CN104998258A - Method for preparing propolis inactivated vaccine applicable to rabbit viral haemorrhagic syndrome - Google Patents
Method for preparing propolis inactivated vaccine applicable to rabbit viral haemorrhagic syndrome Download PDFInfo
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- CN104998258A CN104998258A CN201510478394.6A CN201510478394A CN104998258A CN 104998258 A CN104998258 A CN 104998258A CN 201510478394 A CN201510478394 A CN 201510478394A CN 104998258 A CN104998258 A CN 104998258A
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Abstract
The invention provides a method for preparing propolis inactivated vaccine applicable to the rabbit viral haemorrhagic syndrome. The method comprises the steps of firstly, inoculating rabbit viral haemorrhagic syndrome viruses, namely CVCC AV272 bacterial strains, into rabbits, collecting livers of rabbits which die within 24-96 hours in a sterile mode, and getting rid of connective tissues on the livers; then pounding the livers into pieces, conducting the grinding and filtration, preparing emulsion with the liver pieces, conducting the inactivation on the emulsion, configuring high-quality propolis and astragalus polysaccharide for being mixed with the emulsion, and obtaining the inactivated vaccine. The inactivated vaccine obtained through the method is little in side effect, the immune effect can reach over 90% and is far above 75% which is the existing domestic standard, and the inactivated vaccine has the advantages of being good in safety, long in preservation time, green, environment-friendly and the like.
Description
Technical field
The present invention relates to a kind of preparation method being applicable to rabbit hemorrhagic disease inactivated propolis vaccines, belong to field of biological pharmacy.
Background of invention
Rabbit hemorrhagic disease, also known as rabbit pestilence, is that the one that caused by rabbit hemorrhagic disease virus is acute, high degree in contact sexually transmitted disease, with respiratory system hemorrhage, hepatic necrosis, substantial viscera edema, blood stasis, hemorrhage and high mortality for characteristic.Primary disease is in Late Cambrian in 1984 in Jiangsu Province of China, and rapid spread, to the whole nation, all has generation all over the world afterwards.The normal Outbreak of primary disease, sickness rate and mortality rate high, to rabbit industry cultivation bring significant damage, be one of Infectious Diseases of current Intensive livestock farm.Rabbit hemorrhagic disease virus norwalk virus.This virus only can " O " type erythrocyte of coagulation people, and the erythrocyte of not coagulation horse, cattle, sheep, dog, pig, chicken, duck, rabbit, rat, Cavia porcellus and hamster, this coagulation Property comparison is stablized, within the specific limits not by the impact of temperature, pH value, organic solvent and some inorganic ions, but can be suppressed by RHDV antiserum specificity.This virus only has One serotype.This virus exists in all histoorgans of sick rabbit, body fluid, secretions and Excreta, especially the highest with content in liver,spleen,kidney, lung and blood.At present this virus still can not be bred in various primary or passage cell.And this virus can resist multiple extreme environment, comprise low pH, high temperature and multigelation, can keep stable in natural environment.
1984, teaching and research group of Agricultural University Of Nanjing proposed using-system and goes out and can add aluminium hydroxide or the urgent prophylactic immunization of incomplete Freund's adjuvant to control the method for epidemic situation by Seedling, and have received better effects.In the same year, Jiangsu Agricultural College utilizes rabbit pestilence West China to be strong malicious HC-84 strain inoculation susceptible rabbit, gets the liver,spleen,kidney of the rabbit that dies of illness as seedling material, smash to pieces aseptic for material, filter after diluting with normal saline 1:10, add formalin solution fully mix, go out can, obtained " rabbit pestilence " vaccine.This vaccine immunity phase is 6 months.Preferably preventive measure takes above-mentioned internal organs inactivated vaccine to carry out immunity exactly at present.
Propolis becomes rapidly the Zhi Xin army in current adjuvant exploitation upsurge with the advantage function of the aspects such as its immune defence, immunological homeostasis, immune surveillance.And it declares publicly it in the application of poultry vaccines arts and there is huge market prospect.Propolis contains a large amount of flavonoid (flavone, flavonol and flavanone), aromatic acid, aliphatic acid and ester thereof, and terpenoid substance, it is a kind of natural materials with broad-spectrum biological activity and immunological enhancement, there is antibacterial, antiviral, antitumor, antiinflammatory, enhancing human body immunity function and promote the effects such as tissue regeneration, these characteristics make propolis can quick, efficient, lasting enhancing immune effect of vaccine as adjuvant, for vaccine provides green, environmental protection, safe adjuvant efficacy.Because propolis has good antiseptic and inhibiting bacteria function effect, in the process preparing rabbit hemorrhagic disease inactivated vaccine, strict operations, does not add the antiseptic such as thimerosal in vaccine, reduces the introducing of allogenic material in vaccine, increases the safety of vaccine.The special ultrastructure of Proplis_adjuvant vaccine makes its Stability Analysis of Structures, and heat-resistant is cold, in advance the generation of neutralizing antibody, extends the series of advantages such as immune period.The people such as Mr. Zhang are at the indoor propolis adjuvant of the experiment inactivated vaccine prepared and the inactivated vaccine prepared with other adjuvants immunizing rabbit simultaneously, and carried out challenge test, result shows, the vaccine rabbit adding propolis is all strong alive, then group is all dead, embodies good physiological action.
To sum up, the object of the invention is to be separated and cultivate the stable strong-poison strain of a kind of virulence, and add high-quality propolis and mix with it to prepare inactivated vaccine.
Summary of the invention:
The invention provides a kind of rabbit hemorrhagic disease inactivated propolis vaccines, this vaccine side effect is little, and immune effect can reach more than 90%, has good immunological safety.
For achieving the above object, the present invention is by the following technical solutions:
A kind of rabbit hemorrhagic disease inactivated vaccine, comprise antigen and adjuvant, described antigen is rabbit hemorrhagic disease CVCCAV272 bacterial strain, and described adjuvant is propolis alcohol leaching liquid, when joining Seedling, in every milliliter of vaccine, propolis dry matter content is 8-15mg, also comprises the astragalus polysaccharides of 1-3mg.
According to the present invention, not containing thimerosal in described vaccine.
A preparation method for rabbit poison hemorrhage inactivated propolis vaccines as described in above-mentioned any one, comprises the following steps:
1. the selection-breeding of rabbit hemorrhagic disease strong-poison strain CVCC AV272 bacterial strain;
(1) aseptic collection is exempted from viral hemorrhagic disease and to be died of illness rabbit liver, and put in aseptic glass grinding device, limit is ground, and limit 10% adds physiological saline solution and is prepared into homogenate, with No. 5 sieved filters in mass ratio.After freeze thawing 3 times, add the centrifugal 20min of mycillin each 1000IU/mL, 3000r/min, Aspirate supernatant, is distributed into aseptic vial, preserves in cryogenic refrigerator (-20 DEG C).
(2) viral suspension physiological saline solution continuous doubling dilution on 96 holes " V " template is organized by the Hepar Leporis seu Oryctolagi of preparation to carry out the mensuration of HA-HI test, using the most high dilution of erythrocyte 100% coagulation as judgement terminal
(3) each strain of rabbit hemorrhagic disease will be separated, diluting respectively with normal saline is the viral suspension of 4 HAUs, each virus isolated strain fully mixes with the anti-rabbit viral hemorrhagic disease virus-specific serum of equivalent respectively, 37 DEG C of effect 30min, period jolting 2 ~ 3 times, establishes positive controls and negative control group simultaneously.After neutralization, above each group is respectively added on row 96 hole " V " template, every hole 25 μ L, then add 25 μ L people " O " type erythrocyte in every Kong Zhongzai, fully the rear 37 DEG C of effect 30min of vibration, observed result.
(4) each strain is done 10 respectively
-1, 10
-2, 10
-3, 10
-4, 10
-5doubly dilution, inoculates the healthy susceptible rabbit 5 of 50 ~ 55 ages in days respectively, and only, Continuous Observation 10 days, records challenge test rabbit invasion and death condition to cervical region subcutaneous injection 1.0mL/.
(5) choose virulence comparatively virulent strain in rabbit hemorrhagic disease virus separated strain, prepare inactivated vaccine and carry out Immunity identification.Get the healthy susceptible rabbit 10 of 50 ~ 55 ages in days; cervical region subcutaneous injection inactivated vaccine 1.0mL/ only; after 14 days; together with the contrast rabbit 5 that condition is identical; the rabbit hemorrhagic disease virus separated strain virus liquid 1.0mL/ that each cervical region subcutaneous injection 1:10 dilutes only; observe 10 days, record matched group and immune group test rabbit immunoprotection and morbidity death condition.
The pure strain CVCC AV272 bacterial strain that HA hemagglutinative titer is high, rabbit hemorrhagic disease virus virulence is the strongest and immunogenicity is good is selected, the deactivation tissue vaccine prepared with this strain according to above-mentioned testing sieve.
2. by the strain CVCC AV272 inoculation susceptible rabbit of selection-breeding, take liver organization, add preferred propolis alcohol leaching liquid, conventionally carry out the preparation of inactivated vaccine, concrete steps are as follows: contain rabbit hemorrhagic disease pathological tissue >=0.05g by every milliliter of vaccine, propolis dry matter content is 8 ~ 15mg, the standard of astragalus polysaccharides 1-3mg, according to the total amount of prewired Seedling, the concentration of rabbit hemorrhagic disease pathological tissue amount and propolis alcohol leaching liquid, calculate required rabbit hemorrhagic disease pathological tissue suspension respectively, the amount of propolis alcohol leaching liquid and water for injection.First being put by the water for injection of aequum joins in Seedling emulsion tank, then rabbit hemorrhagic disease pathological tissue suspension low speed (2000rpm) adding aequum stirs 5 minutes, then slowly add propolis alcohol leaching liquid, 2000rpm stirs 15 ~ 20 minutes, makes its abundant mixing and emulsifying.
Rabbit hemorrhagic disease virus CVCC AV272 bacterial strain in the present invention is the natural sense contamination that inventor is separated in the rabbit that dies of illness of the doubtful rabbit hemorrhagic disease of plant of district in all parts of the country, identify from HA-HI test, specificity, virulence, immunogenicity, pure property aspect, the bacterial strain that the virulence filtered out is strong, immunogenicity good, HA-HI test is high.
Preferably, containing 0.05g rabbit hemorrhagic disease hepatic pathology tissue (HA >=1:256) in every milliliter of rabbit hemorrhagic disease inactivated vaccine, propolis dry 8-15mg, astragalus polysaccharides 1-3mg.
Preferably, when manufacturing vaccine, rabbit hemorrhagic disease virus CVCC AV272 bacterial strain normal saline is done 10 times of dilutions, the healthy susceptible rabbit of inoculation 50-55 age in days, every cervical region subcutaneous injection 1.0mL/ only, after aseptic collection inoculation within 12-72 hour the death time rabbit in 1 hour the liver organization with typical pathologic change, reject the connective tissue around liver.
Preferably, after aseptic collection inoculation within 12-72 hour the death time rabbit in 1 hour the liver with typical pathologic change, and reject the connective tissue around liver.
Preferably, rabbit is selected from the rabbit poison hemorrhage antibody of 50-55 age in days is negative rabbit kind.
Preferably, the liver organization sterile saline of the dead rabbit of rejecting connective tissue is smashed to pieces, be made into tissue suspension after grinding, filtration, the ratio of rabbit hemorrhagic disease pathological tissue and sterile saline is 1:9 (W/V), preserves in cryogenic refrigerator (-20 DEG C).
Preferably, add the formaldehyde by tissue suspension weighing scale 0.4%, deactivation 24-96 hour at 20-50 DEG C, stirred once every 2-4 hour time more preferably 37 DEG C of deactivations 24 hours and in deactivation.
Preferably, propolis alcohol leaching liquid, preferred propolis is stored at low temperatures, first propolis is put-15 DEG C of freezing 24-48 hour during use, then grind with freezing crusher and sieve, add 95% ethanol in the ratio of 1:4 (W/V), at 37 DEG C of lixiviate 48-72 hour, cooling, filtration, obtain pure propolis alcohol leaching liquid (transparent maroon solution), the propolis content of leachate must be greater than 50%.
Beneficial effect: the inactivated vaccine side effect that the inventive method obtains is little, and immune effect can reach more than 90%, far above current domestic current standard 75%, and have safety good, preserve the advantages such as long-term and environmental protection.
Detailed description of the invention:
Embodiment 1
The Secondary Culture of rabbit poison hemorrhage CVCC AV272 strain
Rabbit hemorrhagic disease virus CVCC AV272 bacterial strain normal saline after preservation is done 10 times of dilutions, is inoculated in the healthy susceptible of 50-55 age in days and negative antibody rabbit.Every subcutaneous injection 1mL, the virus titer in this rabbit can remain on HA >=more than 1:1024.Rabbit kind dead in 12-72 hour after aseptic collection inoculation, the rabbit that the aseptic collection death time is no more than 1 hour and there is the liver organization of typical pathologic change, and the connective tissue of rejecting on it is as seed culture of viruses.
Adopt the strain CVCC AV272 inoculation in biography 4 ~ 10 generation, the liver of the dead rabbit of rejecting connective tissue is added normal saline and smash to pieces, grind and filter preparation, the ratio of hepatic pathology tissue and sterile saline is 1:9 (W/V).Add the centrifugal 20min of mycillin each 1000IU/mL, 3000r/min, Aspirate supernatant, is distributed into aseptic vial, preserves in cryogenic refrigerator (-20 DEG C).
Embodiment 2:
The preparation of rabbit poison hemorrhage tissue suspension
Rabbit hemorrhagic disease virus CVCC AV272 bacterial strain normal saline after preservation is done 10 times of dilutions, is inoculated in the healthy susceptible of 50-55 age in days and negative antibody rabbit.Every subcutaneous injection 1mL, the virus titer in this rabbit can remain on HA >=more than 1:1024.Rabbit kind dead in 12-72 hour after aseptic collection inoculation, the rabbit that the aseptic collection death time is no more than 1 hour and there is the liver organization of typical pathologic change, and the connective tissue of rejecting on it is as seed culture of viruses.
Adopt the strain CVCC AV272 inoculation in biography 4 ~ 10 generation, the liver of the dead rabbit of rejecting connective tissue is added normal saline and smash to pieces, grind and filter preparation, the ratio of hepatic pathology tissue and sterile saline is 1:9 (W/V).Add formalin according to 0.4% of tissue suspension total amount and carry out deactivation, deactivation condition is: deactivation 24 hours at 37 DEG C, and stirs once every 2-4 hour, adds high-quality propolis alcohol leaching liquid, mixing and emulsifying after deactivation.Steriling test, deactivation inspection and HA-HI test inspection is carried out after deactivation terminates.
Embodiment 3:
Rabbit hemorrhagic disease pathological tissue >=0.05g is contained by every milliliter of vaccine, propolis dry matter content is 8 ~ 15mg, astragalus polysaccharides 1-3mg, according to the total amount (500,000 mL) of prewired Seedling, the concentration (32g/mL) of rabbit hemorrhagic disease pathological tissue amount (0.1g/ml) and propolis alcohol leaching liquid, add required rabbit hemorrhagic disease pathological tissue suspension (250,000 mL) in proportion, the water for injection (250,000 mL) of aequum injects and joins Seedling emulsion tank by the amount of propolis alcohol leaching liquid (125mL) and water for injection, after add aequum rabbit hemorrhagic disease pathological tissue suspension under rotating speed is 2000rpm/min condition, stir 5-10min, then under stirring, propolis alcohol leaching liquid and astragalus polysaccharides is slowly added, continue to stir 15-20 minute, make its abundant mixing and emulsifying, subpackage.
Although above-mentioned to invention has been detailed description; but be not limited thereto; those skilled in the art can principle according to the present invention modify, and therefore, all various amendments carried out according to principle of the present invention all should be understood to fall into protection scope of the present invention.
Claims (9)
1. a rabbit hemorrhagic disease inactivated propolis vaccines, comprise antigen and adjuvant, it is characterized in that, described antigen is rabbit hemorrhagic disease CVCC AV272 bacterial strain, described adjuvant is propolis alcohol leaching liquid, when joining Seedling, in every milliliter of vaccine, propolis dry matter content is 8-15mg, also comprises the astragalus polysaccharides of 1-3mg.
2. as above-mentioned rabbit hemorrhagic disease inactivated propolis vaccines according to claim 1, it is characterized in that, not containing thimerosal in described vaccine.
3. as above-mentioned rabbit hemorrhagic disease inactivated propolis vaccines according to claim 1, it is characterized in that, the preparation method of described propolis alcohol leaching liquid is: to be first placed in by propolis under-15 DEG C of conditions freezing 24 ~ 48 hours, grind with freezing crusher again and sieve, 95% ethanol is added in the ratio of 1:4 (W/V), 37 DEG C of lixiviates 48 ~ 72 hours, cooling, filtration, obtain the propolis alcohol leaching liquid of pure transparent maroon, in leachate, propolis content is greater than 50%.
4. the preparation method of the rabbit hemorrhagic disease inactivated propolis vaccines as described in any one of claims 1 to 3, it is characterized in that, make Emulsion with sick Hepar Leporis seu Oryctolagi tissue, inoculation susceptible rabbit, Isolation and screening goes out strain CVCC AV272 bacterial strain, and the method comprises the following steps:
(1) cultivated by the continuous passage of susceptible rabbit with toxic liver organization suspension, filter out strong-poison strain CVCC AV272 bacterial strain;
(2) using CVCC AV272 bacterial strain as kind of a poison, inoculation susceptible rabbit, the liver of the dead rabbit that falls ill within 12 ~ 72 hours after gathering inoculation, and reject the connective tissue of liver;
(3) by reject the liver of connective tissue smash to pieces, grind, filter after be made into tissue suspension, and carry out deactivation to this tissue suspension, tissue suspension HA >=1:256 after deactivation, for joining Seedling;
(4) the deactivation Emulsion that step (3) obtains is mixed with adjuvant, subpackage after sterilizing.
5. the preparation method of rabbit hemorrhagic disease inactivated propolis vaccines as claimed in claim 4, it is characterized in that, when manufacturing vaccine, inoculation susceptible rabbit material is ground in sterile vessel, grinding limit, limit 10% adds physiological saline solution and is prepared into homogenate, with No. 5 sieved filters in mass ratio; After freeze thawing 3 times, add the centrifugal 40min of mycillin each 1000IU/mL, 6000r/min, Aspirate supernatant, is distributed into aseptic vial, preserves in cryogenic refrigerator (-20 DEG C).
6. the preparation method of rabbit hemorrhagic disease inactivated propolis vaccines as claimed in claim 4, it is characterized in that, described step (2) is: rabbit hemorrhagic disease strain CVCC AV272 bacterial strain normal saline is done 10 times of dilutions, inoculation 50 ~ 55 age in days Shandong each department plant susceptible rabbits, every cervical region subcutaneous injection 1mL, aseptic collection inoculation after susceptible rabbit, gather inoculation after within 12 ~ 72 hours the death time rabbit that dies of illness in 1 hour have typical pathologic change liver organization.
7. the preparation method of rabbit hemorrhagic disease inactivated propolis vaccines as claimed in claim 4, it is characterized in that, add the formaldehyde by Emulsion weighing scale 0.1% ~ 0.6% in step (3), deactivation 24 ~ 96 hours at 37 DEG C, and stirred once every 2 ~ 4 hours when deactivation.
8. the preparation method of rabbit hemorrhagic disease inactivated propolis vaccines as claimed in claim 4, it is characterized in that, wherein step (4) deactivation Emulsion and adjuvant carry out aseptic emulsifying in proportion, and described adjuvant comprises high-quality propolis alcohol leaching liquid, also comprises astragalus polysaccharides.
9. the preparation method of rabbit hemorrhagic disease inactivated propolis vaccines according to claim 4, it is characterized in that, in the rabbit hemorrhagic disease inactivated vaccine that step (4) obtains, every milliliter contains 0.05g rabbit hemorrhagic disease hepatic pathology tissue, high-quality propolis 8 ~ 15mg, also comprises 1 ~ 3mg astragalus polysaccharides.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111686245A (en) * | 2020-06-29 | 2020-09-22 | 江苏省农业科学院 | Inactivated vaccine for rabbit hemorrhagic disease virus type 2 and preparation method thereof |
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| CN1043739A (en) * | 1988-12-28 | 1990-07-11 | 河北省畜牧兽医研究所 | The seed selection of rabbit hemorrhagic disease strong-poison strain and vaccine development method |
| CN102755644A (en) * | 2012-07-31 | 2012-10-31 | 郑州后羿制药有限公司 | Rabbit haemorrhagic disease tissue inactivated vaccine and preparation method thereof |
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- 2015-08-06 CN CN201510478394.6A patent/CN104998258A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1043739A (en) * | 1988-12-28 | 1990-07-11 | 河北省畜牧兽医研究所 | The seed selection of rabbit hemorrhagic disease strong-poison strain and vaccine development method |
| CN102755644A (en) * | 2012-07-31 | 2012-10-31 | 郑州后羿制药有限公司 | Rabbit haemorrhagic disease tissue inactivated vaccine and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111686245A (en) * | 2020-06-29 | 2020-09-22 | 江苏省农业科学院 | Inactivated vaccine for rabbit hemorrhagic disease virus type 2 and preparation method thereof |
| CN111686245B (en) * | 2020-06-29 | 2023-03-14 | 江苏省农业科学院 | Inactivated vaccine for rabbit hemorrhagic disease virus type 2 and preparation method thereof |
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