CN104693291A - Application of H7 subunit antigen in animal model evaluation - Google Patents
Application of H7 subunit antigen in animal model evaluation Download PDFInfo
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- CN104693291A CN104693291A CN201510086104.3A CN201510086104A CN104693291A CN 104693291 A CN104693291 A CN 104693291A CN 201510086104 A CN201510086104 A CN 201510086104A CN 104693291 A CN104693291 A CN 104693291A
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Abstract
The invention relates to an H7 subunit antigen and application of the H7 subunit antigen in animal model evaluation. The H7 subunit antigen has nucleotide sequences shown as SEQ ID NO:1 and SEQ ID NO:2. A recombinant vaccine prepared by virtue of the H7 subunit antigen has the advantages of stability, effectiveness and the like; with mice or ferrets as an animal model, the vaccine prepared by virtue of the H7 subunit antigen can be accurately and rapidly evaluated, is capable of effectively preventing the mice and the ferrets from infecting with H7N9 virus and has good application prospect.
Description
Technical field
The invention belongs to genetically engineered field, particularly relate to a kind of vaccine of being made up of H7 subunit antigen and the application in animal model evaluation.
Background technology
The bird flu of H7N9 type is a kind of novel bird flu, takes the lead in by the end of March, 2013 finding in Shanghai and two places, Anhui.The bird flu of H7N9 type is the new subtype influenza virus of global Late Cambrian, not yet includes China's notifiable infectious diseases Surveillance system in, and not yet has vaccine to release at the beginning of 2013 4 months.Be there is the symptoms such as heating all in early days by this virus infection, not yet confirm whether this viroid has the characteristic that people infects people in April, 2013.In April, 2013, H7N9 avian influenza virus gene came from the gene resortment of East Asia Region wild bird and Chinese Shanghai, Zhejiang, Jiangsu chicken group through investigation.By on 01 25th, 2015,133 people were made a definite diagnosis in the whole nation, and 37 people are dead, 76 people's recoveries from illness.Case is distributed in the ground such as Beijing, Shanghai, Jiangsu, Zhejiang, Anhui, Shandong, Henan, Taiwan, Fujian, Dongguan, Shanwei.
People infects within H7N9 bird flu is generally 7 days latent period.Patient generally shows as influenza-like symptom, and as heating, cough, few phlegm, can with headache, sore muscle and general malaise.Patient with severe symptoms's PD is rapid, and show as severe pneumonia, body temperature continues mostly more than 39 degrees Celsius, occurs expiratory dyspnea, can with hemoptysis phlegm; Adult respiratory distress syndrome, mediastinal emphysema, Sepsis, shock, the disturbance of consciousness and acute injury of kidney etc. can be there are by rapid progress.
Not yet have at present fast and effectively vaccine to prevent avian influenza virus from propagating and complete animal model evaluation method.
Summary of the invention
In view of the existing problems of prior art, technical problem to be solved by this invention is to provide a kind of H7 subunit antigen and the application in animal model evaluation.
The technical scheme that the present invention solves the problems of the technologies described above is as follows:
A kind of H7 subunit antigen, described H7 subunit antigen has nucleotide sequence shown in SEQ ID NO:1.
Further, described H7 subunit antigen has aminoacid sequence shown in SEQ ID NO:2.
The application of H7 subunit antigen in animal model evaluation, described animal model is mouse or ferret.
The invention has the beneficial effects as follows: the present invention has the advantages such as stable, quick, effective with vaccine prepared by H7 subunit antigen.By mouse or ferret as animal model, the vaccine of preparation can be evaluated accurately and rapidly.The present invention effectively can prevent mouse, ferret infection H7N9 virus with vaccine prepared by H7 subunit antigen, has a good application prospect.
Accompanying drawing explanation
Fig. 1 is recombinant fowl influenza subunit vaccine figure, swimming lane M of the present invention is blue plus2marker, and swimming lane 1-2 is the sample before purifying, and swimming lane 3-4 is effluent liquid, and swimming lane 5-14 is the sample after purifying;
Fig. 2 is H7 antigen samples electrophorogram of the present invention, and swimming lane M is blue plus2 marker, swimming lane 1-3 is H7 antigen samples, and swimming lane 4 is 0.5 gag Bovine serum albumin, and swimming lane 5 is 1.0 gag Bovine serum albumin, and swimming lane 6 is 1.5 gag Bovine serum albumin;
Fig. 3 is recombinant fowl influenza subunit vaccine hemagglutination-inhibition test figure of the present invention;
Fig. 4 is that recombinant fowl influenza subunit vaccine of the present invention affects result figure to Mouse Weight.
Embodiment
Be described principle of the present invention and feature below in conjunction with accompanying drawing, example, only for explaining the present invention, is not intended to limit scope of the present invention.
Experimental technique of the present invention all adopts ordinary skill in the art means unless otherwise noted, can see specification sheets or the molecular biology clone guide third edition.The routine that experiment reagent used in the present invention is this area is unless otherwise noted actual, and market can buy.
Embodiment 1 cultivates virus strain A/Anhui/1/2013 (H7N9), extracts the RNA in virus strain
Described A/Anhui/1/2013 (H7N9) is from Chinese Disease Control and Prevention Center (CDC).
Avian influenza strain A/Anhui/1/2013 (H7N9) is got 0.2mL and is inoculated in instar chicken embryo on the 15th, cultivate 40h, be placed in 4 degrees Celsius of refrigerator overnight, sterile collection allantoic fluid for 37 degrees Celsius.Under getting viral allantoic fluid 500mL4 degrees celsius, the centrifugal 20min of 6000rpm collects supernatant, the centrifugal 2h ultracentrifugation of 25000rpm, precipitation 5mLTEN is resuspended, by the sucrose gradient centrifugation of massfraction 10%-40%, the centrifugal 2h of 35000rpm, collects the virus band of massfraction 30%-35%, dialysis desugar, suspend with the TE damping fluid without RNA enzyme, packing, frozen.
Utilize QIANGEN Rneasy Mini Kit test kit to extract RNA, concrete operation step is see specification sheets.
Embodiment 2 utilizes reverse transcription PCR to obtain cDNA
Get RNA bacteria suspension 10 microlitre, add 20pmol primer 1 (as shown in SEQ ID NO:3 nucleotide sequence) mixing to be placed in 70 C water bath and to react 10min, add 5* ThermoScript II damping fluid, 0.1mol/LDTT2 microlitre, 2.5mmol/LdNTP 4 microlitre, RNA enzyme inhibitors 1 microlitre, M-MLV ThermoScript II 10U again, 37 C water bath 1h, 97 degrees Celsius of effect 10min.Obtain cDNA.
Embodiment 3 is with the cDNA obtained for template, and increase HA gene
According to following reaction system configuration reaction solution:
| cDNA(50ng/μL) | 1μL |
| Primer 1 (10 μm of ol/L) | 1μL |
| Primer 1 (10 μm of ol/L) | 1μL |
| Taq archaeal dna polymerase | 1μL |
| 10*Taq DNA polymerase buffer liquid | 5μL |
| dNTP(2.5mmol/L) | 4μL |
| ddH2O | Supply 50 μ L |
According to following reaction conditions amplification HA gene:
Primer 1 has nucleotide sequence as shown in SEQ ID NO:3, i.e. CCG GGTACCATGAACACTCAAATCCTCGT, has KpnI restriction enzyme site.
Primer 2 has nucleotide sequence as shown in SEQ ID NO:4, i.e. AAT AAGCTTTTAGATACAGATTGTGCAGC, has HindIII restriction enzyme site.
The HA gene expanded has nucleotide sequence as shown in SEQ ID NO:1, that is:
ATGAACACTCAAATCCTCGTTTTCGCCCTCATCGCCATCATCCCCACTAACGCTGATAAGATCTGCCTCGGTCACCACGCTGTCTCCAACGGCACCAAGGTTAACACCCTCACTGAGAGAGGAGTGGAAGTGGTCAACGCTACAGAGACCGTGGAACGCACAAACATCCCCAGGATCTGCTCCAAGGGCAAGCGTACCGTCGACTTGGGCCAGTGTGGCCTGCTCGGAACTATCACAGGCCCCCCTCAGTGCGATCAATTCTTGGAGTTCTCTGCCGACCTGATCATCGAGCGTCGCGAAGGAAGCGATGTGTGTTACCCTGGCAAGTTCGTCAACGAGGAAGCTCTGAGACAGATCCTCCGTGAGAGCGGTGGCATCGACAAGGAAGCCATGGGATTCACCTACTCAGGTATCCGCACAAACGGAGCTACCTCGGCTTGCAGGAGATCCGGCTCCTCTTTCTACGCTGAGATGAAGTGGTTGCTGTCTAACACTGACAACGCTGCCTTCCCACAGATGACAAAGAGCTACAAGAACACCAGGAAGTCACCGGCTCTGATCGTTTGGGGTATCCACCACTCTGTGAGCACTGCCGAGCAAACAAAGCTCTACGGCTCCGGAAACAAGTTGGTTACCGTGGGAAGCTCAAACTACCAGCAATCCTTCGTCCCATCTCCGGGTGCTCGCCCACAGGTTAACGGTCTGTCTGGCAGGATCGATTTCCACTGGTTGATGCTGAACCCAAACGACACCGTGACTTTCTCATTCAACGGCGCTTTCATCGCCCCGGACAGAGCTTCGTTCCTGCGTGGCAAGTCTATGGGCATCCAGAGCGGAGTCCAAGTTGATGCCAACTGCGAGGGTGACTGTTACCACTCAGGAGGTACCATCATCTCGAACCTCCCCTTCCAGAACATCGACAGCCGTGCTGTCGGCAAGTGCCCTCGCTACGTTAAGCAAAGGTCTCTCTTGCTGGCCACTGGAATGAAGAACGTCCCCGAGATCCCTAAGGGTCGCGGCTTGTTCGGTGCTATCGCCGGCTTCATCGAGAACGGATGGGAAGGTCTGATCGACGGCTGGTACGGATTCAGGCACCAGAACGCTCAAGGAGAGGGTACTGCTGCCGATTACAAGTCAACACAGTCGGCCATCGACCAAATCACCGGAAAGCTCAACAGATTGATCGAAAAGACTAACCAGCAATTCGAGCTGATCGATAACGAATTCAACGAGGTGGAAAAGCAGATCGGTAACGTCATCAACTGGACCCGTGACTCCATCACTGAAGTGTGGTCTTACAACGCTGAGCTCTTGGTCGCCATGGAAAACCAGCACACCATCGATCTGGCTGACTCCGAGATGGATAAGCTCTACGAACGTGTGAAGCGCCAATTGAGGGAGAACGCCGAGGAAGACGGCACTGGATGTTTCGAAATCTTCCACAAGTGCGACGATGACTGTATGGCTTCGATCCGCAACAACACATACGACCACTCCAAGTACAGAGAGGAAGCCATGCAGAACCGTATCCAAATCGATCCTGTTAAGCTGTCGTCCGGTTACAAGGACGTCATCCTCTGGTTCAGCTTCGGCGCTTCATGCTTCATCCTGCTCGCCATCGTTATGGGACTGGTTTTCATCTGCGTCAAGAACGGCAACATGCGCTGCACAATCTGTATCTAA
The HA gene expanded has aminoacid sequence as shown in SEQ ID NO:2, that is:
Embodiment 4 build containing HA gene recombinant expression plasmid and at Sf9 cells
Utilize KpnI and HindIII to carry out double digestion to the HA gene obtained, 37 degrees Celsius of reactions are spent the night, and reclaim endonuclease bamhi; Utilize KpnI and HindIII to carry out double digestion to expression vector pBlueBAC4.5,37 degrees Celsius of reactions are spent the night simultaneously, reclaim large fragment carrier.
The endonuclease bamhi of HA and large fragment carrier are configured ligation system according to mass ratio 10:1, and add 1 μ LT4 ligase enzyme and 1 μ LT4 ligase enzyme damping fluid, 16 degrees Celsius of reactions are spent the night.Ligation liquid is proceeded in e. coli jm109, obtains the recombinant expression plasmid containing HA gene, i.e. pBlueBAC-HA.After order-checking, consistent with expected sequence.
Recombinant plasmid pBlueBAC-HA is carried out according to Bac-N-Blue transfection reagent box service manual.Obtain the Sf9 cell containing recombinant plasmid pBlueBAC-HA, called after Sf9-HA.
The cell of embodiment 5 cultivation containing recombinant expression plasmid, collection, purification of Recombinant bird flu subunit vaccine
By the Sf9-HA enchylema obtained, infect Sf9 cell, incubated at room temperature 24h according to volume ratio 1%, collect culturing cell and supernatant.By cells rinsed with PBS three times, ultrasonic degradation, to transparent, after centrifugal, gets cleer and peaceful precipitation.Utilize lens culinaris agglutinin enzyme affinity chromatography to carry out purifying, lens culinaris agglutinin enzyme affinity column is selected from lens culinaris agglutinin affinity chromatography gel 4B-CL (Lens culinaris (Lentil) Agglutinin-
4B-CL), purchased from KingMax bio tech ltd, Shanghai, concrete grammar is see specification sheets.Final acquisition recombinant fowl influenza subunit vaccine, electrophoretogram is shown in Fig. 1.By being standard substance with bovine serum albumin respectively, record in vaccine, the concentration of H7 subunit is 212 μ g/mL, sees Fig. 2.
By before freezing and freezingly carry out hemagglutination-inhibition test respectively afterwards, detect the stability of recombinant fowl influenza subunit vaccine of the present invention, test-results is shown in Fig. 3, as can be seen from Figure 3, recombinant fowl influenza subunit vaccine stable in properties of the present invention, has no significant change in freezing front and back.The standard HI measuring method (World Health Organization Gisn.Manual forthe laboratory dagnosis and virological surveillance of influenza [M] .Geneva, Switzerland:World Health Organization.2011:140.) that concrete steps are promulgated see WHO.
Embodiment 6 take mouse as animal model evaluation recombinant fowl influenza subunit vaccine
Choose three groups of mouse, often organize mouse 10.First group of mouse: the recombinant fowl influenza subunit vaccine prepared every injection 5 microgram the present invention in three weeks, injects three times; Second group of mouse: every three weeks injection recombinant fowl influenza subunit vaccines of preparing of 5 microgram the present invention and 5 microgram adjuvants (single phosphinylidyne lipoid A), inject three times; 3rd group of mouse: every injection 5 microgram adjuvants (single phosphinylidyne lipoid A) in three weeks, inject three times.The relatively body weight of three groups of mouse.As shown in Figure 4, as can be seen from Figure 4, recombinant fowl influenza subunit vaccine of the present invention has no side effect for mouse, on body weight without impact result.
Embodiment 7 take ferret as animal model evaluation recombinant fowl influenza subunit vaccine
Choose 8 SPF level ferrets, ferret is purchased from medical faunae institute of the Chinese Academy of Medical Sciences, and at the 7-10 monthly age, the hemagglutination-inhibition test result of the influenza virus of all animal serum pops is negative.
Be divided into two groups, often organize 4.By first group of injection 0.5mL recombinant fowl influenza subunit vaccine, every injection in 30 days once; By second group of injection 0.5mL PBS damping fluid, every injection in 30 days once.
When the 90th day, carry out HAI Indexs measure, detected result is in table 1:
Table 1 HAI detected result
As can be seen from embodiment 6-7, recombinant fowl influenza subunit vaccine prepared by the present invention effectively can prevent the infection of H7N9, and recombinant fowl influenza subunit vaccine prepared by the present invention has good clinical development potentiality.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. a H7 subunit antigen, is characterized in that, described H7 subunit antigen has nucleotide sequence shown in SEQ I D NO:1.
2. a kind of according to claim 1, H7 subunit antigen, is characterized in that, described H7 subunit antigen has aminoacid sequence shown in SEQ ID NO:2.
3. the application of H7 subunit antigen in animal model evaluation described in a claim 1.
4. the application of H7 subunit antigen in animal model evaluation according to claim 3, is characterized in that, described animal model is mouse or ferret.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1746298A (en) * | 2004-09-10 | 2006-03-15 | 中国农业科学院哈尔滨兽医研究所 | H7 subtype influenza virus of artificial recombination and its production and application |
| CN1935995A (en) * | 2005-09-21 | 2007-03-28 | 金宁一 | H7 subtype highly pathogenic avian influenza virus hemagglutinin gene antigen protein |
| CN101732711A (en) * | 2009-12-31 | 2010-06-16 | 中国人民解放军军事医学科学院微生物流行病研究所 | Preparation of nose-spraying flu immunization pentavalent or multivalent inactivated vaccine 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 |
| WO2014127231A1 (en) * | 2013-02-15 | 2014-08-21 | New York Blood Center, Inc. | Oligomeric influenza immunogenic compositions |
| CN104109655A (en) * | 2013-04-18 | 2014-10-22 | 中国动物卫生与流行病学中心 | Preparation method of seed viruses required by H7 subtype avian influenza vaccines and diagnostic reagents |
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2015
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1746298A (en) * | 2004-09-10 | 2006-03-15 | 中国农业科学院哈尔滨兽医研究所 | H7 subtype influenza virus of artificial recombination and its production and application |
| CN1935995A (en) * | 2005-09-21 | 2007-03-28 | 金宁一 | H7 subtype highly pathogenic avian influenza virus hemagglutinin gene antigen protein |
| CN101732711A (en) * | 2009-12-31 | 2010-06-16 | 中国人民解放军军事医学科学院微生物流行病研究所 | Preparation of nose-spraying flu immunization pentavalent or multivalent inactivated vaccine 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 |
| WO2014127231A1 (en) * | 2013-02-15 | 2014-08-21 | New York Blood Center, Inc. | Oligomeric influenza immunogenic compositions |
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