CN102242122B - Nucleic acid aptamer specifically combined with HCV (hepatitis C virus) non-structural protein NS2, and application thereof - Google Patents
Nucleic acid aptamer specifically combined with HCV (hepatitis C virus) non-structural protein NS2, and application thereof Download PDFInfo
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Abstract
The invention discloses a nucleic acid aptamer specifically combined with HCV (hepatitis C virus) non-structural protein NS2, and application thereof. The nucleic acid aptamer disclosed by the invention is a DNA (deoxyribonucleic acid) molecule as shown in the SEQ ID NO: 2. Shown by experiments, the nucleic acid aptamer disclosed by the invention can be specifically combined with the HCV non-structural protein NS2, but can not be combined with non-structural protein NS5B and the structural protein PET. Therefore, the nucleic acid aptamer disclosed by the invention can be used as a molecular drug or targeting vector for preventing and/or treating viral hepatitis C, and has wide application prospects in the therapeutic field of viral hepatitis C.
Description
Technical field
The present invention relates to aptamer and application thereof with HCV virus Nonstructural Protein NS2 specific combination.
Background technology
Hepatitis C is long latent period, is difficult to early diagnosis and therapy, very harmful to human body.Present treatment means is single.Aptamer becomes molecular medicine or the targeting vector of a new generation to specific protein with its unique chemical antibody characteristic.But present also lacking very much based on aptamer to hepatitis C virus (HCV virus) medicine or diagnostic reagent.And screening does not have report as yet to the aptamer of HCV Nonstructural Protein NS2.
Summary of the invention
An object of the present invention is to provide a kind of aptamer.
Aptamer provided by the present invention is dna molecular shown in the SEQ ID NO:2.
Above-mentioned aptamer also belongs to protection scope of the present invention in the application that preparation is used for diagnosing and/or prevent and/or treat the product of hepatitis C.Wherein, said product is medicine or vaccine.
Another object of the present invention provides a kind of product that is used to diagnose and/or prevent and/or treat hepatitis C.
The product that is used to diagnose and/or prevent and/or treat hepatitis C provided by the present invention, its activeconstituents are above-mentioned aptamer.Wherein, said product is medicine or vaccine.
Above-mentioned aptamer also belongs to protection scope of the present invention in the application that preparation is used for diagnosing and/or prevent and/or treat the pharmaceutical carrier of hepatitis C.
Another object of the present invention provides a kind of pharmaceutical carrier that is used to diagnose and/or prevent and/or treat hepatitis C.
The pharmaceutical carrier that is used to diagnose and/or prevent and/or treat hepatitis C provided by the present invention, its activeconstituents are above-mentioned aptamer.
Above-mentioned aptamer also belongs to protection scope of the present invention in the application that preparation suppresses in the viral reagent of assembling and/or discharging of hepatitis C.
Another object of the present invention provides a kind of reagent that suppresses the assembling of hepatitis C virus and/or discharge.
Inhibition hepatitis C virus provided by the present invention assembling and/or the reagent that discharges, its activeconstituents is above-mentioned aptamer.
The application of above-mentioned aptamer in preparation and hepatitis C virus Nonstructural Protein NS2 bonded reagent also belongs to protection scope of the present invention.
Another object of the present invention provides a kind of and hepatitis C virus Nonstructural Protein NS2 bonded reagent.
Provided by the present invention and hepatitis C virus Nonstructural Protein NS2 bonded reagent, its activeconstituents is above-mentioned aptamer.
Experiment showed, that aptamer of the present invention can be specifically combine with HCV virus Nonstructural Protein NS2, and do not combine with Nonstructural Protein NS5B and reference protein PET200 in screening.Therefore, aptamer of the present invention can be used as molecular medicine or the targeting vector that prevents and/or treats hepatitis C, has broad application prospects in the treatment field of hepatitis C.
Description of drawings
Fig. 1 is the UV-Vis spectrum of albumen NS2 solution before and after fixing.
Fig. 2 is programmed screening product and the PET (left side) and NS2 albumen (right side) the effect situation of FITC mark, fluorescence intensity: 100-1000.
Fig. 3 is that product and PET (left side) and NS2 (right side) effect situation, fluorescence intensity: 100-5000 are screened in the 5th time of FITC mark.
Fig. 4 be fit sequence respectively with the proteic situation that combines of NS2, fluorescence intensity: 300-4000.
Fig. 5 be fit sequence respectively with the proteic situation that combines of PET, fluorescence intensity: 200-400.
Fig. 6 be fit sequence respectively with the proteic situation that combines of NS5B, fluorescence intensity: 200-400.
Fig. 7 is assembling or the release that fit sequence suppresses HCV.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The preparation of embodiment 1, GAP-associated protein GAP and related solution
One, with the preparation of histidine-tagged hepatitis C virus NS2 (target proteins)
1, the amplification of HCV NS2 encoding sox
With DNA shown in the SEQ ID NO:4 (being GENBANK ACCESSION NO.AY605046.1) is template, and the primer of forming with primer 1 and primer 2 obtains pcr amplification product to carrying out pcr amplification.
Primer 1 (upstream primer): 5 '-CGCGCGAATTCATGCCGCGTGGAAGC-3 ';
Primer 2 (downstream primer): 5 '-CTGCAGGGATCCTTAGGCCAATCCGTT-3 ';
5 ' end of upstream and downstream primer is introduced EcoR1 restriction enzyme site and BamH1 restriction enzyme site respectively.
Pcr amplification condition: 95 ℃ of 2min; 95 ℃ of 30s, 66 ℃ of 30s, 72 ℃ of 1min, 35 circulations; 72 ℃ of 7min.
2, construction of prokaryotic expression vector
1. use the pcr amplification product of Restriction Enzyme EcoR1 and BamH1 double digestion step 1, obtain enzyme and cut product.
2. use Restriction Enzyme EcoR1 and BamH1 double digestion prokaryotic expression carrier pET28a, reclaim carrier framework.
3. step enzyme is 1. cut product and be connected, obtain connecting product with step carrier framework 2..
4. will connect product transformed into escherichia coli DH5 α competent cell, with the LB plate screening positive colony that contains penbritin (Amp), the extraction plasmid carries out enzyme successively and cuts the evaluation of identifying and check order.
Sequencing result shows; (skeleton plasmid is prokaryotic expression carrier pET28a to have obtained recombinant plasmid pET28a-NS2; Between EcoR1 and BamH1 restriction enzyme site, inserted the dna sequence dna of coding hepatitis C virus NS2; Histidine-tagged encoding sox on NS2 albumen of expressing and the carrier merges, and expresses with histidine-tagged hepatitis C virus NS2 albumen).
3, identify with the proteic prokaryotic expression of histidine-tagged hepatitis C virus NS2
1. with recombinant plasmid pET28a-NS2 transformed into escherichia coli BL21 (DE3) competent cell, with the LB plate screening positive colony that contains penbritin.
2. the picking positive colony is in the LB liquid nutrient medium, and 37 ℃ of joltings are spent the night, and adds IPTG (final concentration 1mmol/L) continuation cultivation and induces 10h.
3. the centrifugal 1min of 13000rpm collects thalline and carries out SDS-PAGE electrophoresis and Western blot.It is anti-his (available from Sigma company) that one of Westernblot resists.The result shows, contains in the thalline with histidine-tagged hepatitis C virus core albumen.
4, with the proteic great expression of histidine-tagged hepatitis C virus core, purifying and detection
1. with recombinant plasmid pET28a-NS2 transformed into escherichia coli BL21 (DE3) competent cell, obtain the bacterium of recombinating.
2. the reorganization bacterium inoculation LB substratum that 1. step is obtained; 37 ℃ of shaking culture are spent the night; Be transferred in the LB substratum that contains the 50ug/ml penbritin by 1: 50 volume ratio; About 2h (making OD600 is 0.6-0.8) is cultivated in 37 ℃ of joltings, adds IPTG (final concentration 1mmol/L) continuation cultivation and induces 12h.
3. the centrifugal collection thalline of 3500rpm is containing the start buffer of 100ug/ml N,O-Diacetylmuramidase (0.02M sodium phosphate, 0.5M NaCl; PH7.4) ultrasonication (frequency 120w in; Ultrasonic 3s stops 3s in each circulation, and 400 circulations are carried out on ice bath); 4 ℃, the centrifugal 10min of 10000rpm collect the cracking supernatant.
4. the cracking supernatant is splined on Ni-NTA link coupled agarose and sticks post (available from GE company), with washing buffer (0.02M sodium phosphate, 0.5M NaCl; 0.05M imidazoles, pH7.4) foreign protein is removed in washing, with elution buffer (0.02M sodium phosphate; 0.5M NaCl; 0.5M imidazoles, pH7.4) wash-out target protein obtains with histidine-tagged hepatitis C virus NS2 albumen.
Two, prepare with histidine-tagged PET proteic (reference protein)
1, the DNA shown in the SEQ ID NO:5 (being GENBANK ACCESSION NO.U46489.1) is inserted between the EcoR1 and BamH1 site of prokaryotic expression carrier pET28a; Obtain recombinant plasmid pET28a-PET (the histidine-tagged encoding sox on proteic encoding sox of PET and the carrier merges, and expresses with histidine-tagged PET albumen).
2, identify with the proteic prokaryotic expression of histidine-tagged PET
1. with recombinant plasmid pET28a-PET transformed into escherichia coli BL21 (DE3) competent cell, with the LB plate screening positive colony that contains penbritin.
2. the picking positive colony is in the LB liquid nutrient medium, and 37 ℃ of joltings are spent the night, and adds IPTG (final concentration 1mmol/L) continuation cultivation and induces 10h.
3. the centrifugal 1min of 13000rpm collects thalline and carries out SDS-PAGE electrophoresis and Western blot.It is anti-his (available from Sigma company) that one of Westernblot resists.
3, replace recombinant plasmid pET28a-NS2 with recombinant plasmid pET28a-PET, other obtains with histidine-tagged PET albumen with the step 4 of experiment one.
Three, with histidine-tagged HCV virus Nonstructural Protein NS5B preparation
DNA shown in the SEQ ID NO:6 (being GENBANK ACCESSION NO.JF51107.1) is inserted between the EcoR1 and BamH1 site of prokaryotic expression carrier pET28a; Obtain recombinant plasmid pET28a-NS5B (the histidine-tagged encoding sox on proteic encoding sox of NS5B and the carrier merges, and expresses with histidine-tagged NS5B albumen).
2, identify with the proteic prokaryotic expression of histidine-tagged PET
1. with recombinant plasmid pET28a-NS5B transformed into escherichia coli BL21 (DE3) competent cell, with the LB plate screening positive colony that contains penbritin.
2. the picking positive colony is in the LB liquid nutrient medium, and 37 ℃ of joltings are spent the night, and adds IPTG (final concentration 1mmol/L) continuation cultivation and induces 10h.
3. the centrifugal 1min of 13000rpm collects thalline and carries out SDS-PAGE electrophoresis and Western blot.It is anti-his (available from Sigma company) that one of Western blot resists.
3, replace recombinant plasmid pET28a-NS2 with recombinant plasmid pET28a-NS5B, other obtains with histidine-tagged NS5B albumen with the step 4 of experiment one.
Four, the preparation of related solution
PBS damping fluid: pH is 7.4, is made up of water and solute; Solute and concentration thereof are: 39mM NaH
2PO
4, 61mM Na
2HPO
4, 150mM NaCl.
Binding buffer liquid: pH is 7.4, is made up of PBS damping fluid and solute; Solute and concentration thereof are: 1mM MgCl
2, 0.2mg/ml yeast transfer RNA(tRNA).
Washings: pH is 7.4, is made up of water and solute; Solute and concentration thereof are: 25mM Tris, 150mM NaCl, 0.1% (quality percentage composition) bovine serum albumin (BSA), 0.05% (volumn concentration) Tween-20.
With embodiment 1 preparation with histidine-tagged hepatitis C virus NS2 albumen (target proteins; Be the solution form; The concentration of target proteins is 100ug/mL), embodiment 1 preparation with histidine-tagged PET albumen (reference protein; Be the solution form, the concentration of reference protein is 100ug/mL) and the various related solution of embodiment 1 preparation be used for embodiment 2 to embodiment 4.
1. the fit screening process of hepatitis C virus NS2 protein nucleic acid
1.1 it is proteic fixing
After getting 300 microlitre Ni-NTA agar microballons washing three times, being scattered in 3 ml concns is in the histidine-tagged HCV albumen of 1.5 mcg/ml bands (or the PET albumen) solution, places shaking table to hatch 1h (100rpm, 25 ℃) then.PBS centrifuge washing three times is scattered in microballon among the 1ml PBS again, adds the small amounts of protease suppressor factor, place 4 ℃ subsequent use.
Protein solution UV, visible light before and after the absorption absorbs (UV-Vis) spectrum and has confirmed that albumen is fixed on (Fig. 1) on the microballon through Ni-Histidine sequestering action.Among Fig. 1, the UV-Vis spectrum of albumen NS2 solution before 1 expression is fixing, the UV-Vis spectrum of albumen NS2 solution after 2 expressions are fixing.
1.2 screening process
1) pre-treatment in DNA library:
After 5nmol DNA library (5 '-ACG CTC GGA TGC CAC TAC AG (40N) CTC ATG GAC GTG CTG GTG AC-3 ') dissolving, through 94 ℃ of heating and cooled on ice processing, place 37 ℃ for use.
2) anti-sieve:
Get the fixedly proteic microballon of PET of 200 microlitres, add pretreated DNA library, (rotating speed 100rpm) hatched 1 hour in 37 ℃ of shaking tables, and be centrifugal through ultrafiltration.
3) just sieve:
Get the fixedly proteic microballon of HCV NS2 of 100 microlitres, add anti-sieve back ultrafiltration centrifugal solution, (rotating speed 100rpm) hatched 1 hour in 37 ℃ of shaking tables.Behind the ultrafiltration centrifuge washing, microballon is transferred in the centrifuge tube, added the aqua sterilisa of 600 microlitres simultaneously.Microballon behind 95 ℃ of heating eluted dnas, is collected the template that supernatant is used for pcr amplification.
Combine the proteic aptamer of HCV in order to obtain high-affinity and specificity, in the process of screening, progressively change anti-sieve and wash number of times and increase screening pressure with just sieving in proteic microballon consumption, screening time, the screening solution tRNA content and just sieving and washing; With the confocal fluorescent microscope every The selection result of taking turns has been carried out preliminary investigation simultaneously.
4) PCR and ssDNA separate:
The PCR reaction system is formed: and 10 μ L 10X PCR buffered soln (100mM Tris-HCl, pH 8.3; 15mM MgCl
2), 3 μ L 1mM dNTPs; Upstream primer of 3 μ L, 25 μ M FITC marks (5 '-ACG CTC GGA TGC CACTAC AG-3 ') and biotin labeled downstream primer (5 '-GTC ACCAGC ACG TCC ATG AG-3 '), 0.25 μ LTaq warm start polysaccharase, 5-15 μ L screening product and 69-79 μ L sterile purified water.Thermal cycle conditions: 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s.Before amplification, investigate different cyclic amplification products, choose suitable cycle index with agargel electrophoresis.
SsDNA separates: the affine absorption of VISOSE microballon that utilizes avidin to encapsulate, the sodium hydroxide of 0.2M separates double-stranded, and the ssDNA of FITC mark is separated with biotin labeled ssDNA.Again the ssDNA of FITC mark is crossed the NAP-5 pillar and carry out desalination, and collect the filtrating between the 0.5-1.5mL.Collect the DNA concentration in the solution with the UV-vis spectrometry.DNA preserves or is used for the lower whorl screening in-20 ℃ after frozen cryodrying.
2. cloning and sequencing
Through behind the multi-turns screen; Observation is with the instant sign of experiment; When fluorescence intensity no longer strengthens; Getting last screening product is that template and unmarked upstream and downstream primer carry out pcr amplification according to above-mentioned PCR reaction conditions and step, the unmarked dna double chain product that obtains is operated according to pMD18-T Vector test kit handbook, then random sequencing.
3. sequential analysis
Through analyzing the primary structure of ssDNA, then according to whether existing common conserved sequence that institute's calling sequence is classified in the stochastic sequence.HCV structural protein and Nonstructural Protein are carried out the aptamer screening, and owing to reasons such as purity of protein, concentration, drawing better result is the screening to core protein and NS2.
4,5 circulations of the aptamer of NS2 screening having carried out altogether.Since second circulation, carry out fit enrichment with experiment and characterize.The result sees Fig. 2, Fig. 3.
5, result: through the clone, choose 104 samples and check order, and classify, obtain seeing table 1 to the highly enriched fit sequence of NS2 according to the DNA primary structure.Characterize discovery through fluorescence co-focusing, three sequences are roughly the same with the proteic effect that combines of NS2.
Table 1
The Performance Detection of embodiment 3, aptamer
1, raw material:
Synthetic aptamer S14, aptamer S6, aptamer S5 and random library sequence, every 5 OD, 5 ' the end equal flag F ITC (fluorescein isothiocyanate) in aptamer and library.
The random library sequence: 5 '-ACG CTC GGA TGC CAC TAC AG-(N40)-C TCA TGG ACG TGC TGG TGA C-3 '.
Be fixed with the preparation of the microballon of target protein: shown in embodiment 2.With histidine-tagged HCV virus Nonstructural Protein NS2 albumen and with histidine-tagged PET albumen, with the proteic preparation of histidine-tagged NS5B shown in embodiment 1.
PBS damping fluid: pH is 7.4, is made up of water and solute; Solute and concentration thereof are: 39mM NaH
2PO
4, 61mM Na
2HPO
4, 150mM NaCl.
Binding buffer liquid: pH is 7.4, is made up of PBS damping fluid and solute; Solute and concentration thereof are: 1mM MgCl
2, 0.2mg/ml yeast transfer RNA(tRNA).The yeast transfer RNA(tRNA) is available from Invitrogen, and catalog number is 15401-011.
2, condition: in the 37 degree water-baths, aptamer and target protein are hatched, after hatching, all, use binding buffer liquid resuspended at last, carry out fluoroscopic examination again with PBS damping fluid washing 3 times.
The 1st group: each aptamer or the random library of 1ul 10uM FITC mark are dissolved (each aptamer or random library final concentration are 100nM) with binding buffer liquid, and the microballon that is fixed with HCV virus Nonstructural Protein NS2 with 70ul is hatched 1h;
The 2nd group: each aptamer or the random library of 1ul 10uM FITC mark are dissolved (each aptamer or random library final concentration are 100nM) with binding buffer liquid, and the microballon that is fixed with the PET reference protein with 70ul is hatched 1h;
The 3rd group: each aptamer or the random library of 1ul 10uM FITC mark are dissolved (each aptamer or random library final concentration are 100nM) with binding buffer liquid, be fixed with the proteic microballon of NS5B with 70ul and hatch 1h;
3, the result is following:
First group, three kinds of aptamers combine the result as shown in Figure 4 with HCV virus Nonstructural Protein NS2's.The result shows, compares with random library, and three fit sequences and NS2 be proteic to be combined significantly, is combining the proteic sepharose 4B sub-surface of NS2 to form bright fluorescence, shows that to screen three fit sequence-specifics that obtain fine;
Second group, three kinds of aptamers combine the result as shown in Figure 5 with reference protein PET's.The result shows that similar with random library, three fit sequences do not combine with PET albumen, and bead surface does not present fluorescence, shows to screen three sequences and the debond of PET albumen that obtains;
The 3rd group, three kinds of aptamers combine the result as shown in Figure 6 with HCV virus Nonstructural Protein NS5B's.The result shows that similar with random library, three fit sequences do not combine with NS5B albumen, and bead surface does not present fluorescence, shows to screen three sequences and the debond of NS5B albumen that obtains.
Comprehensive above detected result shows that three kinds of aptamers of nuclear have excellent specificity to NS2 albumen.
Among the figure, lib representes the random library sequence.
The application of embodiment 4, aptamer
1, experiment material:
1) random library: 5 '-ACG CTC GGA TGC CAC TAC AG-(N40)-C TCA TGG ACG TGC TGG TGA C-3 '.
The synthetic following DNA of precious biotech firm in Dalian: random library, aptamer S14, aptamer S6, aptamer S5;
2) the Huh7.5 cell disclosed in document " Journal of virology, 2002,76:13001-13004. ", and HCV virus disclosed in document " PNAS, 2011,108:4991-4996 ", and the public can obtain from Institute of Chemistry, Academia Sinica.
3) the Huh7.5.5 cell derives from the Huh7.5 cell, is the HCV viral genome is advanced the novel cell that obtains in the Huh7.5 cell by the method transfection of electric shock.But expression of HCV is viral.Concrete experimental technique is following:
A: will contain the virus genomic plasmid pJFH1 of HCV (Nat.Med.2005.11:791-796.) with restriction enzyme Xba I single endonuclease digestion, and make its linearizing.
B: ordinary method is extracted above-mentioned enzyme and is cut the plasmid among the system A: add the 0.5M ethylenediamine-N,N'-diacetic acid(EDDA), and the absolute ethyl alcohol of 3M sodium-acetate and two volumes, subzero 20 degree are placed and are spent the night behind the mixing; High speed centrifugation, removes supernatant by 13000 rev/mins; Use 70% washing with alcohol again, natural air drying adds the water dissolution of nuclease free at last.
C: transcribe: in the linear plasmid pJFH1 that extracts, add Yeast Nucleic Acid triphosphoric acid mixture, the responsive transcription damping fluid, transcriptase was placed 4 hours in 37 degree behind the mixing.
D: collect RNA: in transcribing mixed system, add lithium chloride solution, subzero 20 degree are placed and are spent the night; High speed centrifugation, removes supernatant by 13000 rev/mins; Use 70% washing with alcohol, natural air drying adds the water dissolution of nuclease free at last.
E: electricity changes:
Cultivate Huh7.5 to suitable cell state.
With pancreatin the Huh7.5 cell dissociation is got off, and with the Opti-MEM nutrient solution washed twice that does not contain serum.
With cell precipitation use Opti-MEM resuspended to density be 1 * 10
7Individual/mL.
The cell thorough mixing of 10mg JFH1 RNA and 0.4mL, join in the electric revolving cup of 4mm.
The electroporation parameter is set to 0.27kV, 100ohms, 960 μ F, shocks by electricity then.
Cell after the cultivation transfection is done the positive rate that immunofluorescence detects cell.Obtain the Huh7.5.5 cell of expression of HCV virus.
4) liposome 2000, DMEM, two anti-goat anti-mouse IgG-FITC and DAPI are all available from Invitrogen; 6 porocyte culture plates are available from Costar;
5) preparation method of anti-HCV NS5A protein antibodies: with the NS5A sequence (like SEQ ID NO:7; Promptly see GENBANK ACCESSION NO.AF529366.1); Insert among the carrier for expression of eukaryon pcDNA3.1, be configured to eukaryotic expression recombinant plasmid pcDNA3.1-NS5A.Behind a large amount of amplification recombinant plasmids, with the saline water dilution, the concentration that makes recombinant plasmid is 1ug/ul in intestinal bacteria.With the recombinant plasmid inoculation mouse of 100ug, the injection of Calf muscle bikini.Strengthen once again after one week.After twice inoculation, the 3rd week was collected the serum of inoculation mouse, as the polyvalent antibody of NS5A.
Carrier for expression of eukaryon pcDNA3.1 is available from Invitrogen.
2, experiment condition:
1) the Huh7.5.5 cell cultures is in 6 porocyte culture plates (200,000/hole), behind the 24h with Lipofectamine 2000 transfection random libraries or NS2 aptamer (concentration: 100nM) get into the Huh7.5.5 cell, continue to cultivate that to reclaim cell conditioned medium behind the 48h for use.
2) the Huh7.5 cell cultures is in 6 porocyte culture plates (200,000/hole), infects the Huh7.5 cell of corresponding aperture behind the 24h with the cell conditioned medium of above-mentioned recovery, does immunofluorescence after infecting 72h, detects the positive area that HCV infects the Huh7.5 cell.One anti-is the polyvalent antibody of NS5A, two anti-be goat anti-mouse IgG-FITC.
The substratum of cell cultures is the DMEM+10% foetal calf serum.
3, experimental result:
As shown in Figure 7
Compare with random library, after aptamer S6 treatment group Huh7.5.5 cell conditioned medium liquid inductance dyed the Huh7.5 cell, its positive area (green fluorescence) significance reduced.S14 treatment group, S5 treatment group and random library treatment group come to the same thing.
The result shows that aptamer S6 can suppress assembling or the release of HCV in the Huh7.5.5 cell efficiently.
Claims (9)
1. an aptamer is dna molecular shown in the SEQ ID NO:2.
2. the described aptamer of claim 1 is used for diagnosing and/or prevent and/or treat the application of the product of hepatitis C in preparation.
3. product that is used to diagnose and/or prevent and/or treat hepatitis C, its activeconstituents is the described aptamer of claim 1.
4. the described aptamer of claim 1 is used for diagnosing and/or prevent and/or treat the application of the pharmaceutical carrier of hepatitis C in preparation.
5. pharmaceutical carrier that is used to diagnose and/or prevent and/or treat hepatitis C, its activeconstituents is the described aptamer of claim 1.
6. the described aptamer of claim 1 suppresses the application in the viral reagent of assembling and/or discharging of hepatitis C in preparation.
7. one kind is suppressed the reagent that hepatitis C virus is assembled and/or discharged, and its activeconstituents is the described aptamer of claim 1.
8. the application of the described aptamer of claim 1 in preparation and hepatitis C virus Nonstructural Protein NS2 bonded reagent.
One kind with hepatitis C virus Nonstructural Protein NS2 bonded reagent, its activeconstituents is the described aptamer of claim 1.
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| BELLECAVE P等.Selection of DNA Aptamers That Bind the RNA-Dependent RNA Polymerase of Hepatitis C Virus and Inhibit Viral RNA Synthesis In Vitro.《OLIGONUCLEOTIDES》.2004,第13卷(第6期),第455-463页. * |
| 王长印等.HCV的分子生物学及实验室检查研究进展.《山东医药》.2010,第50卷(第35期),第109-110页. * |
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