CN101053658A - H3 and H9 subtype influenza composite multiepitope bivalent DNA vaccine - Google Patents
H3 and H9 subtype influenza composite multiepitope bivalent DNA vaccine Download PDFInfo
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Abstract
Provided is a hybrid multi-epitope bivalent nucleic acid vaccine for preventing H3, H9 subtype flu, which pertains to the biotechnology field. The invention uses the influenza virus main epitope gene as the base, the Kozac sequence as the adjuvant gene, eukaryon expression vector pIRESlneo as the vector molecule, conducts artificial molecular design on the epitope gene under the computer aided simulation to obtain a safe and effective hybrid multi-epitope DNA vaccine aiming to a variety of serotypes and epidemic strains. The obtained nucleic acid vaccine can stimulate mouse to generate specific humoral immunity and cellular immunity, and the obtained vaccine is safe to the experimental animals, no pathological phenomenon are caused.
Description
Technical field:
The invention belongs to biological technical field.
Background technology:
Influenza (is called for short influenza, Influenza) be to emit virus (to be called for short influenza virus, Influenza virus, a kind of ancient disease that IV) causes by the influenza sexuality, the popular of the influenza sample disease first time takes place 1173 Christian eras, and tens influenza pandemic were arranged in the centuries afterwards.Influenza A virus had once caused for several times popular on a large scale, comprise the spanish influenza (H1N1) in 1918～1919 years, the Asia influenza of nineteen fifty-seven (H2N2), the Hong-Kong influenza (H3N2) of nineteen sixty-eight and Russia's influenza (H1N1) in 1977 are very popular at every turn and have all caused the huge human resources and the loss of material resources.It is human to confirm that fowl source H9N2 infected in 1999.If bird flu strengthens in person-to-person transmission capacity, so therefore human and the dead and situation of catching an illness will be very serious.
Up to the present, immunity inoculation is the most effectively means of anti-system influenza.But because influenza virus easily makes a variation, so still untappedly so far go out permanently effective vaccine.In addition, still there are many problem and shortage in designed and many recombinant vaccines that studying at present.Still contain in the antigenic component of many vaccine design and a large amount of induce irrelevant composition with specific immunity, as immunopathogenesis compositions such as immunosuppressant, enhancing antibody and autoimmune, and these compositions cause the key factor of part or systemic adverse reactions just; Defectives such as in addition, some vaccine in order to reach purity and safe purpose (as the oligopeptide vaccine), often adopts a few antigenic determinant when design, caused antigen single so again, and immunogenicity is weak.
Technology contents:
Based on the problems referred to above, the present invention has made up a kind of multivalence composite multi-epitope DNA vaccine with prevention H3, H9 subtype influenza.
The present invention is with H3, the HA epitope gene of H9 subtype influenza virus, the major antigen NP of influenza virus, NA, the M epitope gene is the basis, with the base sequence of coding base sequence of proline and encoding serine the gene that is connected as each epi-position, connection mode is :-CCT-CCT-TCA-, attached again with the Kozak sequence, and at 5 of multi-epitope gene molecule ' 3 restriction enzyme site SmalI of end introducing, NheI, NotI, introduce 4 restriction enzyme site NheI at 3 ' end, EcoRI, ApaI, XbaI, utilize the board design and the antigenicity forecast function of computer, determine the optimum composition method of each epi-position, the method by synthetic has obtained at H3, the composite multi-epitope genes of interest Epi of one segment length 765bp of H9 subtype influenza virus, composite multi-epitope genes of interest Epi is inserted among the safety nucleic acid vaccine expression vector pIRES1neo, has made up and have prevention H3, the multivalence composite multi-epitope DNA vaccine pIRE-Epi of H9 subtype influenza.
The present invention is by H3, H9 subtype influenza composite multi-epitope bivalence nucleic acid vaccine SEQ ID NO:1 that said method obtained.
Advantage of the present invention and good effect:
But 1, the fusion rotein of the recombinant dna vaccine pIRE-Epi The expressed that obtains of the present invention, can with the HA antibody generation specific reaction of H3, H9 subtype influenza.
2, the nucleic acid vaccine of the present invention's acquisition can stimulate mice to produce special humoral immunization and cellular immunization.
3, the recombinant dna vaccine of the present invention's acquisition is safe to laboratory animal, does not have any pathological phenomena.
4, genes of interest used in the present invention is the composite multi-epitope gene that comprises the HA epitope of H3, H9 subtype influenza, thereby the recombinant dna vaccine that makes up can be used as the dna vaccination of prevention H3, H9 subtype influenza.
5, the present invention is based on influenza virus major antigen epitope gene, with the Kozac sequence is auxiliary gene, with carrier for expression of eukaryon pIRES1neo is carrier molecule, antigen epitope genes is carried out the artificial molecule design and is aided with the new approaches that the new type influenza recombinant vaccine is developed in computer simulation, obtain safe and efficient composite multi-epitope DNA vaccine at different serotypes and different epidemic strains.
Description of drawings:
Accompanying drawing is that influenza compound multi-epitope DNA vaccine pIRE-Epi of the present invention makes up flow chart.
The specific embodiment:
The present invention is with H3, the HA epitope gene of H9 subtype influenza virus, the major antigen NP of influenza virus, NA, the M epitope gene is the basis, with the base sequence of coding base sequence of proline and encoding serine the gene that is connected as each epi-position, connection mode is :-CCT-CCT-TCA-, attached again with the Kozak sequence, and at 5 of multi-epitope gene molecule ' 3 restriction enzyme site SmalI of end introducing, NheI, NotI, introduce 4 restriction enzyme site NheI at 3 ' end, EcoRI, ApaI, XbaI, utilize the board design and the antigenicity forecast function of computer, determine the optimum composition method of each epi-position, the method by synthetic has obtained at H3, the composite multi-epitope genes of interest Epi of one segment length 765bp of H9 subtype influenza virus, composite multi-epitope genes of interest Epi is inserted among the safety nucleic acid vaccine expression vector pIRES1neo, has made up and have prevention H3, the multivalence composite multi-epitope DNA vaccine pIRE-Epi of H9 subtype influenza.To constructed nucleic acid vaccine liposome transfection method transfection HeLa cell, detect through inverse transcription polymerase chain reaction (RT-PCR), the immune protein marking (WesternBlot) and immunofluorescence test, identify the external activity of the expressed exogenous gene of recombinant dna vaccine plasmid.With the recombinant dna vaccine immunity BALB/c mouse that filters out, carry out the detection of amynologic index.Suppress (HA/HI) test method with enzyme linked immune assay (ELISA) method and blood clotting/blood clotting and carry out the humoral immunization detection, carry out cellular immunization with fluidic cell detection method and ELISpot test kit and detect.
Narrate implementation method of the present invention below:
1, the experimental implementation of molecular biology routine
All (Huang Peitang etc. translate with reference to " molecular cloning experiment guide " for the screening of being connected of the recovery of the extraction of the preparation of competent escherichia coli cell and conversion, plasmid and digestion with restriction enzyme, dna fragmentation, linear DNA fragment, recombiant plasmid and evaluation, pcr amplification reaction etc., the third edition, Science Press, 2002) related Sections carries out.
2, the MOLECULE DESIGN of genes of interest and synthetic
2.1 the MOLECULE DESIGN of genes of interest
Select the composition of IV advantage epi-position as vaccine antigen.The principle that epi-position is selected is: 1. both contained the t cell epitope that the energy excitating organism produces ctl response, and also contained antibody response B cell epitope, based on the former; 2. based on the advantage epi-position of antigen conserved region; 3. avoid bringing out the component that produces immunosuppressant, strengthens immunopathogenesis reactions such as antibody and autoimmune; 4. epitope sequences is based on the IV Chinese epidemic strain; 5. based on the epi-position of main protection antigen genes such as influenza virus HA, NA, NP, M.The advantage epi-position of selecting is connected with flexible aminoacid, form one section artificial peptide sequence Epi; By protein homology modeling (Homologicalmodelling), utilize the homology of structure and sequence in the PDB storehouse, to search for the homologous protein template, the proteic three-dimensional conformation of prediction unknown structure Epi.
2.2 the genes of interest molecule is synthetic
2.2.1DNA segmental making
According to the DNA sequence of 2.1 designs, synthesizing single-stranded small pieces segment DNA by PCR method, is spliced into a complete double chain DNA fragment to strand small pieces segment DNA again.
2.2.2 restriction enzyme digestion reaction
1. in the Microtube pipe, prepare following reactant liquor:
| Dna fragmentation 10 * Buffer restriction endonuclease 1 restriction endonuclease 2 H 2O | About 2 μ l, 10 μ l, 3 μ l, 3 μ l fluid infusion to 100 μ l |
2. 37 ℃ are incubated 1 hour.
3. behind ethanol precipitation, be dissolved among the TE Buffer of 20 μ l (Insert DNA).
2.2.3 coupled reaction
1. in the Microtube pipe, prepare following reactant liquor:
| Insert DNA Vector DNA(pMD18-T Simple) H 2O Ligation Solution I * | 2 μ l (about 200ng), 10 μ l (about 50ng), 2 μ l, 5 μ l |
* the component of TaKaRa DNA Ligation Kit Ver.2 (TaKaRa Code.D6022)
2. 16 ℃ are incubated 30 minutes.
3. connect the liquid full dose and be converted into E.coli JM109 Competent Cell.
4. the single bacterium colony of picking extracts plasmid, promptly gets recombiant plasmid pMD18-T-Sim-Epi.
3, at the structure (flow chart sees Appendix 2) of H3, H9 subtype influenza nucleic acid vaccine recombinant
3.1 the double digestion reaction obtains genetic fragment Epitopes with restricted enzyme Not I and EcoR I digested plasmid pMD18-T-Sim-Epi; With restricted enzyme Not I and EcoR I digestion carrier for expression of eukaryon pIRES1neo, obtain linearizing carrier segments.Concrete steps are as follows:
Plasmid DNA and suitable quantity of water mixing with 1.0 μ g, making its cumulative volume is 17 μ l, each adds two kinds of restricted enzyme 2～3U and the corresponding 10 * restriction enzyme reaction buffer of 2 μ l, flick tube wall mixing and centrifugal, put optimal reactive temperature water-bath 2～3h, get 5 μ l reactant liquors and carry out the agarose gel electrophoresis inspection.Behind the complete degestion, it is standby to reclaim fragment.
Be inserted in the pIRES1neo carrier 3.2 coupled reaction will obtain Epitopes genetic fragment orientation, obtain the pIRE-Epi plasmid recombinant.Concrete steps are as follows:
Get the carrier DNA that 0.5 μ g reclaims, the exogenous dna fragment that adds 2-10 times of mole, 2 μ l 10 * connection buffer adds water and is settled to 20ul, add an amount of T4DNA ligase (1weiss unit) at last, mixing and moment are centrifugal so that at the bottom of the droplet congregating pipe, put that 16 ℃ of water-baths are spent the night or 25 ℃ connect 1-4h, get 5 μ l and connect product transformed into escherichia coli competent cell.The last single bacterium colony of picking carries out plasmid and extracts back acquisition DNA recombinant pIRE-Epi.
4, the detection of expression of recombinant plasmid product
4.1SDS-PAGE gel electrophoresis
72h after the transfection, abandon culture fluid, TEN (40mmol/L TrisClpH7.5,1mmol/L EDTA, 150mmol/L NaCl) eluting cell with 37 ℃ of preheatings of 1ml, be collected in the Eppendorf pipe, the centrifugal 5min of 3000rpm abandons supernatant, and cell precipitation is washed once with PBS, add 60 μ l cell pyrolysis liquid (10mmol/L TrisCl pH7.4,1mmol/L MgCl
2, 0.5%NP40,20 μ g/ml DNase I) and cracking, ice bath 30min boils 3min, and the centrifugal 5min of 5000rpm gets supernatant, adds 2 times of sample-loading buffer mixings of equivalent, carries out electrophoresis in 12%SDS-PAGE.
Cell, blank cell and low-molecular-weight standard protein (97kDa, 66kDa, 43kDa, 31kDa, 20kDa, 14kDa) with the blank eukaryotic expression plasmid of transfection compare, get an amount of sample respectively and add 2 times of sample-loading buffers of equivalent, add loading slot with micropipettor, with 8V/cm voltage electrophoresis.After the bromophenol blue forward position enters separation gel, to change with 12v/cm voltage electrophoresis, bromophenol blue is taken out gel to the separation gel bottom, carry out coomassie brilliant blue staining or Western blot.
4.2Western blot detects
After the SDS-PAGE electrophoresis finishes, cut out and contain proteic gel to be transferred, cut 6 onesize Whatman 3mm filter paper and 1 nitrocellulose filter (Millipore HAWP), carry out labelling for one jiao at fibrous membrane with soft pencil.They are floated on ddH
2On the O horizontal plane, be dipped in 5min in the water after the moistening, drive away the bubble on the film, be soaked in again in the transfering buffering liquid (39mmol/L glycine, 48mmol/L Tris, 0.037%SDS, 20% methanol).On plastic stent, stack wetted sponge, 3 metafiltration paper, gel, nitrocellulose filter, 3 metafiltration paper and sponges successively, make filter paper, gel and nitrocellulose filter alignment, and no bubble exists between each layer; Plastic stent is clamped in the insertion electrotransfer groove; Nitrocellulose filter one side joint anode, gel one side joint negative electrode adds and shifts liquid (39mmol/L glycine, 48mmol/L TrisCl, 0.037%SDS, 20% methanol) slightly above gel, and with 30V～35V voltage, 4 ℃ are shifted 14～16h.Transfer finishes, and nitrocellulose filter is dipped in confining liquid (5% defatted milk powder or 3%BSA, 150mmol/L NaCl, about room temperature 2h 0.02%Tween-20), to seal irrelevant protein binding site, simultaneously gel is dyeed, check whether albumen shifts complete.After the sealing, (10mmol/L TrisCl pH7.5,0.15mol/L NaCl 0.05%Tween-20) wash film 3 times, each 5min with cleaning mixture; One anti-(the anti-AIV polyclonal serum of chicken) used antibody diluent (0.01mol/L TBS, 1%BSA, 0.05%Tween-20) suitably dilution drops on the preservative film, then nitrocellulose filter front (adsorption antigen face) covered on the preservative film that adds antibody room temperature reaction 2h downwards; Wash film 3 times with cleaning mixture, each 5min; With the enzyme-added mark two of quadrat method anti-(the anti-chicken IgG of the goat of alkali phosphatase enzyme mark), room temperature reaction 2h; Wash film 3 times with cleaning mixture, each 5min; Film is dried slightly, and enzyme-added substrate reactions liquid reacts.
The preparation of zymolyte reactant liquor:
(1) NBT (nitrogen blue tetrazole) dissolves 0.5g NBT in the dimethylformamide of 10ml 70%;
(2) BCIP (5-bromo-4-chloro-3-indole phosphoric acid) dissolves 0.5g BCIP in 10ml 100% dimethylformamide;
(3) alkali phosphatase buffer: 100mmol/L NaCl, 5mmol/L MgCl
2, 100mmol/L TrisCl pH9.5.Get 66 μ l NBT and 10ml alkali phosphatase buffer mixing, add 33 μ l BCIP, this solution uses in 30min;
Washed film is moved in the shallow pallet, press 0.1ml/cm
2Add substrate reactions liquid, shake gently in room temperature and carry out the incubation colour developing.Nitrocellulose filter is moved in the distilled water color development stopping, observed result behind colour developing 5～20min.
4.3 indirect immunofluorescence experiment (IFA)
4 method has on the slide of 70%HeLa cell monolayer 5%CO with plasmid transfection to long set by step
2, 37 ℃ cultivate 3d after, take out slide, with PBS liquid flushing three times, 100% cold acetone is fixed 10～30min, reuse PBS liquid washing three times; PBS sealing 2h with containing 3%BSA washes each 5min 3 times; Add one anti-(the anti-AIV serum of chicken), behind 37 ℃ of effect 2h, wash 3 times; Add two anti-(the goat-anti chicken IgG of fluorescein isothiocyanate labelling), room temperature lucifuge effect 1h washes 3 times; Blot, Dropwise 5 0% glycerine water solution is inverted on the microscope slide, and (adding two anti-back operating procedures all should lucifuge carry out in fluorescence microscope observation down; Generally speaking, finish within a short period of time as far as possible and take pictures, in order to avoid fluorescent quenching).
5, nucleic vaccine plasmid immune mouse
Prepare plasmid DNA in a large number with alkaline lysis, be dissolved in behind the PEG purification in the aseptic phosphate buffer (PBS), it is quantitative to carry out DNA with ultraviolet spectrophotometry, and final concentration transfers to 1 μ g/ μ L.
5.1 experiment grouping
6-8 age in week, female BALB/c mouse was divided into 9 groups at random, 12 every group, pressed 100ug/ leg muscle injection recombiant plasmid, empty plasmid pVAX1 and PBS solution respectively.Every interval 2 all booster immunizations once, immunity back 10d kills Mus and gets quantity and the Elisapot that serum and splenocyte detect antibody, t lymphocyte subset group and detect unicellular horizontal IFN-γ secretion for the third time.The grouping situation sees Table 1.
5.2 the lymphocytic preparation of spleen T
Disconnected neck is put to death and is got spleen behind the mouse immune, grinds spleen relatively with the microscope slide hair side, adds 4mL PBS, removes fragment of tissue by membrane filtration; Cell suspension is sucked the 10ml centrifuge tube, and the centrifugal 10min of 2000rpm abandons supernatant; Add 3ml erythrocyte cracked liquid suspension cell (the Tris-NH4Cl buffer is got Tris 1.03g and NH4Cl 3.735g, adds twoly to heat up in a steamer water to 500ml, crosses the G-5 funnel, and 4 ℃ store for future use) room temperature and place 10min, the centrifugal 10min of 2000rpm abandons supernatant; Add 4ml Hank ' s liquid washing and carry out cell counting, centrifugal (the same) back adds an amount of 10%1640 culture fluid, adjusts cell number and reaches 1 * 10
7Individual/ml.
Table 1 mouse experiment immunity grouping
| The immunity plasmid | The immunity for the third time of immunity immunity for the second time for the first time | |
| 1234 | PIRES1neo PIRE-Epi inactivated vaccine PBS | 1. every group of 12 mices; 2. continuous three intramuscular injection, each 14d at interval; 3. per injection plasmid 100 μ g/ only; 4. once, separation of serum every the 10d blood sampling. |
5.3 the excretory detection of unicellular horizontal IFN-γ
Undertaken by the test kit description.
5.4 the immune serum antibody titer detects
Eye socket blood sampling back separation of serum, centrifugal collection supernatant is standby.Measure antibody titer with indirect ELISA method, do negative control with non-immune mouse serum simultaneously, main reference " animal virology " second edition carries out.Concrete operations are as follows:
ELISA reagent:
PBS (pH7.4): NaCl 8.0g, Na
2HPO
412H
2O 2.9g, KCl 0.2g is dissolved in the 1000ml distilled water;
Coating buffer (pH7.6): Na
2CO
31.59g, NaHCO
32.93g, be dissolved in the 1000ml water; Or with the PBS of pH9.6 bag quilt;
Confining liquid: join 3%BSA with the PBS buffer;
Cleaning mixture: add 0.05%Tween-20 in the PBS buffer;
Substrate solution: pH5.0 phosphoric acid citrate buffer solution: 0.2M Na
2HPO
4(28.4g/L) 25.7ml takes out 10ml with after 0.1M citric acid 24.3ml mixes, and adds following composition: o-phenylenediamine (OPD) 4mg, 30%H
2O
210 μ L.Use immediately behind the mixing.
Step:
1) bag quilt: with coating buffer equal-volume dilution antigen, add in the every hole of 96 hole ELISA Plate, 37 ℃ of bags of 100 μ L are by 1.5h (also can 4 ℃ of bags spent the night);
2) sealing: add the confining liquid in 100 μ L/ holes, 37 ℃ of sealing 1.5h;
3) washing: wash plate three times with cleaning mixture, each 3min;
4) anti-in conjunction with one: with the PBS buffer doubling dilution antiserum that contains 0.3%BSA, every hole adds 100 μ L, and wherein string adds the contrast positive serum of multiple proportions dilution, every hole 100 μ L.Hatch 1.5h for 37 ℃;
5) washing: wash plate 3 times with cleaning mixture, each 3min;
6) anti-in conjunction with two: as by proper proportion (with reference to description) dilution, to hatch 1.5h for 37 ℃ with the PBS buffer that contains 0.3%BSA;
7) washing: wash plate 5 times with cleaning mixture, each 3min;
8) colour developing: every hole adds 100 μ L substrate solutions (matching while using), and 1～3min is placed in the dark place, and the every hole, back of waiting to develop the color adds 50 μ L30% concentrated sulphuric acid color development stopping;
9) read plate: use microplate reader to read the OD value of 490nm.
5.5 the detection of spleen t lymphocyte subset group quantity
Preparation mice spleen single cell suspension 1-2 * 10
6/ ml, with the fluorescence washing liquid wash twice (fluorescence washing liquid: 100ml 0.15M PBS (pH7.4), 2%NBS).Add the CD4 antibody of PE labelling and each 20 μ l of CD8 antibody of FITC labelling, cumulative volume is that 40ul is mixed, or adds the CD3 antibody 45 μ l of FITC labelling, puts 4 ℃ of lucifuge 30min, takes out the back and washes twice with the fluorescence washing liquid.Adding 0.5ml fluorescence preservation liquid (fluorescence is preserved liquid: 100ml 0.15M PBS (pH7.4), 2% glucose, and 1% formaldehyde, 0.1%NaN3), 4 ℃ of preservations (in the week) are with flow cytometry analysis T cell subsets.FACS detects 10000 cells altogether, obtains the lymphocytic quantity of CD3+ in the splenocyte, CD4+ and CD8+T respectively, and the gained data are carried out statistical procedures.
Testing result:
The influenza compound multi-epitope gene order Epitopes of design and synthetic sees Appendix 1.
With the nucleic acid vaccine pIRE-Epi transfection HeLa cell that obtains, detect with indirect immunofluorescence experiment and Western-blot, the result is all positive.Illustrate that the nucleic acid vaccine that makes up can be at the fusion rotein of vivoexpression biologically active.Behind this nucleic acid vaccine immunity mice, can stimulate spleen T lymphopoiesis, excite to produce anti-H3, H9 hypotype AIV antibody; Each group of pIRE-Epi immune group and other is compared, and the quantity of CD4+ and CD8+ significantly improves (P＜0.05); The IFN-γ level that its immunity produced is apparently higher than other matched group.
＜110〉gold, peaceful one
＜120〉H3, H9 subtype influenza composite multi-epitope bivalence nucleic acid vaccine pIRE-Epi
<160>1
<170>PatentIn version 3.2
<210>1
<211>765
<212>DNA
<213>Influenza A virus
<221>gene
<222>(1)..(765)
<400>1
cccggggcta gcgcggccgc cgccgccacc atgtgttgtt tcatgtgggg catacatcac 60
ccacctactg atactcctcc ttcagcagtt ggtctgagga atgtacctgc tagatcaagt 120
agacctcctt caaaatatgt tggagtaaag agtctcaaat tgcctccttc atgtgtaaat 180
ggctcttgct ttactgtacc tccttcattg aaatacaatg gcataataac agacactatc 240
cctccttcag catatgagag aatgtgcaac atcctccctc cttcaacata ccagagaaca 300
agagctctcg tgcctccttc aatcctgaga ggatccgtag cccataagcc tccttcaatt 360
ttagggtttg tgttcacgct caccgtgcct ccttcaggcc ccctcaaagc cgagatcgcg 420
cagagacttg aacctccttc agggatttta gggtttgtgt tcacgctccc tccttcattt 480
atcactgagg gtttcacttg gactggggtc actcagaatg ggggaagcaa tgctcctcct 540
tcaagatcag atgcacctat tgacacctgc atttctgaat gcatcactcc aaatggaagc 600
atccctcctt caaatagtaa tggaaaccta atcgctcctc ggggctattt caaaatgcgc 660
actgggaaaa gccctccttc acggggctat ttcaaaatgc gcactgggaa aagctcaata 720
atgagatcac cttgtggtgc tagctaagaa ttcgggccct ctaga 765
Claims (2)
1, a kind of H3, H9 subtype influenza composite multi-epitope bivalence nucleic acid vaccine is characterized in that:
With H3, the HA epitope gene of H9 subtype influenza virus, the major antigen NP of influenza virus, NA, the M epitope gene is the basis, with the base sequence of coding base sequence of proline and encoding serine the gene that is connected as each epi-position, connection mode is :-CCT-CCT-TCA-, attached again with the Kozak sequence, and at 5 of multi-epitope gene molecule ' 3 restriction enzyme site SmalI of end introducing, NheI, NotI, introduce 4 restriction enzyme site NheI at 3 ' end, EcoRI, ApaI, XbaI, utilize the board design and the antigenicity forecast function of computer, determine the optimum composition method of each epi-position, the method by synthetic has obtained at H3, the composite multi-epitope genes of interest Epi of one segment length 765bp of H9 subtype influenza virus, composite multi-epitope genes of interest Epi is inserted among the safety nucleic acid vaccine expression vector pIRES1 neo, has made up and have prevention H3, the multivalence composite multi-epitope DNA vaccine pIRE-Epi of H9 subtype influenza.
2, by the H3 that method obtained, the H9 subtype influenza composite multi-epitope bivalence nucleic acid vaccine SEQ ID NO:1 of claim 1.
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|---|---|---|---|
| CN 200610016761 CN101053658A (en) | 2006-04-12 | 2006-04-12 | H3 and H9 subtype influenza composite multiepitope bivalent DNA vaccine |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610016761 CN101053658A (en) | 2006-04-12 | 2006-04-12 | H3 and H9 subtype influenza composite multiepitope bivalent DNA vaccine |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186644B (en) * | 2007-12-05 | 2010-06-02 | 中国药科大学 | H3 type flu virus hemagglutinin space conformation simulation antigen epitope and application thereof |
| WO2013034069A1 (en) * | 2011-09-08 | 2013-03-14 | 中国农业科学院上海兽医研究所 | Recombinant influenza virus highly expressing ha protein and preparation method and use thereof |
-
2006
- 2006-04-12 CN CN 200610016761 patent/CN101053658A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186644B (en) * | 2007-12-05 | 2010-06-02 | 中国药科大学 | H3 type flu virus hemagglutinin space conformation simulation antigen epitope and application thereof |
| WO2013034069A1 (en) * | 2011-09-08 | 2013-03-14 | 中国农业科学院上海兽医研究所 | Recombinant influenza virus highly expressing ha protein and preparation method and use thereof |
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