CN105602989B - A kind of recombinant vector and its application in preparation or screening Tamiflu - Google Patents

A kind of recombinant vector and its application in preparation or screening Tamiflu Download PDF

Info

Publication number
CN105602989B
CN105602989B CN201610139667.9A CN201610139667A CN105602989B CN 105602989 B CN105602989 B CN 105602989B CN 201610139667 A CN201610139667 A CN 201610139667A CN 105602989 B CN105602989 B CN 105602989B
Authority
CN
China
Prior art keywords
cell
sirna
gene
influenza virus
recombinant vector
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN201610139667.9A
Other languages
Chinese (zh)
Other versions
CN105602989A (en
Inventor
马志永
魏建超
史子学
邵东华
李蓓蓓
晏文君
朱紫祥
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Veterinary Research Institute CAAS
Original Assignee
Shanghai Veterinary Research Institute CAAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Veterinary Research Institute CAAS filed Critical Shanghai Veterinary Research Institute CAAS
Priority to CN201610139667.9A priority Critical patent/CN105602989B/en
Publication of CN105602989A publication Critical patent/CN105602989A/en
Application granted granted Critical
Publication of CN105602989B publication Critical patent/CN105602989B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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 present invention provides a kind of recombinant vector and its preparing or screening the application in Tamiflu.The recombinant vector contains the open reading frame of 3 gene of paraoxon acid enzyme, the open reading frame segment of 3 gene of paraoxon acid enzyme obtained after PCR amplification is connected after digestion into carrier, the recombinant vector is made.The invention has the advantages that provided recombinant vector can be used for gene therapy influenza or carry out nucleic acid immunization for influenza virus；Cell model provided by the invention can be used for virus research, especially swine influenza virus pathogenesis, it can also be used to establish screening antiviral drugs model, especially Tamiflu cell model.

Description

A kind of recombinant vector and its application in preparation or screening Tamiflu
The application is that application No. is the 2012100197129, entitled " siRNA of inhibition influenza virus related gene And its application ", the applying date be on January 20th, 2012 patent application divisional application.
Technical field
The invention belongs to field of biotechnology, in particular to inhibition caveolin 2 and Surfactant proteinD gene expression The nucleotide sequence of siRNA, siRNA, the recombinant vector of the codified siRNA and codified transcription factor A and paraoxon acid The purposes of the recombinant vector of enzyme 3 and their own gene in terms of preparing and screening Tamiflu.
Background technique
Poisoning intrusion host processes are expressed along with host gene to be changed, final to determine infection cell and the respective life of virus Fortune.The duplication for needing to complete virus itself after virus infection host using the metabolic process of host cell, in this process virus Will necessarily modulate host genes within cells group expression, inhibit antiviral gene expression, enhance the gene beneficial to virus replication Expression.Therefore, after virus infection host cell, these genes of host cell inner expression variation often have with virus replication closely Relationship.By one in these intracellular genes of biological means modulate host or regulating and controlling multiple gene expressions simultaneously may have Play the role of inhibiting influenza virus duplication, with the potential application value for preventing and treating influenza infection.
Influenza virus is the segmented sub-thread minus-stranded rna virus of orthomyxoviridae family's Influenza Virus.When virion just separates In polymorph, become spherical after passing in vivo, diameter 80-120nm (Palese, et al., 2007).Virion is by 0.8- The carbohydrate of the protein of 1% RNA, 70-75%, 20% lipid and 5-8% forms, and ratio is mass ratio herein.
Influenza virus is divided into tri- type of A, B, C, and wherein A type (A type) is mostly important.Influenza A virus can cause the mankind, The natural infection and morbidity of pig, horse and various birds.Since its surface antigen HA and NA is easy variation, it is known that HA has 16 hypotypes (H1-H16), NA has 9 hypotypes (N1-N9), the various combination between them, makes influenza A virus there are many hypotypes (such as H1N1, H2N2, H3N2, H5N1 etc.), Major Epidemic H1N1 and the H3N2 hypotype in pig body.
Different animals have different influenza viruses, and such as swine flu, human influenza, equine influenza, these influenzas are only under normal circumstances Infect respective category animal.But influenza virus has the adaptability of host's kind.Also hold between the individual of different animal species It easily propagates, forms the new influenza virus of infection many animals.Another feature is that this animal of pig is more special, and pig is to it The influenza virus of its animal is also susceptible, this is the characteristic that other animals do not have.So once pig has infected human influenza, pig simultaneously Influenza, bird flu etc., this variety of virus can recombinate in pig body, and may will come out one kind can infect pig, people, fowl Influenza virus.So the swine flu of narrow sense is not Amphixenosis.Influenza is very limited in interpersonal spread scope, with influenza Route of transmission it is related.Such as Flu-A in 2009, exactly generated after human influenza, swine flu, bird flu co-infection pig New influenza virus, the people for most starting infection, which is substantially, to be contacted or the people in close relations with pig, then had more people's infection Influenza virus.The great outburst of influenza virus all carrys out significant damage to the health care belt of socio-economic development, people in human development history.For Influenza is prevented and treated, each state has all put into a large amount of manpower and material resources, constantly studies treatment method and drug.In recent years, from host The intracellular gene found with antiviral functions or albumen are a popular domains.
The great outburst of influenza virus all carrys out significant damage to the health care belt of socio-economic development, people in human development history.For Influenza is prevented and treated, each state has all put into a large amount of manpower and material resources, constantly studies treatment method and drug.In recent years, for place It is a popular domain that gene or albumen in chief cell with antiviral functions, which research and develop antiviral drugs as target spot,.
There is broad-spectrum antiviral using endocellular function molecule as the target spot of antiviral therapy, be not likely to produce drug resistance strain Advantage is increasingly becoming the hot spot approach of research and development antiviral therapy drug.High-level paper international in recent years is repeatedly reported in There is the molecule of potential antiviral functions to break trough for screening in signal path in host cell.For example, zinc finger is disease-resistant Toxalbumin (Zinc-finger antiviral protein, ZAP) can inhibit the white blood of mouse by degradation particular virus RNA The duplication of a variety of viruses such as virus is proved to be a kind of important antiviral agent (Chen et al. (2008) Proc.Natl.Acad.Sci.USA.105,4352-4357).It is that carrier carries special enzyme blocking t cell using zinc finger protein In CCR5 expression, can promote T cell remove inhibition of HIV (Holt et al. (2010) Nat.Biotechnol.28,839- 847).E3 ubiquitin ligase RNF5 is positioned at mitochondria, by ubiquitination MITA (also referred to as STING) and causes its degradation And I type interferon expression (Zhong et al. (2009) Immunity.30,397-407) after negative regulation virus infection. With dimer anchoring HIV on the cell membrane of its duplication, blocking virus sprouts Tetherin (host cell proteins) from endochylema film It discharges (Perez-Caballero et al. (2009) Cell.139,499-511).APOBEC3G(Apolipoprotein B MRNA-editing enzyme catalytic polypeptide-like 3G) it can induce HIV genetic mutation, make virus not Reproducible (Shirakawa et al. (2008) Nat.Struct.Mol.Biol.15,1184-1191).Cyclin at present Dependent kinase, prostaglandin, heat shock protein are widely used in such as HCMV, HIV, HSV, adenovirus and nipple as medicine target molecule The treatment of the virosis such as shape tumor.
RNAi is a kind of Gene silence of efficient high specificity, is quickly grown in recent years, becomes function soon The powerful of genome research.DsRNA molecule imported by laboratory facilities it is intracellular, specifically degrade into the cell and its The mRNA of sequence homology closes endogenous gene expression, from the angle research mankind of reverse genetic or other biological genome The function of unknown gene.Early stage has separated with regard to someone using technique various in drosophila insulin signal transduction approach access Ingredient.Also there is Experimental report to study each approach involved in lipid within endothelial cells equilibrium process by RNAi recently.Before this, Once inhibited the generation of its phenotype using the antisense RNA characteristic complementary with said target mrna sequence, but since antisense RNA is to endogenous table The gene inhibiting effect reached is weaker, often generates some transition phenotypes, easily causes and judges incorrectly to gene function, has led at present It crosses examination & approval and thinks clinically have a kind of medicative only drug Vitravene.
RNAi specificity is higher, and effect is rapider, and side reaction is small, while effectively silencing of target genes, to cell sheet The regulator control system of body does not also influence.Successfully nearly 20 kinds of gene functions " strike in human somatic cell recently Except ", especially therefore understand the effect that mankind vacuole albumen Tsg101 is proliferated HIV in human body, has further deepened Research to HIV.Leonid etc., using RNAi come the intracellular immune of inducing cell, is generated using poliovirus as model Antiviral effect, in particular for RNA virus.For the virus being easily mutated, a variety of targeting viral gene conserved sequences can be designed DsRNA, reduce its resistance to dsRNA.Maen etc. has also successfully been blocked in MCF-7 breast cancer cell using RNAi technology A kind of function of the nuclear transcription factor-2 gene 21 relevant to cell proliferation and differentiation of unconventionality expression.The application of RNAi technology, not only The development that human post-genome plan (protein science) can be pushed significantly, screens drug target gene with high throughput, detects people one by one The expression inhibiting situation of genoid group carrys out the function of clear gene, and it will also be applied to gene therapy, new drug development, biology The fields such as medical research open new approach with RNAi technology come the unconventionality expression of suppressor to treat various diseases.
Due to the fast development of Protocols in Molecular Biology and cytobiology technology in recent years, molecular pharmacology research is not yet It is disconnected deeply, new drug target, functional protein, gene expression variation, it is medicine that bioactive ingredients etc., which constantly discover, Object screening provides a large amount of new target spots, such as new receptor, enzyme.These new target spots provide new information for new medicament screen And chance.The application of cellular and molecular level medicaments sifting model is laid a good foundation for automatic operation, makes drug screening by tradition Manual screening form be changed by computer control automation Large-scale Screening new technology system, form high-throughput medicine Object screening.
The advantages of high-flux medicaments sifting: the scale of drug screening is realized, medical substance is utilized to large extent Resource, improves the probability of drug discovery, while improving the quality of discovery new drug；Screening experiment is in microscreen system It completes, amount of samples generally at Gamma Magnitude (μ g), saves sample resource, has established the material base of " medicine sieves more ", together When save experimental material, reduce single medicine screening cost；High-flux medicaments sifting is that increasingly automated operation reduces operation The generation of error reduces labor intensity, and improves the efficiency of drug screening and the accuracy of result；With multidisciplinary reason The characteristics of by being combined with technology.
Medicaments sifting model is the essential condition for finding new drug.The foundation of new model will will drive the appearance of newtype drug. The completion of the development of molecular biology, cell biology, computer science, the especially Human Genome Project is medical research Good opportunity is brought, also to establish new medicaments sifting model, provides various advantages such as theory, technology, material Condition.Therefore, we should make full use of the Development Technology of each subject to establish more new screening models, promote the discovery of new drug.
DNA vaccination is the new technology that last century the nineties grow up, i.e., carries exogenous gene cloning to eukaryotic expression On body construct DNA vaccination plasmid, DNA vaccination after various approach are injected into animal matrix, can by host cell absorb and Intake into being transcribed into mRNA in the nucleus of host cell, then translates into protein in endochylema.A portion protein After degradation in conjunction with MHC I class molecule, and by submission to cell surface by the Receptor recognition of CD8 T cell, and active cell The activity of cytotoxic T cell；Another part protein can also be secreted away, then be taken the photograph as foreign protein by antigen presenting cell It takes, polypeptide is degraded into phagolysosome, and further there is antigen presenting cell to offer to thin in conjunction with MHC II class molecule By the Receptor recognition of Th2 cell, the Cytokine then secreted by Th2 cell is stimulated in B cell with antibody cellular surface Humoral immunity based on generation.DNA vaccination not only can induce and generate humoral immunity, but also can induce strong and lasting cell and exempt from Epidemic disease also can avoid outwardly toxin expelling, and DNA vaccination and attenuation and attenuated vaccine are different, be not present that virulence is anti-strong etc. to ask Topic.Its safety and high efficiency are that traditional vaccine is not achieved, so, the technology is caused by virus, the cytozoicus Huge potentiality are shown in infectious disease, helminth and treatment and prevention of tumour.Wolff can express recombinant plasmid in Mice Body Reported, foreign protein can be expressed in Mice Body up to 60 days, prompt Plasmid DNA can in vivo suitable one section when It is interior to be expressed, it may be used as therapeutic effect, and cause extensive research.
Gene therapy is for cancer, atherosclerosis, osteoporosis, arthritis, Alzheimer's because specific The prevention and treatment of disease caused by Gene Activity exception have prospect very much.The therapy be it is a kind of by nucleic acid introduce people's cell with Reach the therapeutic strategy of therapeutic purposes to modify their hereditary capacity.In general, the nucleic acid is can to have encoded therapeutic effect, The double chain DNA molecule (dsDNA) of the protein of destruction or label effect.The nucleic acid be also likely to be in host cell Target sequence combines, the antisense RNA or single stranded DNA for inhibiting specific gene to express by preventing mRNAs or promoter (ssDNA).So far, the gene therapy based on plasmid, cancer vaccine etc. have been over 600, and the gene of Plasmid DNA is exempted from Epidemic disease simulates the Gene Expression Pathway of pathogen in the cell, has successfully applied the ischaemic to treat lower limb, painstaking effort Pipe regeneration, and pole is hopeful to prevent the difficult and complicated cases of modern medicine by nucleic acid vaccine, including malaria, AIDS, hepatitis B and Tuberculosis etc..For gene therapy, plasmid, which cannot be integrated into genomic DNA, may is that a disadvantage, but plasmid can be with episome Form be present in nucleus, can also with continuous expression for quite a long time.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is to design to inhibit to replicate related gene with influenza virus in host SiRNA sequence, building can encode the recombinant vector of the siRNA, and the recombinant vector containing the sequence open reading frame Application in terms of preparing Tamiflu.
The present invention solve technical solution used by above-mentioned technical problem first is that: it is a kind of for 2 gene of caveolin SiRNA, wherein the corresponding target sequence of siRNA of the present invention is the mRNA of 2 gene of caveolin, it is of the present invention The length of siRNA be 16-30bp, and can make through its transfect after mammalian cell mRNA or protein expression decline 60% with On.
Wherein the mammal includes that pig is preferably in the mammals such as pig, dog, mouse, people, the present invention, this The siRNA of invention has the effect of preferable cryptiogene in pig.
Heretofore described siRNA, length 16-30bp are more preferably 18-25bp.Preferably, the siRNA It is indicated containing SEQ ID NO:1 in nucleotide sequence shown in SEQ ID NO:1, or such as sequence table is selected from.More preferably, described 3 ' extended structures of the end containing 2 T or containing 2 U of siRNA.
The present invention solve technical solution used by above-mentioned technical problem second is that: inhibition alveole egg described in a kind of coding The recombinant vector of the siRNA of white 2 expression.
According to the present invention, the carrier that the recombinant vector uses can be the carrier of this field routine, including adenovirus Carrier, such as various adenovirus vectors based on 5 types (Ad5), 2 types (Ad2), slow virus carrier are selected from HIV-1 type carrier, HIV-2 type carrier, SIV type carrier, FIV type carrier etc..The siRNA fragment is cloned into vector plasmid to get the present invention Recombinant vector.
The present invention solve above-mentioned technical problem used by technical solution third is that: a kind of composition, wherein containing The 0.001-99.99wt% siRNA and acceptable carrier, diluent or excipient of the present invention for caveolin 2. Wherein, the carrier, diluent or excipient, are more preferably pharmaceutically acceptable carrier, diluent or excipient, including Carrier materials or powder, dextrin, the Icing Sugar such as water-soluble, slightly solubility or enteric solubility, two hydrate of calcium sulfate, calcium monohydrogen phosphate, oxidation One of magnesium, magnesium carbonate, calcium carbonate, gel aluminum hydroxide powder and active carbon are a variety of.
The present invention solve above-mentioned technical problem used by technical solution fourth is that: the siRNA prepare or screen Purposes in Tamiflu.Wherein, the siRNA can be used as unique anti-influenza virus activity ingredient, can also be with Other anti-influenza virus medicaments are used in combination.
In preparation Tamiflu, on the way, following technical solution more preferably can be used: such as in siRNA of the present invention It is administered by way of sucking, it is not only easy to use, but also it can be increased in the drug concentration of infection site.Also, due to Infection early stage viral load is small, and sufficient siRNA can more effectively inhibit the duplication of virus, to act the effect prevented and treated Fruit；The selection of RNAi drug target is more in influenza, can be not likely to produce drug resistance strain with compound or multiple medication；Last RNAi with Influenza vaccines are different, and not needing user has sound immune system.
The present invention can also be adopted the following technical scheme that more preferably: as passed through intravenous injection or intramuscular injection siRNA sequence Or the modes such as the siRNA recombinant vector are encoded, so that the siRNA is able to efficient, lasting expression in vivo and exempts to reach RNA Epidemic disease or the purpose of gene therapy influenza virus.
The present invention solve above-mentioned technical problem used by technical solution fifth is that: the siRNA inhibit influenza disease Purposes in poison duplication.
The present invention solve above-mentioned technical problem used by technical solution sixth is that: it is a kind of screen Tamiflu side Method, comprising the following steps:
(1) contact drug candidate with the cell of influenza virus infection, the cell is 2 gene silencing of caveolin Cell；
(2) Influenza virus titer is tested；
(3) drug candidate that selection declines Influenza virus titer.
Wherein, more preferably 2 silenced gene expression cell of caveolin described in step (1) is by containing host cell The siRNA of the gene, which either contains coding, has the recombinant vector of the siRNA to make the gene silencing.
Wherein, the cell can (Africa be green using 293T (human renal epithelial cell line), BHK (hamster kidney cell), VERO MK cells), the cell lines such as IBRS22 (porcine kidney cell) or MDCK (canine kidney cells), the preferred mdck cell of the present invention.
Wherein, it is contacted by the cell with influenza virus, observation cell damage is harmful under the microscope after a period of time Degree, compare with Tamiflu handle and check experiment cell in influenza virus virus titer, can know this Whether antiviral drug can be reduced damage of the virus to cell.
Following scheme more preferably can be used in the method for the invention:
The cytotoxicity of drug detects, mdck cell cultivate in 96 porocyte culture plates form single layer after, difference is added The medical fluid of concentration continues culture 3 days, with mtt assay detection drug to the toxicity of mdck cell.
Drug resisiting influenza virus effect detection, mdck cell cultivate in 96 porocyte culture plates form single layer after, use The influenza infection cell of 100TCID50 adds the drug containing culture solution of the series of concentrations under non-toxic concn, continues culture 3 It, the inhibiting effect of drug infected by influenza is determined through CPE method.
The method of the invention can also preferably adopt the following technical scheme that
Influenza virus RNA polymerase (RNA-dependent RNA polymerase, RdRp) is primarily involved in the base of virus Because of group duplication and transcription.For duplication, RdRp is that template catalysis generates the RNA being complementary with virus genome RNA (vRNA) Chain (cRNA), cRNA then can be used as templated synthesis vRNA and be fitted into progeny virus.For transcription, RdRp is in synthesis cRNA On the basis of at its 3 ' end poly A tract is added, cap sequences are added in 5 ' ends, form mature mRNA and carry out protein synthesis.This Experiment gradually decreases ATP concentration in system according to consumption substrate A TP, UTP, CTP and GTP when synthesis RNA chain, in measurement system Remaining ATP content is to reflect reaction carry out degree.
By detecting influenza virus RNA polymerase change level in the cell strain, assessment 2 silenced cell of caveolin confrontation The sensitivity of flu pharmaceutical.
Wherein the virus titer detection method, MTT method, CPE method, the methods of cell culture are that this field is normal Advise experimental method.
The present invention solve above-mentioned technical problem used by technical solution seventh is that: one kind be directed to Surfactant proteinD base The corresponding target sequence of the siRNA of cause, the siRNA is the mRNA of Surfactant proteinD gene, and the length of the siRNA is 16-30bp, and can make through the mRNA of its mammalian cell after transfecting or 60% or more expressing quantity decline.
Wherein the mammal includes that pig is preferably in the mammals such as pig, dog, mouse, people, the present invention, this The siRNA of invention has the effect of preferable cryptiogene in pig.
Heretofore described siRNA length is 16-30bp, is more preferably 18-25bp.Preferably, the siRNA contains Have and be selected from nucleotide sequence shown in SEQ ID NO:2, or such as SEQ ID NO:2 expression in sequence table.The siRNA, more Good ground sequence 3 ' holds the extended structure containing 2 T or containing 2 U.
The present invention solve technical solution used by above-mentioned technical problem eighth is that: inhibition surface described in a kind of coding is living Property protein D expression siRNA recombinant vector.
According to the present invention, the carrier that the recombinant vector uses can be the carrier of this field routine, including adenovirus Carrier, including the various adenovirus vectors based on 5 types (Ad5), 2 types (Ad2), slow virus carrier includes HIV-1 type carrier, HIV-2 type carrier, SIV type carrier, FIV type carrier etc..The siRNA fragment is cloned into vector plasmid to get the present invention Recombinant vector.
The present invention solve above-mentioned technical problem used by technical solution ninth is that: a kind of composition, wherein containing The 0.001-99.99wt% siRNA sequence and acceptable carrier, diluent of the present invention for Surfactant proteinD Or excipient.Wherein carrier, diluent and the excipient, are more preferably pharmaceutically acceptable carrier, diluent or tax Shape agent.Carrier materials, powder, dextrin, Icing Sugar, two hydrate of calcium sulfate, phosphoric acid such as example, water-soluble, slightly solubility or enteric solubility One of hydrogen calcium, magnesia, magnesium carbonate, calcium carbonate, gel aluminum hydroxide powder and nano material are a variety of.
The present invention solve above-mentioned technical problem used by technical solution tenth is that: the inhibition Surfactant proteinD SiRNA prepare or screen Tamiflu in purposes.Wherein, it is susceptible to can be used as unique anti-current by the siRNA Cytotoxic activity ingredient can also be used in combination with other anti-influenza virus medicaments.
Wherein, following technical solution more preferably can be used: being such as administered by way of sucking, it is not only easy to use, but also It can be increased in the drug concentration of infection site.Also, since infection early stage viral load is small, sufficient siRNA can be more The duplication of virus is effectively inhibited, to act the effect prevented and treated；RNAi drug target selects more, Ke Yifu in influenza Square or multiple medication, is not likely to produce drug resistance strain；Last RNAi is different from influenza vaccines, and not needing user has sound exempt from Epidemic disease system.
The present invention can also be adopted the following technical scheme that more preferably: as passed through intravenous injection or intramuscular injection siRNA sequence Or the modes such as the siRNA recombinant vector are encoded, so that the siRNA is able to efficient, lasting expression in vivo and exempts to reach RNA Epidemic disease or the purpose of gene therapy influenza virus.
The present invention solve above-mentioned technical problem used by technical solution ten first is that: it is a kind of screen Tamiflu side Method, which comprises the following steps:
(1) contact drug candidate with the cell of influenza virus infection, the cell is that Surfactant proteinD gene is heavy Silent cell；
(2) Influenza virus titer is tested；
(3) drug candidate that selection declines Influenza virus titer.
Surfactant proteinD silenced gene expression cell is logical described in method of the present invention, more preferably step (1) It crosses the siRNA for making host cell contain the gene or containing coding has the recombinant vector of the siRNA to make The gene silencing.
Wherein, the cell can (Africa be green using 293T (human renal epithelial cell line), BHK (hamster kidney cell), VERO MK cells), the cell lines such as IBRS22 (porcine kidney cell) or MDCK (canine kidney cells), the preferred MDCK of the present invention.
The method of the invention is contacted with influenza virus by the cell, is observed under the microscope after a period of time The degree of cell damage evil compares the survival rate handled with Tamiflu with the cell of check experiment after influenza infection Or growing state, it can know whether the antiviral drug can be reduced damage of the virus to cell.
Wherein, following scheme more preferably can be used:
(1) drug cytotoxicity detection, mdck cell cultivate in 96 porocyte culture plates form single layer after, addition not With the medical fluid of concentration, continue culture 3 days, with mtt assay detection drug to the toxicity of mdck cell.
(2) drug resisiting influenza virus effect detection, mdck cell cultivate in 96 porocyte culture plates form single layer after, use The influenza infection cell of 100TCID50 adds the drug containing culture solution of the series of concentrations under non-toxic concn, continues culture 3 It, the inhibiting effect of drug infected by influenza is determined through CPE method.
The method of the invention can also preferably adopt the following technical scheme that
(1) influenza virus RNA polymerase (RNA-dependent RNA polymerase, RdRp) is primarily involved in virus Genome duplication and transcription.For duplication, RdRp is that template catalysis generation is complementary with virus genome RNA (vRNA) RNA chain (cRNA), cRNA then can be used as templated synthesis vRNA and be fitted into progeny virus.For transcription, RdRp is being synthesized Poly A tract is added at its 3 ' end on the basis of cRNA, cap sequence is added in 5 ' ends, forms mature mRNA and carries out protein conjunction At.This experiment gradually decreases ATP concentration in system according to consumption substrate A TP, UTP, CTP and GTP when synthesis RNA chain, measures Remaining ATP content is in system to reflect reaction carry out degree.
(2) by detecting influenza virus RNA polymerase change level in the cell strain, Surfactant proteinD gene is assessed Sensitivity of the silenced cell to Tamiflu.
Wherein the virus titer detection method, MTT method, CPE method, the methods of cell culture are that this field is normal Advise experimental method.
The present invention solve above-mentioned technical problem used by technical solution ten second is that: a kind of base containing transcription factor A Because of the recombinant vector of open reading frame.Preferably, the carrier is obtained by following preparation method:
(1) the transcription factor A gene order that accession number in Genbank is NM_001130211.1 is input to DNAStar In software (DNASTAR company, the U.S.) EditSeq, choose sequence in from initiation codon " ATG " to terminator codon " TGA ", Longest sequence 741bp base between " TAG " or " TAA "；
(2) respectively in 5 ' and 3 ' end design amplification overall length PCR primers, the end of upstream primer 5 ' addition Hind III digestion position Point, CGG is as protectiveness base, the end of downstream primer 5 ' addition EcoR V restriction enzyme site；
(3) upstream primer 5 '-CGGAAGCTTATGGCGCTTCTCCGGGGCGTGT-3 '；
(4) downstream primer 5 '-CGGGATATCTCAACACTCCTCAGTGTCTTTC-3 '；
(5) reverse transcription product of the total serum IgE extracted using porcine alveolar macrophage is template, with the upstream and downstream primer of design PCR amplification obtains transcription factor A sequence, and the corresponding restriction enzyme site of carrier for expression of eukaryon is then connected to after digestion.
Wherein, carrier for expression of eukaryon can be using pCMVp-NEO-BAN, pEGFP, pEGFT-Actin, pSV2, CMV4 etc. Common carrier for expression of eukaryon, the present invention in, more preferably use pCDNA 3.1 (+) carrier.
The present invention solve above-mentioned technical problem used by technical solution ten third is that: one kind described in contain transcription factor The recombinant vector of A open reading frame is preparing or is screening the purposes in Tamiflu.
The present invention more preferably, which can be used to transfect different cell lines, and the method for transfection can be, DEAE- glucan method, calcium phosphate method, cationic-liposome method, cationic polymer method, virus-mediated methods, biologic grain pass method (particle gun Particle bombardment), microinjection, electroporation etc., the technical program preferred liposome infection protocol.What is transfected is thin Born of the same parents can be various eukaryocytes, the preferred MDCK of the technical program (canine kidney cells).
The recombinant vector can also be used for gene therapy by the present invention, such as can pass through intravenous injection or intramuscular injection side Formula is able to carry out in vivo efficiently, persistently to express to achieve the purpose that DNA immunization or gene therapy influenza virus.
The present invention solve above-mentioned technical problem used by technical solution ten fourth is that: it is a kind of for screening the cell of drug Model contains the recombinant vector containing transcription factor A open reading frame.
Wherein, the cell can be various eukaryocytes, including 293T (human renal epithelial cell line), BHK (hamster kidney Cell), VERO (African green monkey kidney cell), cell lines such as IBRS22 (porcine kidney cell) and MDCK (canine kidney cells) etc..
The present invention solve above-mentioned technical problem used by technical solution ten fifth is that: it is a kind of screen Tamiflu side Method, the method for the invention, comprising the following steps:
(1) contact drug candidate with the cell of influenza virus infection, the cell is to be overexpressed transcription factor A gene Cell；
(2) Influenza virus titer is tested；
(3) drug candidate that selection declines Influenza virus titer.
The method of the invention is overexpressed the cell of the gene described in step (1), is more preferably intracellular by increasing The copy number of the gene or the controlling element for improveing the gene make the gene overexpression.
The method of the invention, the cell can selected from 293T (human renal epithelial cell line), BHK (hamster kidney cell), The cell lines such as VERO (African green monkey kidney cell), IBRS22 (porcine kidney cell) and MDCK (canine kidney cells), the preferred MDCK of the present invention.
The method of the invention is contacted with influenza virus by the cell, is observed under the microscope after a period of time The degree of cell damage evil, compares the virus titer that influenza virus in the cell with check experiment is handled with Tamiflu, just It may know that whether the antiviral drug can be reduced damage of the virus to cell.
Following scheme more preferably can be used in the method for the invention:
(1) drug cytotoxicity detection, mdck cell cultivate in 96 porocyte culture plates form single layer after, addition not With the medical fluid of concentration, continue culture 3 days, with mtt assay detection drug to the toxicity of mdck cell.
(2) drug resisiting influenza virus effect detection, mdck cell cultivate in 96 porocyte culture plates form single layer after, use The influenza infection cell of 100TCID50 adds the drug containing culture solution of the series of concentrations under non-toxic concn, continues culture 3 It, the inhibiting effect of drug infected by influenza is determined through CPE method.
The method of the invention can also preferably adopt the following technical scheme that
(1) influenza virus RNA polymerase (RNA-dependent RNA polymerase, RdRp) is primarily involved in virus Genome duplication and transcription.For duplication, RdRp is that template catalysis generation is complementary with virus genome RNA (vRNA) RNA chain (cRNA), cRNA then can be used as templated synthesis vRNA and be fitted into progeny virus.For transcription, RdRp is being synthesized Poly A tract is added at its 3 ' end on the basis of cRNA, cap sequence is added in 5 ' ends, forms mature mRNA and carries out protein conjunction At.This experiment gradually decreases ATP concentration in system according to consumption substrate A TP, UTP, CTP and GTP when synthesis RNA chain, measures Remaining ATP content is in system to reflect reaction carry out degree.
(2) by detecting influenza virus RNA polymerase change level in the cell strain, assessment transcription factor A gene crosses table Up to cell to the sensitivity of Tamiflu.
Wherein the virus titer detection method, MTT method, CPE method, the methods of cell culture are that this field is normal Advise experimental method.
The present invention solve above-mentioned technical problem used by technical solution ten sixth is that: one kind contain 3 base of paraoxon acid enzyme The recombinant vector of the open reading frame of cause.Preferably, the carrier is obtained by following preparation method:
(1) from accession number in Genbank be NM_001044604.1 paraoxon acid enzyme 3 in, choose from sequence from Beginning codon " ATG " arrives longest sequence 1065bp base between terminator codon " TGA ", " TAG " or " TAA "；
(2) respectively in 5 ' and 3 ' end design amplification overall length PCR primers, HindIII restriction enzyme site is added at the end of upstream primer 5 ', CGG is as protectiveness base, the end of downstream primer 5 ' addition EcoR I restriction enzyme site；
(3) upstream primer 5 '-CGGAAGCTTATGGGGAAGCTGGTGGCTCTGA-3 '；
(4) downstream primer 5 '-CGGGAATTCCTAGAGCACACAGTACAGAGCT-3 '；
(5) reverse transcription product of the total serum IgE extracted using porcine alveolar macrophage is template, with the upstream and downstream primer of design PCR amplification obtains transcription factor A sequence, and the corresponding restriction enzyme site of carrier for expression of eukaryon is then connected to after digestion.
Wherein, carrier for expression of eukaryon can be using pCMVp-NEO-BAN, pEGFP, pEGFT-Actin, pSV2, CMV4 etc. Common carrier for expression of eukaryon, the present invention in, more preferably use pCDNA 3.1 (+) carrier.
The present invention solve ten of technical solution used by above-mentioned technical problem seventh is that: it is a kind of of the present invention containing right The recombinant vector of 3 open reading frame of oxygen phosphatase is preparing or is screening the purposes in Tamiflu.
The present invention more preferably, which can be used to transfect different cell lines, and the method for transfection can be, DEAE- glucan method, calcium phosphate method, cationic-liposome method, cationic polymer method, virus-mediated methods, biologic grain pass method (particle gun Particle bombardment), microinjection, electroporation etc., the technical program preferred liposome infection protocol.What is transfected is thin Born of the same parents can be various eukaryocytes, the preferred MDCK of the technical program (canine kidney cells).
The recombinant vector can also be used for gene therapy by the present invention, such as can pass through intravenous injection or intramuscular injection side Formula is able to carry out in vivo efficiently, persistently to express to achieve the purpose that DNA immunization or gene therapy influenza virus.
The present invention solve above-mentioned technical problem used by technical solution ten eighth is that: it is a kind of for screening the cell of drug Model contains the recombinant vector of the open reading frame containing 3 gene of paraoxon acid enzyme.
Wherein, the cell can be various eukaryocytes, be selected from 293T (human renal epithelial cell line), BHK (hamster kidney Cell), VERO (African green monkey kidney cell), IBRS22 (porcine kidney cell) and MDCK (canine kidney cells) cell line etc..
The present invention solve above-mentioned technical problem used by technical solution ten ninth is that: it is a kind of screen Tamiflu side Method, the method for the invention, comprising the following steps:
(1) contact drug candidate with the cell of influenza virus infection, the cell is to be overexpressed 3 base of paraoxon acid enzyme The cell of cause；
(2) Influenza virus titer is tested；
(3) drug candidate that selection declines Influenza virus titer.
The method of the invention is overexpressed the cell of the gene described in step (1), is more preferably intracellular by increasing The copy number of the gene or the controlling element for improveing the gene make the gene overexpression.
The method of the invention, the cell can using 293T (human renal epithelial cell line), BHK (hamster kidney cell), The cell lines such as VERO (African green monkey kidney cell), IBRS22 (porcine kidney cell) and MDCK (canine kidney cells), the preferred MDCK of the present invention.
The method of the invention is contacted with influenza virus by the cell, is observed under the microscope after a period of time The degree of cell damage evil, compares the virus titer that influenza virus in the cell with check experiment is handled with Tamiflu, just It may know that whether the antiviral drug can be reduced damage of the virus to cell.
Following scheme more preferably can be used in the method for the invention:
(1) drug cytotoxicity detection, mdck cell cultivate in 96 porocyte culture plates form single layer after, addition not With the medical fluid of concentration, continue culture 3 days, with mtt assay detection drug to the toxicity of mdck cell.
(2) drug resisiting influenza virus effect detection, mdck cell cultivate in 96 porocyte culture plates form single layer after, use The influenza infection cell of 100TCID50 adds the drug containing culture solution of the series of concentrations under non-toxic concn, continues culture 3 It, the inhibiting effect of drug infected by influenza is determined through CPE method.
The method of the invention can also preferably adopt the following technical scheme that
(1) influenza virus RNA polymerase (RNA-dependent RNA polymerase, RdRp) is primarily involved in virus Genome duplication and transcription.For duplication, RdRp is that template catalysis generation is complementary with virus genome RNA (vRNA) RNA chain (cRNA), cRNA then can be used as templated synthesis vRNA and be fitted into progeny virus.For transcription, RdRp is being synthesized Poly A tract is added at its 3 ' end on the basis of cRNA, cap sequence is added in 5 ' ends, forms mature mRNA and carries out protein conjunction At.This experiment gradually decreases ATP concentration in system according to consumption substrate A TP, UTP, CTP and GTP when synthesis RNA chain, measures Remaining ATP content is in system to reflect reaction carry out degree.
(2) by detecting influenza virus RNA polymerase change level in the cell strain, 3 gene mistake of paraoxon acid enzyme is assessed Sensitivity of the expression cell to Tamiflu.
Wherein the virus titer detection method, MTT method, CPE method, the methods of cell culture are that this field is normal Advise experimental method.
Compared with the prior art, beneficial effects of the present invention are as follows: invention is disease-resistant to have in host cell The gene or albumen of malicious function are target spot, provide the drug of resisiting influenza virus and inhibit the method for influenza virus duplication.This Invention is achieved by the expression of gene described in regulating cell.It invents to have and prevents and treats influenza infection Application value, to better Tamiflu is developed, the great outburst to control influenza virus reduces the great outburst pair of influenza virus Socio-economic development, people healthy bring significant damage, have great importance.
Detailed description of the invention
Below in conjunction with Detailed description of the invention feature and beneficial effect of the invention.
Fig. 1 is the Western-blot detection expression of silencing canine kidney cells caveolin 2 as a result, wherein 1:siRNA- is small Nest albumen 2；2:siRNA- caveolin 2 compares；3: not connecing malicious control；4: normal cell.
Fig. 2 be in Western-blot detection silencing canine kidney cells Surfactant proteinD expression as a result, wherein 1: SiRNA- Surfactant proteinD；The control of 2:siRNA- Surfactant proteinD；3: not connecing malicious control；4: normal cell.
Fig. 3 is Influenza virus titer figure after silencing canine kidney cells caveolin 2 is expressed.
Fig. 4 be in silencing canine kidney cells Surfactant proteinD expression after Influenza virus titer figure.
Fig. 5 is the pCDNA 3.1- transcription factor A Vector map of building.
Fig. 6 is 3 Vector map of pCDNA 3.1- paraoxon acid enzyme of building.
Fig. 7 is that factors A and 3 immune-blotting method of paraoxon acid enzyme are transcribed in cell as a result, wherein
1:pCDNA 3.1- transcription factor A；3.1 empty vector control of 2:pCDNA；3: normal cell
4:pCDNA 3.1- paraoxon acid enzyme 3；3.1 empty vector control of 5:pCDNA；6: normal cell.
Fig. 8 is to transcribe Influenza virus titer result after factors A in external source raising expression canine kidney cells.
Fig. 9 is that external source improves in expression canine kidney cells Influenza virus titer result after paraoxon acid enzyme 3.
Figure 10 is that Influenza virus titer figure after amantadine is added in the canine kidney cells of 2 silencing of caveolin.
Figure 11 is that Influenza virus titer figure after amantadine is added in the canine kidney cells of Surfactant proteinD silencing.
Figure 12 be external source improve transcription factor A expression canine kidney cells in be added amantadine after Influenza virus titer figure.
Figure 13 be external source improve paraoxon acid enzyme 3 express canine kidney cells in be added amantadine after Influenza virus titer Figure.
Specific embodiment
The present invention is further illustrated with embodiment below, but the present invention is not intended to be limited thereto.
Reagent used in embodiment is commercially available in addition to special instruction.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, or according to manufacturer Proposed condition.Heretofore described " room temperature " refers to the temperature for the operation room tested, generally 25 DEG C.
Swine influenza virus H3N2Swine/Guangdong/1/2006 (SwGD1/05) strain used in embodiment, biology The swine influenza virus feature in China can be represented by learning characteristic, be originated from the Chinese Academy of Agricultural Sciences Shanghai veterinary institute pig Infectious Disease Research department.
6 porocyte culture plates, U.S.'s Corning product.
DMEM culture medium, U.S.'s GIBCO product, REF:16000-044.
Penicillin, U.S.'s GIBCO Products, REF:15140-122.
Streptomysin, U.S.'s GIBCO Products, REF:15140-122.
Canine kidney cells (mdck cell) is purchased from Chinese Academy of Sciences Shanghai life science institute cell bank.
1.5ml centrifuge tube, U.S.'s Axygen Products.
Acetone, Jiangsu Qiangsheng Chemical Co., Ltd., credit number: XK13-201-00227.
PBS is purchased from U.S. GIBCO company, REF:20012-027.
PBST: preparation method refers to " Molecular Cloning:A Laboratory guide " third edition.
The fluorescence antibody of sheep anti-Mouse FITC label, U.S.'s Invitrogen Products, Code:CA11034s.
100% glycerol, Sinopharm Chemical Reagent Co., Ltd..
The duplication of 1 modulate host genes within cells expression inhibiting influenza virus of embodiment
Gene expression inhibition influenza virus is replicated in 1.siRNA (siRNA) silencing host cell
(1) siRNA molecule designs
The present embodiment selects 2 gene of caveolin, GeneID:100125375；Surfactant proteinD, GeneID: 397198, using Dharmacon Photographing On-line software design siRNA molecule, design the siRNA of 2 gene specific of caveolin Interference sequence；And arbitrarily upset sequence using " siRNA- caveolin " as control.Design surface activated protein D gene specific SiRNA interference sequence；And arbitrarily upset sequence using " siRNA- Surfactant proteinD " as control.The siRNA double-strand of design Disturbing molecule subject sequence is classified as 19 bases, and two UU are added at the end of positive-sense strand 3 ', and two TT are added at the end of antisense strand 3 '.
The sequence of design is as follows:
For 2 target sequence of caveolin, 5 '-GCAAATACGTGATCTACAA-3 '；
2 sense strand sequence of siRNA- caveolin, 5 '-GCAAAUACGUGAUCUACAAUU-3 '；
2 antisense strand sequence of siRNA- caveolin, 3 '-TTCGUUUAUGCACUAGAUGUU-5 '；
SiRNA- caveolin 2 compares sense strand sequence 5 '-CGAUAGAUGUACACAUACAUU-3 '；
SiRNA- caveolin 2 compares antisense strand sequence 3 '-TTGCUAUCUACAUGUGUAUGU-5 '；
For 5 '-GAGCAGAAATGAAGACCTA-3 ' of Surfactant proteinD target sequence；
5 '-GAGCAGAAAUGAAGACCUAUU-3 ' of siRNA- Surfactant proteinD sense strand sequence；
3 '-TTCUCGUCUUUACUUCUGGAU-5 ' of siRNA- Surfactant proteinD antisense strand sequence；
SiRNA- Surfactant proteinD compares positive-sense strand 5 '-CAGAGUGACAGAAGACAUAUU-3 '；
SiRNA- Surfactant proteinD compares antisense strand 3 '-TTGUCUCACUGUCUUCUGUAU-5 '.
It is small through NCBI blast Analysis interference sequence and the genetic homology other than " caveolin or Surfactant proteinD " In 50%, control sequence and pig genome all sequences homology are less than 50%.
According to the siRNA sequence of the design, by Dharmacon company artificial synthesized double-strand siRNA in vitro.
(2) siRNA molecule transfects cell
By the DMEM (U.S.'s GIBCO product, REF:16000-044) (100 of the mdck cell serum-free of pancreatin digestion (GIBCO company, the U.S. produces for units of Penicillin (U.S.'s GIBCO Products, REF:15140-122) and 100 unit streptomysins Product, REF:15140-122)) pass the cell density 4 × 10 in 6 porocyte culture plates (U.S.'s Corning product)6Cells/well, Continue to be incubated at 37 DEG C, 5%CO2In incubator.The 80-90%, double-strand siRNA of the present invention at full culture plate bottom are grown to cell With FuGENE HD reagent, (Roche company, the U.S., product number: 04709705001) being transfected into mdck cell molecule, operation Process by specification.The control of transfection control interference sequence sample siRNA- caveolin 2 is set simultaneously, untransfected compares, normal thin Born of the same parents' control.
(3) siRNA transfects cell and connects poison and sample collection
It is inoculated with influenza virus H3N2, dosage of inoculation 1 × 10 for 24 hours after mdck cell transfection siRNA molecule3EID50/ hole (6 holes Plate).After virus inoculation for 24 hours, 48h, 72h collect sample.Method is as follows: training by cell in tissue culture plate, together with cell Nutrient solution freeze thawing 3 times, 5000g/min is centrifuged 10min, retains supernatant, abandons precipitating.Measure Influenza virus titer in supernatant.Virus drop It spends measuring method and refers to " national influenza central standard operating instruction (revised edition), 2007 ".
Experimental result (Fig. 1) shows that mdck cell caveolin 2, Surfactant proteinD expression are sunk by siRNA molecule It is inoculated with influenza virus after silent, Influenza virus titer compares significant decrease, and difference with control group for 24 hours, in 48h, 72h cell Significantly.
Cell caveolin 2 is by (after virus inoculation as above for 24 hours sample) after siRNA molecule silencing, Western- Blot testing result is shown in Fig. 1, the results showed that 2 expression of silencing group caveolin is significantly lower than control.
Surfactant proteinD is by (after virus inoculation as above for 24 hours sample) after siRNA molecule silencing in cell, Western-blot testing result is shown in Fig. 2, the results showed that Surfactant proteinD expression is significantly lower than control in silencing group.
SiRNA- caveolin 2 compared with 2 control group of siRNA- caveolin p value be respectively p=0.0004 (for 24 hours), 0.001(48h),0.015(72h)；SiRNA- Surfactant proteinD compares p value point with siRNA- Surfactant proteinD control group It Wei not p=0.004 (for 24 hours), 0.001 (48h), 0.009 (72h).
To be able to suppress host thin by regulation caveolin 2 and surfactant protein gene expression for above-mentioned the results show The duplication of influenza virus intracellular.
2. gene expression inhibition influenza virus is replicated in exogenous raising host cell
(1) in the 1st group of heterogenous expression expression vector building
By transcription factor A (TFAM) (GeneID:397279；Gene database accession number: NM_001130211.1) connection To pCDNA 3.1 (+) carrier (Invitrogen company, the U.S.；Article No.: V790-20) HindIII and EcoR V restriction enzyme site Between, specific construction method the following steps are included:
(1) the transcription factor A gene order that accession number in Genbank is NM_001130211.1 is input to DNAStar In software (DNASTAR company, the U.S.) EditSeq, choose sequence in from initiation codon " ATG " to terminator codon " TGA ", Longest sequence 741bp base between " TAG " or " TAA "；
(2) respectively in 5 ' and 3 ' end design amplification overall length PCR primers, HindIII restriction enzyme site is added at the end of upstream primer 5 ', CGG is as protectiveness base, the end of downstream primer 5 ' addition EcoR V restriction enzyme site；
(3) 5 '-CGG of upstream primer(double-crossed is HindIII enzyme to ATGGCGCTTCTCCGGGGCGTGT-3 ' Enzyme site)；
(4) 5 '-CGG of downstream primer(double-crossed is EcoR V enzyme to TCAACACTCCTCAGTGTCTTTC-3 ' Enzyme site)；
(5) total serum IgE reverse transcription product is extracted as template, with the upstream and downstream primer PCR amplification of design using pig pulmonary macrophage Transcription factor A sequence is obtained, the corresponding restriction enzyme site of pCDNA 3.1 (+) carrier is then connected to after digestion.
It constructs successful carrier and is named as pCDNA 3.1- transcription factor A, see Fig. 5.
By paraoxon acid enzyme 3 (PON3) (GeneID:733674；Gene database accession number: NM_001044604.1) base Because open reading frame is connected to pCDNA 3.1 (+) carrier (Invitrogen company, the U.S.；Article No.: V790-20) HindIII and Between EcoR I restriction enzyme site, specific construction method the following steps are included:
(1) 3 gene order of paraoxon acid enzyme that accession number in Genbank is NM_001044604.1 is input to In DNAStar software (DNASTAR company, the U.S.) EditSeq, choose close from initiation codon " ATG " to terminating from sequence Longest sequence 1065bp base between numeral " TGA ", " TAG " or " TAA "；
(2) respectively in 5 ' and 3 ' end design amplification overall length PCR primers, HindIII restriction enzyme site is added at the end of upstream primer 5 ', CGG is as protectiveness base, the end of downstream primer 5 ' addition EcoR I restriction enzyme site；
(3) 5 '-CGG of upstream primer(double-crossed is HindIII enzyme to ATGGGGAAGCTGGTGGCTCTGA-3 ' Enzyme site)；
(4) 5 '-CGG of downstream primer(double-crossed is EcoR I enzyme to CTAGAGCACACAGTACAGAGCT-3 ' Enzyme site)；
(5) total serum IgE reverse transcription product is extracted as template, with the upstream and downstream primer PCR amplification of design using pig pulmonary macrophage Transcription factor A sequence is obtained, the corresponding restriction enzyme site of pCDNA 3.1 (+) carrier is then connected to after digestion.
It constructs successful carrier and is named as pCDNA 3.1- paraoxon acid enzyme 3, see Fig. 6.
(2) recombinant vector transfection cell and expressive host albumen
By the DMEM (U.S.'s GIBCO product, REF:16000-044) 100 of the mdck cell serum-free of pancreatin digestion (GIBCO company, the U.S. produces for units of Penicillin (U.S.'s GIBCO Products, REF:15140-122) and 100 unit streptomysins Product, REF:15140-122)) pass the cell density 4 × 10 in 6 porocyte culture plates (U.S.'s Corning product)6Cells/well, Continue to be incubated at 37 DEG C, in 5%CO2 incubator.The 80-90% that full culture plate bottom is grown to cell, with FuGENE HD reagent (Roche company, the U.S., product number: 04709705001) pCDNA 3.1- transcription factor A, pCDNA 3.1- paraoxon acid 3 carrier of enzyme is transfected into mdck cell respectively, operating process by specification.The thin of transfection pCDNA 3.1 (+) empty carrier is set simultaneously Born of the same parents' control.
After cell transfecting for 24 hours, transfection pCDNA 3.1- transcription factor A, pCDNA 3.1- paraoxon acid enzyme 3, pCDNA are collected 3.1 (+) empty carriers and sample of normal cells.Total protein of cell extract: with PBS rinse 6 porocyte plates in cell 2 times, then plus 1ml PBS scrapes cell with cell spatula, and 4 DEG C, 3000g is centrifuged 5min sedimentation cell, abandons supernatant.It is added to cell precipitation The cell pyrolysis liquid (green skies company, product number: P0013) of 100 μ l is resuspended cell, cracks 5min on ice, then with super Sound wave cell crushing instrument (Sonics company, the U.S., model VCX105PB) instantaneously broken (40HZ, 1S), boiling water boiling 5min, 4 DEG C Lower 13000g is centrifuged 10min, discards precipitating.Take 2 μ l albumen supernatant BCA kits (green skies company, product number: P0012 protein concentration) is measured, is operated to specifications.5 × SDS PAGE buffer (preparation method is added in remaining albumen supernatant See " Molecular Cloning:A Laboratory guide " third edition), boiling water boiling 5min, 13000g is centrifuged 5min at 4 DEG C, and supernatant is taken to carry out protein electricity Swimming.
Denaturing SDS-PAGE gel electrophoresis and albumen transfer (step refers to " Molecular Cloning:A Laboratory guide " third edition): each Protein sample applied sample amount is 30 μ g, SDS-PAGE electrophoresis apparatus (Liuyi Instruments Plant, Beijing) 80V voltage lamination albumen, 120V voltage point From albumen, until electrophoresis terminates.Albumen transfer using nitrocellulose filter (Whatman company, the U.S., product type: 10401396).Bole's electrophoresis tank constant pressure 65V, 2h.It is carried out in the TBST- skimmed milk of 5% (v/v) of NC film immersion after transfer Closing.After room temperature acts on 2h, confining liquid is discarded, is rinsed 3 times, is washed with TBST (pH7.6) buffer containing 1% (v/v) polysorbas20 Residual skimmed milk is removed, antibody incubation can be carried out.
Antibody response and colour developing: addition primary antibody, 3 antibody of paraoxon acid enzyme (Abcam, the U.S., product number: ab40969), Transcription factor A antibody (antikoerper-online.de company, Germany, product number: ABIN484435).Jog shakes at 4 DEG C It swings overnight (12h-16h), after then washing 3 times with 100%TBST, each 5min.HRP (horseradish peroxidase) mark is added later The secondary antibody of note, room temperature act on 2h, after being rinsed 3 times with TBST, each 5min.ECL kit (compile by Pierce company, the U.S., product Number: 32106) colour developing in darkroom operate.Operation is according to ECL kit specification.
Detection internal reference control: the film to have developed the color immersion antibody is stripped off in liquid (green skies company, product number: P0025), 50 DEG C of incubation 30min, every 10min concussion is primary, then with TBST buffer (preparation method: with reference to " Molecular Cloning: A Laboratory refers to South " third edition) rinsing 3 times, the re-closed 15min of confining liquid is added, (green skies company, product are compiled using anti-β-actin antibody Number: AA128) as protein content in primary antibody test sample.
Testing result is shown in Fig. 5, shows to transfect pCDNA 3.1- transcription factor A, pCDNA 3.1- paraoxon in mdck cell After sour enzyme 3, the expression quantity of " transcription factor A " and " paraoxon acid enzyme 3 " is higher than transfection pCDNA 3.1 (+) empty carrier group in cell With normal cell group.
(3) " transcription factor A " or " paraoxon acid enzyme 3 " has inhibition influenza virus effect in exogenous raising mdck cell
Cell transfecting pCDNA 3.1- transcription factor A, pCDNA 3.1- paraoxon acid enzyme 3 or pCDNA 3.1 (+) empty carrier Afterwards for 24 hours, dosage of inoculation 1 × 103EID50/ hole (6 orifice plates) swine influenza virus.After virus inoculation for 24 hours, 48h, 72h collect sample Product.Method is as follows: by cell in tissue culture plate, together with cell culture fluid freeze thawing 3 times, 5000 turns/min is centrifuged 10min, retains Supernatant abandons precipitating.Measure Influenza virus titer in supernatant.Virus titer measuring method is with reference to " national influenza central standard operation Regulation (revised edition), 2007 ".
Experimental result (Fig. 6,7) shows that transcription factors A, paraoxon acid enzyme D are expressed by exogenous raising in mdck cell, For 24 hours, in 48h, 72h cell, Influenza virus titer compares significant decrease, and significant difference with control group.Transfect pCDNA 3.1- transcription factor A group p value compared with transfecting pCDNA 3.1 (+) empty carrier group is respectively p=0.002 (for 24 hours), 0.014 (48h),0.0005(72h)；3 groups of transfection pCDNA 3.1- paraoxon acid enzyme are compared p with transfection pCDNA 3.1 (+) empty carrier group Value is respectively p=0.015 (for 24 hours), 0.008 (48h), 0.012 (72h).Transfect pCDNA 3.1 (+) empty carrier group and normal thin Virus titer difference is not significant (p > 0.05) in born of the same parents.
It is above-mentioned the experimental results showed that regulative transcription factor A and 3 gene expression of paraoxon acid enzyme are able to suppress in host cell The duplication of influenza virus.
2 modulate host genes within cells of embodiment express the antiviral effect for significantly improving influenza virus drug
H3N2 swine influenza virus is infected again after protein expression by adjusting in canine kidney cells in the present embodiment, using to cell Amantadine is added in culture solution, albumen is to amantadine resisiting influenza virus reinforcing effect in research regulating cell.Amantadine By interfering influenza virus particles M2 protein ion channel, inhibit the function of the duplication of virus.
1 silencing canine kidney cells caveolin 2 significantly improves amantadine and inhibits influenza virus effect
(1) silencing canine kidney cells caveolin 2 is expressed
The specific steps are the same as those in embodiment 1.
(2) amantadine prepares
Amantadine hydrochloride is purchased from AlfaAesar (Tianjin) Chemical Co., Ltd., with normal saline at 20mg/ml, mistake Filter out bacterium, 4 DEG C of preservations.
(3) siRNA molecule transfects cell
The specific steps are the same as those in embodiment 1.Drug combination group adds amantadine, final concentration to 0.4 μ g/ in cell culture fluid Ml is grouped as follows: siRNA- caveolin+amantadine (0.4 μ g/ml), 2 controls of siRNA- caveolin+amantadine (0.4 μ g/ml), untransfected control+amantadine (0.4 μ g/ml) and normal cell controls+amantadine (0.4 μ g/ml).
(4) siRNA transfects cell and connects poison and sample collection
With embodiment 1.
Experimental result is as shown in Figure 10, shows that the expression of mdck cell caveolin 2 is significant by energy after siRNA molecule silencing Improve the effect that amantadine inhibits influenza virus.For 24 hours, Influenza virus titer siRNA- caveolin+gold in 48h, 72h cell Rigid alkanamine (0.4 μ g/ml) group is substantially less than 2 controls of siRNA- caveolin+amantadine (0.4 μ g/ml) group, and p value is respectively p =0.016 (for 24 hours), 0.001 (48h), 0.001 (72h).
2 gene expression of caveolin can be improved amantadine inhibition influenza disease in above-mentioned the results show regulating cell The effect of poison duplication.
Surfactant proteinD significantly improves amantadine inhibition influenza virus effect in 2 silencing canine kidney cells
(1) Surfactant proteinD is expressed in silencing canine kidney cells
The specific steps are the same as those in embodiment 1.
(2) amantadine prepares
Amantadine hydrochloride is purchased from AlfaAesar (Tianjin) Chemical Co., Ltd., with normal saline at 20mg/ml, mistake Filter out bacterium, 4 DEG C of preservations.
(3) siRNA molecule transfects cell
Specific steps are the same as embodiment 2.Drug combination group adds amantadine, final concentration to 0.4 μ g/ in cell culture fluid Ml is grouped as follows: siRNA- Surfactant proteinD+amantadine (0.4 μ g/ml), siRNA- Surfactant proteinD control+gold Rigid alkanamine (0.4 μ g/ml), untransfected control+amantadine (0.4 μ g/ml) and normal cell controls+amantadine (0.4 μ g/ ml)。
(4) siRNA transfects cell and connects poison and sample collection with embodiment 1.
Experimental result is as shown in figure 11, shows that Surfactant proteinD expression is by energy after siRNA molecule silencing in mdck cell Significantly improve the effect that amantadine inhibits influenza virus.For 24 hours, the surface Influenza virus titer siRNA- is living in 48h, 72h cell Property protein D+amantadine (0.4 μ g/ml) organize be substantially less than siRNA- Surfactant proteinD control+amantadine (0.4 μ g/ Ml) group, p value are respectively p=0.020 (for 24 hours), 0.025 (48h), 0.130 (72h).
Surfactant proteinD gene expression can be improved amantadine and inhibit to flow in above-mentioned the results show regulating cell The effect of Influenza Virus duplication.
Transcription factor A expression significantly improves amantadine inhibition influenza virus effect in 3 exogenous raising host cells
(1) in the 1st group of heterogenous expression expression vector building
The specific steps are the same as those in embodiment 1.
(2) recombinant vector transfection cell and expressive host albumen
The specific steps are the same as those in embodiment 1.
(3) exogenous to improve the effect that amantadine inhibits influenza virus to replicate after transcription factors A in mdck cell
After 3.1 (+) empty carrier of cell transfecting pCDNA 3.1- transcription factor A or pCDNA for 24 hours, dosage of inoculation 1 × 103The hole EID50/, drug combination group add amantadine in cell culture fluid, and following experiment is arranged in final concentration to 0.4 μ g/ml Group: transfection pCDNA 3.1- transcription factor A+ amantadine (0.4 μ g/ml), transfection pCDNA 3.1 (+) empty carrier+amantadine (0.4 μ g/ml), untransfected control+amantadine (0.4 μ g/ml) and normal cell controls+amantadine (0.4 μ g/ml).? After virus inoculation for 24 hours, 48h, 72h collect sample.Method is as follows: freezing by cell in tissue culture plate, together with cell culture fluid Melt 3 times, 5000 turns/min is centrifuged 10min, retains supernatant, abandons precipitating.Measure Influenza virus titer in supernatant.Virus titer measurement Method refers to " national influenza central standard operating instruction (revised edition), 2007 ".
Factors A is transcribed the experimental results showed that shown in Figure 12, in mdck cell to be expressed by exogenous raising, for 24 hours, 48h, PCDNA 3.1- transcription factor A+ amantadine (0.4 μ g/ml) group and transfection pCDNA 3.1 (+) empty carrier are transfected in 72h cell + amantadine (0.4 μ g/ml) is compared to significant decrease, and significant difference, p value are respectively p=0.310 (for 24 hours), 0.013 (48h)、0.007(72h)。
It is above-mentioned to inhibit influenza virus multiple the experimental results showed that regulating and controlling gene expression in the 1st group and significantly increase amantadine System.
The expression of paraoxon acid enzyme 3 significantly improves amantadine inhibition influenza virus effect in 4 exogenous raising host cells
(1) in the 1st group of heterogenous expression expression vector building
Specific steps are the same as embodiment 2.
(2) recombinant vector transfection cell and expressive host albumen
Specific steps are the same as embodiment 2
(3) exogenous to improve the effect that amantadine inhibits influenza virus to replicate after paraoxon acid enzyme 3 in mdck cell
After cell transfecting pCDNA 3.1- paraoxon acid enzyme 3 or pCDNA 3.1 (+) empty carrier for 24 hours, dosage of inoculation 1 × 103The hole EID50/, drug combination group add amantadine in cell culture fluid, and following experiment is arranged in final concentration to 0.4 μ g/ml Group: transfection pCDNA 3.1- paraoxon acid enzyme 3+ amantadine (0.4 μ g/ml), transfection pCDNA 3.1 (+) empty carrier+adamantane Amine (0.4 μ g/ml), untransfected control+amantadine (0.4 μ g/ml) and normal cell controls+amantadine (0.4 μ g/ml). After virus inoculation for 24 hours, 48h, 72h collect sample.Method is as follows: by cell in tissue culture plate, together with cell culture fluid Freeze thawing 3 times, 5000 turns/min is centrifuged 10min, retains supernatant, abandons precipitating.Measure Influenza virus titer in supernatant.Virus titer is surveyed Determine method and refers to " national influenza central standard operating instruction (revised edition), 2007 ".
Experimental result 13 shows in mdck cell that paraoxon acid enzyme 3 is expressed by exogenous raising, for 24 hours, 48h, 72h it is thin PCDNA 3.1- paraoxon acid enzyme 3+ amantadine (0.4 μ g/ml) group and transfection pCDNA 3.1 (+) empty carrier+gold are transfected in born of the same parents Rigid alkanamine (0.4 μ g/ml) is compared to significantly reducing, and significant difference, p value be respectively p=0.042 (for 24 hours), 0.088 (48h), 0.069(72h)。
It is above-mentioned the experimental results showed that in regulating cell 3 gene expression of paraoxon acid enzyme can significantly increase amantadine inhibition Influenza virus duplication.
All references mentioned in the present invention is incorporated herein by reference, and is alone applied just as each piece As with reference to such.In addition, it should also be understood that, those skilled in the art can be to this hair after having read above-mentioned instruction of the invention Bright to make various changes or modifications, these equivalent forms also fall within the scope of the appended claims of the present application.
It should be understood that those skilled in the art can make the present invention various after having read above content of the invention Change or modification, these equivalent forms also fall within the scope of the appended claims of the present application.

Claims (3)

1. the recombinant vector that a kind of gene containing paraoxon acid enzyme 3 puts reading frame is preparing or is screening anti-swine flu H3N2 hypotype Drug in purposes, which is characterized in that the recombinant vector is prepared by method comprising the following steps,
(1) from Genbank accession number be NM_001044604.1 3 gene order of paraoxon acid enzyme in, choose from sequence from Initiation codon " ATG " arrives longest sequence 1065bp base between terminator codon " TGA ", " TAG " or " TAA "；
(2) amplification overall length PCR primer is designed at the end 5' and 3' respectively, III restriction enzyme site of Hind is added at the end upstream primer 5', and CGG makees For protectiveness base, I restriction enzyme site of EcoR is added at the end downstream primer 5'；
(3) upstream primer 5'-CGGAAGCTTATGGGGAAGCTGGTGGCTCTGA-3'；
(4) downstream primer 5'-CGGGAATTCCTAGAGCACACAGTACAGAGCT-3'；
(5) reverse transcription product of the total serum IgE extracted using porcine alveolar macrophage is expanded as template with the upstream and downstream primer PCR of design Increasing obtains 3 sequence of paraoxon acid enzyme, and the corresponding restriction enzyme site of carrier for expression of eukaryon is then connected to after digestion.
2. purposes as described in claim 1, which is characterized in that the carrier for expression of eukaryon in the step (5) is pCDNA 3.1 (+) carrier.
3. a kind of purposes of cell model in screening anti-swine flu H3N2 hypotype drug, which is characterized in that the cell model Contain recombinant vector as claimed in claim 1 or 2.
CN201610139667.9A 2012-01-20 2012-01-20 A kind of recombinant vector and its application in preparation or screening Tamiflu Expired - Fee Related CN105602989B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610139667.9A CN105602989B (en) 2012-01-20 2012-01-20 A kind of recombinant vector and its application in preparation or screening Tamiflu

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610139667.9A CN105602989B (en) 2012-01-20 2012-01-20 A kind of recombinant vector and its application in preparation or screening Tamiflu
CN201210019712.9A CN103215267B (en) 2012-01-20 2012-01-20 Suppress siRNA and its application of influenza virus related gene

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CN201210019712.9A Division CN103215267B (en) 2012-01-20 2012-01-20 Suppress siRNA and its application of influenza virus related gene

Publications (2)

Publication Number Publication Date
CN105602989A CN105602989A (en) 2016-05-25
CN105602989B true CN105602989B (en) 2019-04-02

Family

ID=48813426

Family Applications (3)

Application Number Title Priority Date Filing Date
CN201610139670.0A Expired - Fee Related CN105586344B (en) 2012-01-20 2012-01-20 Inhibit siRNA and its application of influenza virus related gene
CN201610139667.9A Expired - Fee Related CN105602989B (en) 2012-01-20 2012-01-20 A kind of recombinant vector and its application in preparation or screening Tamiflu
CN201210019712.9A Expired - Fee Related CN103215267B (en) 2012-01-20 2012-01-20 Suppress siRNA and its application of influenza virus related gene

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN201610139670.0A Expired - Fee Related CN105586344B (en) 2012-01-20 2012-01-20 Inhibit siRNA and its application of influenza virus related gene

Family Applications After (1)

Application Number Title Priority Date Filing Date
CN201210019712.9A Expired - Fee Related CN103215267B (en) 2012-01-20 2012-01-20 Suppress siRNA and its application of influenza virus related gene

Country Status (1)

Country Link
CN (3) CN105586344B (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105779460A (en) * 2014-12-22 2016-07-20 深圳华大基因研究院 Separated nucleic acid for encoding ACD mutants and applications thereof
CN108192983B (en) * 2018-01-25 2022-02-25 河南农业大学 Method for detecting pig PRKAG1 gene expression quantity and application
CN112750498B (en) * 2020-12-30 2022-06-24 同济大学 Method for inhibiting HIV virus replication by targeting reverse transcription primer binding site

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1854300A (en) * 2005-04-29 2006-11-01 中国人民解放军军事医学科学院毒物药物研究所 Recombinant plasmid containing PON gene and its use
CN101240292A (en) * 2008-03-12 2008-08-13 南京大学 Construction of human paraoxonase 3 gene expression vector
WO2010148065A2 (en) * 2009-06-16 2010-12-23 Curna, Inc. Treatment of paraoxonase 1 (pon1) related diseases by inhibition of natural antisense transcript to pon1
WO2011048387A2 (en) * 2009-10-22 2011-04-28 Cambridge Enterprise Limited Treatment of pre-term neonates

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010134939A2 (en) * 2008-12-19 2010-11-25 Zirus, Inc. Mammalian genes involved in infection

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1854300A (en) * 2005-04-29 2006-11-01 中国人民解放军军事医学科学院毒物药物研究所 Recombinant plasmid containing PON gene and its use
CN101240292A (en) * 2008-03-12 2008-08-13 南京大学 Construction of human paraoxonase 3 gene expression vector
WO2010148065A2 (en) * 2009-06-16 2010-12-23 Curna, Inc. Treatment of paraoxonase 1 (pon1) related diseases by inhibition of natural antisense transcript to pon1
WO2011048387A2 (en) * 2009-10-22 2011-04-28 Cambridge Enterprise Limited Treatment of pre-term neonates

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
Identification of differentially expressed genes in a porcine in vivo model of adipogenesis using suppression subtractive hybridization;Benoit Labrecque et al.;《Comparative Biochemistry and Physiology, Part D》;20081101;第4卷;第32-44页 *
Molecular characterization and expression analysis of the porcine paraoxonase 3 (PON3) gene;Benoit Labrecque et al.;《Gene》;20090505;第443卷;第112页表1，第113页第2.6节，第117页左栏第1段，第119页第4节结论 *
Serum paraoxonase-1 activity and concentration are influenced by human immunodeficiency virus infection;Sandra Parra et al.;《Atherosclerosis》;20060830;第194卷;第175-181页 *
Sus scrofa paraoxonase 3 (PON3), mRNA,Accession number：NM_001044604.1;Labrecque B et al.;《Genbank》;20111112;第1-2页 *
The three-gene paraoxonase family: Physiologic roles, actions and regulation;Louis-Philippe Précourt et al.;《Atherosclerosis》;20101012;第214卷;第20-36页 *
对氧磷酶的某些研究进展;赵赞梅等;《毒理学杂志》;20050331;第19卷(第1期);第64-66页 *
对氧磷酶的生物学特性及与疾病的关系;李园园等;《现代检验医学杂志》;20070531;第22卷(第3期);第91-94页 *

Also Published As

Publication number Publication date
CN103215267A (en) 2013-07-24
CN105586344A (en) 2016-05-18
CN105586344B (en) 2018-08-31
CN105602989A (en) 2016-05-25
CN103215267B (en) 2018-02-13

Similar Documents

Publication Publication Date Title
CN111560354B (en) Recombinant novel coronavirus, preparation method and application thereof
AU2014215025B2 (en) Cell lines for virus production and methods of use
CN103540614B (en) The signal of packaging of influenza virus vectors
KR100979025B1 (en) Production of viruses, viral isolates and vaccines
KR20100019423A (en) Attenuated viruses useful for vaccines
CN105950541B (en) A kind of construction method of hFGF21 gene knockout human liver cell strain
CN103266091A (en) A type foot-and-mouth disease recombinant vaccine strains and preparation method and application thereof
Zhu et al. Mammalian-adaptive mutation NP-Q357K in Eurasian H1N1 swine influenza viruses determines the virulence phenotype in mice
CN105602989B (en) A kind of recombinant vector and its application in preparation or screening Tamiflu
CN113416768B (en) Application of PRKRA gene as target in inhibiting replication of peste des petits ruminants virus
CN105671002B (en) The building of H9N2 subtype avian influenza virus cell high yield vaccine strain and application
CN110205321A (en) A kind of DNA fragmentation and its application in the recombinant influenza of building expression red fluorescent protein gene
CN104592367B (en) Influenza NP protein mutant and its encoding gene and application
CN101880677B (en) siRNA sequence against 2009 new influenza A virus polymerase gene and nucleoprotein gene and application thereof
CN105853998B (en) Osteopetrosis correlation transmembrane protein is treating or preventing the application in EV71 infection medicine
CN104711240B (en) The application of Avianreovirus σ A albumen and its relevant biological material
CN105814203B (en) Avian influenza virus miRNA and its identification, detection and application
CN109943576A (en) A kind of recombinant rabies virus of chimeric canine distemper virus principal immune gene and its application
CN105647938B (en) A kind of recombinant vector and its application in preparation or screening Tamiflu
CN109971888A (en) A kind of detection method replicating controllable type influenza virus
CN104762274B (en) The application of Avianreovirus σ NS albumen and its relevant biological material
CN101768575B (en) Construction of recombinant rabies virus of double-expression G gene and biological property analysis thereof
CN110904056B (en) Infectious bronchitis virus rH120-YZS1 delta 5a and construction method and application thereof
CN100365123C (en) SiRNA sequence directing towards influenza B virus membrane protein gene and its application
CN109957550A (en) Rescue method of influenza virus and combinations thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20190402

Termination date: 20210120

CF01 Termination of patent right due to non-payment of annual fee