CN102349995B - 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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- CN102349995B CN102349995B CN 201110324897 CN201110324897A CN102349995B CN 102349995 B CN102349995 B CN 102349995B CN 201110324897 CN201110324897 CN 201110324897 CN 201110324897 A CN201110324897 A CN 201110324897A CN 102349995 B CN102349995 B CN 102349995B
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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 biological technical field, relate in particular to medicine of a kind of broad-spectrum antiviral and its preparation method and application.
Background technology
Now, the viral pathogen that causes human virus's property disease is more and more concerned, viral as bird flu virus, SARS (coronavirus) etc. comprising the new burst of nature, the virus of clinicing aspect such as hepatitis virus, HIV (HIV (human immunodeficiency virus)) etc., and relevant potential virus such as variola, the Ebola virus etc. of causing biological fear.Regrettably, for above-mentioned virus, lack so far very effective preventative and therapeutic measures, existing main measure can be divided into three major types:
1, interferon and the anti-inflammatory factor: although to part virus effectively, other viroid is lacked specificity, owing to self being the cell class factor, as excessive absorption, the factor interactions such as itself and immune system can cause host cell class factor level unbalance, produce larger side effect.
2, specificity suppressioning factor: virus replication is transcribed etc. in process institute must play inhibiting specificity suppressioning factor (as the hiv protease inhibitive factor) by target spot, but its shortcoming is virus undergos mutation, when changing corresponding target spot, can cause it effectively to escape the inhibitory action of inhibitive factor, so inhibitive factor can not be tackled the viral pathogen of new burst quickly and effectively, the side effect that produces after drug use can't be predicted.
3, vaccine: due to longer for the cycle of different virus development specificity vaccine, prevention and treatment for new 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, its various hypotypes of same virus are complicated, and for example vaccine can only be specific for a kind of virus, and the virus of other types is reacted without cross protection, and the host can not get good protection.
Summary of the invention
Purpose of the present invention just is to overcome the above-mentioned shortcoming and defect that prior art exists, and medicine of a kind of broad-spectrum antiviral and its preparation method and application is provided.
The object of the present invention is achieved like this:
Technical thought:
Design one group of gene recombinant protein, it can specificity, cause quickly and efficiently the apoptosis of the cell that is infected by the virus, 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 copying and transcribing; All the other viral genome are that the virus of DNA can produce typical dsRNA equally by symmetrical transcription, the cell of opposite uninfection can not produce usually, and whether the host cell natural immunology defense produces dsRNA by 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 by reverse autophosphorylation activated protein kinase, the protein kinase of activation 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 born of the same parents, and such as apoptosis protease-activating factor 1, FLICE (death receptor) activates death domain etc.Precursor apoptotic proteins enzyme activates and then activates apoptotic proteins enzyme and a series of intracellular protein in downstream by shearing, thus cell killing.
The protein kinase that relies on due to interferon-induced RNA activates process and has with apoptotic proteins enzyme activation process that in similar approach and each albumen, each domain has good function, so two above-mentioned approach can be in conjunction with the novel approach that forms 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 cell, it will detect dsRNA and combination with it, and then the apoptosis of inducing cell, be not present in cell as dsRNA on the contrary, reaction can not occur.For can be in vivo or the cell in vitro transmission, add the transduction sequence label in the gene recombinant protein sequence, such as transcribing trans-activator, nexin transduction domain territory-4, poly arginine.These transport in label proteins energy transporters or the protein molecular in external all cells type, even can penetrate blood brain barrier.
Specifically:
1, the medicine of broad-spectrum antiviral
The medicine of broad-spectrum antiviral is comprised 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: as shown in SEQ NO.1;
B: nexin transduction domain 4 (Protein Transduction Domain 4),
Aminoacid sequence: as shown in SEQ NO.2;
C: poly arginine (Polyarginine),
Aminoacid sequence: as 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: as shown in SEQ NO.4;
The ribonuclease (2-5A-dependent ribonuclease) that B:2-5A relies on,
Aminoacid sequence: as shown in SEQ NO.5;
C: vaccinia virus E3L albumen (E3L),
Aminoacid sequence: as 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: as shown in SEQ NO.7;
B: apoptotic protease activating factor-1 (Apoptotic protease activating factor-1),
Aminoacid sequence: as shown in SEQ NO.8;
C: Mus apoptotic protease activating factor-1
(murine?Apoptotic?protease?activating?factor-1)，
Aminoacid sequence: as shown in SEQ NO.9.
2, the preparation method of this broad-spectrum antiviral medicament
The preparation method of this broad-spectrum antiviral medicament comprises structure, expression and the purification of gene recombinant protein:
A, collect cultured cells [as Hela cell (deriving from CCTCC) etc.], utilize Trizol method (derive from DaLian, China precious biotech firm) to extract cell total rna;
B, the cell total rna to extracting, utilization (is annotated: subsidiary restriction enzyme site) for the Auele Specific Primer of each zone design of said gene recombiant protein, carry out a step pcr amplification, namely first utilize random primer to carry out reverse transcription, the RNA reverse transcription is become cDNA, and the recycling primer amplified obtains each regional sequence of corresponding gene recombinant protein;
C, cut glue and reclaim the purpose fragment, connect T carrier sequence verification institute extension increasing sequence correctness;
D, the purpose fragment is connected the prokaryotic expression carrier with pET32a respectively) carry out double digestion and be connected with connection and build the gene recombinant protein clone;
E, the clone that will build transform escherichia coli (BL21), and IPTG (isopropyl-β-D-sulfo-galactopyranoside) induces 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 protein purification post, obtain the gene recombinant protein of purification.
3, the application of this broad-spectrum antiviral medicament
1. cell model experiment
A, get cultured cell, as Vero cell (African green monkey kidney cell), minute A, two groups of B also perform labelling;
B, A group compares group, and B winding kind virus is as enterovirus etc.;
C, in A group cell (as 7 bottles), correspondence adds (as 7 kinds) ddH respectively simultaneously
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 respectively each composition in the c step and mark;
After e, A, B group cell add above-mentioned various compositions, be placed in CO
2Incubator is cultivated 3～5 day time;
F, after 3～5 days, with A, B the group cell be placed in the survival rate that the microscopically observed and recorded is respectively organized cell;
G, result judgement:
In A group cell, the i.e. 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, namely gene recombinant protein, gene recombinant protein mutant, escherichia coli extract, each component cell growth of gene recombinant protein have no side effect;
In 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 that adds, 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%, illustrates that thus gene recombinant protein brings into play good effects in antiviral therapy.
2. animal model experiment
A, get and raise mice, as BALB/c mouse, minute A, two groups of B also mark;
B, A group compares group, B group mice intranasal inoculation virus;
In c, while A group mice, respectively the nasal cavity of mice is injected gene recombinant protein and the injection buffer (comparing group) of 100nM;
In d, B group mice, inject successively according to the c step and mark;
E, continuation were raised 5～7 days, recorded later the mouse survival situation and noted down;
F, result judgement:
In A group mice, i.e. the mice of virus inoculation not, control group mice is normal, compares with matched group, and the mouse survival of injection gene recombinant protein illustrates that the gene recombinant protein that adds has no side effect to mice;
In 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 illustrates 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 produces dsRNA during infection cell; As 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 (human immunodeficiency virus) etc.
The present invention has following advantages 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, be 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 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 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 enterovirns type 71 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 enterovirns type 71 in BALB/c mouse;
Wherein: abscissa is the various components that add; Vertical coordinate is the survival number of mice.
The specific embodiment
Describe in detail below in conjunction with drawings and Examples:
One, embodiment 1
Get the influenza virus H1N1 standard substance of cultured mdck cell (Madin-Darby canine kidney(cell line) (MDCK)), 120pfu/well, the gene recombinant protein liquid (being broad-spectrum antiviral medicament) of 100nM and the negative object of reference of 100nM, as each component of gene recombinant protein, gene recombinant protein mutant and escherichia coli extract.
1, choose the medicine of the broad-spectrum antiviral of a type
The medicine of broad-spectrum antiviral is comprised 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: as 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: as shown in SEQ NO.4;
3. apoptosis-inducing domain
Apoptotic protease activating factor-1 (Apoptotic protease activating factor-1),
Aminoacid sequence: as 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, the cell total rna to extracting, utilization designs three pairs of Auele Specific Primers for the Three regions of said gene recombiant protein, carry out a step pcr amplification, namely first utilize random primer to carry out reverse transcription to the RNA that extracts, then the RNA reverse transcription is become cDNA, three pairs of primers of recycling specificity increase respectively and obtain each regional sequence of corresponding gene recombinant protein;
C, cut the fragment that glue reclaims three entries, connect T carrier sequence verification institute extension increasing sequence correctness;
D, the fragment of three entries is connected the prokaryotic expression carrier with pET32a) carry out double digestion and be connected connection, build this gene recombinant protein clone (annotate: the transduction label is positioned at albumen N/C end or NC two ends);
E, the clone that will build transform escherichia coli (BL21), and 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 protein purification post, obtain the gene recombinant protein of purification, dilute and be final concentration 100nM;
G, the top method of foundation, can build respectively the gene recombinant protein component of defective, as the gene recombinant protein in the gene recombinant protein of disappearance protein transduction label, disappearance dsRNA detection architecture territory and the gene recombinant protein of disappearance apoptosis-inducing domain; Utilize simultaneously PCR positional mutation method to build the gene recombinant protein mutant.
3, the application of this broad-spectrum antiviral medicament
1. cell model experiment
A, get totally 14 bottles of cultured mdck cells, two groups of minute A, B, 7 bottles every group and perform labelling;
B, A group compares group, B winding kind 120pfu/well influenza virus H1N1 standard substance 100ul;
In c, 7 bottles of cells of while A group, add respectively ddH
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 marks;
In d, B group 7 bottle graft kind influenza virus H1N1 cells, add respectively each composition in the c step and mark;
After e, A, B group cell add above-mentioned various compositions, be placed in CO
2Incubator is cultivated 3 day time;
F, after three days, with A, B the group cell be placed in the survival rate that the microscopically observed and recorded is respectively organized cell;
G, result judgement:
In A group cell, the i.e. cell of virus inoculation not, the cellular control unit growth is normal, and cell survival rate is high, compares with cellular control unit, all the other 6 bottles of cells, cell survival rate is close, near 100%, sees Fig. 1, all the other six kinds of materials that add are described, namely gene recombinant protein, gene recombinant protein mutant, escherichia coli extract, each component cell growth of gene recombinant protein have no side effect;
In 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 that adds, 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, illustrates 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 mouse of raising for 7 ages in week, two groups of minute A, B, 6 every group and mark;
B, A group compares group, B group mice intranasal inoculation 120pfu/well influenza virus H1N1 standard substance 100ul;
In c, while A group mice, respectively the nasal cavity of 3 mices is injected gene recombinant protein and other 3 each 100ul of injection buffer (comparing group) of 100nM;
In 6 mices of d, B group, inject successively according to the c step and mark;
E, continuation were raised 5 days, recorded later the mouse survival situation and noted down;
F, result judgement:
In A group mice, i.e. the mice of virus inoculation not, control group mice is normal, compares with matched group, and the mouse survival of the gene recombinant protein of injection 100nM is seen Fig. 2, illustrates that the gene recombinant protein that adds has no side effect to mice;
In 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, illustrates 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, as each component of gene recombinant protein, gene recombinant protein mutant and escherichia coli extract.
1, choose the medicine of the broad-spectrum antiviral of a type
The medicine of broad-spectrum antiviral is comprised 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: as 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: as shown in SEQ NO.4;
3. apoptosis-inducing domain
Apoptotic protease activating factor-1 (Apoptotic protease activating factor-1),
Aminoacid sequence: as 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, the cell total rna to extracting, utilization designs three pairs of Auele Specific Primers for the Three regions of said gene recombiant protein, carry out a step pcr amplification, namely first utilize random primer to carry out reverse transcription to the RNA that extracts, then the RNA reverse transcription is become cDNA, three pairs of primers of recycling specificity increase respectively and obtain each regional sequence of corresponding gene recombinant protein;
C, cut the fragment that glue reclaims three entries, connect T carrier sequence verification institute extension increasing sequence correctness;
D, the fragment of three entries is connected the prokaryotic expression carrier with pET32a) carry out double digestion and be connected connection, build this gene recombinant protein clone (annotate: the transduction label is positioned at albumen N/C end or NC two ends);
E, the clone that will build transform escherichia coli (BL21), and 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 protein purification post, obtain the gene recombinant protein of purification, dilute and be final concentration 100nM;
G, the top method of foundation, can build respectively the gene recombinant protein component of defective, as the gene recombinant protein in the gene recombinant protein of disappearance protein transduction label, disappearance dsRNA detection architecture territory and the gene recombinant protein of disappearance apoptosis-inducing domain; Utilize simultaneously PCR positional mutation method to build the gene recombinant protein mutant.
3, the application of this broad-spectrum antiviral medicament
1. cell model experiment
A, get totally 14 bottles, cultured RD cell, two groups of minute A, B, 7 bottles every group and perform labelling;
B, A group compares group, B winding kind 120pfu/well enterovirns type 71 standard substance 100ul;
In c, 7 bottles of cells of while A group, add respectively ddH
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 marks;
In d, B group 7 bottle graft kind enterovirns type 71 cells, add respectively each composition in the c step and mark;
After e, A, B group cell add above-mentioned various compositions, be placed in CO
2Incubator is cultivated 3 day time;
F, after three days, with A, B the group cell be placed in the survival rate that the microscopically observed and recorded is respectively organized cell;
G, result judgement:
In A group cell, the i.e. cell of virus inoculation not, the cellular control unit growth is normal, and cell survival rate is high, compares with cellular control unit, all the other 6 bottles of cells, cell survival rate is close, near 100%, sees Fig. 3, all the other six kinds of materials that add are described, namely gene recombinant protein, gene recombinant protein mutant, escherichia coli extract, each component cell growth of gene recombinant protein have no side effect;
In 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 that adds, 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, illustrates 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 mouse of raising for 7 ages in week, two groups of minute A, B, 6 every group and mark;
B, A group compares group, B group mice intranasal inoculation 120pfu/well enterovirns type 71 standard substance 100ul;
In c, while A group mice, respectively the nasal cavity of 3 mices is injected gene recombinant protein and other 3 each 100ul of injection buffer (comparing group) of 100nM;
In 6 mices of d, B group, inject successively according to the c step and mark;
E, continuation were raised 5 days, recorded later the mouse survival situation and noted down;
F, result judgement:
In A group mice, i.e. the mice of virus inoculation not, control group mice is normal, compares with matched group, and the mouse survival of the gene recombinant protein of injection 100nM is seen Fig. 4, illustrates that the gene recombinant protein that adds has no side effect to mice;
In 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, illustrates that gene recombinant protein brings into play effect in antiviral therapy.
Sequence table
＜110〉Wuhan University
＜120〉medicine of a kind of broad-spectrum antiviral and its preparation method and application
<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 end-MESRDHNNPQEGPTSSSGRRAAVEDNHLLIKAVQNEDVDLVQQLLEGGANVNFQEE EGGWTPLH NAVQMSREDIVELLLRHGADPVLRKKNGATPFILAAIAGSVKLLKLFLSKGADVNE CDFYGFTAFMEAAVYGKVKALKFLYKRGANVNLRRKTKEDQERLRKGGATALMDAA EKGHVEVLKILLDEMGADVNACDNMGRNALIHALLSSDDSDVEAITHLLLDHGADV NVRGERGKTPLILAVEKKHLGLVQRLLEQEHIEINDTDSDGKTALLLAVELKLKKI AELLCKRGASTDCGDLVMTARRNYDHSLVKVLLSHGAKEDFHPPAED-C end;
<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〉apoptotic protease activating factor-1
<400>
N end-MDAKARNCLLQHREALEKDIKTSYIMDHMISDGFLTISEEEKVRNEPTQQQRAAML IKMILKK
The DNDSYVSFYNALLHEGYKDLAALLHDGIPVVSSS-C end;
<210>?9
<211>?97
＜212〉aminoacid sequence
＜213〉Mus apoptotic protease activating factor-1
<400>
N end-MDAKARNCLLQHREALEKDIKTSYIMDHMISNGVLSVIEEEKVKSQATQYQRAAAL IKMILNK
The DNCAYISFYNALLHEGYKDLAALLQSGLPLVSSS-C end.
Claims (2)
1. the medicine of a broad-spectrum antiviral is characterized in that:
The medicine of broad-spectrum antiviral is comprised of protein transduction label, double-stranded RNA detection architecture territory and apoptosis-inducing domain:
1. the protein transduction label is nexin transduction domain 4, and aminoacid sequence is as shown in SEQ NO.2;
2. double-stranded RNA detection architecture territory is the protein kinase that interferon-induced RNA relies on, and aminoacid sequence is as shown in SEQ NO.4;
3. the apoptosis-inducing domain is apoptotic protease activating factor-1, and aminoacid sequence is as shown in SEQ NO.8.
2. press the preparation method of the medicine of a kind of broad-spectrum antiviral claimed in claim 1, it is characterized in that:
The preparation method 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, the cell total rna to extracting, the protein kinase that utilization relies on for nexin transduction domain 4, interferon-induced RNA and the Auele Specific Primer of each zone design of apoptotic protease activating factor-1, carry out a step pcr amplification, namely first utilize random primer to carry out reverse transcription, the RNA reverse transcription is become cDNA, and the recycling primer amplified obtains each regional sequence of corresponding gene recombinant protein;
C, cut glue and reclaim the purpose fragment, connect T carrier sequence verification institute extension increasing sequence correctness;
D, the purpose fragment is carried out double digestion with pET32a respectively and be connected with connection and build the gene recombinant protein clone;
E, the clone that will build transform escherichia coli, and IPTG induces the expression of destination protein;
F, according to marking protein His-Tag, recombiant protein is filtered His fusion protein purification post, obtain the gene recombinant protein of purification.
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| CN 201110324897 CN102349995B (en) | 2011-10-24 | 2011-10-24 | Broad-spectrum antiviral medicament as well as preparation method and application thereof |
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|---|---|---|---|
| CN 201110324897 CN102349995B (en) | 2011-10-24 | 2011-10-24 | Broad-spectrum antiviral medicament as well as preparation method and application thereof |
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| CN103571863A (en) * | 2013-10-10 | 2014-02-12 | 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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| CN102614506B (en) * | 2012-03-05 | 2013-09-25 | 北京工业大学 | Recombinant HPV16L2 protein vaccine and preparation method thereof |
| CN103797742B (en) * | 2013-01-04 | 2017-11-21 | 华为技术有限公司 | The method of processing data, physical chip and ethernet device in Ethernet |
| 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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| DE602006016468D1 (en) * | 2005-11-04 | 2010-10-07 | Forhumantech Co Ltd | PROCESS FOR DELIVERING FUSION POLYPEPTIDE 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 |
| WO2009065618A2 (en) * | 2007-11-22 | 2009-05-28 | Biontex Laboratories Gmbh | Improvement of transfection results of non-viral gene delivery systems by influencing the congenital immune system |
| CN101525388B (en) * | 2009-02-20 | 2012-02-01 | 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 |
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| CN103571863A (en) * | 2013-10-10 | 2014-02-12 | 中山大学 | Novel pharmaceutical protein DRACO and applications thereof in prevention and treatment of porcine reproductive and respiratory syndrome |
| 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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