CN101525388B - Specific double-strand RNA binding protein chimera and application thereof in virus infectious diseases - Google Patents
Specific double-strand RNA binding protein chimera and application thereof in virus infectious diseases Download PDFInfo
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- CN101525388B CN101525388B CN2009100212269A CN200910021226A CN101525388B CN 101525388 B CN101525388 B CN 101525388B CN 2009100212269 A CN2009100212269 A CN 2009100212269A CN 200910021226 A CN200910021226 A CN 200910021226A CN 101525388 B CN101525388 B CN 101525388B
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Abstract
The invention relates to a specific double-strand RNA binding protein chimera and the application thereof in curing and preventing virus infectious diseases, in particular to four protein chimeras formed by double-strand RNA receptor sequences of NF 90, PACT, ADAR1 and PKR and one or a plurality of applications thereof in curing and preventing the virus infectious diseases. The specific double-strand RNA binding protein chimera is characterized in that a protein transduction signal, a double-stranded RNA receptor and a cell apoptosis signal are sequentially connected by a DNA recombinant method to form the specific double-strand RNA binding protein chimera which is respectively expressed in procaryotic cells and eukaryotic cells. The specific double-strand RNA binding protein chimera can be used for curing and preventing the virus infectious diseases and is taken as a protein drug treatment tool on the level of mammalian cells.
Description
Technical field
The present invention relates to the application of double-strand RNA binding protein chimera in treatment or prophylaxis of viral infections property disease, particularly relate to four kinds of albumen mosaic one or more application in treatment and prophylaxis of viral infections property disease of forming by double-stranded RNA susceptor sequence NF90, PACT, ADAR1 and PKR.
Background technology
Virus infection is the common disease that threatens the human life healthy.Along with the development of Molecular Virology,,, have only few part virus infection can obtain making a definite diagnosis and treatment targetedly clinically because viral species is various and the restriction of diagnostic techniques though the mechanism of virus infection has been had deep understanding.The virus infection that some are serious, like AIDS, SARS, bird flu and Prion virus infection (spongiform encephalopathy) etc., the treatment means of present lack of specific.Therefore, antiviral that if can a kind of wide spectrum of development research, to saving this part patient's life, even can be very valuable like AIDS etc.
Double-stranded RNA (dsRNA) is a notable feature of all virus replication.In general, double-stranded RNA is not expressed in mammalian cell duplicates, and only after cell infection virus, just expresses.So double-stranded RNA is the molecule marker of virus infection mammalian cell, it is representing a kind of early stage danger signal of virus infection.Research confirms that all there is the innate immune system to double-stranded RNA in human and all vertebratess.In general, the double-stranded RNA that surpasses 30 base pairs just can effectively (double-strand RNA binding protein DRBP) acts on, and causes the antiviral response of cell with its acceptor.At first; Double-stranded RNA be distributed in immunoreactivity Interferon, rabbit (IFN) the effect protein effect on the double-stranded RNA protein kinase (PKR) that is activated; Activate the antiviral path of IFN mediation; This path and PACT/RAX, TRBP, 2 ', the special adenosine deaminase (ADAR) of 5 ' oligoadenylate synthetase, double-stranded RNA is relevant with the effect of nf 90 DRBP such as (NF90).Can induce quick antiviral response through this machine-processed double-stranded RNA, cause protein translation to suppress or apoptosis.Secondly, double-stranded RNA can combine with rna helicase enzyme RIG-I and MDA-5 in the cell, activated transcription albumen such as interferon regulatory factor 3 (IRF3) and NF-κ B etc., and this activation is very crucial for the generation of RNA viruses inductive Interferon, rabbit.The 3rd, can combine with Toll appearance acceptor 3 (TLR3) with extracellular double-stranded RNA in the cell, activate IFN through IRF3 and NF-κ B and transcribe.In a word, host cell can be through the existence of a series of DRBP double-stranded RNA inside and outside virus infection detects cell in early days, and causes innate immune responses through different mechanism.
Though the human during evolution innate immune system that the inside and outside double-stranded RNA of pair cell is replied, the viral a kind of approach of avoiding double-stranded RNA inductive antiviral response of also having evolved out of having formed.Virus must successfully be duplicated in infected host in order to survive.Research confirms that virus has been evolved and formed multiple path, is used for suppressing the effect of synthesizing, combine and make excretory IFN molecular inactivation, blocking-up IFN activation signals and destruction IFN inductive antiviral protein of IFN.Because double-stranded RNA is an early signal of IFN reaction; A lot of viruses have been evolved and can have been encoded himself DRBP with the double-stranded RNA acceptor among the competition host; Can effectively compete PKR or other double-stranded RNA acceptor like the NS1 of influenza virus, the RS3 of reovirus and the E3L of vaccinia virus, the antiviral response that blocking-up double-stranded RNA inductive IFN produces; Adenovirus and Epstein-Barr virus all can produce inhibitory RNA molecules, in conjunction with PKR and make its inactivation.Therefore, host or virus all can produce double-stranded RNA by all means and reply, and have disclosed the importance of signal conduction during virus infection of double chain RNA mediate.The signal of inferring double chain RNA mediate thus should be the primary goal of successfully preventing and treating virus infection.
Summary of the invention
The purpose of this invention is to provide a kind of with the protein transduction signal; Double-stranded RNA susceptor and apoptosis signal are linked in sequence by above through the method for DNA reorganization; Form specific double-strand RNA binding protein chimera, and the method for in protokaryon and eukaryotic cell, expressing respectively.
A kind of specific double-strand RNA binding protein chimera that relates to four kinds of different double-stranded RNA susceptors is provided.Wherein the double-stranded RNA susceptor can be respectively NF90, PACT, and any among ADAR1 or the PKR can be its part or all of double-stranded RNA calmodulin binding domain CaM.
Another object of the present invention is that said albumen mosaic is used to prepare antiviral drug.
A further object of the present invention is the application of specific double-strand RNA albumen mosaic in treatment and prophylaxis of viral infections property disease.
The objective of the invention is to realize like this; Specific double-strand RNA binding protein chimera; It is characterized in that the protein transduction signal; Double-stranded RNA susceptor and apoptosis signal are linked in sequence by above through the method for DNA reorganization, form specific double-strand RNA binding protein chimera, and in protokaryon and eukaryotic cell, express respectively.
The said specific double-strand RNA binding protein chimera that relates to four kinds of different double-stranded RNA susceptors, wherein the double-stranded RNA susceptor can be respectively NF90, PACT, any among ADAR1 or the PKR can be its part or all of double-stranded RNA calmodulin binding domain CaM.
Said protein transduction signal, apoptosis signal and the different double-stranded RNA susceptor sequences of comprising, the mosaic that specific double-strand RNA binding protein is formed, its cDNA and aminoacid sequence are following:
No.1dsCARE(PKR)
The cDNA sequence:
ATGTACGCCCGTGCCGCCGCCCGTCAGGCCCGTGCCAGTGGTCTCGAGATGGCTGGTGAT
CTTTCAGCAGGTTTCTTCATGGAGGAACTTAATACATACCGTCAGAAGCAGGGAGTAGTA
CTTAAATATCAAGAACTGCCTAATTCAGGACCTCCACATGATAGGAGGTTTACATTTCAAG
TTATAATAGATGGAAGAGAATTTCCAGAAGGTGAAGGTAGATCAAAGAAGGAAGCAAAA
AATGCCGCAGCCAAATTAGCTGTTGAGATACTTAATAAGGAAAAGAAGGCAGTTAGTCCT
TTATTATTGACAACAACGAATTCTTCAGAAGGATTATCCATGGGGAATTACATAGGCCTTAT
CAATAGAATTGCCCAGAAGAAAAGACTAACTGTAAATTATGAACAGTGTGCATCGGGGGT
GCATGGGCCAGAAGGATTTCATTATAAATGCAAAATGGGACAGAAAGAATATAGTATTGGT
ACAGGTTCTACTAAACAGGAAGCAAAACAATTGGCCGCTAAACTTGCATATCTTCAGATAT
TATCAGAAGAAACCTCAGTGAAATCTGACTACCTGTCCTCTGGTTCCGGTACCATGGATGC
AAAAGCTCGAAATTGTTTGCTTCAACATAGAGAAGCTCTGGAAAAGGACATCAAGACAT
CCTACATCATGGATCACATGATTAGTGATGGATTTTTAACAATATCAGAAGAGGAAAAAGT
AAGAAATGAGCCCACTCAACAGCAAAGAGCAGCTATGCTGATTAAAATGATACTTAAAAA
AGATAATGATTCCTACGTATCATTCTACAATGCTCTACTACATGAAGGATATAAAGATCTTG
CTGCCCTTCTCCATGATGGCATTCCTGTTGTCTAA
Protein sequence:
MYARAAARQARASGLEMAGDLSAGFFMEELNTYRQKQGVVLKYQELPNSGPPHDRRFTFQVIID
GREFPEGEGRSKKEAKNAAAKLAVEILNKEKKAVSPLLLTTTNSSEGLSMGNYIGLINRIAQKKRL
TVNYEQCASGVHGPEGFHYKCKMGQKEYSIGTGSTKQEAKQLAAKLAYLQILSEETSVKSDYLS
SGSGTMDAKARNCLLQHREALEKDIKTSYIMDHMISDGFLTISEEEKVRNEPTQQQRAAMLIKMI
LKKDNDSYVSFYNALLHEGYKDLAALLHDGIPVV
No2.dsCARE(ADAR1)
The cDNA sequence:
ATGTACGCCCGTGCCGCCGCCCGTCAGGCCCGTGCCAGTGGTCTCGAGAAGAACCCCATCAG
CGGGCTGTTAGAATATGCCCAGTTCGCTAGTCAAACCTGTGAGTTCAACATGATAGAGCAGAG
TGGACCACCCCATGAACCTCGATTTAAATTCCAGGTTGTCATCAATGGCCGAGAGTTTCCCCC
AGCTGAAGCTGGAAGCAAGAAAGTGGCCAAGCAGGATGCAGCTATGAAAGCCATGACAATTC
TGCTAGAGGAAGCCAAAGCCAAGGACAGTGGAAAATCAGAAGAATCATCCCACTATTCCACA
GAGAAAGAATCAGAGAAGACTGCAGAGTCCCAGACCCCCACCCCTTCAGCCACATCCTTCTT
TTCTGGGAAGAGCCCCGTCACCACACTGCTTGAGTGTATGCACAAATTGGGGAACTCCTGCG
AATTCCGTCTCCTGTCCAAAGAAGGCCCTGCCCATGAACCCAAGTTCCAATACTGTGTTGCAG
TGGGAGCCCAAACTTTCCCCAGTGTGAGTGCTCCCAGCAAGAAAGTGGCAAAGCAGATGGCC
GCAGAGGAAGCCATGAAGGCCCTGCATGGGGAGGCGACCAACTCCATGGCTTCTGATAACCA
GCCTGAAGGTATGATCTCAGAGTCACTTGATAACTTGGAATCCATGATGCCCAACAAGGTCAG
GAAGATTGGCGAGCTCGTGAGATACCTGAACACCAACCCTGTGGGTGGCCTTTTGGAGTACG
CCCGCTCCCATGGCTTTGCTGCTGAATTCAAGTTGGTCGACCAGTCCGGACCTCCTCACGAGC
CCAAGTTCGTTTACCAAGCAAAAGTTGGGGGTCGCTGGTTCCCAGCCGTCTGCGCACACAGC
AAGAAGCAAGGCAAGCAGGAAGCAGCAGATGCGGCTCTCCGTGTCTTGATTGGGGAGAACG
GTACCATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACATAGAGAAGCTCTGGAAAAGGACA
TCAAGACATCCTACATCATGGATCACATGATTAGTGATGGATTTTTAACAATATCAGAAGAGGA
AAAAGTAAGAAATGAGCCCACTCAACAGCAAAGAGCAGCTATGCTGATTAAAATGATACTTAA
AAAAGATAATGATTCCTACGTATCATTCTACAATGCTCTACTACATGAAGGATATAAAGATCTTG
CTGCCCTTCTCCATGATGGCATTCCTGTTGTCTAA
Protein sequence:
MYARAAARQARASGLEKNPISGLLEYAQFASQTCEFNMIEQSGPPHEPRFKFQVVINGREFPPAEA
GSKKVAKQDAAMKAMTILLEEAKAKDSGKSEESSHYSTEKESEKTAESQTPTPSATSFFSGKSPVT
TLLECMHKLGNSCEFRLLSKEGPAHEPKFQYCVAVGAQTFPSVSAPSKKVAKQMAAEEAMKALH
GEATNSMASDNQPEGMISESLDNLESMMPNKVRKIGELVRYLNTNPVGGLLEYARSHGFAAEFKL
VDQSGPPHEPKFVYQAKVGGRWFPAVCAHSKKQGKQEAADAALRVLIGENGTMDAKARNCLLQ
HREALEKDIKTSYIMDHMISDGFLTISEEEKVRNEPTQQQRAAMLIKMILKKDNDSYVSFYNALL
HEGYKDLAALLHDGIPVV
No3.dsCARE(PACT)
The cDNA sequence:
ATGTACGCCCGTGCCGCCGCCCGTCAGGCCCGTGCCAGTGGTCTCGAGAGCGATTACGACATC
CCCACTACTGAGAATCTTTATTTTCAGGGATCCTCCCAGAGCAGGCACCGCGCCGAGGCCCCG
CCGCTGGAGCGCGAGGACAGTGGGACCTTCAGTTTGGGGAAGATGATAACAGCTAAGCCAGG
GAAAACACCGATTCAGGTATTACACGAATACGGCATGAAGACCAAGAACATCCCAGTTTATGA
ATGTGAAAGATCTGATGTGCAAATACACGTGCCCACTTTCACCTTCAGAGTAACCGTTGGTGA
CATAACCTGCACAGGTGAAGGTACAAGTAAGAAGCTGGCGAAACATAGAGCTGCAGAGGCTG
CCATAAACATTTTGAAAGCCAATGCAAGTATTTGCTTTGCAGTTCCTGACCCCTTAATGCCTGA
CCCTTCCAAGCAACCAAAGAACCAGCTTAATCCTATTGGTTCATTACAGGAATTGGCTATTCAT
CATGGCTGGAGACTTCCTGAATATACCCTTTCCCAGGAGGGAGGACCTGCTCATAAGAGAGAA
TATACTACAATTTGCAGGCTAGAGTCATTTATGGAAACTGGAAAGGGGGCATCAAAAAAGCAA
GCCAAAAGGAATGCTGCTGAGAAATTTCTTGCCAAATTTAGTAATATTTCTCCAGAGAACCAC
ATTTCTTTAACAAATGTAGTAGGACATTCTTTAGGATGTACTTGGCATTCCTTGAGGAATTCTCC
TGGTGAAAAGATCAACTTACTGAAAAGAAGCCTCCTCAGTATTCCAAATACAGATTACATCCA
GCTGCTTAGTGAAATTGCCAAGGAACAAGGTTTTAATATAACATATTTGGATATAGATGAACTG
AGCGCCAATGGACAATATCAATGTCTTGCTGAACTGTCCACCAGCCCCATCACAGTCTGTCAT
GGCTCCGGTATCTCCTGTGGCAATGCACAAAGTGATGCAGCTCACAATGCTTTGCAGTATTTAA
AGATAATAGCAGAAAGAAAGGGTACCATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACATA
GAGAAGCTCTGGAAAAGGACATCAAGACATCCTACATCATGGATCACATGATTAGTGATGGAT
TTTTAACAATATCAGAAGAGGAAAAAGTAAGAAATGAGCCCACTCAACAGCAAAGAGCAGCT
ATGCTGATTAAAATGATACTTAAAAAAGATAATGATTCCTACGTATCATTCTACAATGCTCTACT
ACATGAAGGATATAAAGATCTTGCTGCCCTTCTCCATGATGGCATTCCTGTTGTCTAA
Protein sequence:
MYARAAARQARASGLESDYDIPTTENLYFQGSSQSRHRAEAPPLEREDSGTFSLGKMITAKPGKT
PIQVLHEYGMKTKNIPVYECERSDVQIHVPTFTFRVTVGDITCTGEGTSKKLAKHRAAEAAINILK
ANASICFAVPDPLMPDPSKQPKNQLNPIGSLQELAIHHGWRLPEYTLSQEGGPAHKREYTTICRLES
FMETGKGASKKQAKRNAAEKFLAKFSNISPENHISLTNVVGHSLGCTWHSLRNSPGEKINLLKRS
LLSIPNTDYIQLLSEIAKEQGFNITYLDIDELSANGQYQCLAELSTSPITVCHGSGISCGNAQSDAAH
NALQYLKIIAERKGTMDAKARNCLLQHREALEKDIKTSYIMDHMISDGFLTISEEEKVRNEPTQQ
QRAAMLIKMILKKDNDSYVSFYNALLHEGYKDLAALLHDGIPVV
No4.dsCARE(NF90)
The cDNA sequence:
ATGTACGCCCGTGCCGCCGCCCGTCAGGCCCGTGCCAGTGGTCTCGAGATGAATGCCCTGATG
CGGTTGAACCAGCTGAAGCCAGGGCTGCAGTACAAGCTGGTGTCCCAGACTGGGCCCGTCCA
TGCCCCCATCTTTACCATGTCTGTGGAGGTTGATGGCAATTCATTCGAGGCCTCTGGGCCCTCC
AAAAAGACGGCCAAGCTGCACGTGGCCGTTAAGGTGTTACAGGACATGGGCTTGCCGACGGG
TGCTGAAGGCAGGGACTCGAGCAAGGGGGAGGACTCGGCTGAGGAGACCGAGGCGAAGCC
AGCAGTGGTGGCCCCTGCCCCAGTGGTAGAAGCTGTCTCCACCCCTAGTGCGGCCTTTCCCTC
AGATGCCACTGCCGAGAACGTAAAACAGCAGGGGCCGATCCTGACAAAGCACGGCAAGAAC
CCAGTCATGGAGCTGAACGAGAAGAGGCGTGGGCTCAAGTACGAGCTCATCTCCGAGACCGG
GGGCAGCCACGACAAGCGCTTCGTCATGGAGGTCGAAGTGGATGGACAGAAGTTCCAAGGT
GCTGGTTCCAACAAAAAGGTGGGTACCATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACAT
AGAGAAGCTCTGGAAAAGGACATCAAGACATCCTACATCATGGATCACATGATTAGTGATGGA
TTTTTAACAATATCAGAAGAGGAAAAAGTAAGAAATGAGCCCACTCAACAGCAAAGAGCAGC
TATGCTGATTAAAATGATACTTAAAAAAGATAATGATTCCTACGTATCATTCTACAATGCTCTACT
ACATGAAGGATATAAAGATCTTGCTGCCCTTCTCCATGATGGCATTCCTGTTGTCTAA
Protein sequence:
MYARAAARQARASGLEMNALMRLNQLKPGLQYKLVSQTGPVHAPIFTMSVEVDGNSFEASGPS
KKTAKLHVAVKVLQDMGLPTGAEGRDSSKGEDSAEETEAKPAVVAPAPVVEAVSTPSAAFPSDAT
AENVKQQGPILTKHGKNPVMELNEKRRGLKYELISETGGSHDKRFVMEVEVDGQKFQGAGSNK
KVGTMDAKARNCLLQHREALEKDIKTSYIMDHMISDGFLTISEEEKVRNEPTQQQRAAMLIKMIL
KKDNDSYVSFYNALLHEGYKDLAALLHDGIPVV
The object of the invention can realize as follows that the chimeric preparation method of said albumen is with the protein transduction signal; Double-stranded RNA susceptor and apoptosis signal are linked in sequence by above through the method for DNA reorganization; Form the recombinant DNA of specific double-strand RNA binding protein chimera, be cloned on the suitable eucaryon or prokaryotic expression carrier, for example; PRSET B expression vector; Clone's step is: elder generation with identical endonuclease digestion, connects the recombinant DNA of pRSET B and binding protein chimera to them with the T4 dna ligase again according to different ratios, cut with the method for PCR with enzyme at last and identify.
Above-mentioned expression vector is well known to those skilled in the art, and the suitable carriers instance is seen for example Donnelly et al, and Ann.Rev.Immunol. (1997) 15:617 is said, comprises but non-pcDNA3 of being limited to and pcCMV.
Said double-strand RNA binding protein chimera, NF90, PACT, the sequence of ADAR1 and PKR can derive from any Mammals and vertebrates.
Said recombinant DNA with specific double-strand RNA binding protein chimera transforms various protokaryons and the eukaryotic cell expression system is expressed, and expressed albumen is prepared into the antiviral drug of injection, tablet, oral preparation, capsule or spray etc. by means commonly known in the art.
Said specific double-strand RNA albumen mosaic is used for treatment and prevention at disease of viral infection, on the mammalian cell level as the protein drug treatment tool.
Double-strand RNA binding protein chimera of the present invention and medicine thereof can be used for increasing the ability of human or animal's immunity system antagonism virus.Special, it can be used for administration of human or animal target.The preferred non-enteron aisle of said double-strand RNA binding protein chimera and medicine thereof gives, for example through the injection of (intralesional) approach or perfusion or sprinkling or ointment formulation external application in intravenously, intramuscular, intra-arterial or the infringement.Said medicine can be through routine techniques known in the art and medicine acceptable medium or delivery vehicles combination.The method for compositions that preparation can non-enteron aisle gives is well known, and in each data, more detailed description is arranged, and comprises for example Remington ' sPharmaceutical Sciences, Ed.AR Gennaro, 20
ThEdition, 2000, Williams & Wilkins, PA, USA..Said medicine preferably gives with the treatment significant quantity, promptly increases the dosage of the ability of human or animal's immunity system antagonism virus.
The recombinant DNA of double-strand RNA binding protein chimera can be any DNA that is suitable for protein expression, for example cDNA or dsDNA, but non-genomic group DNA or ssDNA.
The albumen mosaic that the present invention produced has the effect of anti-virus infection property disease.Advantage of the present invention is that it makes and can prepare broad-spectrum antiviral medicament.And the instance that we select is a viral encephalitis.This disease is mainly controlled through medicine at present, but the non-constant of effect, mortality ratio is up to 95%.
The present invention can also treat the disease of viral infection that enterovirus, Respirovirus, hsv, ebv infection etc. and not too common viruses such as measles, AIDS, SARS, bird flu and Prion cause.In one embodiment of the invention, comprise the drug alone that a kind of albumen mosaic is formed, also comprise two to four kinds of medicinal composition that the albumen mosaic is arranged in pairs or groups each other.
On the other hand, the invention provides the method for prophylaxis of viral infections through the function of curative drug.Between infection and disease, the regular hour section is arranged.In this case, said medicine works as the pharmacology immune product, and it is through exciting the immunne response treatment disease of the pathology effect of resisting said infection in the host.The difference of curative drug and preventive medicine be curative drug infect or ill patient's body in produce provide protection.
Description of drawings
Fig. 1 is the albumen mosaic that is formed by double-stranded RNA, ADAR1, PKR and NF90.
Fig. 2 is that four kinds of different albumen mosaics of proof have good active and purity.
(a): four kinds of chimeric structures of different albumen.
(b): four kinds of chimeric purity of different albumen.TP with protein electrophoresis confirms that these four kinds different albumen mosaics have good purity.
(c): four kinds of chimeric activity of different albumen.TP with instance 3 confirms that these four kinds different albumen mosaics have good active.
Fig. 3 is at the antivirus action synoptic diagram with the 293T cell that comprises sequence PKR albumen mosaic dsCARE (PKR) treatment adenovirus infection of the purifying of various dose.
(a) and (b), (c): respectively with 0,0.2,0.4,1.0,2.0,4.0, or the purifying protein of 8ug/ml and 10
-4The GFP of MOI
+The male adenovirus is handled cell simultaneously, at the 7th day, and the antiviral result significantly different (P＜0.001) of control group and each treatment group.
(d), (e): respectively with the BSA of the dsCARE of 2ug/ml or 100ug/ml add cultivate 7 days in the 293T cell after, add 10 again
-4The GFP of MOI
+The male adenovirus, at the 10th day, the preventative antiviral result of control group and treatment group is different (P＜0.001) significantly.
Embodiment
Embodiment 1: the research of the antivirus action of double-stranded RNA-ADAR1-PKR-NF90 mixture
(1) effect when ADAR1 and double-stranded RNA-PKR signal combination
1, ADAR1 edits the relation between double-stranded RNA and the double-stranded RNA-PKR Signal Regulation
1) ADAR1+ /+, ADAR1+/-and ADAR1-/-cultivation of neuronal cell: pregnant 11-12dADAR1+ /+, ADAR1+/-and ADAR1-/-mouse, the tire mouse is taken out in conventional anesthesia under the sterile state.Carefully peel off cerebellar cortex, remove meninx and vascular tissue, add 37 ℃ of digestion of 0.125% trypsinase 30min after being cut into the tissue block of 1mm size.Add the DMEM nutrient solution that contains 10% foetal calf serum and stop digestion 10min, blow and beat into cell suspension, be seeded to the Tissue Culture Plate that poly-lysine encapsulates with transfer pipet.Test as follows behind the isolated culture 7d.
2) make up pENTR/U6 (MS2-double-stranded RNA) expression vector: first pcr amplification goes out mouse Alu appearance gene, is cloned in the pCRIITA cloning vector, identifies to have from both direction and inserts segmental clone, thereby positive-sense strand and antisense strand RNA are contrasted; Cut this with the EcoRI enzyme again and insert segment and identify, fragment and carrier ratio are once more to be connected to it in identical carrier at 10: 1 then, make gene repeatedly thereby produce one; Then identify and make gene repeatedly, confirm through order-checking with restriction enzyme; With T7 and SP6 primer amplification positive-sense strand, antisense strand with make gene repeatedly, be cloned into again in the pENTR/U6 carrier at last.Be increased to the MS2RNA sequence back of SP6 primer simultaneously, so each inserts the binding site that segmental ending all has MS2.Be placed on the detection that 5 ' and 3 ' end will help transcript to T7 and SP6 sequence respectively.Insertion fragment in nucleus will receive the control that U6RNA starts the factor, also transcribed by RNA polymerase III simultaneously.After vector construction is accomplished, transfection ADAR1+ /+and ADAR1-/-neurone.
3) transfection neuronal cell: with Lipofectamine 2000 with pENTR/U6 (MS2-double-stranded RNA) carrier of 6 μ g import ready ADAR1+ /+, ADAR1+/-and ADAR1-/-neuronal cell in.
4) Western blotting detects PKR and eIF2a phosphorylation: transfection is lysing cell after 48 hours, and the cracking supernatant is added sample buffer, 95 ℃ of heating 3min, and electrophoresis moves on to protein transduction on the film, detects quantitative analysis with specific antibody.
5) RT-PCR detects the generation of rna editing: transfection was extracted the total RNA of culturing cell after 48 hours, and reverse transcription generates cDNA, carries out sequencing again, observed among the cDNA A at last to the sudden change of G.
2, the double-stranded RNA generation of stress-induced is with ADAR1 with combine its PKR restraining effect relevant
1) set up the neurone Stress model: ADAR1-/-and ADAR1+ /+neuronal cell plants in the petridish of 6-cm and cultivated 7 days with the ordinary culture medium of 10%FCS; Wash cell with the PBS that does not contain Ca2+/Mg2+ then, cultivated in 0,1,2,4,8 and 12 hour with serum free medium at last.
2) tenuigenin and nuclear double-stranded RNA are quantitative: the first step, and model cell washes twice with PBS earlier, uses Buffer A (10mM HEPES pH 7.9,10mM NaCl, 1.5mM MgCl2,0.5mM DTT, 5mM β-mercaptoethanol) to wash once again; Then contain the Buffer A dissolved cell of the 2x volume of 1%NP-40, knock with glass pestle simultaneously with 2 times of volumes; Ice bath 20 minutes; What float over the upper strata after centrifugal is exactly the tenuigenin part, collects supernatant, extracts cytoplasm rna with the Trizol method.The nucleus of lower floor is with Buffer C (20mM HEPES pH7.9,10mM NaC, 1.5mM MgCl2,0.2mM EDTA; 25% glycerine, 0.5mMDTT, 5mM β-mercaptoethanol; 1mM PMSF 1%NP-40) washes once, extracts the cell nRNA with the Trizol method again.In second step, total RNA that the last step was obtained gets about 50ug, is put into 400ul and contains among the digestion buffer of 0.1 RNaseI of unit and DnaseI/ul, hatches 2 hours for 37 ℃.Removed freshen by the RNA mixed solution that digested through agarose G-50, again through the Nucleotide remover remove little oligoribonucleotide (＜20bps).Quantitative with the UV spectrometry at last to the RNA of Rnase tolerance.
3) co-immunoprecipitation detects the effect of ADAR1-PKR-NF90 mixture and double-stranded RNA: at the last suitable antibody that adds 30 μ g in the tenuigenin part that obtains that goes on foot, 4 ℃ are shaken immunoprecipitate 1h; Add a-protein-Sepharose suspension of 0.9ml, 4 ℃ are shaken immunoprecipitate 30min; Then, repeat to wash 5 times, wash the liquid portion of sucking-off mixture for the last time with NETN with the NETN washing protein A-Sepharose mixture that contains 900mmol/LNaCl; 1 * SDS glue the sample loading buffer that adds 800 μ l then boils 4min in ball, sample is joined in the discontinuous SDS-PAGE gradient glue of macropore, and electrophoresis spends the night under the constant current of 10mA; Through Coomassie blue stain observing protein swimming band, downcut object tape again, put it in the Eppendorf tube, wash twice, each 3min with the 1ml50% acetonitrile from glue; Use the protein in the tryptic digestion glue at last,,, the peptide of collecting is carried out the automatic edman degradation order-checking on ABI 477A or 494A machine through narrow hole performance liquid chromatography isolated peptides again with the peptide electroelution.
4) TLC detects rna editing: elder generation is with nucleus or the cytoplasm rna of ethanol sedimentation 10 μ g; Then with the resuspended back 37 ℃ of hatchings of the 10 μ l RNase T2buffer that contain 5 RNaseT2 of unit 6 hours; Come mark 3 ' NTP through gama-32P-ATP (3000Ci/mmol), the T4 enzyme buffer of 1.5 μ l and the T4 polynucleotide kinase of 1 μ l that adds 3 μ l again; The nucleus P1 buffer (25mM sodium-acetate, pH 5.3, contain the nucleus P1 of 5 units) that adds 2 μ l then mixes back 37 ℃ of digestion 2 hours; Through TLC evaluation of markers on Mierocrystalline cellulose PEI culture plate 5 ' of 32p-IMP and 5 ' AMP are arranged again.This photographic developer is made up of saturated (NH4) SO4,100mM sodium-acetate (pH 6.0) and propyl alcohol (79: 19: 2).After the radioautograph, can identify 5 '-IMP, with the resuspended Mierocrystalline cellulose of 20 μ l water; Analyze the supernatant of 5 μ l again with TLC; Once more after the radioautograph, utilize liquid scintillation counter to come the quantitatively intensity of each point through the mensuration of the radioactive intensity that is cut off TLC and orders.
3, ADAR1 and PKR's is combined in the interaction that double-stranded RNA is induced the activation aspect
1) ADAR1+ /+, ADAR1+/-and ADAR1-/-cultivation of neuronal cell: method is the same.
2) transfection neuronal cell: with the ready ADAR1+ of pENTR/U6 (MS2-double-stranded RNA) carrier transfection /+, ADAR1+/-and ADAR1-/-neuronal cell in.
3) co-immunoprecipitation detects PKR or NF90: method is the same.
4) make up the proteic expression vector of MS2:, under the control of the CMV startup factor, be cloned into this cDNA in the pLenti6/V5 carrier, thereby construct the proteic expression vector of MS2 through the cDNA of RT-PCR amplification bacterium MS2 coat protein.
5) pENTR/U6 (MS2-double-stranded RNA) and plenty/V5 (MS2 albumen) cotransfection neuronal cell: method is the same.
6) method of co-immunoprecipitation detects MS2: method is the same.
7) Western blotting detects the expression of ADAR1, PKR, NF90 and pPKR: to 2) with 5) cell harvesting albumen carry out the immunoblotting detection.
(2) effect of research NF90 in the PKR that combines ADAR1 to regulate activates
1, the effect of NF90 in the cell signal of PKR mediation
1) NF90+ /+, NF90+/-and NF90-/-cultivation of neuronal cell: from pregnant 11-12d NF90+ /+, NF90+/-and NF90-/-get former generation neuronal cell the mouse, concrete the same.
2) Western blotting detects PKR, eIF2a, MAPK p38 and JNK: method is the same.
2, NF90 combines mediation PKR to suppress with ADAR1
1) RT-PCR detects MS2-double-stranded RNA editor's generation: method is the same.
2) TLC detects the double-stranded RNA editor: method is the same.
3) pENTR/U6 (MS2-double-stranded RNA) transfection neuronal cell: method is the same.
4) co-immunoprecipitation detects PKR and ADAR1: method is the same.
5) Western blotting detects PKR, ADAR1 and phosphorylation PKR: method is the same.
3, NF90 promotes double-stranded RNA output and it to combine the effect in double-stranded RNA-PKR signal at ADAR1
1) expression vector of structure GFP-MS2: the cDNA of the GFP that from the pEGFP carrier, increases earlier is connected in the pLenti6/V5 carrier at the following MS2 of control of the CMV startup factor and the cDNA of GFP.
2) transfection neuronal cell: with the ready NF90-of the common transfection of the expression vector of MS2-double-stranded RNA and MS2-GFP/-, NF90+ /+, ADAR1-/-or ADAR1+ /+neuronal cell.
3) real time imagery: transfection made the distribution pattern of GFP develop under fluorescent microscope after 4 hours, per hour once, and up to 12 hours; Per then 4 hours once, up to 48 days.
(3) the double chain RNA mediate ADAR1 of cell regulates the PKR activation
1, the endogenous double-stranded RNA is relevant with ADAR1-PKR-NF90 mixture (seeing shown in Figure 1).
1) ADAR1 p150 or ADAR1 p80 expression vector transfection neuronal cell: two target ADAR1 p150 of band or the ready neuronal cell of ADAR1p80 expression vector transfection.
2) ADAR1 mixture co-immunoprecipitation: method is the same.
3) purification double-stranded RNA: the last synthetics that obtains the Proteinase K mixture (5ug/ml, 500mMNaCl, 10mMTrisHCl pH 7, resuspended and 55 ℃ of digestion 2 hours in 0.2%SDS), thereby remove protein part.The recentrifuge pillar extracts the upper strata suspension with PHENOL 99.8 MIN ((CARBOLIC ACID)), and double-stranded RNA partly precipitates with 2.5 volume of ethanol.Obtain to such an extent that throw out includes relevant double-stranded RNA.
4) microarray of double-stranded RNA screening: come from by the experiment RNA of ADAR1 p150 transfectional cell with the Cy3 fluorochrome label; Come from by the contrast RNA of ADAR1 p80 transfectional cell with the Cy5 fluorochrome label.Accomplish mark with random primer through reverse transcription.Observe special double-stranded RNA mark: partially mixed with random primer and double-stranded RNA, be put on ice fast after 2 minutes 94 ℃ of heating, thereby make sex change RNA that better must an annealing arranged when a random primer of lacking.Through the cDNA of CHROMASPIN-200column purifying mark, 68 ℃ of sex change 10 minutes are used in 68 ℃ of the neutralizers and 20 minutes again.In YaleKeck Microarray Facilities, make the cDNA hybridization of mark, scan and obtain the intensity data of positive gene then.Probe from neuronal cell will be hybridized with people's the little lattice point of 15K cDNA.Analyze positive gene with Blast and RT-PCR.Further identify heterogeneic expression characteristic through RT-PCR.
5) the direct clone of double-stranded RNA:, monitor quantity with 8-15%PAGE with T4 polynucleotide kinase mark double-stranded RNA part.Use T4RNA to be connected with the oligonucleotide that a poly (dA) tail p-T7-(A) 17-NH2 forms the antisense strand that starts factor sequence by T7 with double-stranded RNA.When connecting, 3 ' end has will can not being connected with oligonucleotide of NH2.Remove the free oligonucleotide with PCR cloned sequence purification kit.Combine with cDNA with oligo poly (dT) 18 primers.The cDNA of purifying is added to and is heated to 80 ℃ of annealing 5 minutes in the damping fluid that contains 50mM Tris-HCl pH 7.5,100mM NaCl, 10mM MgCl2 and 1mM DTT.65 ℃ of hatchings 16 hours, last 30 ℃ of coolings were above 3 hours again.Pcr amplification is preceding to be the two ends linearizings with Klenow.Different big or small cDNA can use the T7 primer in order to increase.Be cloned into cDNA in the pENTR/U6 carrier again, and after order-checking with the Genbank DB in sequence compare.
6) RT-PCR confirms that with order-checking A is to the sudden change of I in the sequence.
2, identified the characteristic of transcript in double-stranded RNA-PKR signal
1) the MFOLD software analysis is identified the secondary structure of transcript.
2) identified the pENTR/U6 carrier transfection neuronal cell of transcript: method is the same.
3) Northern blotting detects and is identified transcript: extract total RNA with Trizol.In order to analyze the double-stranded RNA part, get RNA and the ethanol sedimentation of 50ug, it is resuspended to contain the digestion buffer of 0.1 unit/ul RNase I and DNase I (Sigma) with 400ul then, 37 ℃ 2 hours; Remove freshen with agarose G-50 again, remove test kit with Nucleotide and remove little oligoribonucleotide.
4) RNase V1 digestion: the RNA with the resuspended Rnase tolerance of the RNase V1 buffer that contains 0.4 unit/μ l RNase V1 37 ℃, 25 minutes, breaks it through centrifugal fast.
5) fluorescence microscope fluor PKR: 2) pass through fluorescence microscope fluor PKR in the cell after the transfection.
3, identified that transcript replenishes formation ADAR1-PKR-NF90 mixture
1) identified the pENTR/U6 carrier transfection neuronal cell of transcript: method is the same.
2) PKR co-immunoprecipitation: the ADAR1-PKR-NF90 mixture that obtains is resuspended in the Proteinase K damping fluid that 200ul comprises the 10ug/ml Proteinase K 50 ℃, 1 hour; Collect after the extracting of supernatant phenylic acid and ethanol sedimentation.
3) RT-PCR detects and to be identified transcript: the throw out that the last step is obtained carries out being identified the RT-PCR of transcript.
4, identified the effect of transcript in stress-induced double-stranded RNA-PKR signal
1) foundation of neurone Stress model: method is the same.
2) Northern blotting detects and identified transcript: method is the same.
3) Western blotting detects fluor PKR and fluor eIF2a: method is the same.
Above test-results has confirmed that double-stranded RNA-ADAR1-PKR-NF90 mixture has tangible antivirus action.
Embodiment 2: make up the chimeric expression vector of albumen and the extracting albumen that include different double-stranded RNA susceptor sequences, natural death of cerebral cells signal and protein transduction domain
1, four kinds of chimeric structures of different albumen: the four kinds of chimeric double-stranded RNA susceptor of different albumen sequences are respectively NF90, PACT, ADAR1 and PKR (seeing shown in Figure 2).Concrete method of attachment is following: from human cDNA gene pool, amplify the cDNA of NF90, PACT, ADAR1 and PKR earlier through the method for RT-PCR, on these four kinds different double-stranded RNA susceptor sequences, increase the restriction enzyme site of Xhol and Kpnl again through PCR; The restriction enzyme site that on the natural death of cerebral cells signal, also adds simultaneously Xhol and Kpnl; Two kinds of oligonucleotide agctt
GgatccTacgcccgtgccgccgcccgtcaggcccgtgccagtggt and ccat
CtcgagAccactggcacgggcctg carries out the cDNA encoding sequence that double digestion obtains protein transduction domain with BamHI and Xhol after through pcr amplification; Use atat
GgatccCatggtctagccagcttgggtctccct and aatc
AagcttCgaacaaaaactcatctcagaa is a primer, and (Invitrogen CA) increases and obtains a BamHI-HindIII sequence to the pcDNA3 carrier; CDNA through obtaining above the BamHI-Xhol-Kpnl-HindIII connection under the effect of the CMV of pcDNA3 promotor; Cut through enzyme at last and order-checking is identified.
2, eukaryon expression: four kinds of chimeric genes of different albumen are connected respectively among the expression vector pcDNA3.1 by above method.Through transfection HEK 293T cell (ATCC, USA), the method for coomassie brilliant blue staining and Western blotting detects four kinds of chimeric expression of different albumen.
3, prokaryotic expression: (Invitrogen, CA) carrier connection after 16 ℃ of connections are spent the night, transform the TOP10 competent cell, and choose the clone and shake bacterium, upgrading grain with pRSET respectively with four kinds of chimeric genes of different albumen by above method; PCR and order-checking are identified.Before the expressing protein mosaic, plasmid is transformed the BL21 competent cell; 37C shakes after bacterium spends the night, and (Qiagen, the Ni-NTA protein purification technology that USA) provides is extracting albumen from bacterium cracking industry to press Qiagen.Proteic purity is seen shown in Figure 2.
3: four kinds of different albumen mosaics of embodiment are to the effect of adenovirus infection HEK293 cell inhibiting
1, adenovirus infection: HEK293 or 293T cell infect 30-60min with having fluorescently-labeled recombinant adenovirus in growth on six orifice plates after 8 hours, cultivate 12h with twice continued of PBS wash-out of temperature with the DMEM nutrient solution that contains 10%FBS.Cell behind the fluorescence microscope virus infection, through the titre that the method for viral gradient dilution is observed the 293T of infective virus, fluorescent microscope is counted GFP positive cell (seeing shown in Figure 3) down behind the infection 24h.
2, extracorporeal antivirus effect analysis: the antiviral protein that above-mentioned four kinds of different albumen mosaics are purified into is respectively before virus infection; Join in the nutrient solution during infection or after infecting, respectively at infecting behind 2 days, a week and 10 days the infection conditions of tracing observation virus under fluorescent microscope.The result finds that the cell of treatment group is still after 10 days at virus infection and (sees shown in Figure 3) normally.
Embodiment 4: the albumen mosaic is to the restraining effect of adenovirus infection neuronal cell
1, tire mouse cortex neurone vitro culture: pregnant 11-12d mouse, the tire mouse is taken out in conventional anesthesia under the sterile state.Carefully peel off cerebellar cortex, remove meninx and vascular tissue, add 37 ℃ of digestion of 0.125% trypsinase 30min after being cut into the tissue block of 1mm size.Add the DMEM nutrient solution that contains 10% foetal calf serum and stop digestion 10min, blow and beat into cell suspension, be seeded to the Tissue Culture Plate that poly-lysine encapsulates with transfer pipet.Test as follows behind the isolated culture 7d.
2, expression vector transfection neuronal cell: utilize Lipofectamine 2000 with in the ready cell of the plasmid transfection to 1 of 6 μ g.
3, these cells of adenovirus infection: using PBS is density 1 * 10
9The adenopathy venom get 10 μ l to be diluted to density respectively be 1 * 10
4, 1 * 10
5, 1 * 10
6Liquid, get in the neurone after 1ml adds transfection, the PBS that gets 1ml again is as negative control, joins viral liquid in the neurone of untransfected and does positive control.
4, fluorescence microscope infection conditions, the flow cytometer check and analysis.
The result shows; Compare with the neurone of untransfected double-stranded RNA complex body plasmid; In all situations, give the neurone of every kind of double-stranded RNA complex body plasmid; After the different concns adenovirus infection, neuron survival rate strengthens, mean survival time increases, cell streaming appearance check and analysis dead cell reduces, and apoptotic cell increases (seeing shown in Figure 3).Show tangible antivirus action, the special plasmid antivirus action that comprises PKR sequence expression vector is the most obvious.
Embodiment 5: observe the result of treatment of these albumen to the viral infection animal model
1, the hypodermic toxicity test of these albumen: subcutaneous nodule, mouse diet and changes of weight situation are observed in the subcutaneous injection of various dose albumen.
2, the mensuration of the medium lethal dose(LD&-{50}) of adenovirus inoculation mouse brain: viral suspension (TCID50=105) dilution is five extent of dilution of 10-5-10-1 with the Hanks damping fluid; Get 3 age in week 40 of mouse, be divided into 5 groups at random, one group of every extent of dilution, 8 every group; With the 1ml syringe in the right side mouse brain inoculation 0.05ml/ only, the left side is not inoculated; Observe 10d, every day the observed and recorded mouse morbidity and death condition.
3, the mensuration of these albumen antagonism adenoviral encephalitis mouse median effective doses: get 3 age in week 250 of mouse, be divided into 25 groups at random, 10 every group, with 103 TCID50 viral suspensions inoculation 0.05ml/ only; Since the 2nd day, with the various albumen subcutaneous injection treatments of 6 kinds of different concns, saline water: 0.1/ (kgd); 0.88,1.67,3.3,4.9,6.6,13.2g/ (kgd) various albumen:.Observe 10d, write down mouse invasion and death condition every day.
4, the method through the part inoculation is injected into adenovirus the animal model of setting up viral infection in the mouse brain: every mouse of 1ml syringe is injected the 0.1ml viral suspension respectively and is expelled to ventriculus dexter cerebri, death and the incidence of 10 days mouse of observed and recorded.
5, be injected into the proteic result of treatment of observation in the mouse body to these albumen through subarachnoid injection, intravenous injection and three kinds of approach of intranasal administration: mouse is divided into sham operated rats, subarachnoid injection group, intravenous injection group, intranasal administration group and blank group at random; Each protein for treatment respectively at treating the back 1,3,5,7,14 day, was put to death 6 for every group after 1,3,5,7,14 days, took out cerebral tissue down in aseptic condition, and each sample part of getting is put into 10% formalin solution, stayed and did histopathologic slide's usefulness; A part is used for the vitro culture neurone, fluorescence microscope and flow cytometer check and analysis; All the other are put into-70 ℃ of Ultralow Temperature Freezers and preserve subsequent use; Each experimental group stays 20 to observe survival rate and mean survival time.The plaque method is surveyed virus titer: sample is inserted in the homogenizer grind repeatedly, behind the low-speed centrifugal, supernatant is done 10 times of dilutions continuously with the Hanks damping fluid; Inoculation grows up to 24 orifice plates of individual layer, adds nutrient agar, after 2 days; Add the agar that contains toluylene red, the numeration plaque.
The result is following, and on survival rate and the mean survival time: the treatment group obviously strengthens (P＜0.01) than sham operated rats and blank group, and the subarachnoid injection group is compared with intravenous injection group and intranasal administration group and acted on more obvious (P＜0.05); The detection of cell streaming appearance, immunohistochemical methods sxemiquantitative, virus titer detection etc. all point out four kinds of different albumen mosaics that tangible antivirus action is arranged, and the plasmid antivirus action that wherein comprises PKR sequence expression vector is the most obvious.
Need to prove; Accompanying drawing provided by the invention is the cell picture of under microscope or fluorescent microscope, gathering, and for medical research picture the most clearly, can not be revised as the black and white contrast picture by appliance computer; Because have only according to different colour developings, the result of ability judgment experiment.
Be not described in detail the known technology that part belongs to the industry or relevant industries among the above embodiment, the equipment of employing is industrial practice equipment.
Sequence table
1.dsCARE(PKR)
The cDNA sequence:
ATGTACGCCCGTGCCGCCGCCCGTCAGGCCCGTGCCAGTGGTCTCGAGATGGCTGGTGAT
CTTTCAGCAGGTTTCTTCATGGAGGAACTTAATACATACCGTCAGAAGCAGGGAGTAGTA
CTTAAATATCAAGAACTGCCTAATTCAGGACCTCCACATGATAGGAGGTTTACATTTCAAG
TTATAATAGATGGAAGAGAATTTCCAGAAGGTGAAGGTAGATCAAAGAAGGAAGCAAAA
AATGCCGCAGCCAAATTAGCTGTTGAGATACTTAATAAGGAAAAGAAGGCAGTTAGTCCT
TTATTATTGACAACAACGAATTCTTCAGAAGGATTATCCATGGGGAATTACATAGGCCTTAT
CAATAGAATTGCCCAGAAGAAAAGACTAACTGTAAATTATGAACAGTGTGCATCGGGGGT
GCATGGGCCAGAAGGATTTCATTATAAATGCAAAATGGGACAGAAAGAATATAGTATTGGT
ACAGGTTCTACTAAACAGGAAGCAAAACAATTGGCCGCTAAACTTGCATATCTTCAGATAT
TATCAGAAGAAACCTCAGTGAAATCTGACTACCTGTCCTCTGGTTCCGGTACCATGGATGC
AAAAGCTCGAAATTGTTTGCTTCAACATAGAGAAGCTCTGGAAAAGGACATCAAGACAT
CCTACATCATGGATCACATGATTAGTGATGGATTTTTAACAATATCAGAAGAGGAAAAAGT
AAGAAATGAGCCCACTCAACAGCAAAGAGCAGCTATGCTGATTAAAATGATACTTAAAAA
AGATAATGATTCCTACGTATCATTCTACAATGCTCTACTACATGAAGGATATAAAGATCTTG
CTGCCCTTCTCCATGATGGCATTCCTGTTGTCTAA
Protein sequence:
MYARAAARQARASGLEMAGDLSAGFFMEELNTYRQKQGVVLKYQELPNSGPPHDRRFTFQVIID
GREFPEGEGRSKKEAKNAAAKLAVEILNKEKKAVSPLLLTTTNSSEGLSMGNYIGLINRIAQKKRL
TVNYEQCASGVHGPEGFHYKCKMGQKEYSIGTGSTKQEAKQLAAKLAYLQILSEETSVKSDYLS
SGSGTMDAKARNCLLQHREALEKDIKTSYIMDHMISDGFLTISEEEKVRNEPTQQQRAAMLIKMI
LKKDNDSYVSFYNALLHEGYKDLAALLHDGIPVV
2.dsCARE(ADAR1)
The cDNA sequence:
ATGTACGCCCGTGCCGCCGCCCGTCAGGCCCGTGCCAGTGGTCTCGAGAAGAACCCCATCAG
CGGGCTGTTAGAATATGCCCAGTTCGCTAGTCAAACCTGTGAGTTCAACATGATAGAGCAGAG
TGGACCACCCCATGAACCTCGATTTAAATTCCAGGTTGTCATCAATGGCCGAGAGTTTCCCCC
AGCTGAAGCTGGAAGCAAGAAAGTGGCCAAGCAGGATGCAGCTATGAAAGCCATGACAATTC
TGCTAGAGGAAGCCAAAGCCAAGGACAGTGGAAAATCAGAAGAATCATCCCACTATTCCACA
GAGAAAGAATCAGAGAAGACTGCAGAGTCCCAGACCCCCACCCCTTCAGCCACATCCTTCTT
TTCTGGGAAGAGCCCCGTCACCACACTGCTTGAGTGTATGCACAAATTGGGGAACTCCTGCG
AATTCCGTCTCCTGTCCAAAGAAGGCCCTGCCCATGAACCCAAGTTCCAATACTGTGTTGCAG
TGGGAGCCCAAACTTTCCCCAGTGTGAGTGCTCCCAGCAAGAAAGTGGCAAAGCAGATGGCC
GCAGAGGAAGCCATGAAGGCCCTGCATGGGGAGGCGACCAACTCCATGGCTTCTGATAACCA
GCCTGAAGGTATGATCTCAGAGTCACTTGATAACTTGGAATCCATGATGCCCAACAAGGTCAG
GAAGATTGGCGAGCTCGTGAGATACCTGAACACCAACCCTGTGGGTGGCCTTTTGGAGTACG
CCCGCTCCCATGGCTTTGCTGCTGAATTCAAGTTGGTCGACCAGTCCGGACCTCCTCACGAGC
CCAAGTTCGTTTACCAAGCAAAAGTTGGGGGTCGCTGGTTCCCAGCCGTCTGCGCACACAGC
AAGAAGCAAGGCAAGCAGGAAGCAGCAGATGCGGCTCTCCGTGTCTTGATTGGGGAGAACG
GTACCATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACATAGAGAAGCTCTGGAAAAGGACA
TCAAGACATCCTACATCATGGATCACATGATTAGTGATGGATTTTTAACAATATCAGAAGAGGA
AAAAGTAAGAAATGAGCCCACTCAACAGCAAAGAGCAGCTATGCTGATTAAAATGATACTTAA
AAAAGATAATGATTCCTACGTATCATTCTACAATGCTCTACTACATGAAGGATATAAAGATCTTG
CTGCCCTTCTCCATGATGGCATTCCTGTTGTCTAA
Protein sequence:
MYARAAARQARASGLEKNPISGLLEYAQFASQTCEFNMIEQSGPPHEPRFKFQVVINGREFPPAEA
GSKKVAKQDAAMKAMTILLEEAKAKDSGKSEESSHYSTEKESEKTAESQTPTPSATSFFSGKSPVT
TLLECMHKLGNSCEFRLLSKEGPAHEPKFQYCVAVGAQTFPSVSAPSKKVAKQMAAEEAMKALH
GEATNSMASDNQPEGMISESLDNLESMMPNKVRKIGELVRYLNTNPVGGLLEYARSHGFAAEFKL
VDQSGPPHEPKFVYQA?KVGGRWFPAVCAHSKKQGKQEAADAALRVLIGENGTMDAKARNCLLQ
HREALEKDIKTSYIMDHMISDGFLTISEEEKVRNEPTQQQRAAMLIKMILKKDNDSYVSFYNALL
HEGYKDLAALLHDGIPVV
3.dsCARE(PACT)
The cDNA sequence:
ATGTACGCCCGTGCCGCCGCCCGTCAGGCCCGTGCCAGTGGTCTCGAGAGCGATTACGACATC
CCCACTACTGAGAATCTTTATTTTCAGGGATCCTCCCAGAGCAGGCACCGCGCCGAGGCCCCG
CCGCTGGAGCGCGAGGACAGTGGGACCTTCAGTTTGGGGAAGATGATAACAGCTAAGCCAGG
GAAAACACCGATTCAGGTATTACACGAATACGGCATGAAGACCAAGAACATCCCAGTTTATGA
ATGTGAAAGATCTGATGTGCAAATACACGTGCCCACTTTCACCTTCAGAGTAACCGTTGGTGA
CATAACCTGCACAGGTGAAGGTACAAGTAAGAAGCTGGCGAAACATAGAGCTGCAGAGGCTG
CCATAAACATTTTGAAAGCCAATGCAAGTATTTGCTTTGCAGTTCCTGACCCCTTAATGCCTGA
CCCTTCCAAGCAACCAAAGAACCAGCTTAATCCTATTGGTTCATTACAGGAATTGGCTATTCAT
CATGGCTGGAGACTTCCTGAATATACCCTTTCCCAGGAGGGAGGACCTGCTCATAAGAGAGAA
TATACTACAATTTGCAGGCTAGAGTCATTTATGGAAACTGGAAAGGGGGCATCAAAAAAGCAA
GCCAAAAGGAATGCTGCTGAGAAATTTCTTGCCAAATTTAGTAATATTTCTCCAGAGAACCAC
ATTTCTTTAACAAATGTAGTAGGACATTCTTTAGGATGTACTTGGCATTCCTTGAGGAATTCTCC
TGGTGAAAAGATCAACTTACTGAAAAGAAGCCTCCTCAGTATTCCAAATACAGATTACATCCA
GCTGCTTAGTGAAATTGCCAAGGAACAAGGTTTTAATATAACATATTTGGATATAGATGAACTG
AGCGCCAATGGACAATATCAATGTCTTGCTGAACTGTCCACCAGCCCCATCACAGTCTGTCAT
GGCTCCGGTATCTCCTGTGGCAATGCACAAAGTGATGCAGCTCACAATGCTTTGCAGTATTTAA
AGATAATAGCAGAAAGAAAGGGTACCATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACATA
GAGAAGCTCTGGAAAAGGACATCAAGACATCCTACATCATGGATCACATGATTAGTGATGGAT
TTTTAACAATATCAGAAGAGGAAAAAGTAAGAAATGAGCCCACTCAACAGCAAAGAGCAGCT
ATGCTGATTAAAATGATACTTAAAAAAGATAATGATTCCTACGTATCATTCTACAATGCTCTACT
ACATGAAGGATATAAAGATCTTGCTGCCCTTCTCCATGATGGCATTCCTGTTGTCTAA
Protein sequence:
MYARAAARQARASGLESDYDIPTTENLYFQGSSQSRHRAEAPPLEREDSGTFSLGKMITAKPGKT
PIQVLHEYGMKTKNIPVYECERSDVQIHVPTFTFRVTVGDITCTGEGTSKKLAKHRAAEAAINILK
ANASICFAVPDPLMPDPSKQPKNQLNPIGSLQELAIHHGWRLPEYTLSQEGGPAHKREYTTICRLES
FMETGKGASKKQAKRNAAEKFLAKFSNISPENHISLTNVVGHSLGCTWHSLRNSPGEKINLLKRS
LLSIPNTDYIQLLSEIAKEQGFNITYLDIDELSANGQYQCLAELSTSPITVCHGSGISCGNAQSDAAH
NALQYLKIIAERKGTMDAKARNCLLQHREALEKDIKTSYIMDHMISDGFLTISEEEKVRNEPTQQ
QRAAMLIKMILKKDNDSYVSFYNALLHEGYKDLAALLHDGIPVV
4.dsCARE(NF90)
The cDNA sequence:
ATGTACGCCCGTGCCGCCGCCCGTCAGGCCCGTGCCAGTGGTCTCGAGATGAATGCCCTGATG
CGGTTGAACCAGCTGAAGCCAGGGCTGCAGTACAAGCTGGTGTCCCAGACTGGGCCCGTCCA
TGCCCCCATCTTTACCATGTCTGTGGAGGTTGATGGCAATTCATTCGAGGCCTCTGGGCCCTCC
AAAAAGACGGCCAAGCTGCACGTGGCCGTTAAGGTGTTACAGGACATGGGCTTGCCGACGGG
TGCTGAAGGCAGGGACTCGAGCAAGGGGGAGGACTCGGCTGAGGAGACCGAGGCGAAGCC
AGCAGTGGTGGCCCCTGCCCCAGTGGTAGAAGCTGTCTCCACCCCTAGTGCGGCCTTTCCCTC
AGATGCCACTGCCGAGAACGTAAAACAGCAGGGGCCGATCCTGACAAAGCACGGCAAGAAC
CCAGTCATGGAGCTGAACGAGAAGAGGCGTGGGCTCAAGTACGAGCTCATCTCCGAGACCGG
GGGCAGCCACGACAAGCGCTTCGTCATGGAGGTCGAAGTGGATGGACAGAAGTTCCAAGGT
GCTGGTTCCAACAAAAAGGTGGGTACCATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACAT
AGAGAAGCTCTGGAAAAGGACATCAAGACATCCTACATCATGGATCACATGATTAGTGATGGA
TTTTTAACAATATCAGAAGAGGAAAAAGTAAGAAATGAGCCCACTCAACAGCAAAGAGCAGC
TATGCTGATTAAAATGATACTTAAAAAAGATAATGATTCCTACGTATCATTCTACAATGCTCTACT
ACATGAAGGATATAAAGATCTTGCTGCCCTTCTCCATGATGGCATTCCTGTTGTCTAA
Protein sequence:
MYARAAARQARASGLEMNALMRLNQLKPGLQYKLVSQTGPVHAPIFTMSVEVDGNSFEASGPS
KKTAKLHVAVKVLQDMGLPTGAEGRDSSKGEDSAEETEAKPAVVAPAPVVEAVSTPSAAFPSDAT
AENVKQQGPILTKHGKNPVMELNEKRRGLKYELISETGGSHDKRFVMEVEVDGQKFQGAGSNK
KVGTMDAKARNCLLQHREALEKDIKTSYIMDHMISDGFLTISEEEKVRNEPTQQQRAAMLIKMIL
KKDNDSYVSFYNALLHEGYKDLAALLHDGIPVV
Claims (3)
1. specific double-strand RNA binding protein chimera; It is characterized in that the protein transduction signal; Double-stranded RNA susceptor and apoptosis signal are linked in sequence by above through the method for DNA reorganization; Form specific double-strand RNA binding protein chimera, and in protokaryon and eukaryotic cell, express respectively; Described specific double-strand RNA binding protein chimera, its cDNA and aminoacid sequence are following:
No.1?dsCARE?PKR
The cDNA sequence:
ATGTACGCCCGTGCCGCCGCCCGTCAGGCCCGTGCCAGTGGTCTCGAGATGGCTG
GTGATCTTTCAGCAGGTTTCTTCATGGAGGAACTTAATACATACCGTCAGAAGCAG
GGAGTAGTACTTAAATATCAAGAACTGCCTAATTCAGGACCTCCACATGATAGGAG
GTTTACATTTCAAGTTATAATAGATGGAAGAGAATTTCCAGAAGGTGAAGGTAGAT
CAAAGAAGGAAGCAAAAAATGCCGCAGCCAAATTAGCTGTTGAGATACTTAATAA
GGAAAAGAAGGCAGTTAGTCCTTTATTATTGACAACAACGAATTCTTCAGAAGGAT
TATCCATGGGGAATTACATAGGCCTTATCAATAGAATTGCCCAGAAGAAAAGACTA
ACTGTAAATTATGAACAGTGTGCATCGGGGGTGCATGGGCCAGAAGGATTTCATTA
TAAATGCAAAATGGGACAGAAAGAATATAGTATTGGTACAGGTTCTACTAAACAGG
AAGCAAAACAATTGGCCGCTAAACTTGCATATCTTCAGATATTATCAGAAGAAACC
TCAGTGAAATCTGACTACCTGTCCTCTGGTTCCGGTACCATGGATGCAAAAGCTCG
AAATTGTTTGCTTCAACATAGAGAAGCTCTGGAAAAGGACATCAAGACATCCTACA
TCATGGATCACATGATTAGTGATGGATTTTTAACAATATCAGAAGAGGAAAAAGTA
AGAAATGAGCCCACTCAACAGCAAAGAGCAGCTATGCTGATTAAAATGATACTTAA
AAAAGATAATGATTCCTACGTATCATTCTACAATGCTCTACTACATGAAGGATATAA
AGATCTTGCTGCCCTTCTCCATGATGGCATTCCTGTTGTCTAA
Protein sequence:
MYARAAARQARASGLEMAGDLSAGFFMEELNTYRQKQGVVLKYQELPNSGPPHDRRFT
FQVIIDGREFPEGEGRSKKEAKNAAAKLAVEILNKEKKAVSPLLLTTTNSSEGLSMGNYIGL
INRIAQKKRLTVNYEQCASGVHGPEGFHYKCKMGQKEYSIGTGSTKQEAKQLAAKLAYL
QILSEETSVKSDYLSSGSGTMDAKARNCLLQHREALEKDIKTSYIMDHMISDGFLTISEEEK
VRNEPTQQQRAAMLIKMILKKDNDSYVSFYNALLHEGYKDLAALLHDGIPVV
No2.dsCARE?ADAR1
The cDNA sequence:
ATGTACGCCCGTGCCGCCGCCCGTCAGGCCCGTGCCAGTGGTCTCGAGAAGAACCCCA
TCAGCGGGCTGTTAGAATATGCCCAGTTCGCTAGTCAAACCTGTGAGTTCAACATGATA
GAGCAGAGTGGACCACCCCATGAACCTCGATTTAAATTCCAGGTTGTCATCAATGGCC
GAGAGTTTCCCCCAGCTGAAGCTGGAAGCAAGAAAGTGGCCAAGCAGGATGCAGCTA
TGAAAGCCATGACAATTCTGCTAGAGGAAGCCAAAGCCAAGGACAGTGGAAAATCAG
AAGAATCATCCCACTATTCCACAGAGAAAGAATCAGAGAAGACTGCAGAGTCCCAGA
CCCCCACCCCTTCAGCCACATCCTTCTTTTCTGGGAAGAGCCCCGTCACCACACTGCTT
GAGTGTATGCACAAATTGGGGAACTCCTGCGAATTCCGTCTCCTGTCCAAAGAAGGCC
CTGCCCATGAACCCAAGTTCCAATACTGTGTTGCAGTGGGAGCCCAAACTTTCCCCAG
TGTGAGTGCTCCCAGCAAGAAAGTGGCAAAGCAGATGGCCGCAGAGGAAGCCATGAA
GGCCCTGCATGGGGAGGCGACCAACTCCATGGCTTCTGATAACCAGCCTGAAGGTATG
ATCTCAGAGTCACTTGATAACTTGGAATCCATGATGCCCAACAAGGTCAGGAAGATTG
GCGAGCTCGTGAGATACCTGAACACCAACCCTGTGGGTGGCCTTTTGGAGTACGCCCG
CTCCCATGGCTTTGCTGCTGAATTCAAGTTGGTCGACCAGTCCGGACCTCCTCACGAG
CCCAAGTTCGTTTACCAAGCAAAAGTTGGGGGTCGCTGGTTCCCAGCCGTCTGCGCAC
ACAGCAAGAAGCAAGGCAAGCAGGAAGCAGCAGATGCGGCTCTCCGTGTCTTGATTG
GGGAGAACGGTACCATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACATAGAGAAGC
TCTGGAAAAGGACATCAAGACATCCTACATCATGGATCACATGATTAGTGATGGATTTTT
AACAATATCAGAAGAGGAAAAAGTAAGAAATGAGCCCACTCAACAGCAAAGAGCAGC
TATGCTGATTAAAATGATACTTAAAAAAGATAATGATTCCTACGTATCATTCTACAATGCT
CTACTACATGAAGGATATAAAGATCTTGCTGCCCTTCTCCATGATGGCATTCCTGTTGTC
TAA
Protein sequence:
MYARAAARQARASGLEKNPISGLLEYAQFASQTCEFNMIEQSGPPHEPRFKFQVVINGREF
PPAEAGSKKVAKQDAAMKAMTILLEEAKAKDSGKSEESSHYSTEKESEKTAESQTPTPSAT
SFFSGKSPVTTLLECMHKLGNSCEFRLLSKEGPAHEPKFQYCVAVGAQTFPSVSAPSKKVA
KQMAAEEAMKALHGEATNSMASDNQPEGMISESLDNLESMMPNKVRKIGELVRYLNTN
PVGGLLEYARSHGEAAEFKLVDQSGPPHEPKFVYQAKVGGRWFPAVCAHSKKQGKQEAA
DAALRVLIGENGTMDAKARNCLLQHREALEKDIKTSYIMDHMISDGFLTISEEEKVRNEPT
QQQRAAMLIKMILKKDNDSYVSFYNALLHEGYKDLAALLHDGIPVV
No3.dsCARE?PACT
The cDNA sequence:
ATGTACGCCCGTGCCGCCGCCCGTCAGGCCCGTGCCAGTGGTCTCGAGAGCGATTACG
ACATCCCCACTACTGAGAATCTTTATTTTCAGGGATCCTCCCAGAGCAGGCACCGCGCC
GAGGCCCCGCCGCTGGAGCGCGAGGACAGTGGGACCTTCAGTTTGGGGAAGATGATA
ACAGCTAAGCCAGGGAAAACACCGATTCAGGTATTACACGAATACGGCATGAAGACCA
AGAACATCCCAGTTTATGAATGTGAAAGATCTGATGTGCAAATACACGTGCCCACTTTC
ACCTTCAGAGTAACCGTTGGTGACATAACCTGCACAGGTGAAGGTACAAGTAAGAAG
CTGGCGAAACATAGAGCTGCAGAGGCTGCCATAAACATTTTGAAAGCCAATGCAAGTA
TTTGCTTTGCAGTTCCTGACCCCTTAATGCCTGACCCTTCCAAGCAACCAAAGAACCA
GCTTAATCCTATTGGTTCATTACAGGAATTGGCTATTCATCATGGCTGGAGACTTCCTGA
ATATACCCTTTCCCAGGAGGGAGGACCTGCTCATAAGAGAGAATATACTACAATTTGCA
GGCTAGAGTCATTTATGGAAACTGGAAAGGGGGCATCAAAAAAGCAAGCCAAAAGGA
ATGCTGCTGAGAAATTTCTTGCCAAATTTAGTAATATTTCTCCAGAGAACCACATTTCTT
TAACAAATGTAGTAGGACATTCTTTAGGATGTACTTGGCATTCCTTGAGGAATTCTCCTG
GTGAAAAGATCAACTTACTGAAAAGAAGCCTCCTCAGTATTCCAAATACAGATTACATC
CAGCTGCTTAGTGAAATTGCCAAGGAACAAGGTTTTAATATAACATATTTGGATATAGAT
GAACTGAGCGCCAATGGACAATATCAATGTCTTGCTGAACTGTCCACCAGCCCCATCA
CAGTCTGTCATGGCTCCGGTATCTCCTGTGGCAATGCACAAAGTGATGCAGCTCACAAT
GCTTTGCAGTATTTAAAGATAATAGCAGAAAGAAAGGGTACCATGGATGCAAAAGCTC
GAAATTGTTTGCTTCAACATAGAGAAGCTCTGGAAAAGGACATCAAGACATCCTACAT
CATGGATCACATGATTAGTGATGGATTTTTAACAATATCAGAAGAGGAAAAAGTAAGAA
ATGAGCCCACTCAACAGCAAAGAGCAGCTATGCTGATTAAAATGATACTTAAAAAAGA
TAATGATTCCTACGTATCATTCTACAATGCTCTACTACATGAAGGATATAAAGATCTTGCT
GCCCTTCTCCATGATGGCATTCCTGTTGTCTAA
Protein sequence:
MYARAAARQARASGLESDYDIPTTENLYFQGSSQSRHREAPPLEREDSGTFSLGKMITAK
PGKTPIQVLHEYGMKTKNIPVYECERSDVQIHVPTFTFRVTVGDITCTGEGTSKKLAKHRA
AEAAINILKANASICFAVPDPLMPDPSKQPKNQLNPIGSLQELAIHHGWRLPEYTLSQEGGP
AHKREYTTICRLESFMETGKGASKKQAKRNAAEKFLAKFSNISPENHISLTNVVGHSLGCT
WHSLRNSPGEKINLLKRSLLSIPNTDYIQLLSEIAKEQGFNITYLDIDELSANGQYQCLAEL
STSPITVCHGSGISCGNAQSDAAHNALQYLKIIAERKGTMDAKARNCLLQHREALEKDIK
TSYIMDHMISDGFLTISEEEKVRNEPTQQQRAAMLIKMILKKDNDSYVSFYNALLHEGYK
DLAALLHDGIPVV
No4.dsCARE?NF90
The cDNA sequence:
ATGTACGCCCGTGCCGCCGCCCGTCAGGCCCGTGCCAGTGGTCTCGAGATGAATGCCC
TGATGCGGTTGAACCAGCTGAAGCCAGGGCTGCAGTACAAGCTGGTGTCCCAGACTG
GGCCCGTCCATGCCCCCATCTTTACCATGTCTGTGGAGGTTGATGGCAATTCATTCGAG
GCCTCTGGGCCCTCCAAAAAGACGGCCAAGCTGCACGTGGCCGTTAAGGTGTTACAG
GACATGGGCTTGCCGACGGGTGCTGAAGGCAGGGACTCGAGCAAGGGGGAGGACTC
GGCTGAGGAGACCGAGGCGAAGCCAGCAGTGGTGGCCCCTGCCCCAGTGGTAGAAGC
TGTCTCCACCCCTAGTGCGGCCTTTCCCTCAGATGCCACTGCCGAGAACGTAAAACAG
CAGGGGCCGATCCTGACAAAGCACGGCAAGAACCCAGTCATGGAGCTGAACGAGAA
GAGGCGTGGGCTCAAGTACGAGCTCATCTCCGAGACCGGGGGCAGCCACGACAAGCG
CTTCGTCATGGAGGTCGAAGTGGATGGACAGAAGTTCCAAGGTGCTGGTTCCAACAA
AAAGGTGGGTACCATGGATGCAAAAGCTCGAAATTGTITGCTTCAACATAGAGAAGCT
CTGGAAAAGGACATCAAGACATCCTACATCATGGATCACATGATTAGTGATGGATTTTT
AACAATATCAGAAGAGGAAAAAGTAAGAAATGAGCCCACTCAACAGCAAAGAGCAGC
TATGCTGATTAAAATGATACTTAAAAAAGATAATGATTCCTACGTATCATTCTACAATGCT
CTACTACATGAAGGATATAAAGATCTTGCTGCCCTTCTCCATGATGGCATTCCTGTTGTC
TAA
Protein sequence:
MYARAAARQARASGLEMNALMRLNQLKPGLQYKLVSQTGPVHAPIFTMSVEVDGNSFE
ASGPSKKTAKLHVAVKVLQDMGLPTGAEGRDSSKGEDSAEETEAKPAVVAPAPVVEAVST
PSAAFPSDATAENVKQQGPILTKHGKNPVMELNEKRRGLKYELISETGGSHDKRFVMEVE
VDGQKFQGAGSNKKVGTMDAKARNCLLQHREALEKDIKTSYIMDHMISDGFLTISEEEK
VRNEPTQQQRAAMLIKMILKKDNDSYVSFYNALLHEGYKDLAALLHDGIPVV
2. according to the described specific double-strand RNA binding protein chimera of claim 1; It is characterized in that said recombinant DNA with specific double-strand RNA binding protein chimera transforms various protokaryons and the eukaryotic cell expression system is expressed, and expressed protein Preparation is become the antiviral drug of injection, tablet, oral preparation, capsule or spray.
3. according to the described specific double-strand RNA binding protein chimera of claim 1; It is characterized in that said specific double-strand RNA albumen mosaic is used for treatment and prevention at disease of viral infection, on the mammalian cell level as the protein drug treatment tool.
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| CN102349995B (en) * | 2011-10-24 | 2013-06-05 | 武汉大学 | Broad-spectrum antiviral medicament as well as preparation method and application thereof |
| CN109266731A (en) * | 2018-08-23 | 2019-01-25 | 窦帅杰 | The interaction of Tim-3 and NF90 is preventing or is treating the application in virus infection product |
| CN113416768B (en) * | 2021-07-06 | 2022-05-17 | 中国农业科学院兰州兽医研究所 | Application of PRKRA gene as target in inhibiting replication of peste des petits ruminants virus |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003062394A2 (en) * | 2002-01-22 | 2003-07-31 | Cold Spring Harbor Laboratory | Methods and compositions for rna interference |
| CN1763106A (en) * | 2005-10-21 | 2006-04-26 | 中国科学院生物物理研究所 | A kind of antiviral fusion protein and encoding gene thereof and application |
| CA2359180C (en) * | 1999-01-30 | 2007-06-12 | Roland Kreutzer | Method and medicament for inhibiting the expression of a given gene |
| CN101081871A (en) * | 2006-05-31 | 2007-12-05 | 华中科技大学 | Protein transduction field 4-apopain fusion protein (PTD4-Apoptin) and preparation method and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2359180C (en) * | 1999-01-30 | 2007-06-12 | Roland Kreutzer | Method and medicament for inhibiting the expression of a given gene |
| WO2003062394A2 (en) * | 2002-01-22 | 2003-07-31 | Cold Spring Harbor Laboratory | Methods and compositions for rna interference |
| CN1763106A (en) * | 2005-10-21 | 2006-04-26 | 中国科学院生物物理研究所 | A kind of antiviral fusion protein and encoding gene thereof and application |
| CN101081871A (en) * | 2006-05-31 | 2007-12-05 | 华中科技大学 | Protein transduction field 4-apopain fusion protein (PTD4-Apoptin) and preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Z. Lichner et al..Double-stranded RNA-binding proteins could suppress RNA interference-mediated antiviral defences.《Journal of General Virology》.2003,第84卷975-980. * |
| 王美霞 等.U1小核核糖核酸嵌合体核酶细胞内抑制丙型肝炎病毒表达的研究.《中华肝脏病杂志》.2006,第14卷(第1期),11-14. * |
| 石华 等.双链RNA依赖性蛋白激酶的结构与作用.《生命的化学》.2006,第26卷(第1期),38-41. * |
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