CN103215267A - siRNA for inhibiting influenza virus-related gene and application - Google Patents

siRNA for inhibiting influenza virus-related gene and application Download PDF

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CN103215267A
CN103215267A CN2012100197129A CN201210019712A CN103215267A CN 103215267 A CN103215267 A CN 103215267A CN 2012100197129 A CN2012100197129 A CN 2012100197129A CN 201210019712 A CN201210019712 A CN 201210019712A CN 103215267 A CN103215267 A CN 103215267A
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sirna
cell
influenza virus
sequence
gene
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马志永
史子学
魏建超
邵东华
王少辉
李蓓蓓
晏文君
朱紫祥
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Shanghai Veterinary Research Institute CAAS
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Abstract

The invention discloses siRNA and a recombinant vector by aiming at caveolin 2 and surfactant protein D; a construction method of a recombinant vector containing a transcription factor A and paraoxonase 3 transcription box, a corresponded cell model and a method for screening medicines. The invention has the advantages that the provided siRNA can reduce mRNA or protein expression of mammal cells after transfection by more than 60%; and the siRNA and the recombinant vector can be used for treating influenza by gene or performing nucleic acid immunization by aiming at influenza virus. The provided cell model can be used for virus research, and especially relates to swine influenza virus pathogenesis researches, and can be used for establishing a model for screening antiviral drugs, especially an anti-influenza medicine cell model.

Description

Suppress the siRNA and the application thereof of influenza virus genes involved
Technical field
The invention belongs to biological technical field, be particularly related to the nucleotide sequence of the siRNA, the siRNA that suppress caveolin 2 and surfactant protein D genetic expression, the recombinant vectors of this siRNA of codified and the recombinant vectors of codified transcription factor A and paraoxon acid enzyme 3, and the purposes of their genes separately aspect preparation and screening Tamiflu.
Background technology
Poisoning intrusion host process is accompanied by host gene and expresses variation, final decision cells infected and virus destiny separately.Need to utilize the metabolic process of host cell to finish duplicating of virus self behind the virus infection host, genomic expression in this process virus will inevitably the modulate host cell suppresses antiviral gene and expresses, and strengthens the genetic expression useful to virus replication.Therefore, behind the virus infection host cell, these genes that host cell inner expression changes often have confidential relation with virus replication.By in these genes in the biological means modulate host cell one or regulate and control a plurality of genetic expressions simultaneously and may have and suppress the effect that influenza virus is duplicated, has the using value of potential prevention and treatment influenza infection.
Influenza virus is the segmented sub-thread minus-stranded rna virus of orthomyxoviridae family's Influenza Virus.Virus particle is polymorph when just having separated, become sphere after going down to posterity in vivo, diameter 80-120nm (Palese, et al., 2007).Virus particle is made up of the protein of RNA, the 70-75% of 0.8-1%, 20% lipid and the carbohydrate of 5-8%, and ratio is mass ratio herein.
Influenza virus is divided into A, B, C three types, and wherein A type (first type) is the most important.Influenza A virus can cause natural infection and the morbidity of the mankind, pig, horse and various birds.Because its surface antigen HA and NA make a variation easily, known HA has 16 hypotypes (H1-H16), NA has 9 hypotypes (N1-N9), various combination between them, make influenza A virus that many hypotypes (as H1N1, H2N2, H3N2, H5N1 etc.), main popular H1N1 and H3N2 hypotype in the pig body be arranged.
Different animals has different influenza viruses, and as porcine influenza, human influenza, equine influenza, these influenzas only infect this genus animal separately generally speaking.But influenza virus has the adaptability that the host plants.Between not homozoic individuality, also propagate easily, form the new influenza virus that infects multiple animal.Another characteristics are that this animal of pig is more special, and pig is to the influenza virus of other animal also susceptible, and this is the characteristic that other animal does not have.In case so pig has infected human influenza, porcine influenza, bird flu etc. simultaneously, this multiple virus can be recombinated in the pig body, possible will come out a kind of can infected pigs, the influenza virus of people, fowl.So the porcine influenza of narrow sense is not the Amphixenosis.Influenza is very limited in interpersonal spread scope, and is relevant with the route of transmission of influenza.Such as influenza A in 2009, be exactly the new influenza virus that produces behind human influenza, porcine influenza, the bird flu co-infected pig, the people who begins to infect most substantially is contacted or follows pig people in close relations, has more people's influenza virus infections subsequently.The great outburst of influenza virus brings great harm all for socio-economic development, people's health on the human development history.Be prevention and treatment influenza, each state has all dropped into a large amount of manpower and materials, constantly studies methods of treatment and medicine.In recent years, seeking gene or albumen with antiviral function in the host cell is a popular domain.
The great outburst of influenza virus brings great harm all for socio-economic development, people's health on the human development history.Be prevention and treatment influenza, each state has all dropped into a large amount of manpower and materials, constantly studies methods of treatment and medicine.In recent years, be a popular domain at gene that has antiviral function in the host cell or albumen as target spot research and development antiviral.
Have the advantage of broad-spectrum antiviral, difficult generation resistance strain with the endocellular function molecule as the target spot of antiviral therapy, become the focus approach of research and development antiviral therapy medicine gradually.International in recent years high-level paper repeatedly is reported in the host cell molecule that screening in the signal path has potential antiviral function and breaks trough.For example, zinc refers to antiviral protein (Zinc-finger antiviral protein, ZAP) can be by degrading special viral RNA and then suppress duplicating of multiple viruses such as murine leukemia virus, be proved to be a kind of important antiviral agent (Chen et al. (2008) Proc.Natl.Acad.Sci.U S A.105,4352-4357).Utilize zinc finger protein for the CCR5 in carrier the carries special enzyme blocking t cell expresses, can promote the T cell remove HIV virus (Holt et al. (2010) Nat.Biotechnol.28,839-847).E3 ubiquitin ligase RNF5 is positioned plastosome, by ubiquitin modification MITA (being also referred to as STING) and cause its degraded and I type interferon expression behind the negative regulation virus infection (Zhong et al. (2009) Immunity.30,397-407).Tetherin (host cell proteins) with dimer grappling HIV on its cytolemma that duplicates, blocking virus from the endochylema film sprout release (Perez-Caballero et al. (2009) Cell.139,499-511).APOBEC3G (Apolipoprotein BmRNA-editing enzyme catalytic polypeptide-like 3G) can induce HIV genovariation, make virus not reproducible (Shirakawa et al. (2008) Nat.Struct.Mol.Biol.15,1184-1191).At present Cyclin Dependent Kinase, prostaglandin(PG), heat shock protein(HSP) are widely used in as, virus diseases such as HCMV, HIV, HSV, adenovirus and papilloma treatment as the medicine target molecule.
RNAi is a kind of gene disruption technology of high specificity efficiently, and development in recent years is rapid, becomes the strong instrument of functional genome research soon.Means are in the dsRNA molecule transfered cell by experiment, specifically in the degradation of cell with the mRNA of its sequence homology, sealing endogenous gene expression, the function of unknown gene from the angle research mankind of reverse genetic or other biological genome.Early stage just someone uses this technical point from the various compositions in the fruit bat insulin signaling transduction pathway path.Experiment report each bar approach by relating in the RNAi research lipid within endothelial cells equilibrium process is also arranged recently.Before this, once utilized sense-rna and said target mrna sequence complementary characteristic to suppress the generation of its phenotype, but because a little less than the gene inhibition effect of sense-rna to endogenous expression, tend to produce some transition phenotypes, easily cause the gene function misjudgment, think to have a kind of medicine Vitravene of only having of therapeutic action clinically by examining at present.
The RNAi specificity is higher, acts on rapidlyer, and side reaction is little, and in reticent target gene effectively, the regulator control system of pair cell itself is not influence also.In human somatocyte, successfully nearly 20 kinds of gene functions have been carried out " knocking out " recently, especially therefore understood human cavity albumen Tsg101, further deepened research HIV to the effect of HIV at people's proliferation in vivo.Leonid etc. are model with the poliovirus, utilize RNAi to come the interior immunity of born of the same parents of inducing cell, produce antiviral effect, especially at RNA viruses.For the virus of easy sudden change, can design the dsRNA of multiple target virogene conserved sequence, reduce its opposing to dsRNA.Maen etc. also use the function that the RNAi technology has successfully been blocked a kind of nuclear transcription factor-2 gene 21 relevant with cell proliferation and differentiation of unconventionality expression in the MCF-7 breast cancer cell.The RNAi The Application of Technology, can not only promote the development of human post genome project (protein science) greatly, high-throughput ground screening of medicaments target gene, detect the function of the next clear and definite gene of expression inhibiting situation of human genome one by one, and it also will be applied to fields such as gene therapy, new drug development, biomedical research, come the unconventionality expression of suppressor gene with the RNAi technology, for the treatment various diseases has been opened up new approach.
Because the fast development of Protocols in Molecular Biology and cytobiology technology in recent years, molecular pharmacology research also deepens continuously, the variation of new drug target, functional protein, genetic expression, bioactive ingredients 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.The automated operation that is applied as of cellular and molecular level medicaments sifting model is laid a good foundation, and makes drug screening by the traditional new technology system of manual screening formal transformation for being screened on a large scale by computer-controlled automatization, has formed high-flux medicaments sifting.
The advantage of high-flux medicaments sifting: realized the mass-producing of drug screening, utilized the medicinal substance resource to large extent, improved the probability of drug discovery, improved the quality of finding new drug simultaneously; Screening experiment is finished in the microscreen system, and amount of samples generally at Gamma Magnitude (μ g), has been saved the sample resource, has established the basic substance of " medicine sieves more ", has saved experiment material simultaneously, has reduced single medicine screening cost; High-flux medicaments sifting is the generation that increasingly automated operation has reduced operate miss, has reduced labour intensity, and has improved the efficient of drug screening and result's accuracy; Have multidisciplinary theory and technology bonded characteristics.
Medicaments sifting model is an essential condition of finding new drug.The foundation of new model will drive the appearance of newtype drug.The development of molecular biology, cytobiology, computer science, particularly the Human Genome Project finishes, for medical research has brought good opportunity,, many-sided superiority conditions such as theory, technology, material are provided also for to set up new medicaments sifting model.Therefore, we should make full use of each subject development technology and set up how new screening model, promote the discovery of new drug.
Dna vaccination is a nineties new developing technology in last century, be about to exogenous gene cloning constructed dna vaccine plasmid to the carrier for expression of eukaryon, after dna vaccination is expelled in the animal matrix through various approach, can be absorbed by host cell and absorb, enter in the nucleus of host cell and be transcribed into mRNA, in endochylema, translate into protein again.Wherein a part of protein combines with the MHCI quasi-molecule after degraded, and is discerned by the acceptor of cd8 t cell to cell surface by submission, and the activating cytotoxic T cell activity; Another part protein also can be secreted away, absorbed by antigen presenting cell as foreign protein again, in phagolysosome, be degraded into polypeptide, and further combine with the mhc class ii molecule, having antigen presenting cell to offer cell surface is discerned by the acceptor of Th2 cell, cytokine by Th2 emiocytosis acts on the B cell then, is produced as main humoral immunization and stimulate with antibody.Dna vaccination not only can be induced the generation humoral immunization, and can induce strong and persistent cellular immunization, also can avoid to extraneous toxin expelling, and dna vaccination and attenuation and attenuated vaccine is different, and there are not problems such as virulence is anti-strong in it.Its security and high efficiency are that traditional vaccine institute is inaccessible, so this technology demonstrates great potential in virus, the caused transmissible disease of cytozoicus, parasite and treatment and prevention of tumour.Wolff can report at the mouse expression in vivo recombinant plasmid, foreign protein can reach 60 days at the mouse expression in vivo, the prompting plasmid DNA is expressed in the quite a while in vivo, can be used as the therapeutic effect, and cause extensive studies.
Gene therapy has prospect for the prevention and treatment of diseases that cancer, atherosclerosis, osteoporosis, sacroiliitis, Alzheimer's cause because specific gene is unusually movable very much.This therapy is a kind of nucleic acid to be introduced people's cell reaches therapeutic purpose to be used for revising their hereditary property therapeutic strategy.Usually, this nucleic acid is the therapeutic action of can having encoded, the proteinic double chain DNA molecule (dsDNA) of destruction or mark effect.This nucleic acid also may be to combine with target sequence in the host cell, suppresses sense-rna or single stranded DNA (ssDNA) that specific gene is expressed by stoping mRNAs or promotor.So far, with the plasmid is that based gene treatment, cancer vaccine etc. have surpassed 600 examples, the genetic immunization of plasmid DNA has been simulated pathogenic agent in intracellular genetic expression approach, successfully should be used for treating the local asphyxia of lower limb, cardiovascular regeneration, and the utmost point is hopeful to prevent by nucleic acid vaccine the difficult and complicated cases of modern medicine, comprises malaria, acquired immune deficiency syndrome (AIDS), hepatitis B and tuberculosis etc.For gene therapy, perhaps plasmid can not be integrated into genomic dna is a shortcoming, but plasmid can episomal form be present in the 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 is exactly the siRNA sequence that design suppresses in the host and influenza virus is duplicated genes involved, the structure recombinant vectors of this siRNA of can encoding, and the application of recombinant vectors aspect the preparation Tamiflu that contains this sequence open reading frame.
The present invention solves the problems of the technologies described above one of technical scheme of being adopted: a kind of siRNA at caveolin 2 genes, the target sequence of siRNA correspondence wherein of the present invention is the mRNA of caveolin 2 genes, the length of siRNA of the present invention is 16-30bp, and can make the mRNA of the mammalian cell after its transfection or protein expression descend more than 60%.
Wherein said Mammals comprises Mammalss such as being selected from pig, dog, mouse, people, preferably pig among the present invention, and siRNA of the present invention has the effect of silencer preferably in pig.
SiRNA described in the present invention, length is 16-30bp, more preferably is 18-25bp.Preferable, described siRNA contains and is selected from the nucleotide sequence shown in the SEQ ID NO:1, or as SEQ ID NO in the sequence table; 1 expression.More preferably, 3 ' of described siRNA hold the extended structure that contains 2 T or contain 2 U.
The present invention solves the problems of the technologies described above two of the technical scheme that adopted: the recombinant vectors of the siRNA that a kind of described inhibition caveolin 2 of encoding is expressed.
According to the present invention, the carrier that described recombinant vectors adopts can be the carrier of this area routine, comprise adenovirus carrier, as various adenovirus carriers based on 5 types (Ad5), 2 types (Ad2), lentiviral vectors is selected from HIV-1 type carrier, HIV-2 type carrier, SIV type carrier, FIV type carrier etc.Described siRNA fragment cloning in vector plasmid, is promptly got recombinant vectors of the present invention.
The present invention solves the problems of the technologies described above three of the technical scheme that adopted: a kind of composition, wherein contain 0.001-99.99wt% siRNA and acceptable carrier, thinner or vehicle at caveolin 2 of the present invention.Wherein, described carrier, thinner or vehicle, more preferably be pharmaceutically acceptable carrier, thinner or vehicle, comprise solid support materials such as water-soluble, insoluble or enteric solubility, or powder, dextrin, Icing Sugar, one or more in calcium sulfate two hydrates, secondary calcium phosphate, magnesium oxide, magnesiumcarbonate, lime carbonate, aluminum hydroxide gel powder and the gac.
The present invention solves the problems of the technologies described above four of the technical scheme that adopted: the purposes of described siRNA in preparation or screening Tamiflu.Wherein, described siRNA can be used as unique anti-influenza virus activity composition, also can unite use with other anti-influenza virus medicaments.
SiRNA of the present invention more preferably can adopt following technical scheme in the purposes of preparation Tamiflu: not only easy to use as mode administration by sucking, and can increase its drug level at infection site.And because it is little to infect early stage viral load, competent siRNA can more effectively suppress duplicating of virus, thereby plays the effect of prevention and treatment; In the influenza RNAi drug target select more, can compound or multiple medication, be difficult for producing the resistance strain; Last RNAi is different with influenza vaccines, does not need the user to have sound immunity system.
The present invention can also more preferably take following technical scheme: as by modes such as intravenous injection or intramuscular injection siRNA sequence or this siRNA recombinant vectorss of encoding, thereby make this siRNA be able to efficiently, reach lastingly the purpose of RNA immunity or gene therapy influenza virus in vivo.
The present invention solves the problems of the technologies described above five of the technical scheme that adopted: described siRNA is suppressing the purposes of influenza virus in duplicating.
The present invention solves the problems of the technologies described above six of the technical scheme that adopted: a kind of method of screening Tamiflu may further comprise the steps:
(1) make the cells contacting of drug candidate and influenza virus infection, described cell is the cell of caveolin 2 gene silencings;
(2) test influenza virus titre;
(3) selection makes the drug candidate that the influenza virus titre descends.
Wherein, more preferably the reticent cell of caveolin 2 genetic expressions described in the step (1) is to have the recombinant vectors of described siRNA to make this gene silencing by making host cell contain the siRNA of described gene or containing coding.
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 clones such as (Madin-Darby canine kidney(cell line)), the preferred mdck cell of the present invention.
Wherein, contact with influenza virus by this cell, after after a while, examine under a microscope the injured degree of cell, relatively, know just whether this antiviral drug can reduce the infringement of viral pair cell with the virus titer of the influenza virus in the cell of Tamiflu processing and controlled trial.
The method of the invention, more preferably can adopt following scheme:
The cytotoxicity of medicine detects, and after mdck cell is cultivated in 96 porocyte culture plates and formed individual layer, adds the soup of different concns, continues to cultivate 3 days, with the toxicity of mtt assay detection of drugs to mdck cell.
The effect of medicine resisiting influenza virus detects, after mdck cell is cultivated in 96 porocyte culture plates and is formed individual layer, influenza infection cell with 100TCID50, the pastille nutrient solution that adds the series concentration under the non-toxic concn again, continue to cultivate 3 days, determine the restraining effect of medicine influenza virus through the CPE method.
The method of the invention preferably can also adopt following technical scheme;
(RNA-dependent RNA polymerase RdRp) mainly participates in viral genome duplication and transcribes influenza virus RNA polymerase.For duplicating, RdRp is that template catalysis generates and its complementary RNA chain (cRNA) with virus genome RNA (vRNA), and cRNA can be used as the synthetic vRNA of template subsequently and is fitted into progeny virus.For transcribing, RdRp adds poly A tract at its 3 ' end on the basis of synthetic cRNA, and 5 ' end adds cap sequence, forms sophisticated mRNA and carries out protein synthesis.Consume substrate A TP, UTP, CTP and GTP during the synthetic RNA chain of this experimental basis and make that ATP concentration reduces gradually in the system, residual A TP content carries out degree to reflect reaction in the mensuration system.
By detecting influenza virus RNA polymerase change level in this cell strain, assessment caveolin 2 reticent cells are to the sensitivity of Tamiflu.
Wherein said virus titer detection method, the MTT method, the CPE method, methods such as cell cultures are this area normal experiment method.
The present invention solves the problems of the technologies described above seven of the technical scheme that adopted: a kind of siRNA at surfactant protein D gene, the target sequence of described siRNA correspondence is the mRNA of surfactant protein D gene, the length of described siRNA is 16-30bp, and can make the mRNA of the mammalian cell after its transfection or expressing quantity descend more than 60%.
Wherein said Mammals comprises Mammalss such as being selected from pig, dog, mouse, people, preferably pig among the present invention, and siRNA of the present invention has the effect of silencer preferably in pig.
SiRNA length described in the present invention is 16-30bp, more preferably is 18-25bp.Preferable, described siRNA contains and is selected from the nucleotide sequence shown in the SEQ ID NO:2, or represents as SEQ ID NO:2 in the sequence table.Described siRNA, more preferably sequence 3 ' is held the extended structure that contains 2 T or contain 2 U.
The present invention solves the problems of the technologies described above eight of the technical scheme that adopted: the recombinant vectors of the siRNA that a kind of described inhibition surfactant protein D that encodes expresses.
According to the present invention, the carrier that described recombinant vectors adopts can be the carrier of this area routine, comprise adenovirus carrier, comprise various adenovirus carriers based on 5 types (Ad5), 2 types (Ad2), lentiviral vectors comprises HIV-1 type carrier, HIV-2 type carrier, SIV type carrier, FIV type carrier etc.。Described siRNA fragment cloning in vector plasmid, is promptly got recombinant vectors of the present invention.
The present invention solves the problems of the technologies described above nine of the technical scheme that adopted: a kind of composition, wherein contain 0.001-99.99wt% siRNA sequence and acceptable carrier, thinner or vehicle at surfactant protein D of the present invention.Wherein said carrier, thinner and vehicle more preferably are pharmaceutically acceptable carrier, thinner or vehicle.For example be in solid support material, powder, dextrin, Icing Sugar, calcium sulfate two hydrates, secondary calcium phosphate, magnesium oxide, magnesiumcarbonate, lime carbonate, aluminum hydroxide gel powder and the nano materials such as water-soluble, insoluble or enteric solubility one or more.
The present invention solves the problems of the technologies described above ten of the technical scheme that adopted: the purposes of the siRNA of described inhibition surfactant protein D in preparation or screening Tamiflu.Wherein, described siRNA can be used as unique anti-influenza virus activity composition, also can unite use with other anti-influenza virus medicaments.
Wherein, more preferably can adopt following technical scheme: not only easy to use as mode administration by sucking, and can increase its drug level at infection site.And because it is little to infect early stage viral load, competent siRNA can more effectively suppress duplicating of virus, thereby plays the effect of prevention and treatment; In the influenza RNAi drug target select more, can compound or multiple medication, be difficult for producing the resistance strain; Last RNAi is different with influenza vaccines, does not need the user to have sound immunity system.
The present invention can also more preferably take following technical scheme: as by modes such as intravenous injection or intramuscular injection siRNA sequence or this siRNA recombinant vectorss of encoding, thereby make this siRNA be able to efficiently, reach lastingly the purpose of RNA immunity or gene therapy influenza virus in vivo.
The present invention solves the problems of the technologies described above 11 of the technical scheme that adopted: a kind of method of screening Tamiflu, it is characterized in that, and may further comprise the steps:
(1) make the cells contacting of drug candidate and influenza virus infection, described cell is the cell of surfactant protein D gene silencing;
(2) test influenza virus titre:
(3) selection makes the drug candidate that the influenza virus titre descends.
Method of the present invention, more preferably the reticent cell of surfactant protein D genetic expression described in the step (1) is to have the recombinant vectors of described siRNA to make this gene silencing by making host cell contain the siRNA of described gene or containing coding.
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 clones such as (Madin-Darby canine kidney(cell line)), the preferred MDCK of the present invention.
The method of the invention, contact with influenza virus by this cell, after after a while, examine under a microscope the injured degree of cell, relatively, know just whether this antiviral drug can reduce the infringement of viral pair cell with the survival rate or the growing state of cell behind influenza infection of Tamiflu processing and controlled trial.
Wherein, more preferably can adopt following scheme:
(1) cytotoxicity of medicine detects, and after mdck cell is cultivated in 96 porocyte culture plates and formed individual layer, adds the soup of different concns, continues to cultivate 3 days, with the toxicity of mtt assay detection of drugs to mdck cell.
(2) effect of medicine resisiting influenza virus detects, after mdck cell is cultivated in 96 porocyte culture plates and is formed individual layer, influenza infection cell with 100TCID50, the pastille nutrient solution that adds the series concentration under the non-toxic concn again, continue to cultivate 3 days, determine the restraining effect of medicine influenza virus through the CPE method.
The method of the invention, preferably can also adopt following technical scheme:
(1) (RNA-dependent RNA polymerase RdRp) mainly participates in viral genome duplication and transcribes influenza virus RNA polymerase.For duplicating, RdRp is that template catalysis generates and its complementary RNA chain (cRNA) with virus genome RNA (vRNA), and cRNA can be used as the synthetic vRNA of template subsequently and is fitted into progeny virus.
For transcribing, RdRp adds poly A tract at its 3 ' end on the basis of synthetic cRNA, and 5 ' end adds cap sequence, forms sophisticated mRNA and carries out protein synthesis.Consume substrate A TP, UTP, CTP and GTP during the synthetic RNA chain of this experimental basis and make that ATP concentration reduces gradually in the system, residual A TP content carries out degree to reflect reaction in the mensuration system.
(2) by detecting influenza virus RNA polymerase change level in this cell strain, assessment surfactant protein D gene silencing cell is to the sensitivity of Tamiflu.
Wherein said virus titer detection method, the MTT method, the CPE method, methods such as cell cultures are this area normal experiment method.
The present invention solves the problems of the technologies described above 12 of the technical scheme that adopted: a kind of recombinant vectors that contains the gene open reading frame of transcription factor A.Preferable, described carrier obtains by following preparation method:
(1) be accession number among the Genbank that the transcription factor A gene order of NM 001130211.1 is input to DNAStar software (DNASTAR company, the U.S.) among the EditSeq, choose in the sequence from initiator codon " ATG " to terminator codon " TGA ", the longest sequence 741bp base " TAG " or " TAA ";
(2) respectively 5 ' and 3 ' end design amplification total length PCR primer, upstream primer 5 ' end adds the HindIII restriction enzyme site, CGG is as the protectiveness base, downstream primer 5 ' end interpolation EcoR V restriction enzyme site;
(3) upstream primer 5 '-CGGAAGCTTATGGCGCTTCTCCGGGGCGTGT-3 ';
(4) downstream primer 5 '-CGGGATATCTCAACACTCCTCAGTGTCTTTC-3 ';
(5) reverse transcription product of the total RNA that extracts with porcine alveolar macrophage is a template, obtains transcription factor A sequence with the upstream and downstream primer PCR amplification of design, passes through being connected to the corresponding restriction enzyme site of carrier for expression of eukaryon after enzyme is cut then.
Wherein, carrier for expression of eukaryon can adopt common carrier for expression of eukaryon such as pCMVp-NEO-BAN, pEGFP, pEGFT-Actin, pSV2, CMV4, among the present invention, more preferably adopts pCDNA 3.1 (+) carrier.
The present invention solves the problems of the technologies described above 13 of the technical scheme that adopted: a kind of described purposes of recombinant vectors in preparation or screening Tamiflu that contains transcription factor A open reading frame.
The present invention more preferably, this recombinant vectors can be used for the different clones of transfection, the method for transfection can be DEAE-dextran method, calcium phosphate method, the cationic-liposome method, cationic polymers method, virus-mediated method, biological particles passes method (particle gun particle bombardment method), microinjection, electroporation etc., the technical program preferred liposome infection protocol.Cells transfected can be various eukaryotic cells, the preferred MDCK of the technical program (Madin-Darby canine kidney(cell line)).
The present invention can also be used for gene therapy with this recombinant vectors, as can be by modes such as intravenous injection or intramuscular injection, thereby be carried out efficiently, reaches lastingly the purpose of dna immunization or gene therapy influenza virus in vivo.
The present invention solves the problems of the technologies described above 14 of the technical scheme that adopted: a kind of cell model that is used for screening of medicaments, it contains the described recombinant vectors that contains transcription factor A open reading frame.
Wherein, described cell can be various eukaryotic cells, comprises 293T (people's renal epithelial cell system), BHK (hamster nephrocyte), VERO (African green monkey kidney cell), IBRS22 (porcine kidney cell) and MDCK clones such as (Madin-Darby canine kidney(cell line)) etc.
The present invention solves the problems of the technologies described above 15 of the technical scheme that adopted: a kind of method of screening Tamiflu, and the method for the invention may further comprise the steps:
(1) make the cells contacting of drug candidate and influenza virus infection, described cell was the cell of expressing transcription factor A gene;
(2) test influenza virus titre;
(3) selection makes the drug candidate that the influenza virus titre descends.
The method of the invention, the described mistake of step (1) is expressed the cell of this gene, more preferably is to make this gene overexpression by copy number that increases this gene in the cell or the controlling element of improveing this gene.
The method of the invention, described cell can 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 clones such as (Madin-Darby canine kidney(cell line)), the preferred MDCK of the present invention.
The method of the invention, contact with influenza virus by this cell, after after a while, examine under a microscope the injured degree of cell, relatively, know just whether this antiviral drug can reduce the infringement of viral pair cell with the virus titer of influenza virus in the cell of Tamiflu processing and controlled trial.
The method of the invention, more preferably can adopt following scheme:
(1) cytotoxicity of medicine detects, and after mdck cell is cultivated in 96 porocyte culture plates and formed individual layer, adds the soup of different concns, continues to cultivate 3 days, with the toxicity of mtt assay detection of drugs to mdck cell.
(2) effect of medicine resisiting influenza virus detects, after mdck cell is cultivated in 96 porocyte culture plates and is formed individual layer, influenza infection cell with 100TCID50, the pastille nutrient solution that adds the series concentration under the non-toxic concn again, continue to cultivate 3 days, determine the restraining effect of medicine influenza virus through the CPE method.
The method of the invention, preferably can also adopt following technical scheme:
(1) (RNA-dependent RNA polymerase RdRp) mainly participates in viral genome duplication and transcribes influenza virus RNA polymerase.For duplicating, RdRp is that template catalysis generates and its complementary RNA chain (cRNA) with virus genome RNA (vRNA), and cRNA can be used as the synthetic vRNA of template subsequently and is fitted into progeny virus.For transcribing, RdRp adds poly A tract at its 3 ' end on the basis of synthetic cRNA, and 5 ' end adds cap sequence, forms sophisticated mRNA and carries out protein synthesis.Consume substrate A TP, UTP, CTP and GTP during the synthetic RNA chain of this experimental basis and make that ATP concentration reduces gradually in the system, residual A TP content carries out degree to reflect reaction in the mensuration system.
(2) by detecting influenza virus RNA polymerase change level in this cell strain, assessment transcription factor A gene overexpression cell is to the sensitivity of Tamiflu.
Wherein said virus titer detection method, the MTT method, the CPE method, methods such as cell cultures are this area normal experiment method.
The present invention solves the problems of the technologies described above 16 of the technical scheme that adopted: a kind of recombinant vectors that contains the open reading frame of paraoxon acid enzyme 3 genes.Preferable, described carrier obtains by following preparation method:
(1) accession number is in the paraoxon acid enzyme 3 of NM 001044604.1 from Genbank, chooses from sequence from initiator codon " ATG " to terminator codon " TGA ", the longest sequence 1065bp base " TAG " or " TAA ";
(2) respectively 5 ' and 3 ' end design amplification total length PCR primer, upstream primer 5 ' end adds the HindIII restriction enzyme site, CGG is as the protectiveness base, downstream primer 5 ' end interpolation EcoR I restriction enzyme site;
(3) upstream primer 5 '-CGGAAGCTTATGGGGAAGCTGGTGGCTCTGA-3 ';
(4) downstream primer 5 '-CGGGAATTCCTAGAGCACACAGTACAGAGCT-3 ';
(5) reverse transcription product of the total RNA that extracts with porcine alveolar macrophage is a template, obtains transcription factor A sequence with the upstream and downstream primer PCR amplification of design, passes through being connected to the corresponding restriction enzyme site of carrier for expression of eukaryon after enzyme is cut then.
Wherein, carrier for expression of eukaryon can adopt common carrier for expression of eukaryon such as pCMVp-NEO-BAN, pEGFP, pEGFT-Actin, pSV2, CMV4, among the present invention, more preferably adopts pCDNA 3.1 (+) carrier.
The present invention solves the problems of the technologies described above 17 of the technical scheme that adopted: a kind of purposes of recombinant vectors in preparation or screening Tamiflu that contains paraoxon acid enzyme 3 open reading frame of the present invention.
The present invention more preferably, this recombinant vectors can be used for the different clones of transfection, the method for transfection can be DEAE-dextran method, calcium phosphate method, the cationic-liposome method, cationic polymers method, virus-mediated method, biological particles passes method (particle gun particle bombardment method), microinjection, electroporation etc., the technical program preferred liposome infection protocol.Cells transfected can be various eukaryotic cells, the preferred MDCK of the technical program (Madin-Darby canine kidney(cell line)).
The present invention can also be used for gene therapy with this recombinant vectors, as can be by modes such as intravenous injection or intramuscular injection, thereby be carried out efficiently, reaches lastingly the purpose of dna immunization or gene therapy influenza virus in vivo.
The present invention solves the problems of the technologies described above 18 of the technical scheme that adopted: a kind of cell model that is used for screening of medicaments, it contains the described recombinant vectors that contains the open reading frame of paraoxon acid enzyme 3 genes.
Wherein, described cell can be various eukaryotic cells, is selected from 293T (people's renal epithelial cell system), BHK (hamster nephrocyte), VERO (African green monkey kidney cell), IBRS22 (porcine kidney cell) and MDCK (Madin-Darby canine kidney(cell line)) clone etc.
The present invention solves the problems of the technologies described above 19 of the technical scheme that adopted: a kind of method of screening Tamiflu, and the method for the invention may further comprise the steps:
(1) make the cells contacting of drug candidate and influenza virus infection, described cell was the cell of expressing paraoxon acid enzyme 3 genes;
(2) test influenza virus titre;
(3) selection makes the drug candidate that the influenza virus titre descends.
The method of the invention, the described mistake of step (1) is expressed the cell of this gene, more preferably is to make this gene overexpression by copy number that increases this gene in the cell or the controlling element of improveing this gene.
The method of the invention, described cell can adopt 293T (people's renal epithelial cell system), BHK (hamster nephrocyte), VERO (African green monkey kidney cell), IBRS22 (porcine kidney cell) and MDCK clones such as (Madin-Darby canine kidney(cell line)), the preferred MDCK of the present invention.
The method of the invention, contact with influenza virus by this cell, after after a while, examine under a microscope the injured degree of cell, relatively, know just whether this antiviral drug can reduce the infringement of viral pair cell with the virus titer of influenza virus in the cell of Tamiflu processing and controlled trial.
The method of the invention, more preferably can adopt following scheme:
(1) cytotoxicity of medicine detects, and after mdck cell is cultivated in 96 porocyte culture plates and formed individual layer, adds the soup of different concns, continues to cultivate 3 days, with the toxicity of mtt assay detection of drugs to mdck cell.
(2) effect of medicine resisiting influenza virus detects, after mdck cell is cultivated in 96 porocyte culture plates and is formed individual layer, influenza infection cell with 100TCID50, the pastille nutrient solution that adds the series concentration under the non-toxic concn again, continue to cultivate 3 days, determine the restraining effect of medicine influenza virus through the CPE method.
The method of the invention, preferably can also adopt following technical scheme:
(1) (RNA-dependent RNA polymerase RdRp) mainly participates in viral genome duplication and transcribes influenza virus RNA polymerase.For duplicating, RdRp is that template catalysis generates and its complementary RNA chain (cRNA) with virus genome RNA (vRNA), and cRNA can be used as the synthetic vRNA of template subsequently and is fitted into progeny virus.For transcribing, RdRp adds poly A tract at its 3 ' end on the basis of synthetic cRNA, and 5 ' end adds cap sequence, forms sophisticated mRNA and carries out protein synthesis.Consume substrate A TP, UTP, CTP and GTP during the synthetic RNA chain of this experimental basis and make that ATP concentration reduces gradually in the system, residual A TP content carries out degree to reflect reaction in the mensuration system.
(2) by detecting influenza virus RNA polymerase change level in this cell strain, assessment paraoxon acid enzyme 3 gene overexpression cells are to the sensitivity of Tamiflu.
Wherein said virus titer detection method, the MTT method, the CPE method, methods such as cell cultures are this area normal experiment method.
Than prior art, beneficial effect of the present invention is as follows: of the present invention is target spot with the gene or the albumen that have antiviral function in the host cell, the medicine of resisiting influenza virus is provided and has suppressed the method that influenza virus is duplicated.The present invention is achieved by described expression of gene in the regulating cell.Invention has the using value of prevention and treatment influenza infection, to developing better Tamiflu, to the great outburst of control influenza virus, the great harm that the great outburst of minimizing influenza virus brings socio-economic development, people's health has great importance.
Description of drawings
Below in conjunction with description of drawings feature of the present invention and beneficial effect.
Fig. 1 is that Western-blot detects the result that reticent Madin-Darby canine kidney(cell line) central foveola albumen 2 is expressed, and wherein the 1:siRNA-caveolin 2; 2 contrasts of 2:siRNA-caveolin; 3: do not connect the poison contrast; 4: normal cell.
Fig. 2 is that Western-blot detects the result that surfactant protein D expresses in the reticent Madin-Darby canine kidney(cell line), wherein 1:siRNA-surfactant protein D; 2:siRNA-surfactant protein D contrast; 3; Do not connect the poison contrast; 4: normal cell.
Fig. 3 is that reticent Madin-Darby canine kidney(cell line) central foveola albumen 2 is expressed back influenza virus titre figure.
Fig. 4 is that surfactant protein D expresses back influenza virus titre figure in the reticent Madin-Darby canine kidney(cell line).
Fig. 5 is the pCDNA 3.1-transcription factor A carrier collection of illustrative plates that makes up.
Fig. 6 is the pCDNA 3.1-paraoxon acid enzyme 3 carrier collection of illustrative plates that make up.
Fig. 7 is transit cell record factors A and paraoxon acid enzyme 3 immunoblotting detected results, wherein
1:pCDNA 3.1-transcription factor A; The contrast of 2:pCDNA 3.1 empty carriers; 3: normal cell
4:pCDNA 3.1-paraoxon acid enzyme 3; The contrast of 5:pCDNA 3.1 empty carriers; 6: normal cell.
Fig. 8 is that external source improve to be expressed and to be transcribed influenza virus titre result after the factors A in the Madin-Darby canine kidney(cell line).
Fig. 9 is that external source improves paraoxon acid enzyme 3 back influenza virus titre results in the expression Madin-Darby canine kidney(cell line).
Figure 10 adds influenza virus titre figure behind the amantadine in the Madin-Darby canine kidney(cell line) of caveolin 2 silences.
Figure 11 adds influenza virus titre figure behind the amantadine in the Madin-Darby canine kidney(cell line) of surfactant protein D silence.
Figure 12 is that external source improves and to add influenza virus titre figure behind the amantadine in the Madin-Darby canine kidney(cell line) that transcription factor A expresses.
Figure 13 is that external source improves and to add influenza virus titre figure behind the amantadine in the Madin-Darby canine kidney(cell line) that paraoxon acid enzyme 3 expresses.
Embodiment
Further specify the present invention with embodiment below, but the present invention is not limited.
Used reagent is except that specifying among the embodiment, all commercially available getting.
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer." room temperature " described in the present invention is meant the temperature of the operation room of testing, and is generally 25 ℃.
Used swine influenza virus H3N2Swine/Guangdong/1/2006 (SwGD1/05) strain among the embodiment, its biological characteristics can be represented the swine influenza virus characteristics of China, is derived from the Chinese Academy of Agricultural Sciences Shanghai veterinary institute pig transmissible disease research department.
6 porocyte culture plates, U.S. Coming product.
The DMEM substratum, U.S. GIBCO product, REF:16000-044.
Penicillin, U.S. GIBCO company product, REF:15140-122.
Streptomycin sulphate, U.S. GIBCO company product, REF:15140-122.
Madin-Darby canine kidney(cell line) (mdck cell) is available from Shanghai life science institute of Chinese Academy of Sciences cell bank.
1.5ml centrifuge tube, U.S. Axygen company product.
Acetone, Jiangsu Qiangsheng Chemical Co., Ltd., credit number: XK13-201-00227.
PBS is available from U.S. GIBCO company, 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.
Genetic expression inhibition influenza virus is duplicated in the embodiment 1 modulate host cell
1.siRNA genetic expression inhibition influenza virus is duplicated in (siRNA) reticent host cell
(1) siRNA molecular designing
Present embodiment is selected caveolin 2 genes, GeneID:100125375; Surfactant protein D, GeneID:397198 uses the online design software design of Dharmacon siRNA molecule, the siRNA interference sequence of design caveolin 2 gene specifics; And arbitrarily upset sequence in contrast with " siRNA-caveolin ".The siRNA interference sequence of design surface activated protein D gene specific; And arbitrarily upset sequence in contrast with " siRNA-surfactant protein D ".The double-stranded disturbing molecule subject sequence of the siRNA of design is classified 19 bases as, and positive-sense strand 3 ' end is added two UU, and antisense strand 3 ' end is added two TT.
The sequence of design is as follows:
At caveolin 2 target sequences 5 '-GCAAATACGTGATCTACAA-3 ';
SiRNA-caveolin 2 positive-sense strand sequence 5 '-GCAAAUACGUGAUCUACAAUU-3 ';
SiRNA-caveolin 2 antisense strand sequence 3 '-TTCGUUUAUGCACUAGAUGUU-5 ';
SiRNA-caveolin 2 contrast positive-sense strand sequence 5 '-CGAUAGAUGUACACAUACAUU-3 ';
SiRNA-caveolin 2 contrast antisense strand sequence 3 '-TTGCUAUCUACAUGUGUAUGU-5 ';
At surfactant protein D target sequence 5 '-GAGCAGAAATGAAGACCTA-3 ';
SiRNA-surfactant protein D positive-sense strand sequence 5 '-GAGCAGAAAUGAAGACCUAUU-3 ';
SiRNA-surfactant protein D antisense strand sequence 3 '-TTCUCGUCUUUACUUCUGGAU-5 ';
SiRNA-surfactant protein D contrasts positive-sense strand 5 '-CAGAGUGACAGAAGACAUAUU-3 ';
SiRNA-surfactant protein D contrasts antisense strand 3 '-TTGUCUCACUGUCUUCUGUAU-5 '.
Analyze interference sequence and " caveolin or surfactant protein D " dna homolog in addition less than 50% through NCBI blast, control sequence and pig genome all sequences homology are less than 50%.
According to the siRNA sequence of this design, by the double-stranded siRNA of the external synthetic of Dharmacon company.
(2) siRNA molecule transfectional cell
The mdck cell of trysinization is used the DMEM (U.S. GIBCO product of serum-free, REF:16000-044) (100 unit penicillin (U.S. GIBCO company products, REF:15140-122) and 100 unit Streptomycin sulphate (U.S. GIBCO company products, REF:15140-122)) pass in 6 porocyte culture plates (U.S. Coming product) cell density 4 * 10 6Cells/well continues to be incubated at 37 ℃, 5%CO 2In the incubator.Treat the 80-90% at the bottom of the full culture plate of cell growth, double-stranded siRNA molecule of the present invention with FuGENE HD reagent (Roche company, the U.S., production code member: 04709705001) transfection in mdck cell, the operating process by specification.Establish 2 contrasts of transfection contrast interference sequence sample siRNA-caveolin, untransfected contrast, normal cell contrast simultaneously.
(3) the siRNA transfectional cell connects poison and sample collection
24h inoculation influenza virus H3N2 behind the mdck cell transfection siRNA molecule, dosage of inoculation 1 * 10 3EID 50/ hole (6 orifice plate).24h, 48h, 72h collect sample behind virus inoculation.Method is as follows: with cell in the Tissue Culture Plate, together with cell culture fluid freeze thawing 3 times, the centrifugal 10min of 5000g/min keeps supernatant, abandons precipitation.Measure influenza virus titre in the supernatant.The virus titer measuring method is with reference to " national influenza central standard working specification (revised edition), 2007 ".
Experimental result (Fig. 1) shows that mdck cell central foveola albumen 2, surfactant protein D express and inoculated influenza virus after the siRNA molecule silence, and the influenza virus titre is compared remarkable reduction with control group in 24h, 48h, 72h cell, and significant difference.
Cell central foveola albumen 2 by siRNA molecule silence after (as above 24h sample behind the virus inoculation), the Western-blot detected result is seen Fig. 1, the result shows that reticent group central foveola albumen 2 expression levels are starkly lower than contrast.
In the cell surfactant protein D by siRNA molecule silence after (as above 24h sample behind the virus inoculation), the Western-blot detected result is seen Fig. 2, the result shows that surfactant protein D expression level is starkly lower than contrast in the reticent group.
SiRNA-caveolin 2 is compared the p value and is respectively p=0.0004 (24h), 0.001 (48h), 0.015 (72h) with siRNA-caveolin 2 control groups; SiRNA-surfactant protein D compares the p value with siRNA-surfactant protein D control group and is respectively p=0.004 (24h), 0.001 (48h), 0.009 (72h).
Above-mentioned experimental result proof can suppress duplicating of influenza virus in the host cell by regulation and control caveolin 2 and surfactant protein genetic expression.
2. genetic expression inhibition influenza virus is duplicated in the exogenous raising host cell
(1) structure of expression vector in the 1st group of the heterogenous expression
With transcription factor A (TFAM) (GeneID:397279; Gene database accession number: NM_001130211.1) be connected to pCDNA 3.1 (+) carrier (Invitrogen company, the U.S.; Article No.: V790-20) between HindIII and EcoR V restriction enzyme site, concrete construction process may further comprise the steps:
(1) be accession number among the Genbank that the transcription factor A gene order of NM_001130211.1 is input to DNAStar software (DNASTAR company, the U.S.) among the EditSeq, choose in the sequence from initiator codon " ATG " to terminator codon " TGA ", the longest sequence 741bp base " TAG " or " TAA ";
(2) respectively 5 ' and 3 ' end design amplification total length PCR primer, upstream primer 5 ' end adds the HindIII restriction enzyme site, CGG is as the protectiveness base, downstream primer 5 ' end interpolation EcoR V restriction enzyme site;
(3) upstream primer 5 '-CGG
Figure BSA00000661346100161
ATGGCGCTTCTCCGGGGCGTGT-3 ' (two HindIII restriction enzyme site that is scribed ss);
(4) downstream primer 5 '-CGG TCAACACTCCTCAGTGTCTTTC-3 ' (two EcoR V restriction enzyme site that is scribed ss);
(5) extracting total RNA reverse transcription product with the pig pulmonary macrophage is template, obtains transcription factor A sequence with the upstream and downstream primer PCR amplification that designs, and is connected to the corresponding restriction enzyme site of pCDNA 3.1 (+) carrier after cutting through enzyme then.The carrier called after pCDNA 3.1-transcription factor A that successfully constructs sees Fig. 5.
With paraoxon acid enzyme 3 (PON3) (GeneID:733674; The gene database accession number: NM_001044604.1) the gene open reading frame is connected to pCDNA 3.1 (+) carrier (Invitrogen company, the U.S.; Article No.: V790-20) between HindIII and EcoR I restriction enzyme site, concrete construction process may further comprise the steps:
(1) be accession number among the Genbank that paraoxon acid enzyme 3 gene orders of NM_001044604.1 are input to DNAStar software (DNASTAR company, the U.S.) among the EditSeq, choose from sequence from initiator codon " ATG " to terminator codon " TGA ", the longest sequence 1065bp base " TAG " or " TAA ";
(2) respectively at 5 ' and 3 ' end design amplification total length PCR primer, upstream primer 5 ' end adds the HindIII restriction enzyme site, and CGG is as the protectiveness base, and downstream primer 5 ' end adds EcoR I restriction enzyme site;
(3) upstream primer 5 '-CGG ATGGGGAAGCTGGTGGCTCTGA-3 ' (two HindIII restriction enzyme site that is scribed ss);
(4) downstream primer 5 '-CGG
Figure BSA00000661346100172
CTAGAGCACACAGTACAGAGCT-3 ' (two EcoR I restriction enzyme site that is scribed ss);
(5) extracting total RNA reverse transcription product with the pig pulmonary macrophage is template, obtains transcription factor A sequence with the upstream and downstream primer PCR amplification that designs, and is connected to the corresponding restriction enzyme site of pCDNA 3.1 (+) carrier after cutting through enzyme then.The carrier called after pCDNA 3.1-paraoxon acid enzyme 3 that successfully constructs is seen Fig. 6.
(2) recombinant vectors transfectional cell and expressive host albumen
The mdck cell of trysinization is used the DMEM (U.S. GIBCO product of serum-free, REF:16000-044) 100 unit penicillin (U.S. GIBCO company products, REF:15140-122) and 100 unit Streptomycin sulphate (U.S. GIBCO company products, REF:15140-122)) pass in 6 porocyte culture plates (U.S. Coming product) cell density 4 * 10 6Cells/well continues to be incubated in 37 ° of C, the 5%CO2 incubator.Treat the 80-90% at the bottom of culture plate is expired in the cell growth, with FuGENE HD reagent (Roche company, the U.S., production code member: 04709705001) pCDNA3.1-transcription factor A, pCDNA 3.1-paraoxon acid enzyme 3 carriers are distinguished transfection in mdck cell, the operating process by specification.Establish the cell contrast of transfection pCDNA 3.1 (+) empty carrier simultaneously.
24h behind the cell transfecting collects transfection pCDNA 3.1-transcription factor A, pCDNA 3.1-paraoxon acid enzyme 3, pCDNA3.1 (+) empty carrier and sample of normal cells.Total protein of cell extracts: cell is 2 times in the usefulness PBS rinsing 6 porocyte plates, adds 1ml PBS then cell is scraped with the cell spatula, and 4 ℃, the centrifugal 5min sedimentation cell of 3000g is abandoned supernatant.Cell pyrolysis liquid (the green skies company that adds 100 μ l to cell precipitation, production code member: P0013), re-suspended cell at cracking 5min on ice, is used ultrasonic cell disruption instrument (Sonics company then, the U.S., instantaneous fragmentation model VCX105PB) (40HZ, 1S), boiling water boils 5min, 4 ℃ of centrifugal 10min of following 13000g discard precipitation.Get 2 μ l albumen supernatants with BCA test kit (green skies company, production code member: P0012) measure protein concentration, to specifications operation.Remaining albumen supernatant adds 5 * SDS PAGE buffer (compound method is seen " molecular cloning experiment guide " third edition), and boiling water boils 5min, and 4 ℃ of centrifugal 5min of following 13000g get supernatant and carry out protein electrophorese.
Sex change SDS-PAGE gel electrophoresis and albumen transfer printing (step is with reference to " molecular cloning experiment 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 protein isolate finishes until electrophoresis.Nitrocellulose filter (Whatman company, the U.S., product type: 10401396) are used in the albumen transfer printing.Bole's electrophoresis chamber constant voltage 65V, 2h.After finishing, transfer printing seals in the TBST-skimming milk of NC film immersion 5% (v/v).Behind the room temperature effect 2h, discard confining liquid, with TBST (pH7.6) the damping fluid rinsing that contains 1% (v/v) polysorbas20 3 times, the residual skimming milk of flush away can carry out antibody incubation.
Antibody response and colour developing: add one and resist, paraoxon acid enzyme 3 antibody (Abcam, the U.S., production code member: ab40969), transcription factor A antibody (antikoerper-online.de company, Germany, production code member: ABIN484435).4 ℃ of following jog shaken overnight (12h-16h), wash 3 times with 100%TBST then after, each 5min.Add two of HRP (horseradish peroxidase) mark afterwards and resist, room temperature effect 2h, after TBST rinsing 3 times, each 5min.The ECL test kit (production code member: 32106) operate in the darkroom for Pierce company, the U.S. by colour developing.Operation is according to ECL test kit specification sheets.
Detection confidential reference items contrasts: the film that will develop the color immerses antibody and strips off liquid (green skies company, production code member: P0025), hatch 30min for 50 ℃, every 10min concussion once, use TBST damping fluid (compound method :) rinsing 3 times then with reference to " molecular cloning experiment guide " third edition, add confining liquid and seal 15min again, use anti-β-actin antibody (green skies company, production code member: AA128) as protein content in the anti-test sample.
Detected result is seen Fig. 5, after showing transfection pCDNA 3.1-transcription factor A in the mdck cell, pCDNA 3.1-paraoxon acid enzyme 3, the expression amount of " transcription factor A " and " paraoxon acid enzyme 3 " is higher than transfection pCDNA 3.1 (+) empty carrier group and normal cell group in the cell.
(3) " transcription factor A " or " paraoxon acid enzyme 3 " has the effect of the influenza virus of inhibition in the exogenous raising mdck cell
24h behind cell transfecting pCDNA 3.1-transcription factor A, pCDNA 3.1-paraoxon acid enzyme 3 or pCDNA 3.1 (+) empty carrier, dosage of inoculation 1 * 10 3EID 50/ hole (6 orifice plate) swine influenza virus.24h, 48h, 72h collect sample behind virus inoculation.Method is as follows: with cell in the Tissue Culture Plate, together with cell culture fluid freeze thawing 3 times, the centrifugal 10min of 5000 commentaries on classics/min keeps supernatant, abandons precipitation.Measure influenza virus titre in the supernatant.The virus titer measuring method is with reference to " national influenza central standard working specification (revised edition), 2007 ".
Experimental result (Fig. 6,7) shows, transcribes factors A in the mdck cell, paraoxon acid enzyme D is expressed by exogenous raising, and the influenza virus titre is compared remarkable reduction with control group in 24h, 48h, 72h cell, and significant difference.Transfection pCDNA 3.1-transcription factor A group is compared the p value with transfection pCDNA 3.1 (+) empty carrier group and is respectively p=0.002 (24h), 0.014 (48h), 0.0005 (72h); Transfection pCDNA 3.1-paraoxon acid enzyme is compared the p value with transfection pCDNA3.1 (+) empty carrier group for 3 groups and is respectively p=0.015 (24h), 0.008 (48h), 0.012 (72h).Virus titer difference not significantly (p＞0.05) in transfection pCDNA3.1 (+) empty carrier group and the normal cell.
Above-mentioned experimental result shows that regulative transcription factor A and 3 genetic expressions of paraoxon acid enzyme can suppress duplicating of the interior influenza virus of host cell.
Genetic expression significantly improves the antiviral effect of influenza virus medicine in the embodiment 2 modulate host cells
Infect the H3N2 swine influenza virus by regulating in the Madin-Darby canine kidney(cell line) again behind the protein expression in the present embodiment, utilize to add amantadine in cell culture fluid, albumen is to amantadine resisiting influenza virus reinforced effects in the research regulating cell.Amantadine suppresses the function of duplicating of virus by disturbing influenza virus particles M2 protein ion passage.
1 reticent Madin-Darby canine kidney(cell line) central foveola albumen 2 significantly improves amantadine and suppresses the influenza virus effect
(1) reticent Madin-Darby canine kidney(cell line) central foveola albumen 2 is expressed
Concrete steps are with embodiment 1.
(2) amantadine is prepared
Amantadine hydrochloride is mixed with 20mg/ml available from the chemical company limited of A Faaisha (Tianjin) with physiological saline, filtration sterilization, 4 ℃ of preservations.
(3) siRNA molecule transfectional cell
Concrete steps are with embodiment 1.The drug combination group adds amantadine in cell culture fluid, final concentration to 0.4 μ g/ml is grouped as follows: siRNA-caveolin+amantadine (0.4 μ g/ml), siRNA-caveolin 2 contrast+amantadines (0.4 μ g/ml), untransfected contrast+amantadine (0.4 μ g/ml) and normal cell contrast+amantadine (0.4 μ g/ml).
(4) the siRNA transfectional cell connects poison and sample collection
With embodiment 1.
Experimental result shows that 2 expression of mdck cell central foveola albumen can be significantly improved the effect of amantadine inhibition influenza virus after the siRNA molecule silence as shown in figure 10.Influenza virus titre siRNA-caveolin+amantadine (0.4 μ g/ml) group significantly is lower than siRNA-caveolin 2 contrast+amantadines (0.4 μ g/ml) group in 24h, 48h, the 72h cell, and the p value is respectively p=0.016 (24h), 0.001 (48h), 0.001 (72h).
Caveolin 2 genetic expressions can improve the effect that amantadine inhibition influenza virus is duplicated in the above-mentioned experimental result proof regulating cell.
Surfactant protein D significantly improves amantadine inhibition influenza virus effect in the 2 reticent Madin-Darby canine kidney(cell line)
(1) surfactant protein D expresses in the reticent Madin-Darby canine kidney(cell line)
Concrete steps are with embodiment 1.
(2) amantadine is prepared
Amantadine hydrochloride is mixed with 20mg/ml available from the chemical company limited of A Faaisha (Tianjin) with physiological saline, filtration sterilization, 4 ℃ of preservations.
(3) siRNA molecule transfectional cell
Concrete steps are with embodiment 2.The drug combination group adds amantadine in cell culture fluid, final concentration to 0.4 μ g/ml is grouped as follows: siRNA-surfactant protein D+ amantadine (0.4 μ g/ml), siRNA-surfactant protein D contrast+amantadine (0.4 μ g/ml), untransfected contrast+amantadine (0.4 μ g/ml) and normal cell contrast+amantadine (0.4 μ g/ml).
(4) the siRNA transfectional cell connects poison and sample collection with embodiment 1.
Experimental result shows that surfactant protein D expresses the effect that can be significantly improved amantadine inhibition influenza virus after the siRNA molecule silence in the mdck cell as shown in figure 11.Influenza virus titre siRNA-surfactant protein D+ amantadine (0.4 μ g/ml) group significantly is lower than siRNA-surfactant protein D contrast+amantadine (0.4 μ g/ml) group in 24h, 48h, the 72h cell, and the p value is respectively p=0.020 (24h), 0.025 (48h), 0.130 (72h).
Surfactant protein D genetic expression can improve the effect that amantadine inhibition influenza virus is duplicated in the above-mentioned experimental result proof regulating cell.
Transcription factor A expresses and significantly improves amantadine inhibition influenza virus effect in the 3 exogenous raising host cells
(1) structure of expression vector in the 1st group of the heterogenous expression
Concrete steps are with embodiment 1.
(2) recombinant vectors transfectional cell and expressive host albumen
Concrete steps are with embodiment 1.
(3) transcribe in the exogenous raising mdck cell that amantadine suppresses the effect that influenza virus is duplicated after the factors A
24h behind cell transfecting pCDNA 3.1-transcription factor A or pCDNA 3.1 (+) empty carrier, dosage of inoculation 1 * 10 3The EID50/ hole, the drug combination group adds amantadine in cell culture fluid, final concentration to 0.4 μ g/ml is provided with following experimental 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 contrast+amantadine (0.4 μ g/ml) and normal cell contrast+amantadine (0.4 μ g/ml).24h, 48h, 72h collect sample behind virus inoculation.Method is as follows: with cell in the Tissue Culture Plate, together with cell culture fluid freeze thawing 3 times, the centrifugal 10min of 5000 commentaries on classics/min keeps supernatant, abandons precipitation.Measure influenza virus titre in the supernatant.The virus titer measuring method is with reference to " national influenza central standard working specification (revised edition), 2007 ".
Experimental result shows, shown in Figure 12, transcribing factors A in the mdck cell is expressed by exogenous raising, dye pCDNA 3.1-transcription factor A+ amantadine (0.4 μ g/ml) group at 24h, 48h, 72h transit cell and compare remarkable reduction with transfection pCDNA 3.1 (+) empty carrier+amantadine (0.4 μ g/ml), and significant difference, p value are respectively p=0.310 (24h), 0.013 (48h), 0.007 (72h).
Above-mentioned experimental result shows in the 1st group of the regulation and control that genetic expression can significantly strengthen amantadine and suppress influenza virus and duplicate.
Paraoxon acid enzyme 3 is expressed and is significantly improved amantadine inhibition influenza virus effect in the 4 exogenous raising host cells
(1) structure of expression vector in the 1st group of the heterogenous expression
Concrete steps are with embodiment 2.
(2) recombinant vectors transfectional cell and expressive host albumen
Concrete steps are with embodiment 2
(3) paraoxon acid enzyme 3 back amantadines suppress the effect that influenza virus is duplicated in the exogenous raising mdck cell
24h behind cell transfecting pCDNA 3.1-paraoxon acid enzyme 3 or pCDNA 3.1 (+) empty carrier, dosage of inoculation 1 * 10 3The EID50/ hole, the drug combination group adds amantadine in cell culture fluid, final concentration to 0.4 μ g/ml is provided with following experimental group: transfection pCDNA 3.1-paraoxon acid enzyme 3+ amantadine (0.4 μ g/ml), transfection pCDNA 3.1 (+) empty carrier+amantadine (0.4 μ g/ml), untransfected contrast+amantadine (0.4 μ g/ml) and normal cell contrast+amantadine (0.4 μ g/ml).24h, 48h, 72h collect sample behind virus inoculation.Method is as follows: with cell in the Tissue Culture Plate, together with cell culture fluid freeze thawing 3 times, the centrifugal 10min of 5000 commentaries on classics/min keeps supernatant, abandons precipitation.Measure influenza virus titre in the supernatant.The virus titer measuring method is with reference to " national influenza central standard working specification (revised edition), 2007 ".
Experimental result 13 shows, paraoxon acid enzyme 3 is expressed by exogenous raising in the mdck cell, dye pCDNA 3.1-paraoxon acid enzyme 3+ amantadine (0.4 μ g/ml) group at 24h, 48h, 72h transit cell and compare remarkable reduction with transfection pCDNA 3.1 (+) empty carrier+amantadine (0.4 μ g/ml), and significant difference, p value are respectively p=0.042 (24h), 0.088 (48h), 0.069 (72h).
Above-mentioned experimental result shows that paraoxon acid enzyme 3 genetic expressions can significantly enhancing amantadine inhibition influenza virus is duplicated in the regulating cell.
All quote in this application as a reference at all documents that the present invention mentions, just use as a reference separately as each piece quilt.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned instruction of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Should be understood that after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims (13)

1. siRNA at caveolin 2 genes, it is characterized in that, the target sequence of described siRNA correspondence is the mRNA of caveolin 2 genes, and the length of described siRNA is 16-30bp, and can make the mRNA of the mammalian cell after its transfection or protein expression descend more than 60%.
2. siRNA as claimed in claim 1 is characterized in that, described siRNA contains the nucleotide sequence shown in the SEQ ID NO:1.
3. siRNA as claimed in claim 1 is characterized in that, SEQ ID NO:1 represents in described siRNA such as the sequence table.
4. siRNA at surfactant protein D gene, it is characterized in that, the target sequence of described siRNA correspondence is the mRNA of surfactant protein D gene, the length of described siRNA is 16-30bp, and can make the mRNA of the mammalian cell after its transfection or expressing quantity descend more than 60%.
5. siRNA as claimed in claim 4 is characterized in that, described siRNA contains and is selected from the nucleotide sequence shown in the SEQ ID NO:2.
6. siRNA as claimed in claim 4 is characterized in that, SEQ IDNO:2 represents in described siRNA sequence such as the sequence table.
7. as claim 2 or 5 described siRNA, it is characterized in that described siRNA sequence 3 ' end also contains 2 T or contains the extended structure of 2 U.
8. as the purposes of each described siRNA of claim 1-7 in preparation or screening Tamiflu, the perhaps purposes in the inhibition influenza virus is duplicated.
9. a recombinant vectors that contains the gene open reading frame of transcription factor A is characterized in that, by the method preparation that may further comprise the steps,
(1) accession number is in the transcription factor A gene order of NM_001130211.1 in Genbank, chooses in the sequence from initiator codon " ATG " to terminator codon " TGA ", the longest sequence 741bp base " TAG " or " TAA ";
(2) respectively 5 ' and 3 ' end design amplification total length PCR primer, upstream primer 5 ' end adds the HindIII restriction enzyme site, CGG is as the protectiveness base, downstream primer 5 ' end interpolation EcoRV restriction enzyme site:
(3) upstream primer 5 '-CGGAAGCTTATGGCGCTTCTCCGGGGCGTGT-3 ';
(4) downstream primer 5 '-CGGGATATCTCAACACTCCTCAGTGTCTTTC-3 ';
(5) reverse transcription product of the total RNA that extracts with porcine alveolar macrophage is a template, obtains transcription factor A with the upstream and downstream primer PCR amplification of design, passes through being connected to the corresponding restriction enzyme site of carrier for expression of eukaryon after enzyme is cut then.
10. a gene that contains paraoxon acid enzyme 3 is put the recombinant vectors of reading frame, it is characterized in that, and by the method preparation that may further comprise the steps,
(1) is paraoxon acid enzyme 3 gene orders of NM_001044604.1 from the Genbank accession number, chooses from sequence from initiator codon " ATG " to terminator codon " TGA ", the longest sequence 1065bp base " TAG " or " TAA ";
(2) respectively 5 ' and 3 ' end design amplification total length PCR primer, upstream primer 5 ' end adds the HindIII restriction enzyme site, CGG is as the protectiveness base, downstream primer 5 ' end interpolation EcoR I restriction enzyme site;
(3) upstream primer 5 '-CGGAAGCTTATGGGGAAGCTGGTGGCTCTGA-3 ';
(4) downstream primer 5 '-CGGGAATTCCTAGAGCACACAGTACAGAGCT-3 ';
(5) reverse transcription product of the total RNA that extracts with porcine alveolar macrophage is a template, obtains paraoxon acid enzyme 3 sequences with the upstream and downstream primer PCR amplification of design, is connected to the corresponding restriction enzyme site of eukaryotic vector after the process enzyme is cut then.
11., it is characterized in that the carrier for expression of eukaryon in the described step (5) is pCDNA 3.1 (+) carrier as claim 9 or 10 described recombinant vectorss.
12. as the purposes of each described recombinant vectors of claim 9-11 in preparation or screening Tamiflu.
13. a cell model that is used for screening of medicaments is characterized in that, contains each described recombinant vectors just like claim 9-11.
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