CN102349995A - Broad-spectrum antiviral medicament as well as preparation method and application thereof - Google Patents
Broad-spectrum antiviral medicament as well as preparation method and application thereof Download PDFInfo
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- CN102349995A CN102349995A CN201110324897XA CN201110324897A CN102349995A CN 102349995 A CN102349995 A CN 102349995A CN 201110324897X A CN201110324897X A CN 201110324897XA CN 201110324897 A CN201110324897 A CN 201110324897A CN 102349995 A CN102349995 A CN 102349995A
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Abstract
The invention discloses a broad-spectrum antiviral medicament as well as a preparation method and application thereof and relates to a creative medicament in the technical field of biology. The broad-spectrum antiviral medicament consists of a protein transduction label, a double-stranded RNA (Ribonucleic Acid) detection domain and an apoptosis induction domain. The preparation method comprises the steps of construction, expression and purification of a gene recombinant protein. The application comprises a cell model experiment, an animal model experiment and antiviral action for DNA (Deoxyribonucleic Acid) or RNA virus of dsRNA generated during infection cell. The broad-spectrum antiviral medicament is designed according to a pure cell antiviral natural mechanism and has the advantages of high reliability, broad-spectrum virus resistance, high specificity, no toxic or side effect, high safety, favorable repeatability of experiment result, easiness in popularization, simpleness and convenience in research and production and low cost.
Description
Technical field
The present invention relates to the original new drug in the biological technical field, relate in particular to a kind of medicine of broad-spectrum antiviral.
Background technology
Now; The viral pathogen that causes human virus's property disease is more and more paid close attention to; Viral comprising the burst of nature New Development like bird flu virus, SARS (coronavirus) etc.; The virus of clinicing aspect such as hepatitis virus, HIV (HIV) etc., and relevant potential cause biological panic virus such as variola, Ebola virus etc.Regrettably, for above-mentioned virus, lack very effective preventative and therapeutic measures so far, existing main measure can be divided into three major types:
1, the interferon and the anti-inflammatory factor: though effectively to part virus, to other viroid lack of specific, owing to self be the cell class factor; Like excessive absorption; Factor interactions such as itself and immune system can cause host cell class factor level unbalance, produce big side effect.
2, specificity suppressioning factor: virus replication is transcribed etc. in the process institute must play inhibiting specificity suppressioning factor (like the hiv protease inhibitive factor) by target spot; But its shortcoming is a virus undergos mutation; When changing corresponding target spot; Can cause its inhibitory action of effectively escaping inhibitive factor, so inhibitive factor can not be tackled the viral pathogen of New Development burst quickly and effectively, the side effect that produces after the drug use can't be predicted.
3, vaccine: because longer to the cycle of different virus development specificity vaccine; Prevention and treatment for New Development burst viral pathogen are not timely and effectively; And the development difficulty of vaccine is also quite huge, and this is mainly reflected in: one, viral species is various; Two, complicated with its various hypotypes of a kind of virus, for example vaccine can only be specific to a kind of virus, and the virus of other types is not had the cross protection reaction, and the host can not get good protection.
Summary of the invention
The object of the invention just is to overcome the above-mentioned shortcoming and defect that prior art exists, and a kind of medicine of broad-spectrum antiviral is provided.
The objective of the invention is to realize like this:
Technical thought:
Design one group of gene recombinant protein, it can specificity, cause the apoptosis of the cell that is infected by the virus quickly and efficiently, and does not damage the host cell of uninfection.
The gene recombinant protein prophylactic treatment is associated with two natural mechanism of cell:
1, the dsRNA (double-stranded RNA) in the interferon approach detects:
The genome of most of virus is double-stranded RNA or single stranded RNA.Virus infected cell produces dsRNA during duplicating and transcribing; All the other viral genome are that the virus of DNA can produce typical dsRNA equally through symmetrical transcription; The cell of opposite uninfection can not produce usually, and whether the host cell natural immunology defense produces dsRNA through above-mentioned cell and detect, catch and eliminate the cell that is infected by the virus.Two domain dsRBM1 that the protein kinase that interferon-induced RNA relies on is comprised by N-terminal and dsRBM2 (dsRNA binding motif 1 and 2) and the kinase domain of C-terminal are formed; The protein kinase that interferon-induced RNA relies on is with after dsRNA combines; Thereby through reverse autophosphorylation activated protein kinase; Activated protein kinase is phosphorylation eIF-2a (transcription factor) again, therefore can suppress the translation process of virus protein.
2, apoptotic pathways:
The apoptosis complex comprises apoptosis signaling molecule in the born of the same parents, and such as apoptosis protease-activating factor 1, FLICE (death receptor) activates death domain etc.Precursor apoptotic proteins enzyme is through the apoptotic proteins enzyme and a series of intracellular protein in shearing activation and then activation downstream, thus cell killing.
Each domain all has excellent function in similar approach and each albumen because the protein kinase that interferon-induced RNA relies on activation process has with apoptotic proteins enzyme activation process, so two above-mentioned approach can combine to form the novel approach of a broad-spectrum antiviral.This gene recombinant protein; Its domain comprises dsRNA land, apoptosis induction district (apoptotic proteins proenzyme land); As there being dsRNA in the cell, it will detect dsRNA and combine with it, and then the apoptosis of inducing cell; Be not present in the cell like dsRNA on the contrary, reaction then can not take place.In order to make it can be in vivo or the cell in vitro transmission, in the gene recombinant protein sequence, add the transduction sequence label, such as transcribing trans-activator, nexin transduction domain territory-4, poly arginine.Protein molecular in the interior perhaps external all cells type of these transhipment label proteins ability transporters, even can penetrate blood brain barrier.
Specifically:
1, the medicine of broad-spectrum antiviral
The medicine of broad-spectrum antiviral is made up of protein transduction label, double-stranded RNA detection architecture territory and apoptosis-inducing domain:
1. protein transduction label
The protein transduction label is following A, B or C
A: transcribe trans-activator (Trans-activator Transcription),
Aminoacid sequence: shown in SEQ NO.1;
B: nexin transduction domain 4 (Protein Transduction Domain 4),
Aminoacid sequence: shown in SEQ NO.2;
C: poly arginine (Polyarginine),
Aminoacid sequence: shown in SEQ NO.1.
2. double-stranded RNA detection architecture territory
Double-stranded RNA detection architecture territory is following A, B or C
A: the protein kinase that interferon-induced RNA relies on
(Interferon-inducible?RNA-dependent?protein?kinase)，
Aminoacid sequence: shown in SEQ NO.4;
The ribonuclease (2-5A-dependent ribonuclease) that B:2-5A relies on,
Aminoacid sequence: shown in SEQ NO.5;
C: vaccinia virus E3L albumen (E3L),
Aminoacid sequence: shown in SEQ NO.6.
3. apoptosis-inducing domain
The apoptosis-inducing domain is following A, B or C
A: growth suppressor gene 3 albumen (Growth-inhibiting gene 3protein),
Aminoacid sequence: shown in SEQ NO.7;
B: apoptosis protease-activating factor-1 (Apoptotic protease activating factor-1),
Aminoacid sequence: shown in SEQ NO.8;
C: Mus apoptosis protease-activating factor-1
(murine?Apoptotic?protease?activating?factor-1)，
Aminoacid sequence: shown in SEQ NO.9.
2, the method for preparing of this broad-spectrum antiviral medicament
The method for preparing of this broad-spectrum antiviral medicament comprises structure, expression and the purification of gene recombinant protein:
A, collect cultured cells [like Hela cell (deriving from CCTCC) etc.], utilize Trizol method (deriving from the precious biotech firm of DaLian, China) to extract cell total rna;
B, cell total rna to extracting; Utilize Auele Specific Primer (to annotate: subsidiary restriction enzyme site) to each zone design of said gene recombiant protein; Carry out a step pcr amplification; Promptly utilize random primer to carry out reverse transcription earlier, the RNA reverse transcription is become cDNA, utilize primer amplified to obtain each regional sequence of corresponding gene recombinant protein again;
C, cut glue and reclaim the purpose fragment, connect T carrier sequence verification institute extension increasing sequence correctness;
D, respectively the purpose fragment is carried out double digestion and is connected with pET32a (prokaryotic expression carrier), make up the gene recombinant protein clone;
E, clone's transformed into escherichia coli (BL21) that will build, IPTG (isopropyl-β-D-sulfo-galactopyranoside) are induced the expression (annotate: the transduction label is positioned at N/C end or NC two ends) of destination protein;
F, according to marking protein His-Tag (histidine-tagged), recombiant protein is filtered His fusion rotein purification column, obtain the gene recombinant protein of purification.
3, the application of this broad-spectrum antiviral medicament
1. cell model experiment
A, get cultured cell,, divide A, two groups of B also to perform labelling like Vero cell (African green monkey kidney cell);
B, A group compares group, and B winding kind virus is like enterovirus etc.;
C, in A group cell (as 7 bottles), corresponding respectively simultaneously (as the 7 kinds) ddH that adds
2O (distilled water compares group), gene recombinant protein, gene recombinant protein mutant (the protein kinase aminoacid sequence F10E that the interferon-induced RNA in dsRNA detection architecture territory relies on), escherichia coli extract, each component of gene recombinant protein (the gene recombinant protein B in the gene recombinant protein A of disappearance protein transduction label, disappearance dsRNA detection architecture territory and the gene recombinant protein C of disappearance apoptosis-inducing domain) are also marked;
D, in B winding kind virocyte, add each composition in the c step respectively and mark;
After e, A, B group cell add above-mentioned various composition, place CO
2Incubator is cultivated 3～5 day time;
F, after 3～5 days, place the microscopically observed and recorded respectively to organize the survival rate of cell in A, B group cell;
G, result judge:
In the A group cell, i.e. the cell of virus inoculation not, the cellular control unit growth is normal; Cell survival rate is high, compares all the other cells with cellular control unit; Cell survival rate is close; Near 100%, all the other six kinds of materials that add are described, promptly gene recombinant protein, gene recombinant protein mutant, escherichia coli extract, each component cell growth of gene recombinant protein have no side effect;
In the B group cell, i.e. the cell of virus inoculation, cellular control unit is all dead, compares with cellular control unit, the composition escherichia coli extract of adding, each component of gene recombinant protein (totally three), cell survival rate all is close to 0; The component gene recombiant protein mutant that adds, the cell survival survival rate is close to 50%, and the component gene recombiant protein cell survival rate that adds is close to 100%, explains that thus gene recombinant protein brings into play good effects in antiviral therapy.
2. animal model experiment
A, get and raise mice,, divide A, two groups of B also to mark like BALB/c mouse;
B, A group compare group, B group mice intranasal inoculation virus;
In c, the while A group mice, the gene recombinant protein and the injection buffer (comparing group) of respectively the nasal cavity of mice being injected 100nM;
In d, the B group mice, mark according to the injection of c steps in sequence;
E, continue to raise 5～7 days, write down the mice survival later and note down;
F, result judge:
In the A group mice, i.e. the mice of virus inoculation not, control group mice is normal, compares with matched group, and the mice survival of injection gene recombinant protein explain that the gene recombinant protein of adding has no side effect to mice;
In the B group mice, i.e. the mice of virus inoculation, control group mice is all dead, compares with matched group, the injection gene recombinant protein, mice all survives, and explains that gene recombinant protein brings into play effect in antiviral therapy.
3. the antiviral scope of this broad-spectrum antiviral medicament
This broad-spectrum antiviral medicament plays antivirus action to DNA or the RNA viruses that during infection cell, produces dsRNA; Like influenza virus, respiratory syncytial virus, adenovirus, parainfluenza virus, metapneumovirus, coronavirus, bocavirus, rhinovirus, enterovirus, rubella virus, Measles virus, encephalitis b virus, mumps virus, herpes simplex virus, rotavirus, hepatitis virus and HIV etc.
The present invention has advantage and good effect:
1. according to the natural Mechanism Design of pure cell anti-virus, reliability is high;
2. broad-spectrum antiviral, specificity is high;
3. have no side effect, safe;
4. the experimental result good reproducibility is easy to promote;
5. development and production are easy, and are with low cost.
Description of drawings
Fig. 1 is the infection coordinate diagram that gene recombinant protein is effectively resisted influenza virus H1N1 in the mdck cell;
Wherein: abscissa is the various components that add; Vertical coordinate is the survival rate of cell.
Fig. 2 is the infection coordinate diagram that gene recombinant protein is effectively resisted influenza virus H1N1 in the BALB/c mouse;
Wherein: abscissa is the various components that add; Vertical coordinate is the survival number of mice.
Fig. 3 is the infection coordinate diagram that gene recombinant protein is effectively resisted Enterovirus 71 type in the RD cell;
Wherein: abscissa is the various components that add; Vertical coordinate is the survival rate of cell.
Fig. 4 is the infection coordinate diagram that gene recombinant protein is effectively resisted Enterovirus 71 type in the BALB/c mouse;
Wherein: abscissa is the various components that add; Vertical coordinate is the survival number of mice.
The specific embodiment
Specify below in conjunction with accompanying drawing and embodiment:
One, embodiment 1
Get the influenza virus H1N1 standard substance of cultured mdck cell (MDCK), 120pfu/well, the gene recombinant protein liquid (being broad-spectrum antiviral medicament) of 100nM and the negative object of reference of 100nM, like each component of gene recombinant protein, gene recombinant protein mutant and escherichia coli extract.
1, chooses the medicine of one type broad-spectrum antiviral
The medicine of broad-spectrum antiviral is made up of protein transduction label, double-stranded RNA detection architecture territory and apoptosis-inducing domain:
1. protein transduction label
Nexin transduction domain 4 (Protein Transduction Domain 4),
Aminoacid sequence: shown in SEQ NO.2;
2. double-stranded RNA detection architecture territory
The protein kinase that interferon-induced RNA relies on
(Interferon-inducible?RNA-dependent?prote?in?kinase)，
Aminoacid sequence: shown in SEQ NO.4;
3. apoptosis-inducing domain
Apoptosis protease-activating factor-1 (Apoptotic protease activating factor-1),
Aminoacid sequence: shown in SEQ NO.8;
2, the preparation of this broad-spectrum antiviral medicament
A, collection cultured Hela cell (deriving from CCTCC) utilize Trizol method (deriving from Japanese precious biotech firm) to extract cell total rna;
B, cell total rna to extracting; Utilize to three pairs of three zone design Auele Specific Primers of said gene recombiant protein; Carry out a step pcr amplification; Promptly utilize random primer that the RNA that extracts is carried out reverse transcription earlier, then the RNA reverse transcription is become cDNA, utilize three pairs of primers of specificity to increase respectively again and obtain each regional sequence of corresponding gene recombinant protein;
C, cut the fragment that glue reclaims three clauses and subclauses, connect T carrier sequence verification institute extension increasing sequence correctness;
D, the fragment and the pET32a (prokaryotic expression carrier) of three clauses and subclauses carried out double digestion and be connected successively, build this gene recombinant protein clone (annotate: the transduction label is positioned at albumen N/C end or NC two ends);
E, clone's transformed into escherichia coli (BL21) that will build, IPTG (isopropyl-β-D-sulfo-galactopyranoside) induces the expression of destination protein;
F, according to marking protein His-Tag (histidine-tagged), gene recombinant protein is filtered His fusion rotein purification column, obtain the gene recombinant protein of purification, dilute and be final concentration 100nM;
G, the top method of foundation; Can make up the gene recombinant protein component of defective respectively, like the gene recombinant protein of disappearance protein transduction label, the gene recombinant protein in disappearance dsRNA detection architecture territory and the gene recombinant protein of disappearance apoptosis-inducing domain; Utilize PCR positional mutation method to make up the gene recombinant protein mutant simultaneously.
3, the application of this broad-spectrum antiviral medicament
1. cell model experiment
A, get totally 14 bottles of cultured mdck cells, divide two groups of A, B, 7 bottles every group and perform labelling;
B, A group compare group, B winding kind 120pfu/well influenza virus H1N1 standard substance 100ul;
In c, 7 bottles of cells of while A group, add ddH respectively
2O (distilled water compares group), 100nM gene recombinant protein, gene recombinant protein mutant (the protein kinase aminoacid sequence F10E that the interferon-induced RNA in dsRNA detection architecture territory relies on), escherichia coli extract, each component of gene recombinant protein (the gene recombinant protein B in the gene recombinant protein A of disappearance protein transduction label, disappearance dsRNA detection architecture territory and the gene recombinant protein C of disappearance apoptosis-inducing domain) each 100ul also mark;
D, B organize in the 7 bottle graft kind influenza virus H1N1 cells, add each composition in the c step respectively and mark;
After e, A, B group cell add above-mentioned various composition, place CO
2Incubator is cultivated 3 day time;
F, after three days, place the microscopically observed and recorded respectively to organize the survival rate of cell in A, B group cell;
G, result judge:
In the A group cell, i.e. the cell of virus inoculation not, the cellular control unit growth is normal; Cell survival rate is high, compares all the other 6 bottles of cells with cellular control unit; Cell survival rate is close, near 100%, sees Fig. 1; All the other six kinds of materials that add are described, promptly gene recombinant protein, gene recombinant protein mutant, escherichia coli extract, each component cell growth of gene recombinant protein have no side effect;
In the B group cell, i.e. the cell of virus inoculation, cellular control unit is all dead, compares with cellular control unit, the composition escherichia coli extract of adding, each component of gene recombinant protein (totally three), totally 4 bottles of cell survival rates all are close to 0; The component gene recombiant protein mutant that adds, the cell survival survival rate is close to 50%, and the component gene recombiant protein cell survival rate that adds is close to 100%, sees Fig. 1, explains that thus gene recombinant protein brings into play good effects in antiviral therapy.
2. animal model experiment
A, get totally 12 of the BALB/c mouses of raising for 7 ages in week, divide two groups of A, B, 6 every group and mark;
B, A group compare group, B group mice intranasal inoculation 120pfu/well influenza virus H1N1 standard substance 100ul;
In c, the while A group mice, respectively the nasal cavity of 3 mices is injected gene recombinant protein and each 100ul of other 3 injection buffer (comparing group) of 100nM;
In d, 6 mices of B group, mark according to the injection of c steps in sequence;
E, continue to raise 5 days, write down the mice survival later and note down;
F, result judge:
In the A group mice, i.e. the mice of virus inoculation not, control group mice is normal, compares with matched group, and Fig. 2 is seen in the mice survival of the gene recombinant protein of injection 100nM, explain that the gene recombinant protein of adding has no side effect to mice;
In the B group mice, i.e. the mice of virus inoculation, control group mice is all dead, compares with matched group, the gene recombinant protein of injection 100nM, mice all survives, and sees Fig. 2, explains that gene recombinant protein brings into play effect in antiviral therapy.
Two, embodiment 2
Get the enterovirns type 71 standard substance of cultured RD cell (human rhabdomyosarcoma's cell), 120pfu/well, the gene recombinant protein liquid (being broad-spectrum antiviral medicament) of 100nM and the negative object of reference of 100nM, like each component of gene recombinant protein, gene recombinant protein mutant and escherichia coli extract.
1, chooses the medicine of one type broad-spectrum antiviral
The medicine of broad-spectrum antiviral is made up of protein transduction label, double-stranded RNA detection architecture territory and apoptosis-inducing domain:
1. protein transduction label
Nexin transduction domain 4 (Protein Transduction Domain 4),
Aminoacid sequence: shown in SEQ NO.2;
2. double-stranded RNA detection architecture territory
The protein kinase that interferon-induced RNA relies on
(Interferon-inducible?RNA-dependent?protein?kinase)，
Aminoacid sequence: shown in SEQ NO.4;
3. apoptosis-inducing domain
Apoptosis protease-activating factor-1 (Apoptotic protease activating factor-1),
Aminoacid sequence: shown in SEQ NO.8;
2, the preparation of this broad-spectrum antiviral medicament
1. the structure of gene recombinant protein, expression and purification:
A, collection cultured Hela cell (deriving from CCTCC) utilize Trizol method (deriving from Japanese precious biotech firm) to extract cell total rna;
B, cell total rna to extracting; Utilize to three pairs of three zone design Auele Specific Primers of said gene recombiant protein; Carry out a step pcr amplification; Promptly utilize random primer that the RNA that extracts is carried out reverse transcription earlier, then the RNA reverse transcription is become cDNA, utilize three pairs of primers of specificity to increase respectively again and obtain each regional sequence of corresponding gene recombinant protein;
C, cut the fragment that glue reclaims three clauses and subclauses, connect T carrier sequence verification institute extension increasing sequence correctness;
D, the fragment and the pET32a (prokaryotic expression carrier) of three clauses and subclauses carried out double digestion and be connected successively, build this gene recombinant protein clone (annotate: the transduction label is positioned at albumen N/C end or NC two ends);
E, clone's transformed into escherichia coli (BL21) that will build, IPTG (isopropyl-β-D-sulfo-galactopyranoside) induces the expression of destination protein;
F, according to marking protein His-Tag (histidine-tagged), gene recombinant protein is filtered His fusion rotein purification column, obtain the gene recombinant protein of purification, dilute and be final concentration 100nM;
G, the top method of foundation; Can make up the gene recombinant protein component of defective respectively, like the gene recombinant protein of disappearance protein transduction label, the gene recombinant protein in disappearance dsRNA detection architecture territory and the gene recombinant protein of disappearance apoptosis-inducing domain; Utilize PCR positional mutation method to make up the gene recombinant protein mutant simultaneously.
3, the application of this broad-spectrum antiviral medicament
1. cell model experiment
A, get totally 14 bottles in cultured RD cell, divide two groups of A, B, 7 bottles every group and perform labelling;
B, A group compare group, B winding kind 120pfu/well enterovirns type 71 standard substance 100ul;
In c, 7 bottles of cells of while A group, add ddH respectively
2O (distilled water compares group), 100nM gene recombinant protein, gene recombinant protein mutant (the protein kinase aminoacid sequence F10E that the interferon-induced RNA in dsRNA detection architecture territory relies on), escherichia coli extract, each component of gene recombinant protein (the gene recombinant protein B in the gene recombinant protein A of disappearance protein transduction label, disappearance dsRNA detection architecture territory and the gene recombinant protein C of disappearance apoptosis-inducing domain) each 100ul also mark;
D, B organize in the 7 bottle graft kind enterovirns type 71 cells, add each composition in the c step respectively and mark;
After e, A, B group cell add above-mentioned various composition, place CO
2Incubator is cultivated 3 day time;
F, after three days, place the microscopically observed and recorded respectively to organize the survival rate of cell in A, B group cell;
G, result judge:
In the A group cell, i.e. the cell of virus inoculation not, the cellular control unit growth is normal; Cell survival rate is high, compares all the other 6 bottles of cells with cellular control unit; Cell survival rate is close, near 100%, sees Fig. 3; All the other six kinds of materials that add are described, promptly gene recombinant protein, gene recombinant protein mutant, escherichia coli extract, each component cell growth of gene recombinant protein have no side effect;
In the B group cell, i.e. the cell of virus inoculation, cellular control unit is all dead, compares with cellular control unit, the composition escherichia coli extract of adding, each component of gene recombinant protein (totally three), totally 4 bottles of cell survival rates all are close to 0; The component gene recombiant protein mutant that adds, the cell survival survival rate is close to 50%, and the component gene recombiant protein cell survival rate that adds is close to 100%, sees Fig. 3, explains that thus gene recombinant protein brings into play good effects in antiviral therapy.
2. animal model experiment
A, get totally 12 of the BALB/c mouses of raising for 7 ages in week, divide two groups of A, B, 6 every group and mark;
B, A group compare group, B group mice intranasal inoculation 120pfu/well enterovirns type 71 standard substance 100ul;
In c, the while A group mice, respectively the nasal cavity of 3 mices is injected gene recombinant protein and each 100ul of other 3 injection buffer (comparing group) of 100nM;
In d, 6 mices of B group, mark according to the injection of c steps in sequence;
E, continue to raise 5 days, write down the mice survival later and note down;
F, result judge:
In the A group mice, i.e. the mice of virus inoculation not, control group mice is normal, compares with matched group, and Fig. 4 is seen in the mice survival of the gene recombinant protein of injection 100nM, explain that the gene recombinant protein of adding has no side effect to mice;
In the B group mice, i.e. the mice of virus inoculation, control group mice is all dead, compares with matched group, the gene recombinant protein of injection 100nM, mice all survives, and sees Fig. 4, explains that gene recombinant protein brings into play effect in antiviral therapy.
Sequence table
< 110>Wuhan University
< 120>a kind of medicine of broad-spectrum antiviral
<140>
<141>
<160>?9
<210>?1
<211>?11
< 212>aminoacid sequence
< 213>transcribe trans-activator
< 400>N end-YGRKKRRQRRR-C end;
<210>?2
<211>?11
< 212>aminoacid sequence
< 213>nexin transduction domain 4
< 400>N end-YARAAARQARA-C end;
<210>?3
<211>?9
< 212>aminoacid sequence
< 213>poly arginine
< 400>N end-RRRRRRRRR-C end;
<210>?4
<211>?181
< 212>aminoacid sequence
< 213>protein kinase of interferon-induced RNA dependence
<400>
N end-MAGDLSAGFFMEELNTYRQKQGVVLKYQELPNSGPPHDRRFTFQVIIDGREFPEGE GRSKKEAK
NAAAKLAVEILNKEKKAVSPLLLTTTNSSEGLSMGNYIGLINRIAQKKRLTVNYEQ CASGVHGPEGFHYKCKMGQKEYSIGTGSTKQEAKQLAAKLAYLQILSEETSVKSDY LSSGS-C end;
<210>?5
<211>?335
< 212>aminoacid sequence
< 213>ribonuclease of 2-5A dependence
<400>
N-terminal-MESRDHNNPQEGPTSSSGRRAAVEDNHLLIKAVQNEDVDLVQQLLEGGANVNFQEEEGGWTPLH? NAVQMSREDIVELLLRHGADPVLRKKNGATPFILAAIAGSVKLLKLFLSKGADVNECDFYGFTAFMEAAVYGKVKALKFLYKRGANVNLRRKTKEDQERLRKGGATALMDAAEKGHVEVLKILLDEMGADVNACDNMGRNALIHALLSSDDSDVEAITHLLLDHGADVNVRGERGKTPLILAVEKKHLGLVQRLLEQEHIEINDTDSDGKTALLLAVELKLKKIAELLCKRGASTDCGDLVMTARRNYDHSLVKVLLSHGAKEDFHPPAED-C terminal;
<210>?6
<211>?179
< 212>aminoacid sequence
< 213>vaccinia virus E3L albumen
<400>
N end-DERSDAEIVCQAIKNIGIEGATAAQLTRQLNMEKRKVNKALYDLQRSAMVYSSDDI PPRWFMTT
EADKPDADAMADVIIDDVSREKSMREDHKSFDDVIPAKKIIDWKSANPVTIINEYCQITRRDWSFRIE
The SVGPSNSPTFYACVDIDGRVFDKADGKSKRDAKNNAAKLAVDKLLGY-C end;
<210>?7
<211>?100
< 212>aminoacid sequence
< 213>growth suppressor gene 3 albumen
<400>
N end-MDPFLVLLHSVSSSLSSSELTELKFLCLGRVGKRKLERVQSGLDLFSMLLEQNDLE PGHTELL
The RELLASLRRHDLLRRVDDFEAGAAAGAAPGEEDLCAA-C end;
<210>?8
<211>?97
< 212>aminoacid sequence
< 213>apoptosis protease-activating factor-1
<400>
N end-MDAKARNCLLQHREALEKDIKTSYIMDHMISDGFLTISEEEKVRNEPTQQQRAAML IKMILKK
The DNDSYVSFYNALLHEGYKDLAALLHDGIPVVSSS-C end;
<210>?9
<211>?97
< 212>aminoacid sequence
< 213>Mus apoptosis protease-activating factor-1
<400>
N end-MDAKARNCLLQHREALEKDIKTSYIMDHMISNGVLSVIEEEKVKSQATQYQRAAAL IKMILNK
The DNCAYISFYNALLHEGYKDLAALLQSGLPLVSSS-C end.
Claims (3)
1. the medicine of a broad-spectrum antiviral is characterized in that:
The medicine of broad-spectrum antiviral is made up of protein transduction label, double-stranded RNA detection architecture territory and apoptosis-inducing domain:
1. the protein transduction label is following A, B or C
A: transcribe trans-activator, aminoacid sequence is shown in SEQ NO.1;
B: nexin transduction domain 4, aminoacid sequence is shown in SEQ NO.2;
C: the poly arginine, aminoacid sequence is shown in SEQ NO.3;
2. double-stranded RNA detection architecture territory is following A, B or C
A: the protein kinase that interferon-induced RNA relies on, aminoacid sequence is shown in SEQ NO.4;
The ribonuclease that B:2-5A relies on, aminoacid sequence is shown in SEQ NO.5;
C: vaccinia virus E3L albumen, aminoacid sequence is shown in SEQ NO.6;
3. the apoptosis-inducing domain is following A, B or C
A: growth suppressor gene 3 albumen, aminoacid sequence is shown in SEQ NO.7;
B: apoptosis protease-activating factor-1, aminoacid sequence is shown in SEQ NO.8;
C: Mus apoptosis protease-activating factor-1, aminoacid sequence is shown in SEQ NO.9.
2. press the method for preparing of the medicine of the described a kind of broad-spectrum antiviral of claim 1, it is characterized in that:
The method for preparing of this broad-spectrum antiviral medicament comprises structure, expression and the purification of gene recombinant protein:
A, collection cultured cells utilize the Trizol method to extract cell total rna;
B, cell total rna to extracting; Utilize Auele Specific Primer to each zone design of said gene recombiant protein; Carry out a step pcr amplification; Promptly utilize random primer to carry out reverse transcription earlier, the RNA reverse transcription is become cDNA, utilize primer amplified to obtain each regional sequence of corresponding gene recombinant protein again;
C, cut glue and reclaim the purpose fragment, connect T carrier sequence verification institute extension increasing sequence correctness;
D, respectively the purpose fragment is carried out double digestion and is connected with pET32a, make up the gene recombinant protein clone;
E, clone's transformed into escherichia coli that will build, IPTG induces the expression of destination protein;
F, according to marking protein His-Tag, recombiant protein is filtered His fusion rotein purification column, obtain the gene recombinant protein of purification.
3. press the application of the medicine of the described a kind of broad-spectrum antiviral of claim 1, it is characterized in that:
1. cell model experiment;
2. animal model experiment;
3. DNA or the RNA viruses that during infection cell, produces dsRNA played antivirus action.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102614506A (en) * | 2012-03-05 | 2012-08-01 | 北京工业大学 | Recombinant HPV16L2 protein vaccine and preparation method thereof |
| WO2014106319A1 (en) * | 2013-01-04 | 2014-07-10 | 华为技术有限公司 | Method for processing data in the ethernet, physical layer chip and ethernet device |
| US20160222072A1 (en) * | 2013-10-23 | 2016-08-04 | University Of Washington Through Its Center For Commercialization | Universal Protein Tag for Double Stranded Nucleic Acid Delivery |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103571863B (en) * | 2013-10-10 | 2015-03-04 | 中山大学 | Novel pharmaceutical protein DRACO and applications thereof in prevention and treatment of porcine reproductive and respiratory syndrome |
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| CN1908016A (en) * | 2006-08-24 | 2007-02-07 | 复旦大学 | Fusion protein with protein transduction structure field TAT-PTD and application thereof |
| CN101355957A (en) * | 2005-11-04 | 2009-01-28 | 为人技术株式会社 | Methods for fusion polypeptide delivery into a cell |
| CN101525388A (en) * | 2009-02-20 | 2009-09-09 | 中国人民解放军第四军医大学 | Specific double-strand RNA binding protein chimera and application thereof in virus infectious diseases |
| CN101918566A (en) * | 2007-11-22 | 2010-12-15 | Biontex实验室有限公司 | By influencing the transfection results that innate immune system improves non-viral delivery system |
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| CN101355957A (en) * | 2005-11-04 | 2009-01-28 | 为人技术株式会社 | Methods for fusion polypeptide delivery into a cell |
| CN1908016A (en) * | 2006-08-24 | 2007-02-07 | 复旦大学 | Fusion protein with protein transduction structure field TAT-PTD and application thereof |
| CN101918566A (en) * | 2007-11-22 | 2010-12-15 | Biontex实验室有限公司 | By influencing the transfection results that innate immune system improves non-viral delivery system |
| CN101525388A (en) * | 2009-02-20 | 2009-09-09 | 中国人民解放军第四军医大学 | Specific double-strand RNA binding protein chimera and application thereof in virus infectious diseases |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102614506A (en) * | 2012-03-05 | 2012-08-01 | 北京工业大学 | Recombinant HPV16L2 protein vaccine and preparation method thereof |
| CN102614506B (en) * | 2012-03-05 | 2013-09-25 | 北京工业大学 | Recombinant HPV16L2 protein vaccine and preparation method thereof |
| WO2014106319A1 (en) * | 2013-01-04 | 2014-07-10 | 华为技术有限公司 | Method for processing data in the ethernet, physical layer chip and ethernet device |
| US20160222072A1 (en) * | 2013-10-23 | 2016-08-04 | University Of Washington Through Its Center For Commercialization | Universal Protein Tag for Double Stranded Nucleic Acid Delivery |
| US9809632B2 (en) * | 2013-10-23 | 2017-11-07 | University Of Washington Through Its Center For Commercialization | Universal protein tag for double stranded nucleic acid delivery |
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