CN106109424A - A kind of porcine circovirus 2 type liposome dilution freeze-dried products and preparation method thereof - Google Patents
A kind of porcine circovirus 2 type liposome dilution freeze-dried products and preparation method thereof Download PDFInfo
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- CN106109424A CN106109424A CN201610678380.3A CN201610678380A CN106109424A CN 106109424 A CN106109424 A CN 106109424A CN 201610678380 A CN201610678380 A CN 201610678380A CN 106109424 A CN106109424 A CN 106109424A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
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- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Abstract
A kind of porcine circovirus 2 type liposome dilution freeze-dried products and preparation method thereof, belongs to veterinary biologics technical field.Every 1000mlPBS solution contains following components: Cordyceps polysaccharide 5 10g, lycopene 5 10g, ginsenoside Rh2 1 5g, liposome 6 12g, sucrose 10 15g and mannitol 10 15g.Liposome dilution freeze-dried products prepared by the present invention can reduce difference between the batch of semi-finished product.Liposome dilution freeze-dried products prepared by the present invention can promote the breeding of porcine circovirus 2 type, improves viral level, strengthens vaccine immunogenicity.
Description
Technical field
The invention belongs to veterinary biologics technical field, be specifically related to a kind of porcine circovirus 2 type liposome dilution
Freeze-dried products and preparation method thereof.
Background technology
Porcine circovirus desease is to be caused pig by porcine circovirus 2 type (Porcine circovirus type 2, PCV2)
A kind of new infectious disease, it is mainly characterized by, and piglet becomes thin, retarded growth, dyspnea, diarrhoea, sow return feelings rate and increase
Add, produce the extensive pathological change of mummy tire or weak son, miscarriage, internal organs and skin, produce additionally, primary disease can also result in swinery
Raw serious immunosuppressant, thus it is easily caused secondary infection or concurrently other infections.This disease causes to whole world pig industry
Huge economic loss, is newfound another important epidemic disease after Porcine reproductive and respiratory syndrome, and vaccine immunity is pre-
One of major measure that anti-PCV-2 infects.
Liposome is that one has a same biomembrane kin phospholipid bilayer structure carrier, and Chinese medicine liposome system
The spherical drug carriers formulations of a kind of superminiature referring to that middle the effective elements of the medicine is encapsulated in lipoids bilayer and make.This medicine
Thing carrier has the advantage of targeting, sustained-release administration, and liposome to normal cell and organizes harmless and inhibitory action, also may be used
To show good cellular affinity and histocompatibility.Liposome is the most degradable, avirulence and non-immunogenicity, its
It is possible not only to improve drug therapeutic indices as a kind of pharmaceutical carrier, additionally it is possible to reduce drug toxicity and reduce poisonous side effect of medicine
Etc. advantage.
The Radix Astragali is the most important Chinese medicine, has benefit and defends the effects such as consolidating superficial resistance, diuretic be swollen clearly, promoting pus discharge and tissue regeneration strengthening, invigorating the spleen and replenishing QI, the people
Between be usually used in treating spontaneous perspiration, night sweat, arthralgia due to stagnation of blood, edema, cellulitis do not burst or burst do not hold back for a long time, interior part band void, diarrhea due to hypofunction of the spleen and all gas
The disease of the blood deficiency that declines.Radix Astragali saponin is one of main active of the Radix Astragali, and Radix Astragali saponin is possible not only to promote lymphocytic hyperplasia and resist
The increase of body, moreover it is possible to promote humoral and cellular immune response responsing reaction.
Polysaccharide is one of active ingredient of Chinese herbs, and numerous studies show, polysaccharide and saccharide complex are not only work in vivo
For energy resource and constituent material, it is often more important that it is present in all membrane structures, participate in cell in biosis
Various activities.Polysaccharose substance is the important component part of all Living organisms, has removing free radical, improves antioxidase
Activity and the ability of anti-lipid peroxidation
One of most important index of veterinary biologics performance rating is exactly the stability between batch, but in actual production process
In be difficult to control to stability between the batch of semi-finished product, mainly ratio when inoculating living body biological of tracing it to its cause is uncertain, also
That is inoculum concentration instability causes producing semi-finished product bigger difference.Present inventive concept is that solving pig circular ring virus increases
The difficult problem that during growing, between batch, difference is big and viral level is too low.
Summary of the invention
The problem existed for prior art, it is an object of the invention to design provides a kind of porcine circovirus 2 type lipid
The technical scheme of body diluent freeze-dried products and preparation method thereof.
Described a kind of porcine circovirus 2 type liposome dilution freeze-dried products, it is characterised in that every 1000mlPBS solution
Containing following components: Cordyceps polysaccharide 5-10g, lycopene 5-10g, ginsenoside Rh2 1-5g, liposome 6-12g, sucrose 10-
15g and mannitol 10-15g.
Described a kind of porcine circovirus 2 type liposome dilution freeze-dried products, it is characterised in that every 1000mlPBS solution
Containing following components: Cordyceps polysaccharide 6-8g, lycopene 6-8g, ginsenoside Rh2 2-4g, liposome 8-10g, sucrose 12-
14g and mannitol 12-14g.
Described a kind of porcine circovirus 2 type liposome dilution freeze-dried products, it is characterised in that described liposome leads to
Cross following steps to prepare: weigh soybean lecithin, cholesterol, dissolve with chloroform, and make its mix homogeneously, existed by this solution
37 DEG C of decompression rotary evaporations, remove organic solvent, form immobilized artificial membrane；Adding the PBS solution of pH value 7.0 in film, water-bath rotates
Aquation, gained suspension carries out supersound process, is prepared as liposome after being finally dehydrated with refiner.
Described a kind of porcine circovirus 2 type liposome dilution freeze-dried products, it is characterised in that described PBS solution is led to
Cross following steps to prepare: take sodium chloride 6-10g, potassium chloride 0.05-0.5g, disodium hydrogen phosphate 1-1.2g, potassium dihydrogen phosphate 0.05-
0.5g, calcium chloride 0.05-0.2g, magnesium chloride 0.05-0.2g containing 6 water of crystallization；After above each composition is mixed, it is dissolved in
In 1000ml distilled water, through 116 DEG C, autoclaving 30 minutes and get final product.
The preparation method of described a kind of porcine circovirus 2 type liposome dilution freeze-dried products, it is characterised in that include
Following steps: after the component of described weight being sufficiently mixed, join vibration aquation in 1000ml pH 7 PBS solution, are formed
Uniform solution；After the filter of 0.22um filters, being first positioned over-40 DEG C of cryogenic refrigerator quick freezing, room temperature is slowly melted
Change, be repeated four times, then be placed in-40 DEG C of pre-freeze 4-8h；Finally it is dried in vacuum freeze-drying machine, i.e. obtains pig circular ring virus 2
Poison 2 type liposome dilution freeze-dried products.
The preparation method of described a kind of porcine circovirus 2 type liposome dilution freeze-dried products, it is characterised in that described
PBS solution by following steps prepare: take sodium chloride 6-10g, potassium chloride 0.05-0.5g, disodium hydrogen phosphate 1-1.2g, phosphoric acid
Potassium dihydrogen 0.05-0.5g, calcium chloride 0.05-0.2g, magnesium chloride 0.05-0.2g containing 6 water of crystallization；Above each composition is mixed
After, it is dissolved in 1000ml distilled water, through 116 DEG C, autoclaving 30 minutes and get final product.
In the present invention, Cordyceps polysaccharide far is become to create Science and Technology Ltd.'s production and sales by Wuhan, and lycopene is green climing by Hunan
Bio tech ltd's production and sales, ginsenoside Rh2 is by the production and sales of lark prestige Science and Technology Ltd..
The method have the advantages that
1, the liposome dilution freeze-dried products prepared by the present invention can reduce difference between the batch of semi-finished product.
2, the liposome dilution freeze-dried products prepared by the present invention can promote the breeding of porcine circovirus 2 type, improves
Viral level, strengthens vaccine immunogenicity.
Detailed description of the invention
In order to make the present invention easier to understand, below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that
These embodiments are merely to illustrate the present invention rather than limit the scope of the present invention, NM concrete reality in the following example
Proved recipe method, experimental technique is carried out the most routinely.
The preparation of embodiment 1:PBS solution
Take sodium chloride 6-10g, potassium chloride 0.05-0.5g, disodium hydrogen phosphate 1-1.2g, potassium dihydrogen phosphate 0.05-0.5g, calcium chloride
0.05-0.2g, magnesium chloride 0.05-0.2g containing 6 water of crystallization；After above each composition is mixed, it is dissolved in 1000ml distilled water,
Through 116 DEG C, autoclaving 30 minutes and get final product.
Embodiment 2: the preparation of liposome
Weigh soybean lecithin 6g, cholesterol 2g, dissolve with 500ml chloroform, and make its mix homogeneously, by this solution 37
DEG C decompression rotary evaporation, remove organic solvent, formed immobilized artificial membrane；Add 200ml in film, the PBS solution of pH value 7.0 (is implemented
Example 1 prepares), water-bath rotates aquation, and gained suspension carries out supersound process, is prepared as liposome after being finally dehydrated with refiner.
Embodiment 3: the preparation of liposome
Weigh soybean lecithin 4g, cholesterol 4g, dissolve with 500ml chloroform, and make its mix homogeneously, by this solution 37
DEG C decompression rotary evaporation, remove organic solvent, formed immobilized artificial membrane；Add 200ml in film, the PBS solution of pH value 7.0 (is implemented
Example 1 prepares), water-bath rotates aquation, and gained suspension carries out supersound process, is prepared as liposome after being finally dehydrated with refiner.
Embodiment 4: the preparation of porcine circovirus 2 type liposome dilution freeze-dried products
Insect-taking grass polysaccharide 5g, lycopene 5g, ginsenoside Rh2 1g, liposome (embodiment 2 prepare) 6g, sucrose 10g and sweet
Dew alcohol 10g；
After the component of described weight being sufficiently mixed, join vibration in the PBS solution (embodiment 1 prepares) of 1000ml pH 7
Aquation, forms uniform solution；After the filter of 0.22um filters, first it is positioned over-40 DEG C of cryogenic refrigerator quick freezing, room
Temperature is slowly melted, and is repeated four times, then is placed in-40 DEG C of pre-freeze 6h；Finally it is dried in vacuum freeze-drying machine, i.e. obtains pig
Circovurus type 2 liposome dilution freeze-dried products.
Embodiment 5: the preparation of porcine circovirus 2 type liposome dilution freeze-dried products
Insect-taking grass polysaccharide 8g, lycopene 8g, ginsenoside Rh2 4g, liposome (embodiment 3 prepare) 10g, sucrose 12g and sweet
Dew alcohol 14g；
After the component of described weight being sufficiently mixed, join vibration in the PBS solution (embodiment 1 prepares) of 1000ml pH 7
Aquation, forms uniform solution；After the filter of 0.22um filters, first it is positioned over-40 DEG C of cryogenic refrigerator quick freezing, room
Temperature is slowly melted, and is repeated four times, then is placed in-40 DEG C of pre-freeze 4h；Finally it is dried in vacuum freeze-drying machine, i.e. obtains pig
Circovurus type 2 liposome dilution freeze-dried products.
Embodiment 6: the preparation of porcine circovirus 2 type liposome dilution freeze-dried products
Insect-taking grass polysaccharide 10g, lycopene 10g, ginsenoside Rh2 5g, liposome (embodiment 3 prepare) 12g, sucrose 15g and
Mannitol 15g；
After the component of described weight being sufficiently mixed, join vibration in the PBS solution (embodiment 1 prepares) of 1000ml pH 7
Aquation, forms uniform solution；After the filter of 0.22um filters, first it is positioned over-40 DEG C of cryogenic refrigerator quick freezing, room
Temperature is slowly melted, and is repeated four times, then is placed in-40 DEG C of pre-freeze 8h；Finally it is dried in vacuum freeze-drying machine, i.e. obtains pig
Circovurus type 2 liposome dilution freeze-dried products.
The application of test example 1 present invention
1 material
1.1 porcine circovirus 2 type liposome dilution freeze-dried products
Prepare by embodiment of the present invention 4-6
1.2 porcine circovirus 2 types, PK15-B1 cell
1.3 5-6 week old Healthy female cleaning grade Balb/c mices, PCV2 ELISA antibody test is negative (≤1:50)
2 methods
The mensuration of 2.1 envelop rates
Take freeze-dried lipidosome and the lipidosome freeze-dried powder of entrapped drug of blank respectively, redissolve in phosphate buffer,
Ultracentrifugation, takes after supernatant dilutes by a certain percentage, measures its absorbance (with reference to veterinary drug allusion quotation), and average each sample is surveyed 3 times, calculates
Its meansigma methods.
2.2 morphologic observations and particle diameter
Liposome dilution lyophilized powder, after PBS redissolves, is observed under transmission electron microscope (× 20000)
2.3 cells are cultivated and connect poison
With the MEM cell nutrient solution containing 10% calf serum, it is placed at 37 DEG C in the CO2 incubator containing 5% and carries out cultivating 20-26h,
Treat that cell grows up to monolayer, discard culture fluid, add EDTA-trypsinization liquid, act on several minutes, pass in 1:2-1:3 ratio, all
Divide 4 groups, often group 3 bottles.Liposome dilution freeze-dried products embodiment of the present invention 4-6 prepared is placed in PH7 phosphate buffer
In redissolve after, respectively 14160723,14160724, the virus of 14,160,725 3 kind of different batches be respectively diluted to 1000
TCID50/ml、1500 TCID50/ml、2000 TCID503 kinds of different concentration of/ml.A group, uses the embodiment of the present invention 4 to prepare
Liposome dilution the PCV-2 of 3 kinds of different batches is diluted inoculation；B group, uses the fat of the embodiment of the present invention 5 preparation
Plastid diluent is diluted inoculation to 3 kinds of different batches PCV-2；C group, the liposome using the embodiment of the present invention 6 preparation is dilute
Release liquid and 3 different batches PCV-2 are diluted inoculation；D group, matched group, use the cell culture fluid of serum-free to 3 different batches
PCV-2 is diluted inoculation, often organizes and is all inoculated in cell monolayer by 5%, puts 37 DEG C of absorption 30min, adds containing 4% calf serum thin
Born of the same parents maintain liquid, in 37 DEG C, 5% CO2 cell culture incubator cultivates 72h, after freeze thawing 2-3 time, carry out receiving malicious.
2.4 TCID50Measure
By four papova liquid of results, 10 times of serial dilutions respectively, take 10-5、10-6、10-7Three dilution factors, it is inoculated in respectively
In the Tissue Culture Plate of PK15-B1 cell monolayer, inoculate by 0.1ml/ hole, be placed in 37 DEG C, the incubator of 5% is cultivated
24h, maintains liquid to carry out changing liquid with MEM, is cultivated for 24h, fixes cell with cold acetone, use indirect immunofluorescence assay
(IFA) measure each dilution factor and contain the hole count of PCV2 positive cell (in green), calculate virus TCID50 according to Karber method.
2.5 immunogenicity determining
The cultivation 72h 14160723 batch four papova liquid mentioned of results 2.3 are qualified through inspection, carry out respectively inactivateing, emulsifying
After be fabricated to vaccine finished product for measuring the immunogenicity of vaccine.5-6 week old Balb/c mice is divided into 4 groups, often group 5, the Ith
The vaccine that group is made after using the PCV-2 inactivation emulsifying that the liposome dilution prepared through the embodiment of the present invention 4 processed is carried out
Immunity；Make after the PCV-2 inactivation emulsifying that IIth group uses the liposome dilution prepared through the embodiment of the present invention 5 to process
Vaccine immunity；After IIIth group is the PCV-2 inactivation emulsifying using the liposome dilution prepared through the embodiment of the present invention 6 to process
The vaccine immunity made；IVth group, vaccine matched group, the vaccine immunity made after inactivateing without liposome-treated；Successively immunity 2
Secondary, every 0.2ml, carry out immunity by identical approach and dosage after two weeks.After head exempts from 5 weeks, start blood sampling, separate serum, measure
PCV-2 ELISA antibody in serum.
3 results
3.1 envelop rate results
According to formula: envelop rate (%)=system is encapsulated and non-encapsulated total dose (A2) in dose (the A1)/system of encapsulating
× 100%, the results are shown in Table 1.
Table 1 entrapment efficiency determination result
3.2 morphologic observations and particle size determination
Liposome dilution freeze-dried products is unilamelar liposome, and granularity rule, for circle, particle diameter is basically identical, the results are shown in Table 2.
Table 2 morphologic observation and particle size determination result
3.3 TCID50Measurement result
Table 3 TCID50Measurement result
3.4 Serum Antibody Detection results
Table 4 TPPA result
4 conclusions
In sum, pig circular ring virus liposome dilution freeze-dried products of the present invention has good envelop rate, and granularity rule,
Particle diameter is basically identical；Can be good at ensureing the stability of semi-finished product inoculum concentration, reduce pig circular ring virus inactivated vaccine semi-finished product
Between batch wise differences；Improve the serum antibody titer of vaccine, strengthen the immunogenicity of vaccine.
Claims (6)
1. a porcine circovirus 2 type liposome dilution freeze-dried products, it is characterised in that every 1000mlPBS solution contains following
Component: Cordyceps polysaccharide 5-10g, lycopene 5-10g, ginsenoside Rh2 1-5g, liposome 6-12g, sucrose 10-15g and sweet
Dew alcohol 10-15g.
2. a kind of porcine circovirus 2 type liposome dilution freeze-dried products as claimed in claim 1, it is characterised in that every
1000mlPBS solution contains following components: Cordyceps polysaccharide 6-8g, lycopene 6-8g, ginsenoside Rh2 2-4g, liposome 8-
10g, sucrose 12-14g and mannitol 12-14g.
3. a kind of porcine circovirus 2 type liposome dilution freeze-dried products as claimed in claim 1 or 2, it is characterised in that institute
The liposome stated is prepared by following steps: weighs soybean lecithin, cholesterol, dissolves with chloroform, and makes it mix all
Even, by this solution at 37 DEG C of rotary evaporations that reduce pressure, remove organic solvent, form immobilized artificial membrane；The PBS of pH value 7.0 is added in film
Solution, water-bath rotates aquation, and gained suspension carries out supersound process, is prepared as liposome after being finally dehydrated with refiner.
4. a kind of porcine circovirus 2 type liposome dilution freeze-dried products as claimed in claim 1 or 2, it is characterised in that institute
The PBS solution stated is prepared by following steps: take sodium chloride 6-10g, potassium chloride 0.05-0.5g, disodium hydrogen phosphate 1-1.2g, phosphorus
Acid dihydride potassium 0.05-0.5g, calcium chloride 0.05-0.2g, magnesium chloride 0.05-0.2g containing 6 water of crystallization；Above each composition is mixed
After conjunction, it is dissolved in 1000ml distilled water, through 116 DEG C, autoclaving 30 minutes and get final product.
The preparation method of a kind of porcine circovirus 2 type liposome dilution freeze-dried products the most as claimed in claim 1 or 2, its
It is characterised by comprising the following steps: after the component of described weight being sufficiently mixed, joins in 1000ml pH 7 PBS solution and shake
Swing aquation, form uniform solution；After the filter of 0.22um filters, first it is positioned over-40 DEG C of cryogenic refrigerator quick freezing,
Room temperature is slowly melted, and is repeated four times, then is placed in-40 DEG C of pre-freeze 4-8h；Finally it is dried in vacuum freeze-drying machine, to obtain final product
To porcine circovirus 2 type liposome dilution freeze-dried products.
The preparation method of a kind of porcine circovirus 2 type liposome dilution freeze-dried products the most as claimed in claim 1 or 2, its
The PBS solution being characterised by described is prepared by following steps: take sodium chloride 6-10g, potassium chloride 0.05-0.5g, phosphoric acid hydrogen two
Sodium 1-1.2g, potassium dihydrogen phosphate 0.05-0.5g, calcium chloride 0.05-0.2g, magnesium chloride 0.05-0.2g containing 6 water of crystallization；Will
After the mixing of the most each composition, it is dissolved in 1000ml distilled water, through 116 DEG C, autoclaving 30 minutes and get final product.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106821966A (en) * | 2016-11-29 | 2017-06-13 | 浙江美保龙生物技术有限公司 | A kind of live vaccines of hog cholera dilution |
| CN107475205A (en) * | 2017-08-31 | 2017-12-15 | 浙江美保龙生物技术有限公司 | A kind of pig circular ring virus separation method |
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| WO2012006378A1 (en) * | 2010-07-06 | 2012-01-12 | Novartis Ag | Liposomes with lipids having an advantageous pka- value for rna delivery |
| CN105505887A (en) * | 2015-12-29 | 2016-04-20 | 浙江美保龙生物技术有限公司 | Porcine reproductive and respiratory syndrome virus diluent and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2012006378A1 (en) * | 2010-07-06 | 2012-01-12 | Novartis Ag | Liposomes with lipids having an advantageous pka- value for rna delivery |
| CN105505887A (en) * | 2015-12-29 | 2016-04-20 | 浙江美保龙生物技术有限公司 | Porcine reproductive and respiratory syndrome virus diluent and preparation method thereof |
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| 崔尚金: "《猪圆环病毒病及其防治》", 30 June 2007 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106821966A (en) * | 2016-11-29 | 2017-06-13 | 浙江美保龙生物技术有限公司 | A kind of live vaccines of hog cholera dilution |
| CN107475205A (en) * | 2017-08-31 | 2017-12-15 | 浙江美保龙生物技术有限公司 | A kind of pig circular ring virus separation method |
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Application publication date: 20161116 |