CN103215267B - Suppress siRNA and its application of influenza virus related gene - Google Patents

Suppress siRNA and its application of influenza virus related gene Download PDF

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CN103215267B
CN103215267B CN201210019712.9A CN201210019712A CN103215267B CN 103215267 B CN103215267 B CN 103215267B CN 201210019712 A CN201210019712 A CN 201210019712A CN 103215267 B CN103215267 B CN 103215267B
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sirna
influenza virus
gene
virus
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CN103215267A (en
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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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Priority to CN201610139670.0A priority patent/CN105586344B/en
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Abstract

The invention discloses the siRNA and recombinant vector for caveolin 2, Surfactant proteinD；Contain transcription factor A, the construction method of the transcription box recombinant vector of paraoxon acid enzyme 3 and corresponding cell model and the method for screening medicine.The invention has the advantages that the siRNA provided makes mRNA or protein expression through its mammalian cell after transfecting decline more than 60%；The siRNA and recombinant vector can be used for gene therapy influenza or carry out nucleic acid immunization for influenza virus；Cell model provided by the invention, available for virus research, particularly swine influenza virus pathogenesis, it can also be used to establish screening antiviral drugs model, particularly Tamiflu cell model.

Description

Suppress siRNA and its application of influenza virus related gene
Technical field
The invention belongs to biological technical field, more particularly to suppress caveolin 2 and Surfactant proteinD gene expression The recombinant vector and codified transcription factor A and paraoxon acid of siRNA, siRNA nucleotide sequence, the codified siRNA The recombinant vector of enzyme 3, and purposes of their own gene in terms of preparing and screening Tamiflu.
Background technology
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.Need to complete the viral duplication of itself using the metabolic process of host cell after viral infection host, in this process virus Will necessarily modulate host genes within cells group expression, suppress antiviral gene expression, strengthen the gene beneficial to virus replication Expression.Therefore, after viral host cells infected, these genes of host cell inner expression change often have closely with virus replication Relation.Regulate and control by one in these intracellular genes of biological means modulate host or simultaneously multiple gene expressions and may have Play the role of to suppress influenza virus duplication, there is the application 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.When virion just separates In polymorph, become globulate after passing in vivo, diameter 80-120nm (Palese, et al., 2007).Virion is by 0.8- The carbohydrate composition of 1% RNA, 70-75% protein, 20% lipid and 5-8%, ratio is mass ratio herein.
Influenza virus is divided into the type of A, B, C tri-, and wherein A types (A type) are mostly important.Influenza A virus can cause the mankind, The natural infection and morbidity of pig, horse and various birds.Because its surface antigen HA and NA easily make a variation, it is known that HA has 16 hypotypes (H1-H16), NA has 9 hypotypes (N1-N9), the various combination between them, influenza A virus is had 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, generally these influenzas are only Infect respective category animal.But influenza virus has the adaptability that host plants.Also hold between the individual of different animal species Easily propagate, form 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, and 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, will may come out it is a kind of can infected pigs, 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 relevant.Such as Flu-A in 2009, it is exactly caused by after human influenza, swine flu, bird flu co-infection pig New influenza virus, the people for most starting to infect, which is substantially, to be contacted or with pig people in close relations, then had more people's infection Influenza virus.Health care belt of the great outburst of influenza virus all to socio-economic development, people carrys out significant damage in human development history.For Influenza is prevented and treated, each state has all put into a large amount of manpower and materials, constantly studies treatment method and medicine.In recent years, from host The intracellular gene found with antiviral functions or albumen are a popular domains.
Health care belt of the great outburst of influenza virus all to socio-economic development, people carrys out significant damage in human development history.For Influenza is prevented and treated, each state has all put into a large amount of manpower and materials, constantly studies treatment method and medicine.In recent years, for place It is a popular domain that gene or albumen in chief cell with antiviral functions research and develop antiviral drugs as target spot.
Target spot using endocellular function molecule as antiviral therapy has broad-spectrum antiviral, is not likely to produce resistance strain Advantage, it is increasingly becoming the focus approach of research and development antiviral therapy medicine.High-level paper international in recent years is repeatedly reported in Molecule of the screening with potential antiviral functions breaks trough in signal path in host cell.For example, zinc finger is disease-resistant Toxalbumin (Zinc-finger antiviral protein, ZAP) can suppress the white blood of mouse by degrading particular virus RNA A variety of viral duplications such as virus, it is proved to be a kind of important antiviral agent (Chen et al. (2008) Proc.Natl.Acad.Sci.U S A.105,4352-4357).It is that the special enzyme of carrier carrying blocks T thin using zinc finger protein CCR5 expression in born of the same parents, can promote T cell to remove inhibition of HIV (Holt et al. (2010) Nat.Biotechnol.28,839- 847).E3 ubiquitin ligases RNF5 is positioned at mitochondria, by ubiquitination MITA (also referred to as STING) and causes its degraded And I types interferon expression (Zhong et al. (2009) Immunity.30,397-407) after negative regulation virus infection. With dimer grappling HIV on the cell membrane that it is replicated, blocking virus sprout Tetherin (host cell proteins) from endochylema film Discharge (Perez-Caballero et al. (2009) Cell.139,499-511).APOBEC3G(Apolipoprotein BmRNA-editing enzyme catalytic polypeptide-like 3G) HIV genetic mutations are can induce, 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 virosis such as shape knurl are treated.
RNAi is a kind of Gene silence of efficient high specificity, is quickly grown in recent years, soon as function The powerful of genome research.DsRNA molecules are imported by laboratory facilities it is intracellular, specifically in degradation of cell and its The mRNA of sequence homology, endogenous gene expression is closed, from the angle research mankind of reverse genetic or other biological genome The function of unknown gene.Early stage has separated various in drosophila insulin signal transduction approach path with regard to someone using technique Composition.Recently Experimental report also studies each bar approach being related in lipid within endothelial cells equilibrium process by RNAi.Before this, Once the generation of its phenotype was suppressed using the complementary characteristic of antisense RNA and said target mrna sequence, but because antisense RNA is to endogenous table The gene inhibitory action reached is weaker, often produces some transition phenotypes, easily causes to gene function misjudgment, led at present Cross examination ＆ approval and think clinically have a kind of medicative only medicine 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 mankind vacuole albumen Tsg101 therefore has been understood to HIV in the effect of human body internal breeding, further in-depth Research to HIV.Leonid etc. is immunized using poliovirus as model, using RNAi come the intracellular of inducing cell, is produced 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 cells using RNAi technology A kind of function of the nuclear transcription factor-2 gene 21 related to cell proliferation and differentiation of unconventionality expression.The application of RNAi technology, not only The development of human post-genome plan (protein science) can be promoted significantly, screened drug target gene with high throughput, detected people one by one The expression inhibiting situation of genoid group carrys out the function of clear and definite gene, and it will also be applied to gene therapy, new drug development, biology The fields such as medical research, with RNAi technology come the unconventionality expression of suppressor, new approach is opened 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 Break deeply, new drug target, functional protein, the change of gene expression, bioactive ingredients etc. are constantly found, are medicine Thing screening provides a large amount of new target spots, such as new acceptor, 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 automation mechanized operation, makes drug screening by tradition Manual screening form be changed into by computer control automation Large-scale Screening new technology system, form high flux medicine Thing screens.
The advantages of high-flux medicaments sifting：The scale of drug screening is realized, make use of medical substance to large extent Resource, improves the probability of drug discovery, while improves the quality for finding new drug；Screening experiment is in microscreen system Complete, amount of samples typically at Gamma Magnitude (μ g), saves sample resource, has established the material base of " medicine sieves more ", together When save experiment material, reduce single medicine screening cost；High-flux medicaments sifting reduces operation for increasingly automated operation The generation of error, labor intensity is reduced, and improve 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 drive the appearance of newtype drug. The completion of the development of molecular biology, cell biology, computer science, the particularly Human Genome Project, it is medical research Good opportunity is brought, also to establish new medicaments sifting model, there is provided many 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 Constructed dna vaccine plasmid on body, DNA vaccination after various approach are expelled in animal matrix, can by host cell absorb and Intake, is transcribed into mRNA, then protein is translated into endochylema into the nucleus of host cell.A portion protein Combined after degradation with MHCI quasi-molecules, and by submission to cell surface by the Receptor recognition of cd8 t cell, and active cell is malicious The activity of property T cell；Another part protein can also be secreted away, then be taken the photograph as foreign protein by antigen presenting cell Take, polypeptide is degraded into phagolysosome, and further combined with mhc class ii molecule, there is antigen presenting cell to offer to thin Cellular surface is by the Receptor recognition of Th2 cells, then by the Cytokine that Th2 cells are secreted in B cell, and stimulates with antibody Humoral immunity based on generation.DNA vaccination, which not only can induce, produces humoral immunity, and can induce strong and lasting cell and exempt from Epidemic disease, can also avoid outwardly toxin expelling, and DNA vaccination and attenuation and attenuated vaccine are different, and it is not present, and virulence is anti-strong etc. to ask Topic.Its security and high efficiency are that traditional vaccine is not reached, so, the technology is caused by virus, cytozoicus Huge potentiality are shown in infectious disease, parasite 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 DNA can in vivo suitable one section when It is interior to be expressed, therapeutic effect is may be used as, and cause extensive research.
Gene therapy is for cancer, atherosclerosis, osteoporosis, arthritis, Alzheimer's because specific Prevention and treatment of diseases caused by Gene Activity exception has 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 change their hereditary capacity.Generally, the nucleic acid is can to have encoded therapeutic action, The double chain DNA molecule (dsDNA) of the protein of destruction or mark effect.The nucleic acid be also likely to be with host cell Target sequence combine, by prevent mRNAs either promoter come suppress specific gene express antisense RNA or single stranded DNA (ssDNA).So far, the gene therapy based on plasmid, cancer vaccine etc. have been over 600, and the gene of DNA is exempted from Epidemic disease simulates the Gene Expression Pathway of pathogen in the cell, successfully applies to treat the ischaemic of lower limb, painstaking effort Pipe regenerates, 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 can not be integrated into genomic DNA and may is that a shortcoming, but plasmid can be with episome Form be present in nucleus, can also continuous expression for quite a long time.
The content of the invention
Therefore, the technical problem to be solved in the present invention is exactly that design suppresses to replicate related gene with influenza virus in host SiRNA sequence, structure can encode the recombinant vector of the siRNA, and the recombinant vector containing the sequence open reading frame Application in terms of Tamiflu is prepared.
One of technical scheme is used by present invention solution above-mentioned technical problem：It is a kind of for the gene of caveolin 2 SiRNA, wherein target sequence corresponding to siRNA of the present invention is the mRNA of the gene of caveolin 2, it is of the present invention SiRNA length is 16-30bp, and can make through its transfect after mammalian cell mRNA or protein expression decline 60% with On.
Wherein described mammal includes being preferably pig 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, it is more preferably 18-25bp.Preferably, described siRNA Containing selected from SEQ ID NO：Nucleotide sequence shown in 1, or such as SEQ ID NO in sequence table；1 represents.More preferably, it is described SiRNA 3 ' extended structures of the end containing 2 T or containing 2 U.
The two of technical scheme are used by present invention solution above-mentioned technical problem：A kind of suppression alveole egg described in coding The siRNA of white 2 expression recombinant vector.
According to the present invention, carrier that described recombinant vector uses can be the conventional carrier in this area, including adenovirus Carrier, such as various adenovirus vectors based on 5 types (Ad5), 2 types (Ad2), slow virus carrier, selected from HIV-1 type carriers, HIV-2 type carriers, SIV type carriers, FIV type carriers etc..The siRNA fragment is cloned into vector plasmid, produces the present invention Recombinant vector.
The three of technical scheme are used by present invention solution above-mentioned technical problem：A kind of composition, wherein containing 0.001-99.99wt% siRNA and acceptable carrier, the diluent or excipient of the present invention for caveolin 2. Wherein, described carrier, diluent or excipient, are more preferably pharmaceutically acceptable carrier, diluent or excipient, including The carrier material such as water-soluble, slightly solubility or enteric solubility, or powder, dextrin, Icing Sugar, the hydrate of calcium sulfate two, calcium monohydrogen phosphate, oxidation One or more in magnesium, magnesium carbonate, calcium carbonate, gel aluminum hydroxide powder and activated carbon.
The four of technical scheme are used by present invention solution above-mentioned technical problem：Described siRNA is being prepared or screened Purposes in Tamiflu.Wherein, described siRNA can be used as unique anti-influenza virus activity composition, can also be with Other anti-influenza virus medicaments are used in combination.
SiRNA of the present invention can more preferably be adopted the following technical scheme that in the purposes for preparing Tamiflu：Such as It is administered by way of suction, it is not only easy to use, and its drug concentration in infection site can be increased.Also, due to Infection early stage viral load is small, and sufficient siRNA can more effectively suppress the duplication of virus, so as to play an effect for prevention and treatment Fruit；The selection of RNAi drug targets is more in influenza, can be not likely to produce resistance strain with compound or multiple medication；Last RNAi with Influenza vaccines are different, it is not necessary to which user has sound immune system.
The present invention can also be adopted the following technical scheme that more preferably：Such as pass through intravenous injection or intramuscular injection siRNA sequence Or encode the modes such as the siRNA recombinant vectors, make the siRNA be able in vivo efficiently, persistent table is so as to reach RNA up to exempting from Epidemic disease or the purpose of gene therapy influenza virus.
The five of technical scheme are used by present invention solution above-mentioned technical problem：Described siRNA is suppressing influenza disease Purposes in poison duplication.
The six of technical scheme are used by present invention solution above-mentioned technical problem：A kind of side for screening Tamiflu Method, comprise the following steps：
(1) cells contacting of drug candidate and influenza virus infection is made, described cell is the gene silencing of caveolin 2 Cell；
(2) Influenza virus titer is tested；
(3) selection makes the drug candidate that Influenza virus titer declines.
Wherein, the silenced gene expression cell of caveolin 2 is by containing host cell more preferably described in step (1) The recombinant vector that the siRNA of the gene either has the siRNA containing coding makes the gene silencing.
Wherein, the cell can use 293T (people's renal epithelial cell system), BHK (hamster kidney cell), VERO (African green MK cells), the cell line such as IBRS22 (porcine kidney cell) or MDCK (MDCK), the preferred mdck cell of the present invention.
Wherein, contacted by the cell with influenza virus, through observing cell damage evil under the microscope after a period of time Degree, compare with Tamiflu handle and check experiment cell in influenza virus virus titer, this can be known Whether antiviral drug can reduce infringement of the virus to cell.
The method of the invention, it can more preferably use following scheme：
Medicine cytotoxicity detection, mdck cell cultivate in 96 porocyte culture plates form individual layer after, add difference The decoction of concentration, continue culture 3 days, toxicity of the medicine to mdck cell is detected with mtt assay.
Medicine resisiting influenza virus effect detection, mdck cell cultivate in 96 porocyte culture plates form individual layer after, use 100TCID50 influenza infection cell, the drug containing nutrient solution of the series concentration added under non-toxic concn, continues culture 3 My god, the inhibitory action of medicine infected by influenza is determined through CPE methods.
The method of the invention, it 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 replicating, RdRp is that template catalysis generates the RNA being complementary to virus genome RNA (vRNA) Chain (cRNA), cRNA then can be fitted into progeny virus as templated synthesis vRNA.For transcription, RdRp is in synthesis cRNA On the basis of its 3 ' end add poly A tract, 5 ' end add cap sequences, formed maturation mRNA carry out protein synthesis.This Experiment makes ATP concentration in system gradually reduce according to consumption substrate A TP, UTP, CTP and GTP during synthesis RNA chains, in measure system Remaining ATP contents react carry out degree to reflect.
By detecting influenza virus RNA polymerase change level in the cell line, the confrontation of the silenced cell of caveolin 2 is assessed The sensitivity of flu pharmaceutical.
Wherein described virus titer detection method, MTT methods, CPE methods, it is that this area is normal the methods of cell culture Advise experimental method.
The seven of technical scheme are used by present invention solution above-mentioned technical problem：One kind is directed to Surfactant proteinD base The siRNA of cause, target sequence corresponding to described siRNA are the mRNA of Surfactant proteinD gene, and the length of the siRNA is 16-30bp, and mRNA or expressing quantity through its mammalian cell after transfecting can be made to decline more than 60%.
Wherein described mammal includes being preferably pig 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, described siRNA contains Have and be selected from SEQ ID NO：Nucleotide sequence shown in 2, or such as SEQ ID NO in sequence table：2 represent.Described siRNA, more Good ground sequence 3 ' holds the extended structure containing 2 T or containing 2 U.
The eight of technical scheme are used by present invention solution above-mentioned technical problem：Live on a kind of suppression surface described in coding Property protein D expression siRNA recombinant vector.
According to the present invention, carrier that described recombinant vector uses can be the conventional carrier in this area, including adenovirus Carrier, including the various adenovirus vectors based on 5 types (Ad5), 2 types (Ad2), slow virus carrier include HIV-1 type carriers, HIV-2 type carriers, SIV type carriers, FIV type carriers etc...The siRNA fragment is cloned into vector plasmid, produces the present invention Recombinant vector.
The nine of technical scheme are used by present invention solution above-mentioned technical problem：A kind of composition, wherein containing The 0.001-99.99wt% siRNA sequences and acceptable carrier, diluent of the present invention for Surfactant proteinD Or excipient.Wherein described carrier, diluent and excipient, more preferably it is pharmaceutically acceptable carrier, diluent or tax Shape agent.Carrier material, powder, dextrin, Icing Sugar, the hydrate of calcium sulfate two, the phosphoric acid such as example, water-soluble, slightly solubility or enteric solubility One or more in hydrogen calcium, magnesia, magnesium carbonate, calcium carbonate, gel aluminum hydroxide powder and nano material.
The ten of technical scheme are used by present invention solution above-mentioned technical problem：Described suppression Surfactant proteinD SiRNA prepare or screen Tamiflu in purposes.Wherein, described siRNA can be used as unique anti-current susceptible Cytotoxic activity composition, it can also be used in combination with other anti-influenza virus medicaments.
Wherein, can more preferably adopt the following technical scheme that：Such as it is administered by way of suction, it is not only easy to use, and Its drug concentration in infection site can be increased.Also, because infection early stage viral load is small, sufficient siRNA can be more Effectively suppress the duplication of virus, so as to play the effect of prevention and treatment；RNAi drug targets select more, Ke Yifu in influenza Square or multiple medication, is not likely to produce resistance strain；Last RNAi is different from influenza vaccines, it is not necessary to which user has sound exempt from Epidemic disease system.
The present invention can also be adopted the following technical scheme that more preferably：Such as pass through intravenous injection or intramuscular injection siRNA sequence Or encode the modes such as the siRNA recombinant vectors, make the siRNA be able in vivo efficiently, persistent table is so as to reach RNA up to exempting from Epidemic disease or the purpose of gene therapy influenza virus.
The 11 of technical scheme are used by present invention solution above-mentioned technical problem：A kind of side for screening Tamiflu Method, it is characterised in that comprise the following steps：
(1) cells contacting of drug candidate and influenza virus infection is made, described cell is that Surfactant proteinD gene sinks Silent cell；
(2) Influenza virus titer is tested：
(3) selection makes the drug candidate that Influenza virus titer declines.
Surfactant proteinD silenced gene expression cell is logical described in method of the present invention, more preferably step (1) Cross the siRNA for making host cell contain the gene or make containing the recombinant vector that coding has the siRNA The gene silencing.
Wherein, the cell can use 293T (people's renal epithelial cell system), BHK (hamster kidney cell), VERO (African green MK cells), the cell line such as IBRS22 (porcine kidney cell) or MDCK (MDCK), the preferred MDCK of the present invention.
The method of the invention, contacted by the cell with influenza virus, through observing under the microscope after a period of time The degree of cell damage evil, compare 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 reduce infringement of the virus to cell.
Wherein, following scheme can more preferably be used：
(1) medicine cytotoxicity detection, mdck cell cultivate in 96 porocyte culture plates form individual layer after, addition not With the decoction of concentration, continue culture 3 days, toxicity of the medicine to mdck cell is detected with mtt assay.
(2) medicine resisiting influenza virus effect detection, mdck cell cultivate in 96 porocyte culture plates form individual layer after, use 100TCID50 influenza infection cell, the drug containing nutrient solution of the series concentration added under non-toxic concn, continues culture 3 My god, the inhibitory action of medicine infected by influenza is determined through CPE methods.
The method of the invention, it 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 replicating, RdRp is that template catalysis generation is complementary to virus genome RNA (vRNA) RNA chains (cRNA), cRNA then can be fitted into progeny virus as templated synthesis vRNA.
For transcription, RdRp adds poly A tract on the basis of cRNA is synthesized at its 3 ' end, and 5 ' ends add cap sequence, Form ripe mRNA and carry out protein synthesis.This experiment makes according to consumption substrate A TP, UTP, CTP and GTP during synthesis RNA chains ATP concentration gradually reduces in system, and remaining ATP contents react carry out degree to reflect in measure system.
(2) by detecting influenza virus RNA polymerase change level in the cell line, Surfactant proteinD gene is assessed Sensitivity of the silenced cell to Tamiflu.
Wherein described virus titer detection method, MTT methods, CPE methods, it is that this area is normal the methods of cell culture Advise experimental method.
The 12 of technical scheme are used by present invention solution above-mentioned technical problem：A kind of base containing transcription factor A Because of the recombinant vector of ORFs.Preferably, the carrier is obtained by following preparation method：
(1) the transcription factor A gene orders that accession number in Genbank is NM 001130211.1 are input to DNAStar In software (DNASTAR companies, the U.S.) EditSeq, choose sequence in from initiation codon " ATG " to terminator codon " TGA ", Most long sequence 741bp bases between " TAG " or " TAA "；
(2) HindIII restriction enzyme sites are added at 5 ' and 3 ' end design amplification total length PCR primers, the end of sense primer 5 ' respectively, CGG is as protectiveness base, the end of anti-sense primer 5 ' addition EcoR V restriction enzyme sites；
(3)-the CGGAAGCTTATGGCGCTTCTCCGGGGCGTGT-3 ' of sense primer 5 '；
(4)-the CGGGATATCTCAACACTCCTCAGTGTCTTTC-3 ' of anti-sense primer 5 '；
(5) using the reverse transcription product of the total serum IgE of porcine alveolar macrophage extraction as template, with the upstream and downstream primer of design PCR is expanded to obtain transcription factor A sequences, 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 use pCMVp-NEO-BAN, pEGFP, pEGFT-Actin, pSV2, CMV4 etc. Common carrier for expression of eukaryon, in of the invention, more preferably using pCDNA 3.1 (+) carrier.
The 13 of technical scheme are used by present invention solution above-mentioned technical problem：Contain transcription factor described in a kind of Purposes of the recombinant vector of A ORFs in preparing or screening Tamiflu.
The recombinant vector more preferably, can be used to transfect different cell lines by the present invention, and the method for transfection can be, DEAE- glucan methods, 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 eukaryotics, the preferred MDCK of the technical program (MDCK).
The recombinant vector can also be used for gene therapy by the present invention, can such as pass through intravenous injection or intramuscular injection side Formula, be able to carry out in vivo efficiently, persistent table reached so as to reaching the purpose of DNA immunization or gene therapy influenza virus.
The 14 of technical scheme are used by present invention solution above-mentioned technical problem：A kind of cell for being used to screen medicine Model, it contains the described recombinant vector containing transcription factor A ORFs.
Wherein, described cell can be various eukaryotics, including 293T (people's renal epithelial cell system), BHK (hamster kidneys Cell), VERO (African green monkey kidney cell), the cell line such as IBRS22 (porcine kidney cell) and MDCK (MDCK) etc..
The 15 of technical scheme are used by present invention solution above-mentioned technical problem：A kind of side for screening Tamiflu Method, the method for the invention, comprise the following steps：
(1) cells contacting of drug candidate and influenza virus infection is made, described cell is to be overexpressed transcription factor A genes Cell；
(2) Influenza virus titer is tested；
(3) selection makes the drug candidate that Influenza virus titer declines.
The method of the invention, the cell for being overexpressed the gene described in step (1), more preferably it is intracellular by increasing The copy number of the gene improves the controlling element of the gene and makes the gene overexpression.
The method of the invention, the cell can be selected from 293T (people's renal epithelial cell system), BHK (hamster kidney cell), The cell lines such as VERO (African green monkey kidney cell), IBRS22 (porcine kidney cell) and MDCK (MDCK), the preferred MDCK of the present invention.
The method of the invention, contacted by the cell with influenza virus, through observing 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 reduce infringement of the virus to cell.
The method of the invention, it can more preferably use following scheme：
(1) medicine cytotoxicity detection, mdck cell cultivate in 96 porocyte culture plates form individual layer after, addition not With the decoction of concentration, continue culture 3 days, toxicity of the medicine to mdck cell is detected with mtt assay.
(2) medicine resisiting influenza virus effect detection, mdck cell cultivate in 96 porocyte culture plates form individual layer after, use 100TCID50 influenza infection cell, the drug containing nutrient solution of the series concentration added under non-toxic concn, continues culture 3 My god, the inhibitory action of medicine infected by influenza is determined through CPE methods.
The method of the invention, it 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 replicating, RdRp is that template catalysis generation is complementary to virus genome RNA (vRNA) RNA chains (cRNA), cRNA then can be fitted into progeny virus as templated synthesis vRNA.For transcription, RdRp is being synthesized Poly A tract is added at its 3 ' end on the basis of cRNA, 5 ' ends add cap sequence, and the mRNA for forming maturation carries out protein conjunction Into.This experiment makes ATP concentration in system gradually reduce according to consumption substrate A TP, UTP, CTP and GTP during synthesis RNA chains, determines Remaining ATP contents react carry out degree to reflect in system.
(2) by detecting influenza virus RNA polymerase change level in the cell line, transcription factor A genes is assessed and cross table Sensitivity up to cell to Tamiflu.
Wherein described virus titer detection method, MTT methods, CPE methods, it is that this area is normal the methods of cell culture Advise experimental method.
The 16 of technical scheme are used by present invention solution above-mentioned technical problem：One kind contains the base of paraoxon acid enzyme 3 The recombinant vector of the ORFs of cause,.Preferably, the carrier is obtained by following preparation method：
(1) accession number is in NM 001044604.1 paraoxon acid enzyme 3 from Genbank, is chosen from sequence from Beginning codon " ATG " arrives most long sequence 1065bp bases between terminator codon " TGA ", " TAG " or " TAA "；
(2) HindIII restriction enzyme sites are added at 5 ' and 3 ' end design amplification total length PCR primers, the end of sense primer 5 ' respectively, CGG is as protectiveness base, the end of anti-sense primer 5 ' addition EcoR I restriction enzyme sites；
(3)-the CGGAAGCTTATGGGGAAGCTGGTGGCTCTGA-3 ' of sense primer 5 '；
(4)-the CGGGAATTCCTAGAGCACACAGTACAGAGCT-3 ' of anti-sense primer 5 '；
(5) using the reverse transcription product of the total serum IgE of porcine alveolar macrophage extraction as template, with the upstream and downstream primer of design PCR is expanded to obtain transcription factor A sequences, 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 use pCMVp-NEO-BAN, pEGFP, pEGFT-Actin, pSV2, CMV4 etc. Common carrier for expression of eukaryon, in of the invention, more preferably using pCDNA 3.1 (+) carrier.
The 17 of technical scheme are used by present invention solution above-mentioned technical problem：It is a kind of of the present invention containing right Purposes of the recombinant vector of the ORFs of oxygen phosphatase 3 in preparing or screening Tamiflu.
The recombinant vector more preferably, can be used to transfect different cell lines by the present invention, and the method for transfection can be, DEAE- glucan methods, 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 eukaryotics, the preferred MDCK of the technical program (MDCK).
The recombinant vector can also be used for gene therapy by the present invention, can such as pass through intravenous injection or intramuscular injection side Formula, be able to carry out in vivo efficiently, persistent table reached so as to reaching the purpose of DNA immunization or gene therapy influenza virus.
The 18 of technical scheme are used by present invention solution above-mentioned technical problem：A kind of cell for being used to screen medicine Model, it contains the recombinant vector of the described ORFs containing the gene of paraoxon acid enzyme 3.
Wherein, described cell can be various eukaryotics, selected from 293T (people's renal epithelial cell system), BHK (hamster kidneys Cell), VERO (African green monkey kidney cell), IBRS22 (porcine kidney cell) and MDCK (MDCK) cell line etc..
The 19 of technical scheme are used by present invention solution above-mentioned technical problem：A kind of side for screening Tamiflu Method, the method for the invention, comprise the following steps：
(1) cells contacting of drug candidate and influenza virus infection is made, described cell is to be overexpressed the base of paraoxon acid enzyme 3 The cell of cause；
(2) Influenza virus titer is tested；
(3) selection makes the drug candidate that Influenza virus titer declines.
The method of the invention, the cell for being overexpressed the gene described in step (1), more preferably it is intracellular by increasing The copy number of the gene improves the controlling element of the gene and makes the gene overexpression.
The method of the invention, the cell can use 293T (people's renal epithelial cell system), BHK (hamster kidney cell), The cell lines such as VERO (African green monkey kidney cell), IBRS22 (porcine kidney cell) and MDCK (MDCK), the preferred MDCK of the present invention.
The method of the invention, contacted by the cell with influenza virus, through observing 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 reduce infringement of the virus to cell.
The method of the invention, it can more preferably use following scheme：
(1) medicine cytotoxicity detection, mdck cell cultivate in 96 porocyte culture plates form individual layer after, addition not With the decoction of concentration, continue culture 3 days, toxicity of the medicine to mdck cell is detected with mtt assay.
(2) medicine resisiting influenza virus effect detection, mdck cell cultivate in 96 porocyte culture plates form individual layer after, use 100TCID50 influenza infection cell, the drug containing nutrient solution of the series concentration added under non-toxic concn, continues culture 3 My god, the inhibitory action of medicine infected by influenza is determined through CPE methods.
The method of the invention, it 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 replicating, RdRp is that template catalysis generation is complementary to virus genome RNA (vRNA) RNA chains (cRNA), cRNA then can be fitted into progeny virus as templated synthesis vRNA.For transcription, RdRp is being synthesized Poly A tract is added at its 3 ' end on the basis of cRNA, 5 ' ends add cap sequence, and the mRNA for forming maturation carries out protein conjunction Into.This experiment makes ATP concentration in system gradually reduce according to consumption substrate A TP, UTP, CTP and GTP during synthesis RNA chains, determines Remaining ATP contents react carry out degree to reflect in system.
(2) by detecting influenza virus RNA polymerase change level in the cell line, the gene mistake of paraoxon acid enzyme 3 is assessed Sensitivity of the expression cell to Tamiflu.
Wherein described virus titer detection method, MTT methods, CPE methods, it is that this area is normal the methods of cell culture Advise experimental method.
It is as follows compared to prior art, beneficial effects of the present invention：Invention is disease-resistant to have in host cell The gene or albumen of malicious function are target spot, there is provided the medicine of resisiting influenza virus and the method for suppressing influenza virus duplication.This Invention is achieved by the expression of the gene in regulating cell.Invention is with prevention and treatment influenza infection Application value, to developing more preferable Tamiflu, the great outburst to controlling influenza virus, reduce the great outburst pair of influenza virus The significant damage that socio-economic development, the health care belt of people come, has great importance.
Brief description of the drawings
Below in conjunction with the feature and beneficial effect of the brief description of the drawings present invention.
Fig. 1 is the result of the expression of Western-blot detection silence MDCKs caveolin 2, wherein 1：SiRNA- is small Nest albumen 2；2：SiRNA- caveolins 2 compare；3：Malicious control is not connect；4：Normal cell.
Fig. 2 is the result that Surfactant proteinD is expressed in Western-blot detection silence MDCKs, wherein 1： SiRNA- Surfactant proteinDs；2：SiRNA- Surfactant proteinDs compare；3；Malicious control is not connect；4：Normal cell.
Fig. 3 is Influenza virus titer figure after silence MDCK caveolin 2 is expressed.
Fig. 4 be in silence MDCK Surfactant proteinD expression after Influenza virus titer figure.
Fig. 5 is the pCDNA 3.1- transcription factor A Vector maps of structure.
Fig. 6 is the Vector map of pCDNA 3.1- paraoxon acid enzyme 3 of structure.
Fig. 7 is that factors A and the immune-blotting method result of paraoxon acid enzyme 3 are transcribed in cell, wherein
1：PCDNA 3.1- transcription factors A；2：The empty vector controls of pCDNA 3.1；3：Normal cell
4：PCDNA 3.1- paraoxon acid enzyme 3；5：The empty vector controls of pCDNA 3.1；6：Normal cell.
Fig. 8 is to transcribe Influenza virus titer result after factors A in external source raising expression MDCK.
Fig. 9 is that external source improves in expression MDCK Influenza virus titer result after paraoxon acid enzyme 3.
Figure 10 is to add Influenza virus titer figure after amantadine in the MDCK of the silence of caveolin 2.
Figure 11 is to add Influenza virus titer figure after amantadine in the MDCK of Surfactant proteinD silence.
Figure 12 is to add Influenza virus titer figure after amantadine in the MDCK of external source raising transcription factor A expression.
Figure 13 is to add Influenza virus titer after amantadine in the MDCK of the expression of external source raising paraoxon acid enzyme 3 Figure.
Embodiment
The present invention is further illustrated with embodiment below, but the present invention is not intended to be limited thereto.
Reagent used is commercially available in addition to special instruction in embodiment.
The experimental method of unreceipted actual conditions in the following example, generally 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, its biology The swine influenza virus feature in China can be represented by learning characteristic, from the Chinese Academy of Agricultural Sciences's Shanghai veterinary institute pig Infectious Disease Research department.
6 porocyte culture plates, U.S.'s Coming products.
DMEM culture mediums, U.S.'s GIBCO products, REF：16000-044.
Penicillin, U.S.'s GIBCO Products, REF：15140-122.
Streptomysin, U.S.'s GIBCO Products, REF：15140-122.
MDCK (mdck cell), purchased from Shanghai life science institute of Chinese Academy of Sciences cell bank.
1.5ml centrifuge tubes, U.S.'s Axygen Products.
Acetone, Jiangsu Qiangsheng Chemical Co., Ltd., credit number：XK13-201-00227.
PBS, purchased from GIBCO companies of the U.S., REF：20012-027.
PBST：Compound method refers to《Molecular Cloning:A Laboratory guide》The third edition.
The fluorescence antibody of sheep anti-Mouse FITC marks, U.S.'s Invitrogen Products, Code：CA11034s.
100% glycerine, Chemical Reagent Co., Ltd., Sinopharm Group.
The modulate host genes within cells expression inhibiting influenza virus of embodiment 1 is replicated
Gene expression inhibition influenza virus is replicated in 1.siRNA (siRNA) silence host cell
(1) siRNA molecule designs
The present embodiment selects the gene of caveolin 2, GeneID：100125375；Surfactant proteinD, GeneID： 397198, use Dharmacon Photographing On-line Software for Design siRNA molecules, the siRNA of the design gene specific of caveolin 2 Interference sequence；And arbitrarily upset sequence using " siRNA- caveolins " as control.Design surface activated protein D gene specifics SiRNA interference sequences；And arbitrarily upset sequence using " siRNA- Surfactant proteinDs " 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-the GCAAATACGTGATCTACAA-3 ' of 2 target sequence of caveolin 5 '；
- the GCAAAUACGUGAUCUACAAUU-3 ' of 2 sense strand sequence of siRNA- caveolins 5 '；
- the TTCGUUUAUGCACUAGAUGUU-5 ' of 2 antisense strand sequence of siRNA- caveolins 3 '；
SiRNA- caveolins 2 compare the-CGAUAGAUGUACACAUACAUU-3 ' of sense strand sequence 5 '；
SiRNA- caveolins 2 compare the-TTGCUAUCUACAUGUGUAUGU-5 ' of antisense strand sequence 3 '；
For-the GAGCAGAAATGAAGACCTA-3 ' of Surfactant proteinD target sequence 5 '；
- the GAGCAGAAAUGAAGACCUAUU-3 ' of siRNA- Surfactant proteinDs sense strand sequence 5 '；
- the TTCUCGUCUUUACUUCUGGAU-5 ' of siRNA- Surfactant proteinDs antisense strand sequence 3 '；
SiRNA- Surfactant proteinDs control positive-sense strand 5 '-CAGAGUGACAGAAGACAUAUU-3 '；
SiRNA- Surfactant proteinDs control antisense strand 3 '-TTGUCUCACUGUCUUCUGUAU-5 '.
It is small through NCBI blast Analysis interferences sequences and the genetic homology beyond " caveolin or Surfactant proteinD " In 50%, control sequence is less than 50% with pig genome all sequences homology.
According to the siRNA sequence of the design, by Dharmacon companies artificial synthesized double-strand siRNA in vitro.
(2) siRNA molecule transfectional cell
By DMEM (U.S.'s GIBCO products, the REF of the mdck cell serum-free of pancreatin digestion：16000-044) (100 Units of Penicillin (U.S.'s GIBCO Products, REF：15140-122) (GIBCO companies of the U.S. produce with 100 unit streptomysins Product, REF：15140-122)) pass in 6 porocyte culture plates (U.S.'s Coming products), cell density 4 × 106Cells/well, Continue to be incubated at 37 DEG C, 5%CO2In incubator.Treat the 80-90%, double-strand siRNA of the present invention at the full culture plate bottom of cell growth Molecule FuGENE HD reagent (Roche companies, the U.S., production code member：04709705001) it is transfected into mdck cell, operates Process by specification.Transfection control interference sequence sample siRNA- caveolins 2 are set simultaneously to compare, be untransfected control, normal thin Born of the same parents compare.
(3) siRNA transfectional cells connect poison and sample collection
24h inoculation influenza virus H3N2, dosage of inoculation 1 × 10 after mdck cell transfection siRNA molecule3EID50/ hole (6 holes Plate).24h, 48h, 72h collect sample after virus inoculation.Method is as follows：Trained by cell in Tissue Culture Plate, together with cell Nutrient solution freeze thawing 3 times, 5000g/min centrifugation 10min, retains supernatant, abandons precipitation.Determine Influenza virus titer in supernatant.Virus drop Spend assay method reference《National influenza central standard operational procedure (revised edition), 2007》.
Experimental result (Fig. 1) shows that mdck cell caveolin 2, Surfactant proteinD expression are sunk by siRNA molecule Influenza virus is inoculated with after silent, Influenza virus titer is compared with control group and significantly reduced in 24h, 48h, 72h cell, and difference Significantly.
Cell caveolin 2 is by (as above 24h samples after virus inoculation), Western- after siRNA molecule silence Blot testing results are shown in Fig. 1, the results showed that the expression of silence group caveolin 2 is significantly lower than control.
Surfactant proteinD is by (as above 24h samples after virus inoculation) after siRNA molecule silence in cell, Western-blot testing results are shown in Fig. 2, the results showed that Surfactant proteinD expression is significantly lower than control in silence group.
SiRNA- caveolins 2 compared with the control group of siRNA- caveolins 2 p value be respectively p=0.0004 (24h), 0.001(48h)、0.015(72h)；SiRNA- Surfactant proteinDs compare p value point with siRNA- Surfactant proteinD control groups Wei not p=0.004 (24h), 0.001 (48h), 0.009 (72h).
Above-mentioned the results show by regulate and control caveolin 2 and surfactant protein gene expression can to suppress host thin The duplication of intracellular influenza virus.
2. gene expression inhibition influenza virus is replicated in exogenous raising host cell
(1) in the 1st group of heterogenous expression expression vector structure
By transcription factor A (TFAM) (GeneID：397279；Gene database accession number：NM_001130211.1) connect To pCDNA 3.1 (+) carrier (Invitrogen companies, the U.S.；Article No.：V790-20) HindIII and EcoR V restriction enzyme sites Between, specific construction method comprises the following steps：
(1) the transcription factor A gene orders that accession number in Genbank is NM_001130211.1 are input to DNAStar In software (DNASTAR companies, the U.S.) EditSeq, choose sequence in from initiation codon " ATG " to terminator codon " TGA ", Most long sequence 741bp bases between " TAG " or " TAA "；
(2) HindIII restriction enzyme sites are added at 5 ' and 3 ' end design amplification total length PCR primers, the end of sense primer 5 ' respectively, CGG is as protectiveness base, the end of anti-sense primer 5 ' addition EcoR V restriction enzyme sites；
(3)-the CGG of sense primer 5 '(double-crossed is HindIII enzymes to ATGGCGCTTCTCCGGGGCGTGT-3 ' Enzyme site)；
(4)-the CGG of anti-sense primer 5 '(double-crossed is EcoR V digestions to TCAACACTCCTCAGTGTCTTTC-3 ' Site)；
(5) total serum IgE reverse transcription product is extracted as template using pig pulmonary macrophage, expanded with the upstream and downstream primer PCR of design Transcription factor A sequences are obtained, the corresponding restriction enzyme site of pCDNA 3.1 (+) carrier is then connected to after digestion.Successfully construct Carrier is named as pCDNA 3.1- transcription factor A, sees Fig. 5.
By paraoxon acid enzyme 3 (PON3) (GeneID：733674；Gene database accession number：NM_001044604.1) base Because ORFs is connected to pCDNA 3.1 (+) carrier (Invitrogen companies, the U.S.；Article No.：V790-20) HindIII and Between EcoR I restriction enzyme sites, specific construction method comprises the following steps：
(1) gene order of paraoxon acid enzyme 3 that accession number in Genbank is NM_001044604.1 is input to In DNAStar softwares (DNASTAR companies, the U.S.) EditSeq, choose close from initiation codon " ATG " to terminating from sequence Most long sequence 1065bp bases between numeral " TGA ", " TAG " or " TAA "；
(2) HindIII restriction enzyme sites are added at 5 ' and 3 ' end design amplification total length PCR primers, the end of sense primer 5 ' respectively, CGG is as protectiveness base, the end of anti-sense primer 5 ' addition EcoR I restriction enzyme sites；
(3)-the CGG of sense primer 5 '(double-crossed is HindIII enzymes to ATGGGGAAGCTGGTGGCTCTGA-3 ' Enzyme site)；
(4)-the CGG of anti-sense primer 5 '(double-crossed is EcoR I digestions to CTAGAGCACACAGTACAGAGCT-3 ' Site)；
(5) total serum IgE reverse transcription product is extracted as template using pig pulmonary macrophage, expanded with the upstream and downstream primer PCR of design Transcription factor A sequences are obtained, the corresponding restriction enzyme site of pCDNA 3.1 (+) carrier is then connected to after digestion.Successfully construct Carrier is named as pCDNA 3.1- paraoxon acid enzyme 3, sees Fig. 6.
(2) recombinant vector transfectional cell and expressive host albumen
By DMEM (U.S.'s GIBCO products, the REF of the mdck cell serum-free of pancreatin digestion：16000-044) 100 Units of Penicillin (U.S.'s GIBCO Products, REF：15140-122) (GIBCO companies of the U.S. produce with 100 unit streptomysins Product, REF：15140-122)) pass in 6 porocyte culture plates (U.S.'s Coming products), cell density 4 × 106Cells/well, Continue to be incubated at 37 °C, in 5%CO2 incubators.The 80-90% at the full culture plate bottom of cell growth is treated, with FuGENE HD reagents (Roche companies, the U.S., production code member：04709705001) pCDNA3.1- transcription factor A, pCDNA 3.1- paraoxon acid The carrier of enzyme 3 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 compare.
24h after cell transfecting, collection 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 2 times in 6 porocyte plates are rinsed with PBS, so Afterwards plus 1ml PBS scrape cell with cell spatula, 4 DEG C, 3000g centrifugation 5min sedimentation cells, abandon supernatant.To cell precipitation plus Enter 100 μ l cell pyrolysis liquid (green skies company, production code member：P0013), cell is resuspended, 5min, Ran Houyong are cracked on ice Ultrasonic cell disruption instrument (Sonics companies, the U.S., model VCX105PB) is instantaneously broken (40HZ, 1S), boiling water boiling 5min, and 4 13000g centrifuges 10min at DEG C, discards precipitation.Take 2 μ l albumen supernatant BCA kit (green skies company, production code members： P0012 protein concentration) is determined, is operated to specifications.Remaining albumen supernatant adds 5 × SDS PAGE buffer (compound methods See《Molecular Cloning:A Laboratory guide》The third edition), boiling water boiling 5min, 13000g centrifugation 5min at 4 DEG C, take supernatant to carry out protein electricity Swimming.
(step refers to for denaturing SDS-PAGE gel electrophoresis and albumen transfer《Molecular Cloning:A Laboratory guide》The third edition)：Each Protein sample applied sample amount is 30 μ g, SDS-PAGE electrophoresis apparatus (Liuyi Instruments Plant, Beijing) 80V voltage lamination albumen, and 120V voltages divide From albumen, until electrophoresis terminates.Albumen transfer uses nitrocellulose filter (Whatman companies, the U.S., product type： 10401396).Bole's electrophoresis tank constant pressure 65V, 2h.Transfer terminates to carry out in 5% (v/v) of rear NC films immersion TBST- skimmed milks Closing.After room temperature effect 2h, confining liquid is discarded, is rinsed 3 times, washed with TBST (pH7.6) buffer solution containing 1% (v/v) polysorbas20 Go to remain skimmed milk, you can carry out antibody incubation.
Antibody response and colour developing：Add primary antibody, paraoxon acid enzyme 3 antibody (Abcam, the U.S., production code member：Ab40969), Transcription factor A antibody (antikoerper-online.de companies, Germany, production code member：ABIN484435).Jog shakes at 4 DEG C Swing overnight (12h-16h), after then washing 3 times with 100%TBST, each 5min.HRP (horseradish peroxidase) marks are added afterwards The secondary antibody of note, room temperature effect 2h, after being rinsed 3 times with TBST, each 5min.ECL kits (compile by Pierce companies, the U.S., product Number：32106) colour developing operates in darkroom.Operation is according to ECL kit specifications.
Detect internal reference control：The film to have developed the color immersion antibody is stripped off into liquid (green skies company, production code member：P0025 in), 50 DEG C of incubation 30min, shake once per 10min, then with TBST buffer solution (compound methods：With reference to《Molecular Cloning: A Laboratory refers to South》The third edition) rinsing 3 times, the addition re-closed 15min of confining liquid, using anti-β-actin antibody, (green skies company, product are compiled Number：AA128) as protein content in primary antibody detection 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, in cell " transcription factor A " and " expression quantity of paraoxon acid enzyme 3 " higher than transfection pCDNA 3.1 (+) empty carrier group With normal cell group.
(3) it is exogenous raising mdck cell in " transcription factor A " or " paraoxon acid enzyme 3 " have suppress influenza virus effect
Cell transfecting pCDNA 3.1- transcription factor A, pCDNA 3.1- paraoxon acid enzyme 3 or pCDNA 3.1 (+) empty carrier 24h afterwards, dosage of inoculation 1 × 103EID50/ hole (6 orifice plate) swine influenza virus.24h, 48h, 72h collect sample after virus inoculation Product.Method is as follows：By cell in Tissue Culture Plate, together with cell culture fluid freeze thawing 3 times, 5000 turns/min centrifugation 10min, retain Supernatant, abandon precipitation.Determine Influenza virus titer in supernatant.Virus titer assay method refers to《National influenza central standard operation Code (revised edition), 2007》.
Experimental result (Fig. 6,7) shows that transcription factors A, paraoxon acid enzyme D are expressed by exogenous improve in mdck cell, Influenza virus titer is compared with control group and significantly reduced in 24h, 48h, 72h cell, and significant difference.Transfect pCDNA 3.1- transcription factor A groups p value compared with transfecting pCDNA 3.1 (+) empty carrier group is respectively p=0.002 (24h), 0.014 (48h)、0.0005(72h)；3 groups of transfection pCDNA 3.1- paraoxon acid enzyme compares p value with transfection pCDNA3.1 (+) empty carrier group Respectively p=0.015 (24h), 0.008 (48h), 0.012 (72h).Transfect in pCDNA3.1 (+) empty carrier group and normal cell Virus titer difference is not notable (p ＞ 0.05).
It is above-mentioned test result indicates that regulative transcription factor A and the gene expression of paraoxon acid enzyme 3 can suppress in host cell The duplication of influenza virus.
The modulate host genes within cells of embodiment 2 express the antiviral effect for significantly improving influenza virus medicine
H3N2 swine influenza viruses are infected in MDCK after protein expression again by adjusting, in the present embodiment using to cell Amantadine is added in nutrient solution, albumen is to amantadine resisiting influenza virus enhancing effect in research regulating cell.Amantadine By disturbing influenza virus particles M2 protein ion channels, suppress the function of the duplication of virus.
1 silence MDCK caveolin 2 significantly improves amantadine and suppresses influenza virus effect
(1) silence MDCK caveolin 2 is expressed
Specific steps are the same as embodiment 1.
(2) amantadine prepares
Amantadine hydrochloride is purchased from AlfaAesar (Tianjin) Chemical Co., Ltd., with normal saline into 20mg/ml, mistake Filter out bacterium, 4 DEG C of preservations.
(3) siRNA molecule transfectional cell
Specific steps are the same as embodiment 1.Drug combination group adds amantadine, final concentration to 0.4 μ g/ in cell culture fluid Ml, it is grouped as follows：SiRNA- caveolins+amantadine (0.4 μ g/ml), siRNA- caveolins 2 compare+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 transfectional cells connect poison and sample collection
With embodiment 1.
Experimental result is as shown in Figure 10, shows that mdck cell caveolin 2 is expressed by energy after siRNA molecule silence significantly Improve the effect that amantadine suppresses influenza virus.Influenza virus titer siRNA- caveolins+gold in 24h, 48h, 72h cell Firm alkanamine (0.4 μ g/ml) group is substantially less than siRNA- caveolins 2 and compares+amantadine (0.4 μ g/ml) group, and p value is respectively p =0.016 (24h), 0.001 (48h), 0.001 (72h).
The gene expression of caveolin 2 can improve amantadine suppression influenza disease in above-mentioned the results show regulating cell The effect that poison replicates.
Surfactant proteinD significantly improves amantadine suppression influenza virus effect in 2 silence MDCKs
(1) Surfactant proteinD is expressed in silence MDCK
Specific steps are the same as embodiment 1.
(2) amantadine prepares
Amantadine hydrochloride is purchased from AlfaAesar (Tianjin) Chemical Co., Ltd., with normal saline into 20mg/ml, mistake Filter out bacterium, 4 DEG C of preservations.
(3) siRNA molecule transfectional 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, it is grouped as follows：SiRNA- Surfactant proteinDs+amantadine (0.4 μ g/ml), siRNA- Surfactant proteinDs control+gold Firm 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 transfectional cells connect 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 silence in mdck cell Significantly improve the effect that amantadine suppresses influenza virus.Influenza virus titer siRNA- surfaces are lived in 24h, 48h, 72h cell Property protein D+amantadine (0.4 μ g/ml) group be substantially less than siRNA- Surfactant proteinDs control+amantadine (0.4 μ g/ Ml) group, p value are respectively p=0.020 (24h), 0.025 (48h), 0.130 (72h).
Surfactant proteinD gene expression can improve amantadine and suppress to flow in above-mentioned the results show regulating cell The effect that Influenza Virus replicates.
Transcription factor A expression significantly improves amantadine suppression influenza virus effect in 3 exogenous raising host cells
(1) in the 1st group of heterogenous expression expression vector structure
Specific steps are the same as embodiment 1.
(2) recombinant vector transfectional cell and expressive host albumen
Specific steps are the same as embodiment 1.
(3) it is exogenous to improve the effect that amantadine suppression influenza virus is replicated after transcription factors A in mdck cell
24h after (+) empty carrier of cell transfecting pCDNA 3.1- transcription factors A or pCDNA 3.1, dosage of inoculation 1 × 103EID50/ holes, drug combination group add amantadine in cell culture fluid, final concentration to 0.4 μ g/ml, set following test Group：Transfect pCDNA 3.1- transcription factor A+ amantadines (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). 24h, 48h, 72h collect sample after virus inoculation.Method is as follows：Freeze by cell in Tissue Culture Plate, together with cell culture fluid Melt 3 times, 5000 turns/min centrifugation 10min, retain supernatant, abandon precipitation.Determine Influenza virus titer in supernatant.Virus titer determines Method refers to《National influenza central standard operational procedure (revised edition), 2007》.
Factors A is transcribed test result indicates that shown in Figure 12, in mdck cell to be expressed by exogenous improve, 24h, 48h, PCDNA 3.1- transcription factor A+ amantadines (0.4 μ g/ml) group and transfection pCDNA 3.1 (+) empty carrier are transfected in 72h cells + amantadine (0.4 μ g/ml) is compared and significantly reduced, and significant difference, p value are respectively p=0.310 (24h), 0.013 (48h)、0.007(72h)。
It is above-mentioned to be answered test result indicates that regulating and controlling gene expression in the 1st group and significantly increase amantadine suppression influenza virus System.
The expression of paraoxon acid enzyme 3 significantly improves amantadine suppression influenza virus effect in 4 exogenous raising host cells
(1) in the 1st group of heterogenous expression expression vector structure
Specific steps are the same as embodiment 2.
(2) recombinant vector transfectional cell and expressive host albumen
Specific steps are the same as embodiment 2
(3) it is exogenous to improve the effect that amantadine suppression influenza virus is replicated after paraoxon acid enzyme 3 in mdck cell
24h after cell transfecting pCDNA 3.1- paraoxon acid enzyme 3 or pCDNA 3.1 (+) empty carrier, dosage of inoculation 1 × 103EID50/ holes, drug combination group add amantadine in cell culture fluid, final concentration to 0.4 μ g/ml, set following test Group：Transfect pCDNA 3.1- paraoxon acid enzyme 3+ amantadines (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). 24h, 48h, 72h collect sample after virus inoculation.Method is as follows：By cell in Tissue Culture Plate, together with cell culture fluid Freeze thawing 3 times, 5000 turns/min centrifugation 10min, retains supernatant, abandons precipitation.Determine Influenza virus titer in supernatant.Virus titer is surveyed Determine method reference《National influenza central standard operational procedure (revised edition), 2007》.
Experimental result 13 shows that paraoxon acid enzyme 3 is expressed by exogenous improve in mdck cell, thin in 24h, 48h, 72h PCDNA 3.1- paraoxon acid enzyme 3+ amantadines (0.4 μ g/ml) groups and transfection pCDNA 3.1 (+) empty carrier+gold are transfected in born of the same parents Firm alkanamine (0.4 μ g/ml) is compared to significantly reducing, and significant difference, p value be respectively p=0.042 (24h), 0.088 (48h), 0.069(72h)。
It is above-mentioned test result indicates that in regulating cell the gene expression of paraoxon acid enzyme 3 can significantly increase amantadine suppression Influenza virus is replicated.
All it is incorporated as referring in this application in all documents that the present invention refers to, is alone applied just as each piece As with reference to such.In addition, it is to be understood that after the above-mentioned instruction of the present invention has been read, those skilled in the art can be to this hair Bright to make various changes or modifications, these equivalent form of values equally fall within the application appended claims limited range.
It should be understood that after the above of the present invention has been read, those skilled in the art can make various to the present invention Change or modification, these equivalent form of values equally fall within the application appended claims limited range.

Claims (3)

  1. A kind of 1. siRNA for the gene of pig caveolin 2, it is characterised in that SEQ ID in described siRNA such as sequence table NO:1 represents.
  2. 2. siRNA as claimed in claim 1, it is characterised in that also containing 2 T or contain 2 in the described end of siRNA sequence 3 ' Individual U extended structure.
  3. 3. purposes of the siRNA as claimed in claim 1 or 2 in anti-swine flu H3N2 medicines are prepared.
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