CN110117579A - Express recombinant virus and its construction method and the application of 16 type blue tongue virus VP2 genes - Google Patents
Express recombinant virus and its construction method and the application of 16 type blue tongue virus VP2 genes Download PDFInfo
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Abstract
The present invention relates to a kind of recombinant virus for expressing 16 type blue tongue virus VP2 genes and its construction method and applications, recombinant virus includes viral vectors and the recombinant shuttle plasmid with 16 type blue tongue virus VP2 genes that is packaged in the viral vectors, and the sequence of the 16 type blue tongue virus VP2 gene is as shown in SEQ ID NO.1.The present invention at home and abroad constructs for the first time and has screened the recombinant virus rVTT-VP2 of 16 type blue tongue virus (BTV-16) VP2 genes of expression, and is used for molecular biology method and is identified.By 10 plaque purifications repeatedly, continuous to pass 20 generations, PCR qualification result shows that VP2 gene has been integrated into vaccinia virus recombinant genome, and fails to amplify TK gene, shows that vaccinia virus recombinant rVTT-VP2 genetic stability is good, and virus is purified.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to a kind of recombination for expressing 16 type blue tongue virus VP2 genes
Virus and its construction method and application.
Background technique
Blue tongue virus (bluetongue virus, BTV) is Reoviridae Orbivirus member.It infects master
It will be by arthropod such as Storehouse midge, sand fly etc..Blue tongue disease is World Organization for Animal Health (Office International Des
Epizooties, OIE) provide the animal infection epidemic disease that must be reported, China is also defined as a kind of dynamic after accession to the WTO
Object epidemic disease.The significant damage of blue tongue disease is mainly reflected in the high lethality rate of sheep and goat, and annual caused economic loss is general
At 300,000,000 dollars or more, manpower and material resources consumption is huge.To 2019, blue tongue disease had 27 kinds of serotypes, China at least 7 kinds of BTV
Serotype, but Major Epidemic serotype is BTV-1 type and 16 types.Blue tongue virus VP2 as blue tongue virus capsid protein,
It plays a significant role during viruses adsorption, is the specific antigen for determining serotype.It is special that the antigen can stimulate body to generate
Property neutralizing antibody to protect animal, be blue tongue disease novel gene engineered vaccine research preferred antigen.Commercial vaccine includes at present
Attenuated vaccine and inactivated vaccine not yet carry out the research of 16 type recombinant vaccine of blue tongue disease both at home and abroad.
Summary of the invention
Based on this, it is necessary to provide a kind of recombinant virus and its construction method for expressing 16 type blue tongue virus VP2 genes
And application.
A kind of recombinant virus for expressing 16 type blue tongue virus VP2 genes, including viral vectors and it is packaged in the virus
The shuttle plasmid with 16 type blue tongue virus VP2 genes in carrier, the sequence of the 16 type blue tongue virus VP2 gene is such as
Shown in SEQ ID NO.1.
The viral vectors is vaccinia virus vector in one of the embodiments, and the shuttle plasmid is vaccinia virus
Shuttle plasmid pSTKE.
The upstream of the 16 type blue tongue virus VP2 gene also has kozak sequence in one of the embodiments,.
A kind of construction method of recombinant virus that expressing 16 type blue tongue virus VP2 genes, includes the following steps: that offer contains
There is the plasmid of 16 type blue tongue virus VP2 genes, PCR amplification carried out to 16 type blue tongue virus VP2 genes using amplimer,
The 16 type blue tongue virus VP2 genes that amplification obtains are inserted into shuttle plasmid and obtain recombinant shuttle plasmid, by the recombination
Shuttle plasmid and virus transfection are into cell, and screening collection obtains 16 type blue tongue virus VP2 genes of the expression after 2 ~ 3 days
Recombinant virus.
The amplimer includes upstream primer and downstream primer in one of the embodiments, the upstream primer
Sequence is as shown in SEQ ID NO.2, and the sequence of the downstream primer is as shown in SEQ ID NO.3.
The 16 type blue tongue virus VP2 genes that amplification is obtained are inserted into shuttle matter in one of the embodiments,
Step in grain includes: to connect to obtain pEasy- with pEasy cloning vector by the 16 type blue tongue virus VP2 genes that amplification obtains
Then 16 type blue tongue virus VP2 genes are connected in shuttle plasmid by double digestion and connection and obtain recombination shuttle matter by VP2
Grain.
The enzyme that the double digestion uses in one of the embodiments, is Xba I and Sal I.
The virus is vaccinia virus Tiantan strain in one of the embodiments, and the shuttle plasmid is worn for vaccinia virus
Shuttle plasmid pSTKE.
The cell is BHK-21 cell in one of the embodiments,.
A kind of application of the recombinant virus of 16 type blue tongue virus VP2 gene of above-mentioned expression in preparation blue tongue disease vaccine.
Blue tongue disease has 27 serotypes, and numerous serotypes lack cross protection.The VP2 of BTV L2 gene coding is BTV
Major capsid protein plays important work during viruses adsorption and intrusion host cell positioned at the outermost layer of no cyst membrane BTV
With induction body generates specific serotype antibody, neutralizes blue tongue virus, and the inactivation of VP2 will lead to blue tongue virus mistake
Remove hemagglutination activity.For now, the research of state's intradermal vaccine mainly 1 type inactivated vaccine of blue tongue disease, fowl pox virus vectors vaccine,
Horse herpes virus carrier, VLP, the preparation of blue tongue disease monoclonal antibody etc., but still without carrying out grinding for 16 types recombination blue tongue disease vaccine
Study carefully.The present invention at home and abroad constructs for the first time and has screened the recombinant virus of 16 type blue tongue virus (BTV-16) VP2 genes of expression
RVTT-VP2, and be used for molecular biology method and identified.It is continuous to pass for 20 generations by 10 plaque purifications repeatedly
Secondary, PCR qualification result shows that VP2 gene has been integrated into vaccinia virus recombinant genome, and fails to amplify TK gene, shows
Vaccinia virus recombinant rVTT-VP2 genetic stability is good, and virus is purified.
Vaccinia virus vector is a kind of novel vaccine carrier, and vaccinia virus host range is wide, and research background is more clear, peace
Overall height effect, genome is huge, still has the ability of infection while can be inserted into more huge exogenous genetic fragment.In addition,
The present invention is added to before VP2 gene when carrying out the building of recombinant virus of 16 type blue tongue virus VP2 genes of expression
The expression of target gene can be enhanced in kozak sequence.And 3 vaccinia virus termination signal sequences in VP2 gene have been mutated it
It (TTTTNT), can be against the interference of other transcription of foreign genes.Identification and genetic stability analysis knot are carried out from gene level
Fruit shows that the rVTT-VP2 recombinant virus genetic stability of this research and establishment is good, can stablize expression blue tongue disease capsid protein
VP2, rVTT-VP2 can the largely stable expression on BHK-21 cell, established solid foundation for follow-up immunization zoopery, with
Phase provides safely and effectively vaccine, prevalence and the outburst of prevention and control blue tongue disease for 16 type blue tongue diseases.
Detailed description of the invention
Fig. 1 is the digestion qualification figure of recombinant shuttle plasmid pSTKE-VP2, wherein swimming lane M is I digest of λ-EcoT14
I/Sal of pSTKE-VP2 Xba I, swimming lane 2 are pSTKE-VP2；DNA Marker, swimming lane 1 are plasmid
Fig. 2 is the structural schematic diagram of recombinant shuttle plasmid pSTKE-VP2；
Fig. 3 is that the fluorescence microscopy of vaccinia virus recombinant screens figure, and amplification factor is 200 times；
Marker, swimming lane 1 are rVTT-VP2
Fig. 4 is PCR the and RT-PCR qualification figure of vaccinia virus recombinant, wherein swimming lane M is I digest DNA of λ-EcoT14
GDNA, swimming lane 2 are rVTT-VP2 cDNA, and swimming lane 3 is negative control, and swimming lane 4 is negative control, and swimming lane 5 is VTT TK, swimming lane 6
For TK positive control, swimming lane 7 is rVTT-VP2 gDNA, and swimming lane 8 is rVTT-VP2 cDNA；
Fig. 5 is the genetic stability qualification figure of vaccinia virus recombinant, wherein swimming lane M is Wide Range DNA Marker, swimming
Road 1 ~ 5 is respectively rVTT-VP2 1st generation, the 5th generation, the 10th generation, the 15th generation and the 20th generation, and swimming lane 6 is negative control, and swimming lane 7 is
Positive control.
Specific embodiment
It is further described below in conjunction with attached drawing and example, unless otherwise indicated, this field routine can be used in the present invention
Technology.Unless otherwise explained, whole technical and scientific term used herein have with by the common skill of disclosure fields
The usually clear identical meaning of art personnel.
The recombinant virus of the 16 type blue tongue virus VP2 gene of expression of one embodiment of the invention, including viral vectors and packet
Loaded on the shuttle plasmid with 16 type blue tongue virus VP2 genes in viral vectors, the sequence of 16 type blue tongue virus VP2 genes
Column are as shown in SEQ ID NO.1.
Preferably, viral vectors is vaccinia virus vector, and shuttle plasmid is vaccinia virus shuttle plasmid pSTKE.
Preferably, the upstream of 16 type blue tongue virus VP2 genes also has kozak sequence.
The construction method of the recombinant virus of the 16 type blue tongue virus VP2 gene of expression of one embodiment of the invention, including such as
Lower step: the plasmid containing 16 type blue tongue virus VP2 genes is provided, using amplimer to 16 type blue tongue virus VP2 bases
Because carrying out PCR amplification, the 16 type blue tongue virus VP2 genes that amplification obtains are inserted into shuttle plasmid and obtain recombination shuttle matter
Grain, screening, which is collected, by recombinant shuttle plasmid and virus transfection into cell, after 2 ~ 3 days obtains 16 type blue tongue virus VP2 of expression
The recombinant virus of gene.
Preferably, amplimer includes upstream primer and downstream primer, the sequence of upstream primer such as SEQ ID NO.2 institute
Show, the sequence of downstream primer is as shown in SEQ ID NO.3.
Preferably, will step that the obtained 16 type blue tongue virus VP2 genes of amplification are inserted into shuttle plasmid include: by
It expands 16 obtained type blue tongue virus VP2 genes to connect to obtain pEasy-VP2 with pEasy cloning vector, then passes through double enzymes
It cuts and connects for 16 type blue tongue virus VP2 genes to be connected in shuttle plasmid and obtain recombinant shuttle plasmid.Preferably, double digestion
The enzyme used is Xba I and Sal I.
Preferably, virus is vaccinia virus Tiantan strain, and shuttle plasmid is vaccinia virus shuttle plasmid pSTKE.Preferably, carefully
Born of the same parents are BHK-21 cell.
The following are specific embodiments.
One, material and method
1.1 plasmids, bacterial strain, virus and cell strain
Plasmid containing BTV-16 VP2 gene is provided by Animal &. Plant Inspection and Quarantine Techn Center, Shenzhen Bureau of Impor,
Competence Trans-T1 is enough from Quan Shi King Company, and vaccinia virus shuttle vector pSTKE, vaccinia virus Tiantan strain (VTT plants) are by army
Military affairs veterinary institute in the thing Academy of Medical Sciences provides, and BHK-21 cell is stored in liquid nitrogen container, it will be understood that can also buy in market
It obtains.
The building of 1.2 target gene design of primers and shuttle plasmid
To construct vaccinia virus recombinant shuttle plasmid, BTV-16 VP2 primer is designed, restriction enzyme site Xba I and kozak is added in upstream
Restriction enzyme site Sal I is added in sequence, downstream, and primer sequence is as follows.
BTV-16 VP2-F:ATCTAGAGCCACCATGGCTGCTCAGAA；
BTV-16 VP2-R:GTCGACTTTTGCTAGCCTACACAGTCGGCGCA.
The another TK primer designed for amplification TK gene, sequence are as follows.
TK-F:GGACGCGTATTGATTCACACCGTATTACAG；
TK-R:CGCCCGGGTTCTCCTAATAAGTTACACCGTTTG.
Recombination, screening and the purifying of 1.3 vaccinia virus
With 5 MOI VTT and pSTKE-VP2 cotransfection 2 × 106 Bhk cell is observed after 48h, marks and scrape plaque.It uses
DMEM is cleaned and is transferred in 1.5mL EP pipe, freezes -80 DEG C.It repeats above operation, plaque to be formed and green fluorescence are complete
Portion coincide, and its plaque peripheral region is without lesion.
The PCR and RT-PCR of 1.4 vaccinia virus recombinants are identified
RVTT-VP2 virus is expanded, collects sick cell after 48h, and extract genomic DNA and RNA.With BTV-16 VP2 primer
With TK primer, VP2 and the nonessential TK gene of bovine vaccine are expanded.
The analysis of 1.5 vaccinia virus recombinant genetic stabilities
Continuous passage 20 times in BHK-21 cell by rVTT-VP2, take 1,5,10,15,20 generation sick cells, and extract gene
Group DNA, with the genetic stability of PCR method detection rVTT-VP2.
Two, result
Building, the identification of 2.1 shuttle plasmids
The plasmid containing BTV-16 VP2 gene is expanded using BTV-16 VP2 primer BTV-16 VP2-F and BTV-16 VP2-R
2706 segment can be successfully obtained, Song Kumei company is sequenced, and target gene is consistent with theoretical sequence.It is connected to
PEasy-VP2 is obtained in pEasy-blunt-simple-T1, later using fast enzyme cutting Xba I, the Sal I of Sai Mofei company in 37 DEG C
VP2 is connected in the pSTKE by same digestion and obtains pSTKE-VP2 by double digestion plasmid pEasy-VP2.Use Xba I, Sal I
The target fragment of 2.7kb or so and the carrier segments of 4.5kb or so can be successfully cut out, as shown in Figure 1, it is consistent with expected results,
Prove that recombinant shuttle plasmid pSTKE-VP2 is constructed successfully, structure is as shown in Figure 2.
2.2 the recombination of vaccinia virus, screening and purifying
Vaccinia virus recombinant is bitten by observing green fluorescence plaque under No. 1 recombination, fluorescence microscope channel in light under the microscope
Spot.Screening and multigelation connect malicious purifying, ten instead of after, vaccinia virus is successful on the whole, and the screening of rVTT-VP2 matches (merge)
As a result as shown in Figure 3.
2.3 vaccinia virus recombinant PCR and RT-PCR identification
The genomic DNA and RNA for screening successful recombinant virus rVTT-VP2 are extracted, BTV-16 VP2 primer and TK primer are used
BTV-VP2 gene, TK gene are expanded respectively, carry out the identification of genome conformity and transcription.As a result as shown in figure 4, recombinant vaccinia
Virus genom DNA and cDNA amplify BTV-16 VP2 segment, do not amplify VTT TK gene specific band, it was demonstrated that VP2
Gene is successfully integrated into vaccinia virus recombinant and successfully transcribes vaccinia virus recombinant and purified.
2.4 genetic stabilities analyze result
Successful rVTT-VP2 will be screened and continuously passed for 20 generations, it is right using BTV-16 VP2 primer and TK primer amplification its genome
Its structural stability is detected, as a result as shown in figure 5,1,5,10,15,20 vaccinia virus recombinant rVTT-VP2 can be amplified
The target fragment of 2706bp shows that vaccinia virus recombinant rVTT-VP2 is passed in 20 times with good genetic stability.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
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<120>recombinant virus of 16 type blue tongue virus VP2 genes of expression and its construction method and application
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<170> SIPOSequenceListing 1.0
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atggaggagc tagttatacc agtcataact cgtcaatttg ataaaaaact agttgggcgt 60
tacgattacg tcattgaact gacggaacca gaggatggcg agtggagtgg ccacgatgtt 120
acagagattc caaatagacg catgtttgat attaagcagc aaaatatcag agaggcaata 180
gaatacaaac ctgtagataa tgatggggaa gttttaccgc gcattctaga tatgtccgtt 240
gcgtgttacg atatgaaaaa gagtatgatg aagaaggatg gggtcgattt tgtatcaaac 300
acaaaatggt tagaatggat gataggagat tcgatggacg tgcaacctct aaaagttcaa 360
ctgaaagaag atcacagtac aatacaatat gggatgttct caaacacgtt acacatcgat 420
tcgaggaaag cggataccac gtcatatcat acaattgctg tagaatcaaa agggggacgt 480
gggtgctgtc acgttcacac ggcaatttgg aatcacatgg tacgtaatca cttgtttaac 540
gccgtccaag aggcgtgtta cgtgtttaag ccgacgtatg atttaatagt gattggtgaa 600
aagcagaatc gtgaggatga atttaggatt ggcgaacata atttctatac cataacacgg 660
aatcaccaca tgcgtttagg cgataacgcg tataatcaat tcatgaaggg tttggttcag 720
ttgcgcgtgg cgggagtgac accgaacgtg atacgagagg aaatggccgc tttagatgcg 780
ataagagata cttggatagg agggaacttc gagcgaacac acattaaatc tcttgaaata 840
tgtaagttat tatccagcat tggaagaaag atggttaaca tggaggagga accaaaggat 900
gaaagagacc tatcagttaa gtttcaattt aaactcgacg acaaattttc aacaaccgat 960
ccggaaagaa acgtcatctt tacacataaa acacaccgta cgaatcaaga tcgtttctat 1020
gtgttgctaa tgattgcggc gtcggacaca aataacggta gagtatggtg gtcaaaccct 1080
tatccatgtt tacgtggcgc gttgatcgcg tctgaatgca agcttggtga cgtgtaccat 1140
aaattacggg cgtggtatga gtggagcgta agacctgaat ataaaccgcg agatttggag 1200
cgagaacaag aaaaatacat cgttgggcgc gttaacctct ttgacttaga aggggagcct 1260
gcaacgaagg tgtttcactg ggagtacgaa ttgattacta aagtgtatca gataacgaat 1320
catgcgggga atcattgcga tttgtatcct gacgatgtag agatcacggc caaatttgat 1380
gaggaaaaat acggagagat gattcaaacg ataattaacg aaggatggaa gcacggtgac 1440
tttaagatgt ttaagattct gaaggaggag ggtaacccct tactatatga tctagagaag 1500
gacattaggt tagatagtag atcacaagtt atatttccac catatttcaa caaatggacg 1560
cacgcaccaa tgtttaatgc aaaagtgaaa ccatgcgaag tcgagttagc gcaacgaaag 1620
aacgaagacc cttacgtaaa acgaacggta aagcctataa gtgcggattg tgtcgatctg 1680
ttaagatatc acatgtcgca ttacatggat atgagagtgt caatgaaggg gctaagttta 1740
gcggtcaaac agacgccatc cagtattcat caagaattgg cgaaggatcc attatatcca 1800
ggttttctgc agaggagaga tgaaaattta gatcataagt cggtatgtcc aatcgttaca 1860
aactatttct tattggagaa gttctatact ttggttttaa ctataatgga aaagcattat 1920
tgggatttag atgatagtga tgttacgtat gagtttcccg cattggatca ttcagcttac 1980
aagaccgagg gaacgttgta tgatgtctcg caagtggtag ttcatatgat tgatagattt 2040
tttgagaaga ggcgtttctt gcgctcagtc gatgagtgca ggtggatatt gcatttagta 2100
aggtccactc aaagtcggga caggctgagc gttttcaagc gcttcttccc aacctttggt 2160
gagggattgt gtgtaaacaa ctttcgtaaa gtaaaagata ttatgctatt aaattttctg 2220
cctttcttct ttttaatcgg tgacaatata gcgtatgaac atagacagtg ggcggtgcct 2280
ttgctcttct acgcggataa aataagggtt atacccgctg aggtgggtgc ttattataat 2340
cgctttggat tggtgagcat cttagagctg ctaacattct ttccctcgta tgaaatgcgt 2400
gataataagg ttgaggagga tgtgcacgca tgtgcgagcg cgattctgga tttctatctg 2460
acgacaacca tttcaaatgg aggtatacaa gctagcatag tttcaactaa ggcgttactt 2520
tatgagacgt atctatcctc ggtgtgtggc ggtttctctg aagcgattct gtggtacctc 2580
ccgattaccc atccagttaa gtgtttagtt gcgcttgagg tgtcggactc gctagtagat 2640
ccaaacgtac gtatcgacaa gatcaaacga agatttcctt tatcccacag gcatttacga 2700
ggcattgtcc agatctctgt acggccagat cgcaccttcg gcgtgacgac gtgtggtatt 2760
gtaaagcata aaatatgtaa aaagactctg ctgaggcagc ggtgtgatgt aattttaatc 2820
cagatcccgg gttacgtttt cggtaatgat gaattactca cgaagcttct aaatatttaa 2880
<210> 2
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atctagagcc accatggctg ctcagaa 27
<210> 3
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gtcgactttt gctagcctac acagtcggcg ca 32
Claims (10)
1. a kind of recombinant virus for expressing 16 type blue tongue virus VP2 genes, which is characterized in that including viral vectors and be packaged in
The shuttle plasmid with 16 type blue tongue virus VP2 genes in the viral vectors, the 16 type blue tongue virus VP2 gene
Sequence as shown in SEQ ID NO.1.
2. recombinant virus according to claim 1, which is characterized in that the viral vectors is vaccinia virus vector, described
Shuttle plasmid is vaccinia virus shuttle plasmid pSTKE.
3. recombinant virus according to claim 1, which is characterized in that the upstream of the 16 type blue tongue virus VP2 gene
Also there is kozak sequence.
4. a kind of construction method for the recombinant virus for expressing 16 type blue tongue virus VP2 genes, which is characterized in that including walking as follows
It is rapid: provide the plasmid containing 16 type blue tongue virus VP2 genes, using amplimer to 16 type blue tongue virus VP2 genes into
The 16 type blue tongue virus VP2 genes that amplification obtains are inserted into shuttle plasmid and obtain recombinant shuttle plasmid by row PCR amplification,
Screening, which is collected, by the recombinant shuttle plasmid and virus transfection into cell, after 2 ~ 3 days obtains the 16 type blue tongue disease disease of expression
The recombinant virus of malicious VP2 gene.
5. construction method according to claim 4, which is characterized in that the amplimer includes that upstream primer and downstream are drawn
Object, the sequence of the upstream primer is as shown in SEQ ID NO.2, and the sequence of the downstream primer is as shown in SEQ ID NO.3.
6. construction method according to claim 4, which is characterized in that the 16 type blue tongue virus for obtaining amplification
It includes: the 16 type blue tongue virus VP2 genes for obtaining amplification and pEasy grams that VP2 gene, which is inserted into the step in shuttle plasmid,
Grand carrier connects to obtain pEasy-VP2, and 16 type blue tongue virus VP2 genes are then connected to shuttle by double digestion and connection
Recombinant shuttle plasmid is obtained in plasmid.
7. construction method according to claim 6, which is characterized in that the enzyme that the double digestion uses is Xba I and Sal I.
8. construction method according to claim 4, which is characterized in that the virus is vaccinia virus Tiantan strain, described to wear
Shuttle plasmid is vaccinia virus shuttle plasmid pSTKE.
9. construction method according to claim 4, which is characterized in that the cell is BHK-21 cell.
10. a kind of recombinant virus of 16 type blue tongue virus VP2 genes of described in any item expression of claim 1 ~ 3 is blue in preparation
Application in glossopathy vaccine.
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| CN201910454911.4A CN110117579B (en) | 2019-05-29 | 2019-05-29 | Recombinant virus for expressing 16 type bluetongue virus VP2 gene and construction method and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110819629A (en) * | 2019-12-24 | 2020-02-21 | 军事科学院军事医学研究院军事兽医研究所 | Primer combination and detection method for detecting blue tongue 8 type and/or blue tongue 16 type viruses |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0098867A1 (en) * | 1982-01-19 | 1984-01-25 | The Texas A&M University System | Blue tongue virus vaccine and method of producing same |
-
2019
- 2019-05-29 CN CN201910454911.4A patent/CN110117579B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0098867A1 (en) * | 1982-01-19 | 1984-01-25 | The Texas A&M University System | Blue tongue virus vaccine and method of producing same |
Non-Patent Citations (1)
| Title |
|---|
| AUR´ELIE PERRIN等: "Recombinant capripoxviruses expressing proteins of bluetongue virus:Evaluation of immune responses and protection in small ruminants", 《VACCINE》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110819629A (en) * | 2019-12-24 | 2020-02-21 | 军事科学院军事医学研究院军事兽医研究所 | Primer combination and detection method for detecting blue tongue 8 type and/or blue tongue 16 type viruses |
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| CN110117579B (en) | 2021-12-14 |
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