CN103387996A - Canine parvovirus-like particles and preparation method and application thereof - Google Patents
Canine parvovirus-like particles and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides canine parvovirus-like particles. The VP2 gene of canine parvovirus is optimized according to the preferred codons of insect cells, the optimized VP2 gene is connected with an insect expression vector, and the canine parvovirus-like particles are produced by use of a rhabdovirus/insect cell expression system. The canine parvovirus-like particles are combined with a preservative and an adjuvant to prepare a canine parvovirus enteritis vaccine. By adopting the canine parvovirus-like particles provided by the invention, the prepared canine parvovirus enteritis vaccine has the advantages of high output, low production cost, high immunogenicity, good safety, convenience in large-scale production and the like, and has good immunization and prevention effects on canine parvovirus enteritis.
Description
Technical field
The present invention relates to microorganism field, genetically engineered field and animal doctor's field of biological pharmacy, specifically, relate to canine parvovirus virus sample particle, its preparation method and application.
Background technology
1978, Australian Kelly and the Thomson of Canadian etc. was separated to the canine parvovirus cause of disease from the sick dog that suffers from hemorrhage enteritis, in order with the superfine small virus of dog, to distinguish, with this isolated cause of disease called after CPV-2.1979 and 1984, there are again subtype C PV-2a, the CPV-2b of two kinds of canine parvovirus CPV-2 in succession to occur and replace, be widely current in the world.Report about CPV-2c appearred in 2002.CPV infects Canis animals, has the height contagiousness, mainly causing bleeding property diarrhoea and myocarditis, and in pup, M ﹠ M is all very high.This disease has become a kind of Important Infectious Diseases of Canis animals, causes great harm for the economic animal aquaculture.
Canine parvovirus belongs to the Parvoviridae parvovirus and belongs to the member, and viral outward appearance is sexangle, without cyst membrane, and the rotational symmetry such as icosahedron, diameter is about 20nm, and capsid is comprised of 32 capsomeres.This viral hemoagglutination characteristic is stronger, can the aggegation pig and the red corpuscle of rhesus monkey.The CPV genome is about 5.2kb, comprises two open reading frame, encode respectively Nonstructural Protein and structural protein.Wherein virus structural protein VP1 and VP2 by identical mRNA by alternative splicing after translation come, VP1 comprises the full sequence of VP2 albumen, VP2 is the chief component albumen of viral capsid, and determines viral Main Biological.
The vaccine that is used at present canine parvovirus is mainly attenuated vaccine and inactivated vaccine, but existent defect separately, for example, there is the danger of virulence reversion and restructuring in attenuated vaccine, the inactivated vaccine less immunogenic, and can not cause cellullar immunologic response etc.Therefore, the CPV vaccine of development of new becomes the technical problem that needs to be resolved hurrily.
Summary of the invention
The purpose of this invention is to provide a kind of canine parvovirus virus sample particle, its preparation method and application.
Another object of the present invention is to provide a kind of canine parvovirus viral enteritis vaccine, its preparation method and application.
In order to realize the object of the invention, the present invention utilizes baculovirus/insect cell expression system to produce canine parvovirus disease poison sample particle.
At first the present invention provides a kind of canine parvovirus VP2 protein gene of optimization, namely according to the insect cell preference codon, the canine parvovirus VP2 protein gene is optimized, the canine parvovirus VP2 protein gene that is optimized.Before optimizing, the nucleotide sequence of canine parvovirus VP2 protein gene is as shown in SEQ ID No.1, and the nucleotide sequence of the canine parvovirus VP2 protein gene after optimization is as shown in SEQ ID No.2.The aminoacid sequence of the VP2 gene coded protein after optimization is as shown in SEQ ID No.3.
The present invention also provides a kind of preparation method of canine parvovirus virus sample particle, comprise the following steps: the structure of (1) recombinant baculovirus Bacmid plasmid: the VP2 gene of aforementioned optimization is inserted in insect cell expression carrier pFastBac1, transform intestinal bacteria DH10Bac competent cell, extract positive plasmid and be recombinant baculovirus Bacmid plasmid; (2) rescue of recombinant baculovirus: with recombinant baculovirus Bacmid plasmid transfection insect cell, rescue obtains recombinant baculovirus; (3) preparation of canine parvovirus virus sample particle: the recombinant baculovirus that will obtain is pressed MOI=0.1 inoculation insect cell, gathers in the crops supernatant after 4-5 days, obtains canine parvovirus virus sample particle.
In preceding method, the insect cell that uses in step (2)-(3) is the Sf9 cell.
Reach 80% until the cell degree of converging of Sf9 cell in step (2) and carry out transfection when above.The present invention also provides the canine parvovirus virus sample particle by the aforesaid method preparation.
The present invention also provides the application of described canine parvovirus virus sample particle in preparing canine parvovirus viral enteritis vaccine.
The present invention also provides a kind of canine parvovirus viral enteritis vaccine, and it is the canine parvovirus of above-mentioned preparation virus sample particle to be aided with sanitas and adjuvant is prepared from.Wherein, described sanitas is sodium azide etc., and preferred final concentration is 0.01%; Described adjuvant is aluminum phosphate etc., and preferred final concentration is 0.4%.
Canine parvovirus viral enteritis vaccine of the present invention is intramuscular injection vaccine or oral vaccine.
The present invention further provides the application of canine parvovirus viral enteritis vaccine in preventing canine/feline minute viral enteritis of above-mentioned preparation.
The present invention utilizes baculovirus/insect cell expression system to produce canine parvovirus disease poison sample particle, use baculovirus as expression vector, use the Sf9 cell as bio-reactor, the canine parvovirus viral enteritis vaccine of preparation has that output is high, production cost is low, immunogenicity is high, security is good, be convenient to the advantage such as scale operation, and the canine parvovirus viral enteritis is had good immunity and preventive effect.
Description of drawings
Fig. 1 is the structure schematic diagram of recombinant baculovirus expression plasmid Bac-VP2 in the embodiment of the present invention 1.
Fig. 2 is the situation after recombinant expression plasmid Bac-VP2 transfection Sf 9 insect cell in the embodiment of the present invention 1; Wherein, A is the normal cell control group, and B is recombinant expression plasmid transfectional cell group.
Fig. 3 is the Electronic Speculum detected result of recombinate shape virus infection cell conditioned medium in the embodiment of the present invention 1.
Fig. 4 is indirect immunofluorescene assay result after recombinate shape virus infection Sf9 cell in the embodiment of the present invention 1; Wherein, A is the recombinate shape virus infection group, and B is the normal cell group.
Fig. 5 is HI antibody titer in body after canine parvovirus viral enteritis vaccine immunity cavy in the embodiment of the present invention 2.
Fig. 6 is HI antibody titer growth and decline situation in body after canine parvovirus viral enteritis vaccine immunity dog in the embodiment of the present invention 2.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art, the raw materials used commercial goods that is.
Preparation and the evaluation of embodiment 1 canine parvovirus virus sample particle
1 material
Donor plasmid pFastBac1 is available from Invitrogen company, and bacterial classification E.coli DH10Bac, Insect cells Sf9, canine parvovirus are provided by Xinuo Biological Science ﹠ Technology Co., Ltd., Changchun.
pEASY-blunt simple, IPTG, X-gal is available from the precious biotech firm in Dalian, the Phusion archaeal dna polymerase, the T4DNA ligase enzyme, restriction enzyme is available from NEB company, competent cell is available from Takata company, genome DNA extracting reagent kit, gel reclaims test kit, plasmid extraction kit is available from Axygen company, liposome Lipofectin2000 is available from Invitrogen company, penbritin, tetracycline, kantlex, gentamicin is available from Sigma company, foetal calf serum is that Xinuo Biological Science ﹠ Technology Co., Ltd., Changchun produces, TC-100 insect cell substratum is available from Applichem company, rabbit anti-dog parvovirus polyclonal antibody is available from Xinuo Biological Science ﹠ Technology Co., Ltd., Changchun, the goat antirabbit two of FITC mark is anti-available from shirt company in Beijing.
2 methods
2.1 virus genomic extraction
Canine parvovirus is synchronously inoculated the F81 cell, inoculate multigelation after 4 days and receive afterwards poison 3 times, with commercial kit, extract total DNA, working method is referring to the test kit specification sheets.
2.2CPV the mensuration of VP2 gene order and optimization
, with reference to the upper canine parvovirus VP2 protein gene order (FJ435347.1) of announcing of GenBank, utilize Primer5.0 software design PCR primer VP2-F1, VP2-R1(table 1).Pass through pcr amplification, the product (CPV-VP2) that obtains is spent the night and is connected with 4 ℃, pEASY-blunt simple carrier, connect product transformed competence colibacillus cell Trans5 α, select positive colony and carry out sequencing analysis, the canine parvovirus VP2 protein gene order that obtains is as shown in SEQ ID NO.1.
The canine parvovirus VP2 protein gene order that obtains is optimized, namely, in the situation that the aminoacid sequence (SEQ ID NO.3) of assurance coding is constant,, by Substitution, obtains the codon of insect cell preference.VP2 gene order after optimization as shown in SEQ ID NO.2, is cloned on carrier pMD18, the recombinant vectors called after opti-CPV-VP2 that obtains.
2.3 the structure of restructuring donor plasmid pF-CPV-VP2
Take opti-CPV-VP2 as template, VP2-F2, VP2-R2 are primer (table 1), carry out pcr amplification, the amplified production called after SF-CPV-VP2 that obtains.By NotI and HindIII double digestion, SF-CPV-VP2 is connected in insect expression vector pFastBac1 the recombinant vectors called after pF-CPV-VP2 that obtains.
Table 1PCR primer sequence
2.4 the structure of recombinant baculovirus expression plasmid Bac-VP2
with pF-CPV-VP2 transformed competence colibacillus DH10Bac: get 1 μ L pF-CPV-VP2 and join in 50 μ L competence DH10Bac cells, ice bath 30min, 42 ℃ of heat shock 45s, ice bath 2min, add 1mL non-resistant LB substratum, 37 ℃ of 200rpm/min4h, bacterium liquid is got 80 μ L to be coated with the LB flat board and (to contain kantlex, gentamicin, three kinds of microbiotic of tsiklomitsin), cultivate 36-48h for 37 ℃, select the hickie bacterium colony, add LB substratum (kantlex, gentamicin, the tetracycline final concentration is respectively 50 μ g/mL, 7 μ g/mL, 10 μ g/mL), 37 ℃ of 200rpm/min shaking tables are cultivated 12h.Extract plasmid DNA, carry out PCR and identify.Positive recombinant plasmid called after Bac-VP2 will be accredited as.
2.5 the rescue of recombinant baculovirus and evaluation
With recombinant baculovirus expression plasmid Bac-VP2 transfection insect cell: transfection the day before yesterday, the Sf9 cell is placed in 12 orifice plates, 27 ℃ of cultivations in the 10%TC-100 substratum, carry out transfection when above when cell density reaches 80%.The Bac-VP2 of 1 μ g is added to the two without softly mixing in (serum-free, unparalleled anti-) TC-100 nutrient solution of 100 μ L; 4 μ L liposomes are joined 100 μ L two without softly mixing in the TC-100 nutrient solution; Plasmid and liposome are mixed the standing 15min of room temperature.Wash 12 orifice plate cells twice with two without the TC-100 nutrient solution around here, then 800 μ L plasmids and liposome mixture are added in the Sf9 cell, be placed in 27 ℃ of incubators and cultivate, treat cellular swelling, become circle, after coming off, get supernatant, be labeled as P1 for recombinant baculovirus.With the Sf9 cell of the P1 of rescue acquisition for the recombinate shape virus infection normal growth, cultivate 4-5 days, treat cellular swelling, become circle, after coming off, get supernatant, extract DNA, take VP2-F2 and VP2-R2(table 1), as primer, carry out the PCR evaluation.
2.6 indirect immunofluorescence
With 1 * 10
6Individual/hole Sf9 cell paving 6 porocyte culture plates, connect poison with 0.1 MOI, after infecting 30h, pre-cold acetone with 80% fixedly spends the night in-20 ℃, with PBST, washes 3 times, each 3min, add the primary antibodie (rabbit anti-dog parvovirus polyclonal antibody) of PBST1:200 dilution, hatch 1h for 37 ℃, with PBST, wash 2 times, each 3min, add the goat antirabbit two of the FITC mark of PBST1:500 dilution to resist, hatch 1h for 37 ℃, with PBST, wash 3 times, each 3min, observe fluorescence.
2.7 electron microscopic observation
Results produce cytopathic insect cell, multigelation 3 times, and the centrifugal 5min of 2000rpm/min, get supernatant, with phospho-wolframic acid dyeing, the Packing Condition of electron microscopic observation virus like particle.
2.8 canine parvovirus virus sample particle content measuring
With the recombinant baculovirus inoculation Sf9 cell that rescue obtains, gather in the crops virus liquid after cytopathy to be generated, multigelation 3 times, the centrifugal 5min of 2000rpm/min, get supernatant, detects the canine parvovirus virus sample granule content that forms with hemagglutination test (HA).
3 results
3.1VP2 gene optimization result
Through Sequence analysis, canine parvovirus VP2 protein gene open reading frame total length 1755bp, 584 amino acid of encoding.According to the preferences of insect cell to codon, the canine parvovirus VP2 protein sequence that obtains is optimized, optimization principles follow following some: the aminoacid sequence of coding is constant; Rare codon is removed; Destroy affecting mRNA stability and affecting the loop-stem structure of with rrna, being combined.Canine parvovirus VP2 protein gene order after optimization is as shown in SEQ ID NO.2.
3.2 recombinant baculovirus expression plasmid construction result
After donor plasmid pF-CPV-VP2 transfection competence DH10Bac, can carry out by Tn7R, the Tn7L of polyclone restriction enzyme site both sides the swivel base restructuring, obtain recombinant baculovirus expression plasmid Bac-VP2(structure schematic diagram and see Fig. 1), Bac-VP2 comprises the canine parvovirus VP2 protein gene.
3.3 the rescue of recombinant baculovirus
, with recombinant expression plasmid Bac-VP2 transfection Sf 9 insect cell, carry out the rescue of recombinant virus.After transfection 72h, same normal cell (Fig. 2 A) control wells is compared, and transfection porocyte (Fig. 2 B) starts to come off, swelling, cracking, and form is irregular, show the recombinant expression plasmid transfectional cell after packing produced recombinant baculovirus, cause cytopathy.
3.4 electron microscopic observation result
With the virus liquid of results, after phospho-wolframic acid dyeing, through the Packing Condition of electron microscope observation virus-like particle.Result shows: the CPV viral particle morphology that packing forms is the spheroidal particle of icosahedron, and size is about 25nm(Fig. 3), illustrate that recombinant baculovirus successfully assembles and formed virus-like particle at the VP2 of insect cell inner expression albumen.
3.5 indirect immunofluorescence
, with recombinate shape virus infection Sf9 cell, detect the expression of canine parvovirus VP2 protein albumen with indirect immunofluorescence.As shown in Fig. 4 A, visible a large amount of green fluorescences, show VP2 great expression (the negative cell contrast of Fig. 4 B).
3.6 canine parvovirus virus sample particle content measuring
, with the virus liquid of results, detect the content that wherein forms virus-like particle with hemagglutination test (HA).Result shows: the canine parvovirus virus sample particle that after recombinate shape virus infection Sf9 cell, packing forms can successful aggegation swine erythrocyte, and its hemagglutinative titer is 1:2
12.
Preparation and the animal immune experiment of embodiment 2 canine parvovirus viral enteritis vaccines
1 material
After aluminum phosphate was made into proper concn with isotonic saline solution, ultrasonication 20min, be mixed with suspension, and was standby after high-temperature sterilization.Female cavy (about 250g), (at the 2-4 monthly age, the CPV HI antibody titer is less than 1:2 for Chinese rural area dog
3) available from Changchun Biological Products Institute.
2 methods
2.1 the preparation of canine parvovirus viral enteritis vaccine
To express the recombinant baculovirus inoculation Sf9 cell of canine parvovirus VP2 protein gene, 27 ℃ of cultured continuously 4-5 days, after pathology appears in cell, the harvested cell suspension, multigelation 3 times, the centrifugal 10min of 2000rpm, the results supernatant liquor, obtain canine parvovirus virus sample particle.The canine parvovirus of results virus sample particle is added sodium azide (0.01%) and aluminum phosphate (0.4%), prepare canine parvovirus viral enteritis vaccine.
2.2 hemagglutination-inhibition test
, through 56 ℃ of deactivation 30min, add 1/10 volume pig red blood corpuscle to mix the dog that gathers and guinea pig serum, room temperature is placed 2h, then with the centrifugal 10min of 2000rpm.Get clean V-shaped 96 orifice plates, every hole adds 25 μ L PBS, to adding serum 25 μ L after deactivation and absorption in the first hole, and doubling dilution to the ten holes continuously from left to right, 11-holes is the positive control of 8 unit antigens, the 12 negative contrast in hole.Then add the 8 antigen 25 μ L to the of unit ten holes, add the 8 antigen 25 μ L of unit in 11-holes, and at these row, carry out two times of doubling dilutions downwards, then to the 11,12 holes, add PBS25 μ L.After vibration mixes, hatch 1h for 37 ℃, then add 1% pig red blood corpuscle suspension (containing 0.5% rabbit anteserum) 50 μ L, after vibration mixes, place 1h for 4 ℃, observe blood clotting inhibition (HI) and tire.
2.3 cavy immunization experiment
Get 25 of female cavys (250g left and right) and be divided at random 5 groups, every group 5 (table 2).Wherein M1, M2 group is carried out intramuscular injection, and O3, O4 group is carried out oral immunity, and the C5 group is normal control.M1, O3 immunity 1 time, M2, O4 immunity 2 times, 14 days, interval.After immunity, blood sampling in the 0th, 7,14,21,28 day, carry out the detection of hemagglutination inhibition antibody titer.
2.4 dog immunization experiment
Get 12 Chinese rural area dogs, label is M1, O2, O3, C4 successively, and specifically grouping and immune programme for children are in Table 3.Wherein the M1 group is carried out intramuscular injection, and O2, O3 group is carried out oral immunity, and head exempted from the 0th day, the 15th day booster immunization.Blood samplings in every 15 days after the immunity same day and immunity, carry out the detection of hemagglutination inhibition antibody titer.The C4 group is as normal control.
Table 2 cavy immunity grouping
Table 3 dog immunity grouping and immune programme for children
3 results
3.1 cavy immune result
By table 2 immune programme for children, cavy is carried out immunity, after the 14th day booster immunization, HI antibody titer progressively raises, and the lower intramuscular injection group antibody titer of same time point is higher than the oral immunity group, and 2 times immune effect is better than 1 immunity (Fig. 5).Wherein, after intramuscular injection, HI antibody titer reaches as high as 1:2
9, after oral immunity, HI antibody titer reaches as high as 1:2
7.
3.2 dog immune result
By table 3 immune programme for children, dog is carried out canine parvovirus viral enteritis vaccine immunity.Result shows: immunity was respectively organized dog in rear 15 days all can detect the canine parvovirus hemagglutination inhibition antibody, and the intramuscular injection group is higher than the oral immunity group.After oral immunity 2 times (booster immunization), in the dog body, HI antibody titer obviously increases, and reaches the level identical with the intramuscular injection group.Wherein, after intramuscular injection or oral immunity, HI antibody titer all reaches as high as 1:2
12(Fig. 6).Immune duration test shows: intramuscular injection or oral immunity are organized each dog for 2 times in immunity rear 12 months, and in body, the parvovirus HI antibody titer is still higher than 1:2
5, i.e. the antibody titer of canine parvovirus viral enteritis boosting vaccine body generation can be kept 12 months at least.
The protection test of embodiment 3 canine parvovirus viral enteritis vaccines to dog
1 material
Canine parvovirus viral enteritis vaccine is pressed the preparation of method described in embodiment 2, and (at the 2-4 monthly age, the CPV HI antibody titer is less than 1:2 for Chinese rural area dog
3) available from Changchun Biological Products Institute.
2 methods
2.1 animal immune
Get 12 Chinese rural area dogs, label is M1, O2, O3, C4 successively, by table 3 in embodiment 2, divides into groups and immunity.Wherein the M1 group is carried out intramuscular injection, and O2, O3 group is carried out oral immunity, and head exempted from the 0th day, the 15th day booster immunization.
2.2 zoogenetic infection
The canine parvovirus strong virus attack was carried out in immunity in rear 21 days.Concrete grammar is as follows: the strong malicious 2mL(viral level of oral vaccination canine parvovirus is 10
6TCID
50), appetite, the mental status, the body temperature of observing dog after inoculation every day change and death condition.Gather simultaneously ight soil every day, detect CPV viral level in ight soil, determine its toxin expelling situation.
3 results
Control group (C4) dog is 3-5 days after attacking poison, typical canine parvovirus viral enteritis clinical symptom in succession occurs, and main manifestations is: appetite stimulator, One's spirits are drooping, oligoleukocythemia, have loose bowels etc., canine parvovirus can be detected in ight soil.Attack the rear 8-10 days of poison, 3 contrast dogs die off.Oral 1 (O2) immune group, have 1 dog appetite stimulator, One's spirits are drooping symptom to occur, virus detected in attacking the poison ight soil of rear 4 days, but symptom faded away in infection in rear 6 days, and eventual rehabilitation.Clinical symptom does not all appear in intramuscular injection (M1) and oral 2 (O3) immune group dogs, parvovirus do not detected in ight soil yet.Result shows: the canine parvovirus viral enteritis vaccine of preparation is to dog safety, no pathogenicity, and canine parvovirus viral enteritis poison is had good immunity and preventive effect.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. the preparation method of canine parvovirus virus sample particle, is characterized in that, comprises the following steps:
(1) according to the insect cell preference codon, the canine parvovirus VP2 protein gene is optimized the canine parvovirus VP2 protein gene that is optimized;
(2) structure of recombinant baculovirus Bacmid plasmid: the VP2 gene that will optimize is inserted in insect cell expression carrier pFastBac1, transforms intestinal bacteria DH10Bac competent cell, extracts positive plasmid and is recombinant baculovirus Bacmid plasmid;
(3) rescue of recombinant baculovirus: with recombinant baculovirus Bacmid plasmid transfection insect cell, rescue obtains recombinant baculovirus;
(4) preparation of canine parvovirus virus sample particle: the recombinant baculovirus that will obtain is pressed MOI=0.1 inoculation insect cell, gathers in the crops supernatant after 4-5 days, obtains canine parvovirus virus sample particle.
2. method according to claim 1, is characterized in that, the nucleotide sequence of the canine parvovirus VP2 protein gene of optimizing in step (1) is as shown in SEQ ID No.2.
3. method according to claim 1, is characterized in that, the insect cell that uses in step (3)-(4) is the Sf9 cell.
4. method according to claim 3, is characterized in that, reaches 80% until the cell degree of converging of Sf9 cell in step (3) and carry out transfection when above.
5. the canine parvovirus virus sample particle of according to claim 1-4 described methods of any one preparations.
6. the application of canine parvovirus virus sample particle claimed in claim 5 in preparing canine parvovirus viral enteritis vaccine.
7. canine parvovirus viral enteritis vaccine, is characterized in that, canine parvovirus claimed in claim 5 virus sample particle is aided with sanitas and adjuvant is prepared from.
8. vaccine according to claim 7, is characterized in that, described sanitas is sodium azide, and final concentration is 0.01%.
9. vaccine according to claim 7, is characterized in that, described adjuvant is aluminum phosphate, and final concentration is 0.4%.
10. vaccine according to claim 7, is characterized in that, described vaccine is intramuscular injection vaccine or oral vaccine.
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| CN107868131A (en) * | 2017-10-13 | 2018-04-03 | 长春西诺生物科技有限公司 | A kind of porcine parvovirus subunit vaccine and preparation method thereof |
| CN108348594A (en) * | 2015-09-29 | 2018-07-31 | 梅里亚股份有限公司 | Canid parvovirus (CPV) virus-like particle (VLP) vaccine and application thereof |
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| CN110907642A (en) * | 2019-12-02 | 2020-03-24 | 青岛农业大学 | Kit and method for rapidly detecting canine parvovirus antigen and canine parvovirus antibody |
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| CN108348594B (en) * | 2015-09-29 | 2022-09-09 | 勃林格殷格翰动物保健美国公司 | Canine Parvovirus (CPV) virus-like particle (VLP) vaccines and uses thereof |
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| CN110907642A (en) * | 2019-12-02 | 2020-03-24 | 青岛农业大学 | Kit and method for rapidly detecting canine parvovirus antigen and canine parvovirus antibody |
| CN114317459A (en) * | 2021-12-31 | 2022-04-12 | 长春西诺生物科技有限公司 | Canine distemper virus strain, bivalent vaccine based on canine distemper virus and canine parvovirus and application |
| CN114317459B (en) * | 2021-12-31 | 2024-02-23 | 长春西诺生物科技有限公司 | Canine distemper virus strain, bivalent vaccine based on canine distemper virus and canine parvovirus and application |
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