has the effect of good cryptiogene in pig.
SiRNA length described in the present invention is 16-30bp, is more preferably 18-25bp. Preferably,Described siRNA contains and is selected from the nucleotide sequence shown in SEQIDNO:2, or as SEQ in sequence tableIDNO:2 represents. Described siRNA, more preferably sequence 3 ' end contains 2 T or contains 2 UExtended structure.
The present invention solves the problems of the technologies described above eight of adopted technical scheme: a kind of described pressing down of encodingThe recombinant vector of the siRNA that control surface activated protein D expresses.
According to the present invention, the carrier that described recombinant vector adopts can be the carrier of this area routine, bagDraw together adenovirus vector, comprise taking 5 types (Ad5), 2 types (Ad2) as basic various adenovirus vectors,Slow virus carrier comprises HIV-1 type carrier, HIV-2 type carrier, SIV type carrier, FIV type carrier etc. .Described siRNA fragment is cloned in vector plasmid, obtains recombinant vector of the present invention.
The present invention solves the problems of the technologies described above nine of adopted technical scheme: a kind of composition, whereinContain the 0.001-99.99wt% siRNA sequence for Surfactant proteinD of the present invention and canCarrier, diluent or the excipient accepted. Wherein said carrier, diluent and excipient, more preferablyPharmaceutically acceptable carrier, diluent or excipient. Be for example water-soluble, slightly solubility or enteric solubilityIn carrier material, powder, dextrin, Icing Sugar, calcium sulfate two hydrates, calcium monohydrogen phosphate, magnesia, carbonOne or more in acid magnesium, calcium carbonate, gel aluminum hydroxide powder and nano material.
The present invention solves the problems of the technologies described above ten of adopted technical scheme: live in described inhibition surfaceProperty protein D siRNA in preparation or screening the purposes in Tamiflu. Wherein, described siRNACan be used as unique anti-influenza virus activity composition, also can combine and make with other anti-influenza virus medicamentsWith.
Wherein, more preferably can adopt following technical scheme: as by the mode administration sucking, not only useConvenient, and can increase its drug concentration at infection site. And, owing to infecting early stage viral numberMeasure little, sufficient siRNA and can more effectively suppress copying of virus, thereby play the effect of prevention and treatmentReally; In influenza RNAi drug target select more, can compound or multiple medication, be difficult for producing resistance poisonStrain; Last RNAi is different from influenza vaccines, does not need user to have sound immune system.
The present invention can also more preferably take following technical scheme: as by intravenous injection or intramuscular injectionSiRNA sequence or the modes such as this siRNA recombinant vector of encoding, be able to this siRNA in vivoThereby efficiently, reach lastingly the object of RNA immunity or gene therapy influenza virus.
The present invention solves the problems of the technologies described above 11 of adopted technical scheme: a kind of screening anti influenzaThe method of medicine, is characterized in that, comprises the following steps:
(1) make the cells contacting of drug candidate and influenza virus infection, described cell is surface-active eggThe cell of white D gene silencing;
(2) test influenza virus titre;
(3) selection makes the drug candidate that influenza virus titre declines.
Method of the present invention, more preferably Surfactant proteinD gene expression described in step (1)Reticent cell is to have by making the siRNA that host cell contains described gene or contain codingThe recombinant vector of described siRNA makes this gene silencing.
Wherein, described cell can adopt 293T (people's renal epithelial cell system), BHK (hamster nephrocyte),VERO (African green monkey kidney cell), IBRS22 (porcine kidney cell) or MDCK (MDCK) etc.Clone, the preferred MDCK of the present invention.
The method of the invention, contacts with influenza virus by this cell, through after a period of time micro-The injured degree of Microscopic observation cell, relatively process with Tamiflu and the cell of check experiment through flowingThe metainfective survival rate of Influenza Virus or growing state, just can know whether this antiviral drug can reduce virus rightThe infringement of cell.
Wherein, more preferably can adopt following scheme:
(1) cytotoxicity of medicine detects, and mdck cell is cultivated formation in 96 porocyte culture platesAfter individual layer, add the liquid of variable concentrations, continue to cultivate 3 days, by mtt assay detection of drugs pairThe toxicity of mdck cell.
(2) effect of medicine resisiting influenza virus detects, and mdck cell is cultivated in 96 porocyte culture platesForm after individual layer, with the influenza infection cell of 100TCID50, then add and under non-toxic concn beThe pastille nutrient solution of row concentration, continues to cultivate 3 days, determines pressing down of medicine infected by influenza through CPE methodMake and use.
The method of the invention, preferably can also adopt following technical scheme:
(1) influenza virus RNA polymerase (RNA-dependentRNApolymerase, RdRp) is mainParticipate in viral genome duplication and transcribe. For copying, RdRp is with virus genome RNA (vRNA)For template catalysis generates the RNA chain (cRNA) complementary with it, cRNA can be used as subsequently template and closesBecome vRNA to be fitted into progeny virus. For transcribing, RdRp on the basis of synthetic cRNA at it3 ' end adds poly A tract, and 5 ' end adds cap sequence, forms ripe mRNA and carries out protein and closeBecome. When the synthetic RNA chain of this experiment basis, consuming substrate A TP, UTP, CTP and GTP makes in systemATP concentration reduces gradually, and in mensuration system, remaining ATP content carries out degree to reflect reaction.
(2) by detecting influenza virus RNA polymerase change level in this cell line, live in assessment surfaceThe sensitivity of property protein D gene silencing cell to Tamiflu.
Wherein said virus titer detection method, MTT method, CPE method, the methods such as cell cultivation,Be this area normal experiment method.
The present invention solves the problems of the technologies described above 12 of adopted technical scheme: one contains transcribesThe recombinant vector of the gene ORFs of factors A. Preferably, described carrier is by following preparation methodObtain:
(1) the transcription factor A gene order that is NM_001130211.1 accession number in Genbank is defeatedEnter in DNAStar software (DNASTAR company, the U.S.) EditSeq, choose in sequence fromBeginning codon " ATG " is to the longest order between terminator codon " TGA ", " TAG " or " TAA "Row 741bp base;
(2) respectively 5 ' and 3 ' end design amplification total length PCR primer, upstream primer 5 ' end interpolation HindIII restriction enzyme site, CGG is as protectiveness base, and downstream primer 5 ' end adds EcoRV restriction enzyme site;
(3) upstream primer 5 '-CGGAAGCTTATGGCGCTTCTCCGGGGCGTGT-3 ';
(4) downstream primer 5 '-CGGGATATCTCAACACTCCTCAGTGTCTTTC-3 ';
(5) reverse transcription product of the total RNA extracting taking porcine alveolar macrophage is as template, with what designThe amplification of upstream and downstream primer PCR obtains transcription factor A sequence, then after enzyme is cut, is connected to eucaryon tableReach the corresponding restriction enzyme site of carrier.
Wherein, carrier for expression of eukaryon can adopt pCMVp-NEO-BAN, pEGFP, pEGFT-Actin,The common carrier for expression of eukaryon such as pSV2, CMV4, in the present invention, more preferably adopts pCDNA3.1 (+)Carrier.
The present invention solves the problems of the technologies described above 13 of adopted technical scheme: a kind of described containingThe purposes of the recombinant vector that has a transcription factor A ORFs in preparation or screening Tamiflu.
The present invention more preferably, can be by this recombinant vector for the different clone of transfection, and the method for transfection canTo be, DEAE-glucan method, calcium phosphate method, cationic-liposome method, cationic polymer method, diseasePoison mediated method, biologic grain passes method (particle gun Particle bombardment), microinjection, electroporation etc.,The technical program preferred liposome infection protocol. The cell of transfection can be various eukaryotics, this technical sideThe preferred MDCK of case (MDCK).
The present invention can also be by this recombinant vector for gene therapy, as can be by intravenous injection or muscleThe modes such as injection, thus carried out efficiently in vivo, reached lastingly DNA immunization or baseBecause of the object for the treatment of influenza virus.
The present invention solves the problems of the technologies described above 14 of adopted technical scheme: one is used for screening medicineThe cell model of thing, it contains the described recombinant vector that contains transcription factor A ORFs.
Wherein, described cell can be various eukaryotics, comprise 293T (people's renal epithelial cell system),BHK (hamster nephrocyte), VERO (African green monkey kidney cell), IBRS22 (porcine kidney cell) and MDCKClones such as (MDCKs) etc.
The present invention solves the problems of the technologies described above 15 of adopted technical scheme: a kind of screening anti influenzaThe method of medicine, the method for the invention, comprises the following steps:
(1) make the cells contacting of drug candidate and influenza virus infection, described cell was to express to transcribeThe cell of factors A gene;
(2) test influenza virus titre;
(3) selection makes the drug candidate that influenza virus titre declines.
The method of the invention, the described mistake of step (1) is expressed the cell of this gene, is more preferably by increasingThe controlling element that adds the copy number of this gene in cell or improve this gene makes this gene overexpression.
The method of the invention, described cell can be selected from 293T (people's renal epithelial cell system), BHK (hamsterNephrocyte), VERO (African green monkey kidney cell), IBRS22 (porcine kidney cell) and MDCK (dog kidneyCell) etc. clone, the preferred MDCK of the present invention.
The method of the invention, contacts with influenza virus by this cell, through after a period of time micro-The injured degree of Microscopic observation cell, relatively flows in the cell with Tamiflu processing and check experimentThe virus titer of Influenza Virus, just can know whether this antiviral drug can reduce the infringement of virus to cell.
The method of the invention, more preferably can adopt following scheme:
(1) cytotoxicity of medicine detects, and mdck cell is cultivated formation in 96 porocyte culture platesAfter individual layer, add the liquid of variable concentrations, continue to cultivate 3 days, by mtt assay detection of drugs pairThe toxicity of mdck cell.
(2) effect of medicine resisiting influenza virus detects, and mdck cell is cultivated in 96 porocyte culture platesForm after individual layer, with the influenza infection cell of 100TCID50, then add and under non-toxic concn beThe pastille nutrient solution of row concentration, continues to cultivate 3 days, determines pressing down of medicine infected by influenza through CPE methodMake and use.
The method of the invention, preferably can also adopt following technical scheme:
(1) influenza virus RNA polymerase (RNA-dependentRNApolymerase, RdRp) is mainParticipate in viral genome duplication and transcribe. For copying, RdRp is with virus genome RNA (vRNA)For template catalysis generates the RNA chain (cRNA) complementary with it, cRNA can be used as subsequently template and closesBecome vRNA to be fitted into progeny virus. For transcribing, RdRp on the basis of synthetic cRNA at it3 ' end adds poly A tract, and 5 ' end adds cap sequence, forms ripe mRNA and carries out protein and closeBecome. When the synthetic RNA chain of this experiment basis, consuming substrate A TP, UTP, CTP and GTP makes in systemATP concentration reduces gradually, and in mensuration system, remaining ATP content carries out degree to reflect reaction.
(2) by detecting influenza virus RNA polymerase change level in this cell line, assessment transcribe because ofThe sensitivity of sub-A gene overexpression cell to Tamiflu.
Wherein said virus titer detection method, MTT method, CPE method, the methods such as cell cultivation,Be this area normal experiment method.
The present invention solves the problems of the technologies described above 16 of adopted technical scheme: one contains paraoxonThe recombinant vector of the ORFs of acid enzyme 3 genes. Preferably, described carrier is by following preparation methodObtain:
(1), in the paraoxon acid enzyme 3 that accession number is NM_001044604.1 from Genbank, chooseFrom sequence from initiation codon " ATG " to terminator codon " TGA ", " TAG " or " TAA "Between the longest sequence 1065bp base;
(2) respectively 5 ' and 3 ' end design amplification total length PCR primer, upstream primer 5 ' end interpolation HindIIIRestriction enzyme site, CGG is as protectiveness base, and downstream primer 5 ' end adds EcoRI restriction enzyme site;
(3) upstream primer 5 '-CGGAAGCTTATGGGGAAGCTGGTGGCTCTGA-3 ';
(4) downstream primer 5 '-CGGGAATTCCTAGAGCACACAGTACAGAGCT-3 ';
(5) reverse transcription product of the total RNA extracting taking porcine alveolar macrophage is as template, upper with what designDownstream primer pcr amplification obtains transcription factor A sequence, then after enzyme is cut, is connected to eukaryotic expressionThe corresponding restriction enzyme site of carrier.
Wherein, carrier for expression of eukaryon can adopt pCMVp-NEO-BAN, pEGFP, pEGFT-Actin,The common carrier for expression of eukaryon such as pSV2, CMV4, in the present invention, more preferably adopts pCDNA3.1 (+)Carrier.
The present invention solves the problems of the technologies described above 17 of adopted technical scheme: a kind of institute of the present inventionThe recombinant vector that contains paraoxon acid enzyme 3 ORFs of stating is in preparation or the screening TamifluPurposes.
The present invention more preferably, can be by this recombinant vector for the different clone of transfection, and the method for transfection canTo be, DEAE-glucan method, calcium phosphate method, cationic-liposome method, cationic polymer method, diseasePoison mediated method, biologic grain passes method (particle gun Particle bombardment), microinjection, electroporation etc.,The technical program preferred liposome infection protocol. The cell of transfection can be various eukaryotics, this technical sideThe preferred MDCK of case (MDCK).
The present invention can also be by this recombinant vector for gene therapy, as can be by intravenous injection or muscleThe modes such as injection, thus carried out efficiently in vivo, reached lastingly DNA immunization or baseBecause of the object for the treatment of influenza virus.
The present invention solves the problems of the technologies described above 18 of adopted technical scheme: one is used for screening medicineThe cell model of thing, its restructuring that contains the described ORFs that contains paraoxon acid enzyme 3 genes is carriedBody.
Wherein, described cell can be various eukaryotics, be selected from 293T (people's renal epithelial cell system),BHK (hamster nephrocyte), VERO (African green monkey kidney cell), IBRS22 (porcine kidney cell) and MDCK(MDCK) clone etc.
The present invention solves the problems of the technologies described above 19 of adopted technical scheme: a kind of screening anti influenzaThe method of medicine, the method for the invention, comprises the following steps:
(1) make the cells contacting of drug candidate and influenza virus infection, described cell be express rightThe cell of oxygen phosphatase 3 genes;
(2) test influenza virus titre;
(3) selection makes the drug candidate that influenza virus titre declines.
The method of the invention, the cell that the described mistake of step (1) is expressed this gene, more preferably passes throughThe controlling element that increases the copy number of this gene in cell or improve this gene makes this gene overexpression.
The method of the invention, described cell can adopt 293T (people's renal epithelial cell system), BHK (storehouseHamster kidney cell), VERO (African green monkey kidney cell), IBRS22 (porcine kidney cell) and MDCK (dogNephrocyte) etc. clone, the preferred MDCK of the present invention.
The method of the invention, contacts with influenza virus by this cell, through after a period of time micro-The injured degree of Microscopic observation cell, relatively flows in the cell with Tamiflu processing and check experimentThe virus titer of Influenza Virus, just can know whether this antiviral drug can reduce the infringement of virus to cell.
The method of the invention, more preferably can adopt following scheme:
(1) cytotoxicity of medicine detects, and mdck cell is cultivated shape in 96 porocyte culture platesBecome after individual layer, add the liquid of variable concentrations, continue to cultivate 3 days, by mtt assay detection of drugs pairThe toxicity of mdck cell.
(2) effect of medicine resisiting influenza virus detects, and mdck cell is trained in 96 porocyte culture platesSupport and form after individual layer, with the influenza infection cell of 100TCID50, then add under non-toxic concnThe pastille nutrient solution of series concentration, continues to cultivate 3 days, determines medicine infected by influenza through CPE methodInhibitory action.
The method of the invention, preferably can also adopt following technical scheme:
(1) influenza virus RNA polymerase (RNA-dependentRNApolymerase, RdRp) is mainParticipate in viral genome duplication and transcribe. For copying, RdRp is with virus genome RNA (vRNA)For template catalysis generates the RNA chain (cRNA) complementary with it, cRNA can be used as subsequently template and closesBecome vRNA to be fitted into progeny virus. For transcribing, RdRp on the basis of synthetic cRNA at it3 ' end adds poly A tract, and 5 ' end adds cap sequence, forms ripe mRNA and carries out protein and closeBecome. When the synthetic RNA chain of this experiment basis, consuming substrate A TP, UTP, CTP and GTP makes in systemATP concentration reduces gradually, and in mensuration system, remaining ATP content carries out degree to reflect reaction.
(2) by detecting influenza virus RNA polymerase change level in this cell line, assessment paraoxonThe sensitivity of acid enzyme 3 gene overexpression cells to Tamiflu.
Wherein said virus titer detection method, MTT method, CPE method, the methods such as cell cultivation,Be this area normal experiment method.
Than prior art, beneficial effect of the present invention is as follows: of the present invention with in host cellGene or the albumen with antiviral functions are target spot, the medicine of resisiting influenza virus is provided and has suppressed streamThe method that Influenza Virus copies. The present invention is achieved by the expression of described gene in regulating cell.Invention has the using value of prevention and treatment influenza infection, to developing better Tamiflu,To controlling the great outburst of influenza virus, reduce great outburst being good for socio-economic development, people of influenza virusThe great harm that health is brought, has great importance.
Brief description of the drawings
Below in conjunction with brief description of the drawings feature of the present invention and beneficial effect.
Fig. 1 is that Western-blot detects the result that reticent MDCK central foveola albumen 2 is expressed, wherein1:siRNA-caveolin 2; 2:siRNA-caveolin 2 contrasts; 3: do not connect poison contrast; 4: justNormal cell.
Fig. 2 is that Western-blot detects the result that in reticent MDCK, Surfactant proteinD is expressed,Wherein 1:siRNA-Surfactant proteinD; The contrast of 2:siRNA-Surfactant proteinD; 3: do not connectPoison contrast; 4: normal cell.
Fig. 3 is influenza virus titre figure after reticent MDCK central foveola albumen 2 is expressed.
Fig. 4 is that in reticent MDCK, Surfactant proteinD is expressed rear influenza virus titre figure.
Fig. 5 is the pCDNA3.1-transcription factor A carrier collection of illustrative plates building.
Fig. 6 is the pCDNA3.1-paraoxon acid enzyme 3 carrier collection of illustrative plates that build.
Fig. 7 is transit cell record factors A and paraoxon acid enzyme 3 Western blotting testing results, wherein
1:pCDNA3.1-transcription factor A; The contrast of 2:pCDNA3.1 empty carrier; 3: normal cell
4:pCDNA3.1-paraoxon acid enzyme 3; The contrast of 5:pCDNA3.1 empty carrier; 6: normally thinBorn of the same parents.
Fig. 8 is influenza virus titre result after external source raising expression MDCK transcription factor A.
Fig. 9 is that external source improves the rear influenza virus titre of paraoxon acid enzyme 3 result in expression MDCK.
Figure 10 adds influenza virus titre figure after amantadine in the MDCK of caveolin 2 silences.
Figure 11 is that after adding amantadine in the MDCK of Surfactant proteinD silence, influenza virus is drippedDegree figure.
Figure 12 is that external source improves and in the MDCK that transcription factor A expresses, adds influenza disease after amantadinePoison titre figure.
Figure 13 is that external source improves and in the MDCK that paraoxon acid enzyme 3 expresses, adds influenza after amantadineVirus titer figure.
Detailed description of the invention
Further illustrate the present invention with embodiment below, but the present invention is not limited.
In embodiment, reagent used is except special instruction, all commercially available obtaining.
The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or according toThe condition that manufacturer advises. " room temperature " described in the present invention refers to the temperature of the operation room of testingDegree, is generally 25 DEG C.
Swine influenza virus H3N2Swine/Guangdong/1/2006 used (SwGD1/05) poison in embodimentStrain, its biological characteristics performance represents the swine influenza virus feature of China, is derived from the Shanghai animal doctor of the Chinese Academy of Agricultural SciencesPig infectious disease research department of research institute.
6 porocyte culture plates, U.S. Corning product.
DMEM culture medium, U.S. GIBCO product, REF:16000-044.
Penicillin, GIBCO company of U.S. product, REF:15140-122.
Streptomysin, GIBCO company of U.S. product, REF:15140-122.
MDCK (mdck cell), purchased from Shanghai life science institute of Chinese Academy of Sciences cell bank.
1.5ml centrifuge tube, Axygen company of U.S. product.
Acetone, Jiangsu Qiangsheng Chemical Co., Ltd., credit number: XK13-201-00227.
PBS, purchased from GIBCO company of the U.S., REF:20012-027.
PBST: compound method is with reference to " molecular cloning experiment guide " third edition.
The fluorescence antibody of goat-anti mouse FITC mark, American I nvitrogen company product, Code:CA11034s。
100% glycerine, Chemical Reagent Co., Ltd., Sinopharm Group.
In embodiment 1 modulate host cell, gene expression inhibition influenza virus is copied
In the reticent host cell of 1.siRNA (siRNA), gene expression inhibition influenza virus is copied
(1) siRNA MOLECULE DESIGN
The present embodiment is selected caveolin 2 genes, GeneID:100125375; Surfactant proteinD,GeneID:397198, uses the online design software design of Dharmacon siRNA molecule, designs littleThe siRNA interference sequence of nest albumen 2 gene specifics; And arbitrarily upset with " siRNA-caveolin "Sequence in contrast. The siRNA interference sequence of design surface activated protein D gene specific; And with" siRNA-Surfactant proteinD " arbitrarily upsets sequence in contrast. The siRNA of design is double-stranded dryDisturb molecule subject sequence and classify 19 bases as, positive-sense strand 3 ' end is added two UU, and antisense strand 3 ' end is addedTwo TT.
The sequence of design is as follows:
For caveolin 2 target sequences 5 '-GCAAATACGTGATCTACAA-3 ';
SiRNA-caveolin 2 positive-sense strand sequences 5 '-GCAAAUACGUGAUCUACAAUU-3 ';
SiRNA-caveolin 2 antisense strand sequences 3 '-TTCGUUUAUGCACUAGAUGUU-5 ';
SiRNA-caveolin 2 contrasts positive-sense strand sequence 5 '-CGAUAGAUGUACACAUACAUU-3′；
SiRNA-caveolin 2 contrasts antisense strand sequence 3 '-TTGCUAUCUACAUGUGUAUGU-5′；
For Surfactant proteinD target sequence 5 '-GAGCAGAAATGAAGACCTA-3 ';
SiRNA-Surfactant proteinD positive-sense strand sequence 5 '-GAGCAGAAAUGAAGACCUAUU- 3′；
SiRNA-Surfactant proteinD antisense strand sequence 3 '-TTCUCGUCUUUACUUCUGGAU-5′；
SiRNA-Surfactant proteinD contrast positive-sense strand 5 '-CAGAGUGACAGAAGACAUAUU-3′；
SiRNA-Surfactant proteinD contrast antisense strand 3 '-TTGUCUCACUGUCUUCUGUAU-5′。
Through NCBIblast Analysis interference sequence and " caveolin or Surfactant proteinD " base in additionBecause homology is less than 50%, control sequence and pig genome all sequences homology are less than 50%.
According to the siRNA sequence of this design, by the external artificial synthetic double-stranded siRNA of Dharmacon company.
(2) siRNA molecule transfectional cell
By the DMEM of serum-free for the mdck cell of trypsinization (U.S. GIBCO product,REF:16000-044) (100 unit penicillin (GIBCO company of U.S. product, REF:15140-122) and 100 unit streptomysins (GIBCO company of U.S. product, REF:15140-122)) passIn 6 porocyte culture plates (U.S. Corning product), cell density 4 × 106Cells/well, continuesBe incubated at 37 DEG C, 5%CO2In incubator. Treat the 80-90% at the bottom of Growth of Cells is expired culture plate, institute of the present inventionState double-stranded siRNA molecule FuGENEHD reagent (Roche company, the U.S., production code member:04709705001) be transfected in mdck cell operating process by specification. Establish transfection contrast simultaneouslyInterference sequence sample siRNA-caveolin 2 contrasts, untransfected contrasts, normal cell contrast.
(3) siRNA transfectional cell connects poison and sample collection
24h inoculation influenza virus H3N2 after mdck cell transfection siRNA molecule, dosage of inoculation 1×103EID50/ hole (6 orifice plate). After virus inoculation, 24h, 48h, 72h collect sample. MethodAs follows: by cell in Tissue Culture Plate, together with cell culture fluid freeze thawing 3 times, the centrifugal 10min of 5000g/min,Retain supernatant, abandon precipitation. Measure influenza virus titre in supernatant. Virus titer assay method is with reference to " stateFamily's influenza central standard operational procedure (revised edition), 2007 ".
Experimental result (Fig. 1) shows, mdck cell central foveola albumen 2, Surfactant proteinD tableReach and inoculated influenza virus after siRNA molecule silence, influenza virus in 24h, 48h, 72h cellTitre is compared remarkable reduction with control group, and significant difference.
Cell central foveola albumen 2 by siRNA molecule silence after (as above 24h sample after virus inoculation),Western-blot testing result is shown in Fig. 1, and result shows that reticent group central foveola albumen 2 expressions are obviously lowIn contrast.
In cell Surfactant proteinD by siRNA molecule silence after (as above 24h after virus inoculationSample), Western-blot testing result is shown in Fig. 2, result shows Surfactant proteinD table in reticent groupThe level of reaching is starkly lower than contrast.
SiRNA-caveolin 2 is compared p value with siRNA-caveolin 2 control groups and is respectively p=0.0004(24h), 0.001 (48h), 0.015 (72h); SiRNA-Surfactant proteinD and siRNA-tableFace activated protein D control group is compared p value and is respectively p=0.004 (24h), 0.001 (48h), 0.009(72h)。
Above-mentioned the results show can press down by regulation and control caveolin 2 and surfactant protein gene expressionIn host cell processed, influenza virus copies.
2. in exogenous raising host cell, gene expression inhibition influenza virus is copied
(1) structure of expression vector in the 1st group of heterogenous expression
By transcription factor A (TFAM) (GeneID:397279; Gene database accession number:NM_001130211.1) be connected to pCDNA3.1 (+) carrier (Invitrogen company, the U.S.; Article No.:V790-20), between HindIII and EcoRV restriction enzyme site, concrete construction method comprises the following steps:
(1) the transcription factor A gene order that is NM_001130211.1 accession number in Genbank is defeatedEnter in DNAStar software (DNASTAR company, the U.S.) EditSeq, choose sequenceIn from initiation codon " ATG " to terminator codon " TGA ", " TAG " or " TAA "Between the longest sequence 741bp base;
(2) respectively 5 ' and 3 ' end design amplification total length PCR primer, upstream primer 5 ' end interpolation HindIIIRestriction enzyme site, CGG is as protectiveness base, and downstream primer 5 ' end adds EcoRV enzyme and cutsSite;
(3) upstream primer 5 '-CGGATGGCGCTTCTCCGGGGCGTGT-3 ' is (twoBe scribed ss HindIII restriction enzyme site);
(4) downstream primer 5 '-CGG(two strokes of TCAACACTCCTCAGTGTCTTTC-3 'Line is EcoRV restriction enzyme site);
(5) extract total RNA reverse transcription product as template taking pig pulmonary macrophage, with design upstream and downstream drawThing pcr amplification obtains transcription factor A sequence, then after enzyme is cut, is connected to pCDNAThe corresponding restriction enzyme site of 3.1 (+) carrier.
The carrier called after pCDNA3.1-transcription factor A successfully constructing, is shown in Fig. 5.
By paraoxon acid enzyme 3 (PON3) (GeneID:733674; Gene database accession number:NM_001044604.1) gene ORFs is connected to pCDNA3.1 (+) carrier (Invitrogen public affairsDepartment, the U.S.; Article No.: V790-20) between HindIII and EcoRI restriction enzyme site, concrete construction method bagDraw together following steps:
(1) paraoxon that is NM_001044604.1 accession number in Genbank acid enzyme 3 gene ordersBe input in DNAStar software (DNASTAR company, the U.S.) EditSeq, choose fromIn sequence from initiation codon " ATG " to terminator codon " TGA ", " TAG " or " TAA "Between the longest sequence 1065bp base;
(2) respectively at 5 ' and 3 ' end design amplification total length PCR primer, upstream primer 5 ' end adds HindIII restriction enzyme site, CGG is as protectiveness base, and downstream primer 5 ' end adds EcoRI enzymeCut site;
(3) upstream primer 5 '-CGGATGGGGAAGCTGGTGGCTCTGA-3’(two HindIII restriction enzyme site that is scribed ss);
(4) downstream primer 5 '-CGGCTAGAGCACACAGTACAGAGCT-3 ' (two EcoRI restriction enzyme site that is scribed ss);
(5) extract total RNA reverse transcription product as template taking pig pulmonary macrophage, with design upstream and downstream drawThing pcr amplification obtains transcription factor A sequence, then after enzyme is cut, is connected to pCDNAThe corresponding restriction enzyme site of (3.1+) carrier.
The carrier called after pCDNA3.1-paraoxon acid enzyme 3 successfully constructing, is shown in Fig. 6.
(2) recombinant vector transfectional cell and expressive host albumen
By the DMEM of serum-free for the mdck cell of trypsinization (U.S. GIBCO product, REF:16000-044) 100 unit penicillin (GIBCO company of U.S. product, REF:15140-122)With 100 unit streptomysins (GIBCO company of U.S. product, REF:15140-122)) pass in 6 holesIn Tissue Culture Plate (U.S. Corning product), cell density 4 × 106Cells/well, continues to be incubated atIn 37 DEG C, 5%CO2 incubator. Treat the 80-90% at the bottom of Growth of Cells is expired culture plate, use FuGENEHDReagent (Roche company, the U.S., production code member: 04709705001) pCDNA3.1-transcribe because ofSub-A, pCDNA3.1-paraoxon acid enzyme 3 carriers are transfected into respectively in mdck cell, operating processBy specification. Establish the cell contrast of transfection pCDNA3.1 (+) empty carrier simultaneously.
24h after cell transfecting, collects transfection pCDNA3.1-transcription factor A, pCDNA3.1-paraoxonAcid enzyme 3, pCDNA3.1 (+) empty carrier and sample of normal cells. Total protein of cell extracts: float with PBSWash in 6 porocyte plates cell 2 times, then add 1mlPBS cell cell spatula is scraped, 4 DEG C,The centrifugal 5min sedimentation cell of 3000g, abandons supernatant. Add the cell pyrolysis liquid of 100 μ l to cell precipitation(green the skies company, production code member: P0013), re-suspended cell, at cracking 5min on ice, then with superThe instantaneous fragmentation of sound wave cell crushing instrument (Sonics company, the U.S., model VCX105PB) (40HZ,1S), boiling water boiling 5min, the centrifugal 10min of 13000g at 4 DEG C, discards precipitation. Get 2 μ l albumen supernatantsMeasure protein concentration with BCA kit (green the skies company, production code member: P0012), to specificationsOperation. Remaining albumen supernatant adds 5 × SDSPAGEbuffer, and (compound method is shown in " molecular cloning experimentGuide " third edition), boiling water boiling 5min, the centrifugal 5min of 13000g at 4 DEG C, gets supernatant and carries out proteinElectrophoresis.
(step is with reference to " molecular cloning experiment guide " for sex change SDS-PAGE gel electrophoresis and albumen transfer printingThe third edition): each protein sample applied sample amount is 30 μ g, SDS-PAGE electrophoresis apparatus (Beijing 61 instrumentsFactory) 80V voltage lamination albumen, 120V voltage protein isolate, until electrophoresis finishes. Albumen transfer printing is usedNitrocellulose filter (Whatman company, the U.S., product type: 10401396). Bole's electrophoresis tankConstant voltage 65V, 2h. Transfer printing finishes to seal in TBST-skimmed milk that rear NC film immerses 5% (v/v)Close. After room temperature effect 2h, discard confining liquid, with the TBST (pH7.6) containing 1% (v/v) polysorbas20Buffer solution rinsing 3 times, washes away residual skimmed milk, can carry out antibody incubation.
Antibody response and colour developing: add primary antibodie, paraoxon acid enzyme 3 antibody (Abcam, the U.S., productNumbering: ab40969), transcription factor A antibody (antikoerper-online.de company, Germany, productNumbering: ABIN484435). At 4 DEG C, jog shaken overnight (12h-16h), then washes with 100%TBSTAfter 3 times, each 5min. Add afterwards two of HRP (horseradish peroxidase) mark to resist, room temperature is doneWith 2h, with after TBST rinsing 3 times, each 5min. ECL kit (Pierce company, the U.S.,Production code member: 32106) colour developing operates in darkroom. Operation is according to ECL kit description.
Detection internal reference contrast: the film having developed the color is immersed to antibody and strip off liquid (green skies company, product is compiledNumber: P0025), hatch 30min for 50 DEG C, every 10min shakes once, then uses TBST buffer solution(compound method: with reference to " molecular cloning experiment guide " third edition) rinsing 3 times, adds confining liquid againSealing 15min, uses anti-β-actin antibody (green the skies company, production code member: AA128) as primary antibodieDetect protein content in sample.
Testing result is shown in Fig. 5, show transfection pCDNA3.1-transcription factor A in mdck cell,After pCDNA3.1-paraoxon acid enzyme 3, in cell " transcription factor A " and " the sour enzyme 3 of paraoxon "Expression is higher than transfection pCDNA3.1 (+) empty carrier group and normal cell group.
(3) " transcription factor A " or " paraoxon acid enzyme 3 " tool in exogenous raising mdck cellThere is the effect of the influenza virus of inhibition
Cell transfecting pCDNA3.1-transcription factor A, pCDNA3.1-paraoxon acid enzyme 3 or pCDNA24h after 3.1 (+) empty carrier, dosage of inoculation 1 × 103EID50/ hole (6 orifice plate) swine influenza virus. ConnecingAfter kind virus, 24h, 48h, 72h collect sample. Method is as follows: by cell in Tissue Culture Plate,Together with cell culture fluid freeze thawing 3 times, the centrifugal 10min of 5000 turn/min, retains supernatant, abandons precipitation. SurveyDetermine influenza virus titre in supernatant. Virus titer assay method is with reference to " national influenza central standard operation ruleJourney (revised edition), 2007 ".
Experimental result (Fig. 6,7) shows, mdck cell transcription factor A, paraoxon acid enzyme DExpressed by exogenous raising, in 24h, 48h, 72h cell, influenza virus titre is compared with control groupSignificantly reduce, and significant difference. Transfection pCDNA3.1-transcription factor A group and transfection pCDNA3.1 (+) empty carrier group is compared p value and is respectively p=0.002 (24h), 0.014 (48h), 0.0005 (72H); 3 groups of transfection pCDNA3.1-paraoxon acid enzymes are compared p with transfection pCDNA3.1 (+) empty carrier groupValue is respectively p=0.015 (24h), 0.008 (48h), 0.012 (72h). Transfection pCDNA3.1 (+)Not remarkable (p > 0.05) of virus titer difference in empty carrier group and normal cell.
Above-mentioned experimental result shows that regulative transcription factor A and 3 gene expressions of paraoxon acid enzyme can suppressIn host cell, influenza virus copies.
In embodiment 2 modulate host cells, gene expression significantly improves the disease-resistant toxic effect of influenza virus medicineReally
In the present embodiment, after protein expression, infect again H3N2 swine influenza virus by regulating in MDCK,Utilize and add amantadine in cell culture fluid, in research regulating cell, albumen is to amantadine anti influenzaVirus strengthens effect. Amantadine, by disturbing influenza virus particles M2 protein ion passage, suppresses sickThe function copying of poison.
1 reticent MDCK central foveola albumen 2 significantly improves amantadine and suppresses influenza virus effect
(1) reticent MDCK central foveola albumen 2 is expressed
Concrete steps are with embodiment 1.
(2) amantadine is prepared
Amantadine hydrochloride is purchased from A Faaisha (Tianjin) Chemical Co., Ltd., is mixed with physiological saline20mg/ml, filtration sterilization, 4 DEG C of preservations.
(3) siRNA molecule transfectional cell
Concrete steps are with embodiment 1. Drug combination group adds amantadine, final concentration in cell culture fluidTo 0.4 μ g/ml, be grouped as follows: siRNA-caveolin+amantadine (0.4 μ g/ml), siRNA-are littleContrast+amantadine of nest albumen 2 (0.4 μ g/ml), untransfected contrast+amantadine (0.4 μ g/ml) andNormal cell contrast+amantadine (0.4 μ g/ml).
(4) siRNA transfectional cell connects poison and sample collection
With embodiment 1.
As shown in figure 10, show that mdck cell central foveola albumen 2 is expressed by siRNA divides experimental resultAfter sub-silence, can significantly improve the effect that amantadine suppresses influenza virus. 24h, 48h, 72h cellMiddle influenza virus titre siRNA-caveolin+amantadine (0.4 μ g/ml) is organized significantly lower than siRNA-Contrast+amantadine of caveolin 2 (0.4 μ g/ml) group, p value is respectively p=0.016 (24h), 0.001(48h)、0.001(72h)。
Interior caveolin 2 gene expressions of above-mentioned the results show regulating cell can improve amantadine and press downThe effect that influenza virus processed is copied.
In 2 reticent MDCKs, Surfactant proteinD significantly improves amantadine inhibition influenza virus effectReally
(1) in reticent MDCK, Surfactant proteinD is expressed
Concrete steps are with embodiment 1.
(2) amantadine is prepared
Amantadine hydrochloride is purchased from A Faaisha (Tianjin) Chemical Co., Ltd., is mixed with physiological saline20mg/ml, filtration sterilization, 4 DEG C of preservations.
(3) siRNA molecule transfectional cell
Concrete steps are with embodiment 2. Drug combination group adds amantadine, final concentration in cell culture fluidTo 0.4 μ g/ml, be grouped as follows: siRNA-Surfactant proteinD+amantadine (0.4 μ g/ml), siRNA-Surfactant proteinD contrast+amantadine (0.4 μ g/ml), untransfected contrast+amantadine (0.4 μ g/ml)With normal cell contrast+amantadine (0.4 μ g/ml).
(4) siRNA transfectional cell connects poison and sample collection with embodiment 1.
Experimental result as shown in figure 11, shows that in mdck cell, Surfactant proteinD is expressed quiltAfter siRNA molecule silence, can significantly improve the effect that amantadine suppresses influenza virus. 24h, 48h,Influenza virus titre siRNA-Surfactant proteinD+amantadine (0.4 μ g/ml) group in 72h cellSignificantly, lower than siRNA-Surfactant proteinD contrast+amantadine (0.4 μ g/ml) group, p value respectivelyFor p=0.020 (24h), 0.025 (48h), 0.130 (72h).
In above-mentioned the results show regulating cell, Surfactant proteinD gene expression can improve Buddha's warrior attendantAlkanamine suppresses the effect that influenza virus is copied.
In 3 exogenous raising host cells, transcription factor A expresses and significantly improves amantadine inhibition influenzaVirus effect
(1) structure of expression vector in the 1st group of heterogenous expression
Concrete steps are with embodiment 1.
(2) recombinant vector transfectional cell and expressive host albumen
Concrete steps are with embodiment 1.
(3) after exogenous raising mdck cell transcription factor A, amantadine suppresses influenza virus againThe effect of system
24h after cell transfecting pCDNA3.1-transcription factor A or pCDNA3.1 (+) empty carrier, inoculationDosage 1 × 103EID50/ hole, drug combination group adds amantadine in cell culture fluid, and final concentration is extremely0.4 μ g/ml, arranges following experimental group: transfection pCDNA3.1-transcription factor A+ amantadine(0.4 μ g/ml), transfection pCDNA3.1 (+) empty carrier+amantadine (0.4 μ g/ml), untransfected pairAccording to+amantadine (0.4 μ g/ml) and normal cell contrast+amantadine (0.4 μ g/ml). Sick in inoculationAfter poison, 24h, 48h, 72h collect sample. Method is as follows: by cell in Tissue Culture Plate, together withCell culture fluid freeze thawing 3 times, the centrifugal 10min of 5000 turn/min, retains supernatant, abandons precipitation. In mensurationInfluenza virus titre in clear. Virus titer assay method is with reference to " national influenza central standard operational procedure (is repaiiedOrder version), 2007 ".
Experimental result shows, shown in Figure 12, mdck cell transcription factor A is shown by exogenous raisingReach, dye pCDNA3.1-transcription factor A+ amantadine (0.4 μ g/ml) at 24h, 48h, 72h transit cellGroup is compared remarkable reduction with transfection pCDNA3.1 (+) empty carrier+amantadine (0.4 μ g/ml), andSignificant difference, p value is respectively p=0.310 (24h), 0.013 (48h), 0.007 (72h).
Above-mentioned experimental result shows to regulate and control that in the 1st group, gene expression can significantly strengthen amantadine inhibition streamInfluenza Virus copies.
In 4 exogenous raising host cells, paraoxon acid enzyme 3 is expressed and is significantly improved amantadine inhibition influenzaVirus effect
(1) structure of expression vector in the 1st group of heterogenous expression
Concrete steps are with embodiment 2.
(2) recombinant vector transfectional cell and expressive host albumen
Concrete steps are with embodiment 2
(3) in exogenous raising mdck cell, the rear amantadine of paraoxon acid enzyme 3 suppresses influenza virusThe effect copying
24h after cell transfecting pCDNA3.1-paraoxon acid enzyme 3 or pCDNA3.1 (+) empty carrier, connectsPlant dosage 1 × 103EID50/ hole, drug combination group adds amantadine in cell culture fluid, and final concentration is extremely0.4 μ g/ml, arranges following experimental group: transfection pCDNA3.1-paraoxon acid enzyme 3+ amantadine(0.4 μ g/ml), transfection pCDNA3.1 (+) empty carrier+amantadine (0.4 μ g/ml), untransfected pairAccording to+amantadine (0.4 μ g/ml) and normal cell contrast+amantadine (0.4 μ g/ml). Sick in inoculationAfter poison, 24h, 48h, 72h collect sample. Method is as follows: by cell in Tissue Culture Plate, together withCell culture fluid freeze thawing 3 times, the centrifugal 10min of 5000 turn/min, retains supernatant, abandons precipitation. In mensurationInfluenza virus titre in clear. Virus titer assay method is with reference to " national influenza central standard operational procedure (is repaiiedOrder version), 2007 ".
Experimental result 13 shows, in mdck cell, paraoxon acid enzyme 3 is expressed by exogenous raising,24h, 48h, 72h transit cell dye pCDNA3.1-paraoxon acid enzyme 3+ amantadine (0.4 μ g/ml)Group is compared remarkable reduction with transfection pCDNA3.1 (+) empty carrier+amantadine (0.4 μ g/ml), andSignificant difference, p value is respectively p=0.042 (24h), 0.088 (48h), 0.069 (72h).
Above-mentioned experimental result shows that in regulating cell, 3 gene expressions of paraoxon acid enzyme can significantly strengthen Buddha's warrior attendantAlkanamine suppresses influenza virus to be copied.
All documents of mentioning in the present invention are all quoted as a reference in this application, just as each section of quiltApplication as a reference separately. In addition should be understood that after having read above-mentioned instruction of the present invention, thisThose skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within this Shen equallyPlease appended claims limited range.
Should be understood that, after having read foregoing of the present invention, those skilled in the art can be to thisBright making various changes or modifications, these equivalent form of values fall within equally the application's appended claims and limitScope.

Claims (4)

1. the gene that contains paraoxon acid enzyme 3 is put a recombinant vector for reading frame, it is characterized in that,Prepared by the method comprising the following steps,
(1) paraoxon that is NM_001044604.1 from Genbank accession number acid enzyme 3 gene ordersIn, choose from sequence from initiation codon " ATG " to terminator codon " TGA ", " TAG "Or the longest sequence 1065bp base between " TAA ";
(2) respectively 5 ' and 3 ' end design amplification total length PCR primer, upstream primer 5 ' end interpolation HindIII restriction enzyme site, CGG is as protectiveness base, and downstream primer 5 ' end adds EcoRI enzyme and cutsSite;
(3) upstream primer 5 '-CGGAAGCTTATGGGGAAGCTGGTGGCTCTGA-3 ';
(4) downstream primer 5 '-CGGGAATTCCTAGAGCACACAGTACAGAGCT-3 ';
(5) reverse transcription product of the total RNA extracting taking porcine alveolar macrophage is as template, with what designThe amplification of upstream and downstream primer PCR obtains paraoxon acid enzyme 3 sequences, then after enzyme is cut, connectsTo the corresponding restriction enzyme site of eukaryotic vector.
2. recombinant vector as claimed in claim 1, is characterized in that, true in described step (5)Nuclear expression carrier is pCDNA3.1 (+) carrier.
3. the use of recombinant vector as claimed in claim 1 or 2 in preparation or screening TamifluOn the way.
4. for screening a cell model for medicine, it is characterized in that, contain just like claim 1 or 2Described recombinant vector.
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