CN105214082A - Newcastle disease, bird flu bivalent inactivated vaccine and preparation method thereof - Google Patents
Newcastle disease, bird flu bivalent inactivated vaccine and preparation method thereof Download PDFInfo
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- CN105214082A CN105214082A CN201410317579.4A CN201410317579A CN105214082A CN 105214082 A CN105214082 A CN 105214082A CN 201410317579 A CN201410317579 A CN 201410317579A CN 105214082 A CN105214082 A CN 105214082A
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Abstract
The invention provides a kind of newcastle disease, bird flu bivalent inactivated vaccine, comprise NDV La Sota Strain inactivation antigen, H9N2 subtype avian influenza virus WF strain inactivation antigen, water-soluble vaccines adjuvant Gel01ST and pH6.8-7.2PBS solution.The present invention also provides the preparation method of described inactivated vaccine, comprises the preparation of NDV La Sota Strain inactivation antigen and H9N2 subtype avian influenza virus WF strain inactivation antigen, purification, and the preparation of bivalent inactivated vaccine.Bivalent inactivated vaccine product provided by the invention is water soluble preparation, can produce immunity fast after injection, effectively can prevent newcastle disease and H9N2 subtype avian influenza simultaneously.
Description
Technical field
The present invention relates to vaccine preparation field, specifically, relate to newcastle disease, bird flu bivalent inactivated vaccine and preparation method thereof.
Background technology
Newcastle disease and H9N2 subtype avian influenza are two kinds of main viral infectious of chicken, and various age in days chicken can be caused to fall ill.The respiratory symptom that morbidity chicken group the lighter occurrence degree differs, cause fertility performance to decline, severe one likely causes whole chicken group dead, causes serious financial consequences, causes bio-safety crisis.
Vaccine prevention is the effective ways of prevention and control two kinds of epidemic diseases, and the bivalent inactivated vaccine containing newcastle disease and H9N2 subtype avian influenza two kinds of inactivation antigens, can pass through immunity inoculation, prevents two kinds of epidemic diseases simultaneously.
At present; newcastle disease, bird flu bivalent inactivated vaccine all adopt mineral oil adjuvant to prepare; in preparation, inactivation antigen is wrapped up by mineral oil; after vaccine immunity injection; antigenic component rate of release in body is slow; vaccinated flock can not produce protective immunity antibody in time, newcastle disease and the H9N2 subtype avian influenza Blank immunization phase long, easily cause two kinds of epidemic diseases to occur.
Do not see the newcastle disease, the bird flu bivalent inactivated vaccine water soluble preparation that adopt Gel01ST adjuvant to prepare at present.
Summary of the invention
The object of this invention is to provide and a kind ofly produce immunity fast after injection, effectively can prevent the bivalent inactivated vaccine of newcastle disease and H9N2 subtype avian influenza.
Another object of the present invention is to provide the preparation method of above-mentioned bivalent inactivated vaccine.
In order to realize the object of the invention, a kind of newcastle disease of the present invention, bird flu bivalent inactivated vaccine, comprise newcastle disease LaSota strain inactivation antigen, H9N2 subtype avian influenza WF strain inactivation antigen, water-soluble vaccines adjuvant Gel01ST (Montanide
tM) and pH6.8-7.2PBS solution.
Wherein, the preparation method of described NDV La Sota Strain inactivation antigen is: by newcastle disease LaSota strain virus seed culture of viruses inoculation 10-11 day instar chicken embryo, dosage of inoculation is 1000EID
50, cultivate 96-120 hour for 36-37 DEG C, results Embryo Gallus domesticus liquid, adds formalin wherein, makes formaldehyde final concentration reach 0.2%, and in 37 DEG C of deactivations 24 hours after mixing, through steriling test and deactivation after the assay was approved, 2-8 DEG C saves backup.
The preparation method of described H9N2 subtype avian influenza virus WF strain inactivation antigen is: by H9N2 subtype avian influenza WF strain virus seed culture of viruses inoculation 10-11 day instar chicken embryo, dosage of inoculation is 1000EID
50, cultivate 72-96 hour for 36-37 DEG C, results Embryo Gallus domesticus liquid, adds formalin wherein, makes formaldehyde final concentration reach 0.2%, and in 37 DEG C of deactivations 24 hours after mixing, through steriling test and deactivation after the assay was approved, 2-8 DEG C saves backup.
In described inactivated vaccine, each component by weight: Avian pneumo-encephalitis virus LaSota strain inactivation antigen 5-10 part, H9N2 subtype avian influenza virus WF strain inactivation antigen 5-10 part, water-soluble vaccines adjuvant Gel01ST5-20 part, 0.01mol/LPBS solution 60-85 part.
The present invention also provides the preparation method of above-mentioned inactivated vaccine, comprises the following steps:
1) preparation of NDV La Sota Strain inactivation antigen and H9N2 subtype avian influenza WF strain inactivation antigen:
By newcastle disease LaSota strain virus seed culture of viruses inoculation 10-11 day instar chicken embryo, dosage of inoculation is 1000EID
50, cultivate 96-120 hour for 36-37 DEG C, results Embryo Gallus domesticus liquid, add formalin wherein, make formaldehyde final concentration reach 0.2%, in 37 DEG C of deactivations 24 hours after mixing, through steriling test and deactivation after the assay was approved, obtain NDV La Sota Strain inactivation antigen, 2-8 DEG C saves backup;
By H9N2 subtype avian influenza WF strain virus seed culture of viruses inoculation 10-11 day instar chicken embryo, every embryonic breeding kind 0.2 milliliter, dosage of inoculation is 1000EID
50, cultivate 72-96 hour for 36-37 DEG C, results Embryo Gallus domesticus liquid, add formalin wherein, make formaldehyde final concentration reach 0.2%, in 37 DEG C of deactivations 24 hours after mixing, through steriling test and deactivation after the assay was approved, obtain H9N2 subtype avian influenza WF strain inactivation antigen, 2-8 DEG C saves backup;
2) adopt chemical precipitation method or hyperfiltration process respectively to step 1) in the inactivation antigen of preparation carry out purification;
3) the NDV La Sota Strain inactivation antigen after purification and H9N2 subtype avian influenza virus WF strain inactivation antigen, vaccine adjuvant Gel01ST and PBS solution are mixed in proportion, 200-500 rev/min is stirred 20-30 minute, subpackage.
Aforesaid method, step 2) middle employing chemical precipitation method purification, be specially: the sodium chloride adding PEG6000 and 2-6% of antigen weight 4-10% in inactivation antigen respectively, after stirring and dissolving, room temperature leaves standstill 2-4 hour, the centrifugal 30-60 minute of 5000-10000g, abandon supernatant, in precipitation, add PBS solution suspension precipitation according to 1/4 of purification pro-antigen volume, obtain the inactivation antigen of purification, 2-8 DEG C saves backup.
Aforesaid method, step 2) middle employing hyperfiltration process purification, be specially: the molecular cut off specification selected is that the film bag of 300-1000KD carries out cross-flow ultrafiltration to inactivation antigen, in ultra-filtration process, add PBS solution to equilibrium in inactivation antigen, make antigen volume keep constant; When filtrate volume is the 2-3 times of antigen volume, stops adding PBS solution, continue ultrafiltration, until antigen volume reach pure precursor long-pending 1/4.
Aforesaid method, after purification the volume of inactivation antigen be pure precursor long-pending 1/4.
Novel chicken newcastle provided by the invention, bird flu bivalent inactivated vaccine are water soluble preparation, can produce immunity fast after injection, effectively can prevent newcastle disease and H9N2 subtype avian influenza simultaneously.
Detailed description of the invention
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art.The NDV La Sota Strain related in embodiment is purchased from China Veterinery Drug Inspection Office.H9N2 subtype avian influenza virus WF strain is separated from laying hen chicken group for 2012, is identified and preserve by Qianyuanhao Biological Co., Ltd..Other is raw materials used is commercial goods.
Embodiment 1 newcastle disease, bird flu bivalent inactivated vaccine and preparation method thereof
Comprise the following steps:
1, the preparation of NDV La Sota Strain inactivation antigen: by newcastle disease LaSota strain virus seed culture of viruses inoculation 10-11 day instar chicken embryo, dosage of inoculation is 1000EID
50, cultivate 96-120 hour for 36-37 DEG C, results Embryo Gallus domesticus liquid, adds formalin wherein, makes formaldehyde final concentration reach 0.2%, and in 37 DEG C of deactivations 24 hours after mixing, through steriling test and deactivation after the assay was approved, 2-8 DEG C saves backup.
2, the preparation of H9N2 subtype avian influenza virus WF strain inactivation antigen: by H9N2 subtype avian influenza WF strain virus seed culture of viruses inoculation 10-11 day instar chicken embryo, dosage of inoculation is 1000EID
50, cultivate 72-96 hour for 36-37 DEG C, results Embryo Gallus domesticus liquid, adds formalin wherein, makes formaldehyde final concentration reach 0.2%, and in 37 DEG C of deactivations 24 hours after mixing, through steriling test and deactivation after the assay was approved, 2-8 DEG C saves backup.
3, chemical precipitation method or hyperfiltration process is adopted to carry out purification to the inactivation antigen of above-mentioned preparation respectively.
Adopt chemical precipitation method purification, be specially: the sodium chloride adding PEG6000 and 2-6% of antigen weight 4-10% in inactivation antigen respectively, after stirring and dissolving, room temperature leaves standstill 2-4 hour, the centrifugal 30-60 minute of 5000-10000g, abandons supernatant, adds PBS solution suspension precipitation according to 1/4 of purification pro-antigen volume in precipitation, obtain the inactivation antigen of purification, 2-8 DEG C saves backup.
Adopt hyperfiltration process purification, be specially: the molecular cut off specification selected is that the film bag of 1000KD carries out cross-flow ultrafiltration to inactivation antigen, in ultra-filtration process, add PBS solution to equilibrium in inactivation antigen, make antigen volume keep constant; When filtrate volume is the 2-3 times of antigen volume, stops adding PBS solution, continue ultrafiltration, until antigen volume reach pure precursor long-pending 1/4.
After purification the volume of inactivation antigen be pure precursor long-pending 1/4.
4, by the NDV La Sota Strain inactivation antigen after purification and H9N2 subtype avian influenza virus WF strain inactivation antigen, PBS solution (0.01mol/L, pH6.8-7.2) and vaccine adjuvant Montanide
tMgel01ST presses the weight ratio mixing of 1:1:7:1, and 200 revs/min are stirred 20 minutes, and namely subpackage obtains newcastle disease, bird flu bivalent inactivated vaccine finished product.
The application of embodiment 2 newcastle disease, bird flu bivalent inactivated vaccine
By the newcastle disease of preparation in embodiment 1, bird flu bivalent inactivated vaccine according to dose immunization 10 28 age in days SPF chickens of every chicken 20 microlitre, respectively at 7,14,28 days blood sampling separation of serum after immunity, detect newcastle disease HI antibody in serum, calculate HI antibody meansigma methods.Immunity 7 days HI antibody meansigma methodss reach 1:6.96; Within 14 days, reach 1:18.38; Within 21 days, reach 1:90.51.Duration of test, test chicken does not observe immune untoward reaction.Seizure test chicken is cutd open in experiment end, does not observe any pathological change.
The application of embodiment 3 newcastle disease, bird flu bivalent inactivated vaccine
By the newcastle disease of preparation in embodiment 1, bird flu bivalent inactivated vaccine according to dose immunization 10 28 age in days SPF chickens of every chicken 0.3 milliliter, respectively at 7,14,28 days blood sampling separation of serum after immunity, detect newcastle disease HI antibody in serum, calculate HI antibody meansigma methods.Immunity 7 days HI antibody meansigma methodss reach 1:11.31; Within 14 days, reach 1:157.59; Within 21 days, reach 1:3565.78.Duration of test, test chicken does not observe immune untoward reaction.Seizure test chicken is cutd open in experiment end, does not observe any pathological change.
The application of comparative example 1 newcastle disease, bird flu bigeminy mineral oil adjuvant inactivated vaccine
By commercially available newcastle disease, bird flu bigeminy mineral oil adjuvant inactivated vaccine according to dose immunization 10 28 age in days SPF chickens of every chicken 20 microlitre, respectively at 7,14,28 days blood sampling separation of serum after immunity, detect newcastle disease HI antibody in serum, calculate HI antibody meansigma methods.Immunity 7 days HI antibody meansigma methodss reach 1:2.64; Within 14 days, reach 1:7.46; Within 21 days, reach 1:27.86.Duration of test, test chicken does not observe immune untoward reaction.Seizure test chicken is cutd open in experiment end, does not observe any pathological change.
The application of comparative example 2 newcastle disease, bird flu bigeminy mineral oil adjuvant inactivated vaccine
By commercially available newcastle disease, bird flu bigeminy mineral oil adjuvant inactivated vaccine according to dose immunization 10 28 age in days SPF chickens of every chicken 0.3 milliliter, respectively at 7,14,28 days blood sampling separation of serum after immunity, detect newcastle disease HI antibody in serum, calculate HI antibody meansigma methods.Immunity 7 days HI antibody meansigma methodss reach 1:3.48; Within 14 days, reach 1:13.93; Within 21 days, reach 1:294.07.Duration of test, test chicken does not observe immune untoward reaction.Seizure test chicken is cutd open in experiment end, does not observe any pathological change.
By by embodiment 2 and 3, compare with comparative example 1 and 2, result shows, the newcastle disease prepared according to the inventive method, bird flu bivalent inactivated vaccine have good safety and immunogenicity.Compared with commercially available newcastle disease, bird flu bigeminy mineral oil adjuvant inactivated vaccine, this vaccine product can make immune chicken produce qualified immunoprotection antibody ahead of time.
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 without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (7)
1. newcastle disease, a bird flu bivalent inactivated vaccine, is characterized in that: comprise newcastle disease LaSota strain inactivation antigen, H9N2 subtype avian influenza WF strain inactivation antigen, water-soluble vaccines adjuvant Gel01ST and pH6.8-7.2PBS solution.
2. inactivated vaccine according to claim 1, is characterized in that, the preparation method of described NDV La Sota Strain inactivation antigen is: by newcastle disease LaSota strain virus seed culture of viruses inoculation 10-11 day instar chicken embryo, dosage of inoculation is 1000EID
50, cultivate 96-120 hour for 36-37 DEG C, results Embryo Gallus domesticus liquid, adds formalin wherein, makes formaldehyde final concentration reach 0.2%, and in 37 DEG C of deactivations 24 hours after mixing, through steriling test and deactivation after the assay was approved, 2-8 DEG C saves backup;
The preparation method of described H9N2 subtype avian influenza virus WF strain inactivation antigen is: by H9N2 subtype avian influenza WF strain virus seed culture of viruses inoculation 10-11 day instar chicken embryo, dosage of inoculation is 1000EID
50, cultivate 72-96 hour for 36-37 DEG C, results Embryo Gallus domesticus liquid, adds formalin wherein, makes formaldehyde final concentration reach 0.2%, and in 37 DEG C of deactivations 24 hours after mixing, through steriling test and deactivation after the assay was approved, 2-8 DEG C saves backup.
3. inactivated vaccine according to claim 1 and 2, it is characterized in that, in described inactivated vaccine, each component by weight: Avian pneumo-encephalitis virus LaSota strain inactivation antigen 5-10 part, H9N2 subtype avian influenza virus WF strain inactivation antigen 5-10 part, water-soluble vaccines adjuvant Gel01ST5-20 part, 0.01mol/LPBS solution 60-85 part.
4. the preparation method of inactivated vaccine described in any one of claim 1-3, is characterized in that, comprise the following steps:
1) preparation of NDV La Sota Strain inactivation antigen and H9N2 subtype avian influenza WF strain inactivation antigen:
By newcastle disease LaSota strain virus seed culture of viruses inoculation 10-11 day instar chicken embryo, dosage of inoculation is 1000EID
50, cultivate 96-120 hour for 36-37 DEG C, results Embryo Gallus domesticus liquid, add formalin wherein, make formaldehyde final concentration reach 0.2%, in 37 DEG C of deactivations 24 hours after mixing, through steriling test and deactivation after the assay was approved, obtain NDV La Sota Strain inactivation antigen, 2-8 DEG C saves backup;
By H9N2 subtype avian influenza WF strain virus seed culture of viruses inoculation 10-11 day instar chicken embryo, dosage of inoculation is 1000EID
50, cultivate 72-96 hour for 36-37 DEG C, results Embryo Gallus domesticus liquid, add formalin wherein, make formaldehyde final concentration reach 0.2%, in 37 DEG C of deactivations 24 hours after mixing, through steriling test and deactivation after the assay was approved, obtain H9N2 subtype avian influenza WF strain inactivation antigen, 2-8 DEG C saves backup;
2) adopt chemical precipitation method or hyperfiltration process respectively to step 1) in the inactivation antigen of preparation carry out purification;
3) the NDV La Sota Strain inactivation antigen after purification and H9N2 subtype avian influenza virus WF strain inactivation antigen, vaccine adjuvant Gel01ST and PBS solution are mixed in proportion, 200-500 rev/min is stirred 20-30 minute, subpackage.
5. method according to claim 4, it is characterized in that, step 2) middle employing chemical precipitation method purification, be specially: the sodium chloride adding PEG6000 and 2-6% of antigen weight 4-10% in inactivation antigen respectively, after stirring and dissolving, room temperature leaves standstill 2-4 hour, the centrifugal 30-60 minute of 5000-10000g, abandons supernatant, adds PBS solution suspension precipitation according to 1/4 of purification pro-antigen volume in precipitation, obtain the inactivation antigen of purification, 2-8 DEG C saves backup.
6. method according to claim 4, it is characterized in that, step 2) middle employing hyperfiltration process purification, be specially: the molecular cut off specification selected is that the film bag of 300-1000KD carries out cross-flow ultrafiltration to inactivation antigen, in ultra-filtration process, add PBS solution to equilibrium in inactivation antigen, make antigen volume keep constant; When filtrate volume is the 2-3 times of antigen volume, stops adding PBS solution, continue ultrafiltration, until antigen volume reach pure precursor long-pending 1/4.
7. the method according to claim 5 or 6, is characterized in that, after purification the volume of inactivation antigen be pure precursor long-pending 1/4.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107779441A (en) * | 2017-11-10 | 2018-03-09 | 河南后羿生物工程股份有限公司 | A kind of concentrating and purifying process of avian influenza antigen, avian influenza antigen |
| CN110800875A (en) * | 2019-12-04 | 2020-02-18 | 河南牧业经济学院 | Preparation method of additive for improving nonspecific immunity of laying hens |
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| CN102302775A (en) * | 2011-07-06 | 2012-01-04 | 普莱柯生物工程股份有限公司 | Combined inactivated vaccine of Newcastle disease and H9 subtype avian influenza and preparation method thereof |
| CN103083655A (en) * | 2011-11-02 | 2013-05-08 | 普莱柯生物工程股份有限公司 | Vaccine composition for preventing and treating porcine circovirus type 2, haemophilus parasuis and mycoplasma hyopneumoniae infection and preparation method thereof |
| CN103223164A (en) * | 2013-04-15 | 2013-07-31 | 华南农业大学 | Water-in-oil-in-water adjuvant vaccine and preparation method thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101491672A (en) * | 2009-02-24 | 2009-07-29 | 乾元浩生物股份有限公司 | Chicken new castle disease-bird influenza dual oil-emulsion inactivated vaccine and preparation method thereof |
| CN102302775A (en) * | 2011-07-06 | 2012-01-04 | 普莱柯生物工程股份有限公司 | Combined inactivated vaccine of Newcastle disease and H9 subtype avian influenza and preparation method thereof |
| CN103083655A (en) * | 2011-11-02 | 2013-05-08 | 普莱柯生物工程股份有限公司 | Vaccine composition for preventing and treating porcine circovirus type 2, haemophilus parasuis and mycoplasma hyopneumoniae infection and preparation method thereof |
| CN103223164A (en) * | 2013-04-15 | 2013-07-31 | 华南农业大学 | Water-in-oil-in-water adjuvant vaccine and preparation method thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107779441A (en) * | 2017-11-10 | 2018-03-09 | 河南后羿生物工程股份有限公司 | A kind of concentrating and purifying process of avian influenza antigen, avian influenza antigen |
| CN107779441B (en) * | 2017-11-10 | 2021-04-16 | 河南后羿生物工程股份有限公司 | Concentration and purification method of avian influenza antigen and avian influenza antigen |
| CN110800875A (en) * | 2019-12-04 | 2020-02-18 | 河南牧业经济学院 | Preparation method of additive for improving nonspecific immunity of laying hens |
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