CN109223716A - A kind of porcine epidemic diarrhea resisting, swine fever, pseudo- mad dog and tetrad Yolk antibody soluble powder of transmissible gastroenteritis and preparation method thereof - Google Patents
A kind of porcine epidemic diarrhea resisting, swine fever, pseudo- mad dog and tetrad Yolk antibody soluble powder of transmissible gastroenteritis and preparation method thereof Download PDFInfo
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- CN109223716A CN109223716A CN201811020727.0A CN201811020727A CN109223716A CN 109223716 A CN109223716 A CN 109223716A CN 201811020727 A CN201811020727 A CN 201811020727A CN 109223716 A CN109223716 A CN 109223716A
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- tetrad
- yolk antibody
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- epidemic diarrhea
- pig
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- 210000002969 egg yolk Anatomy 0.000 title claims abstract description 116
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- 206010037660 Pyrexia Diseases 0.000 title claims abstract description 41
- 239000000843 powder Substances 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 208000009305 pseudorabies Diseases 0.000 claims abstract description 36
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 17
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 claims abstract description 14
- PUKLDDOGISCFCP-JSQCKWNTSA-N 21-Deoxycortisone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(=O)C)(O)[C@@]1(C)CC2=O PUKLDDOGISCFCP-JSQCKWNTSA-N 0.000 claims abstract description 14
- FCYKAQOGGFGCMD-UHFFFAOYSA-N Fulvic acid Natural products O1C2=CC(O)=C(O)C(C(O)=O)=C2C(=O)C2=C1CC(C)(O)OC2 FCYKAQOGGFGCMD-UHFFFAOYSA-N 0.000 claims abstract description 14
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 14
- 229930006000 Sucrose Natural products 0.000 claims abstract description 14
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- 235000019425 dextrin Nutrition 0.000 claims abstract description 12
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- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 claims description 28
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- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
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- DEXFNLNNUZKHNO-UHFFFAOYSA-N 6-[3-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperidin-1-yl]-3-oxopropyl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1CCN(CC1)C(CCC1=CC2=C(NC(O2)=O)C=C1)=O DEXFNLNNUZKHNO-UHFFFAOYSA-N 0.000 description 1
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- 241001135549 Porcine epidemic diarrhea virus Species 0.000 description 1
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- 241000607479 Yersinia pestis Species 0.000 description 1
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Classifications
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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
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- 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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Immunology (AREA)
- Animal Behavior & Ethology (AREA)
- Genetics & Genomics (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Epidemiology (AREA)
- Biotechnology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention belongs to immunology and antibody production techniques field, a kind of porcine epidemic diarrhea resisting, swine fever, pseudo- mad dog and tetrad Yolk antibody soluble powder of transmissible gastroenteritis and preparation method thereof are specifically disclosed.The present invention is using dry dextrin and maltodextrin as carrier, the tetrad Yolk antibody of the anti-swine fever for being added with fulvic acid, sucrose, potassium sorbate and Sodium azide, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine is sprayed onto carrier surface, high-purity is made, high stability, high curative effect, and the soluble powder for being easy to save, be it is a kind of have that specificity, therapeutic effect be good, a kind of rapid preparation of curative effect for swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine, the above-mentioned four kinds of infectious diseases of pig can be effectively prevented.
Description
Technical field
The invention belongs to immunologys and antibody production techniques field, specifically, being related to a kind of porcine epidemic diarrhea resisting, pig
Pest, pseudo- mad dog and tetrad Yolk antibody soluble powder of transmissible gastroenteritis and preparation method thereof.
Background technique
Swine fever is commonly called as " rinderpest ", is that one kind caused by swine fever virus is acute, generates heat, contagious infection infectious disease.It is various
The pig at age can all infect, and can occur throughout the year.With highly infectious and lethal.It is the main biography for threatening pig breeding industry
One of catch an illness.Porcine pseudorabies are a kind of infectious diseases as caused by Pseudorabies virus.Pseudorabies virus can infect the various ages
Pig, suckling pig are most sensitive.Cause oestrus of sow obstacle after boar infection Pseudorabies virus, miscarriage, produce stillborn foetus, the mummification of fetus
With weak son；Pseudorabies virus congenital infection or early infection cause the nervous symptoms and high mortality of suckling pig；To growing and fattening pigs
Pseudorabies virus is the important pathogen of respiratory system immune complex, causes the Respiratory symptoms of growing-finishing pig and growth slow
It is slow；Pig farm will cause huge economic loss once the pseudoabies positive is presented.Transmissible gastroenteritis of swine and epidemic diarrhea
It is a kind of high degree in contact intestines problem of pig.Respectively as caused by infectious gastroenteritis virus and epidemic diarrhea virus, with
The high degree in contact enteric infectious disease that vomiting, severe diarrhea and dehydration are characterized.
Effective therapeutic agent currently there is no to swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine, connect
Kind of vaccine prevention is the above-mentioned pathogenetic important means of disease of control, but currently due to immunosupress, feed mold, it is various stress,
It is immunized that lack of standardization, vaccine quality is irregular, immune intensity and immune the density reasons such as not enough, causes swinery extensive occult band malicious
And persistent latent infection, moment threaten swinery health.When leakage is exempted from or above-mentioned four kinds of diseases are infected on the pig of immuning failure individual or pig farm
Clinically mainly prevent the generation of complication after poison using antiviral drugs symptomatic treatment and antibiotics, but as new life
Above-mentioned four kinds of virus is once infected when piglet fails to obtain the maternal antibody of sufficient amount from breast milk since morbidity is anxious, lethality is high
Etc. reasons cause the therapeutic effect of above-mentioned treatment method unsatisfactory.Therefore be badly in need of develop one kind can for above-mentioned four boar it is normal
See that special, the special efficacy of infectious disease, therapeutic effect rapidly treat preparation, and is applicable to the disease treatment of newborn piglet.
Summary of the invention
In order to solve the problems in the existing technology, the present invention is intended to provide a kind of anti-swine fever, pseudorabies, pig are popular
Property diarrhea and the tetrad Yolk antibody soluble powder of transmissible gastroenteritis of swine and preparation method thereof, can effectively prevent and treat
Four kinds of infectious diseases are stated, while having that normal temperature condition stability inferior is good, long shelf-life transports and save convenient advantage.
In order to achieve the object of the present invention, technical scheme is as follows:
The present invention provides the tetrad ovum of a kind of anti-swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine
Yellow antibody-soluble powder, using dry dextrin and maltodextrin as carrier, with anti-swine fever, pseudorabies, pig epidemic diarrhea and
The tetrad Yolk antibody of transmissible gastroenteritis of swine is as active constituent.
Further, the preparation method of the tetrad Yolk antibody soluble powder includes the following steps:
(1) the tetrad Yolk antibody for preparing anti-swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine is molten
Liquid；
(2) fulvic acid, sucrose, potassium sorbate and Sodium azide are added into the resulting tetrad Yolk antibody solution of step (1),
Obtain tetrad Yolk antibody combination solution；
(3) on the carrier by the resulting tetrad Yolk antibody combination solution spray of step (2).
Further, in the step (2), the fulvic acid, sucrose, the addition quality of potassium sorbate and Sodium azide are opposite
It is respectively as follows: 3-5%, 15-20%, 0.01-0.03% and 0.01-0.02% in the volume ratio of the tetrad Yolk antibody solution；
Preferably volume ratio is respectively as follows: 5%, 15%, 0.02% and 0.02%.
Further, in the step (3), the mass ratio of the tetrad Yolk antibody combination solution and the carrier is 1:
4-1:5。
Further, in the carrier, the mass ratio of dry dextrin and maltodextrin is 2:1-1:2, preferably 1:1.
Further, the step (1) includes the following steps:
S1, laid eggs peak youth with swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine vaccine immunity
Chicken, the height for then taking chicken group to produce exempt from egg；
S2, the separation height exempt from the yolk of egg, remove grease with the poloxamer precipitation method, collect containing the upper of Yolk antibody
Clear liquid；
S3, take S2 obtain supernatant by concentration and separation ultrafiltration system be concentrated by ultrafiltration to get.
Preferably, the immunization strategy in S1 are as follows: popular using swine fever attenuated vaccine, pseudorabies attenuated vaccine and pig
Diarrhea-transmissible gastroenteritis of swine Bi-combined attenuated Vaccine is each 3 times immune, recycles pig epidemic diarrhea-transmissible gastroenteritis of swine two
It is 2 times immune to join inactivated vaccine.
The present invention furthermore provides a kind of anti-swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine
The preparation method of tetrad Yolk antibody soluble powder, includes the following steps:
(1) the tetrad Yolk antibody for preparing anti-swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine is molten
Liquid；
(2) fulvic acid, sucrose, potassium sorbate and Sodium azide are added into the resulting tetrad Yolk antibody solution of step (1),
Obtain tetrad Yolk antibody combination solution；
(3) on the carrier by the resulting tetrad Yolk antibody combination solution spray of step (2).
Further, the addition quality of the fulvic acid, sucrose, potassium sorbate and Sodium azide is relative to the tetrad yolk
The volume ratio of antibody-solutions is respectively as follows: 3-5%, 15-20%, 0.01-0.03% and 0.01-0.02%；Preferably volume score
Not are as follows: 5%, 15%, 0.02% and 0.02%；
And/or
The mass ratio of the tetrad Yolk antibody combination solution and the carrier is 1:4-1:5；
And/or
In the carrier, the mass ratio of dry dextrin and maltodextrin is 2:1-1:2, preferably 1:1.
More preferably, the step (1) includes the following steps:
S1, laid eggs peak youth with swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine vaccine immunity
Chicken, the height for then taking chicken group to produce exempt from egg；
It is preferred that immunization strategy are as follows: infected using swine fever attenuated vaccine, pseudorabies attenuated vaccine and pig epidemic diarrhea-pig
Property gastroenteritis Bi-combined attenuated Vaccine each immune 3 times, recycle pig epidemic diarrhea-transmissible gastroenteritis of swine bivalent inactivated vaccine to exempt from
Epidemic disease 2 times；
S2, the separation height exempt from the yolk of egg, remove grease with the poloxamer precipitation method, collect containing the upper of Yolk antibody
Clear liquid；
S3, take S2 obtain supernatant by concentration and separation ultrafiltration system be concentrated by ultrafiltration to get.
Optionally, vaccine live vaccines of hog cholera, pseudorabies disease live-vaccine used in S1 are purchased from Shandong Green City biotechnology
Co., Ltd；Transmissible gastroenteritis of swine and pig epidemic diarrhea bigeminal live vaccine are purchased from Da Bei Nong；Transmissible gastroenteritis of swine and pig
Epidemic diarrhea bivalent inactivated vaccine is purchased from Wuhan Ke Qian Biological Co., Ltd..
Preferably, S2 specifically: isolated yolk and deionized water are stirred to being thoroughly mixed, yolk aqueous solution is obtained；
Poloxamer solution is added, poloxamer concentration in mixed solution is made to be no more than 1.0w/v%-1.2w/v%, it is heavy to stand after stirring
Shallow lake yolk grease, separation and Extraction supernatant；After supernatant liquid filtering is removed impurity, Poloxamer solution is added again, makes to mix
Poloxamer concentration is 0.5w/v% in solution, and remaining grease is stood in precipitation and separation yolk again after stirring, is slightly mentioned point
From Yolk antibody solution, as containing the supernatant of Yolk antibody.
Further, the environment temperature stood after Poloxamer solution is added for the first time and is no more than 10 DEG C, time of repose is not
Lower than 8 hours.
More preferably, S2 removes grease using the poloxamer precipitation method, collects the supernatant containing Yolk antibody and specifically wraps
Include following steps:
A) 10% (w/v) Poloxamer solution is prepared with deionized water, such as 1 kilogram of poloxamer is added to 10 liters
Stirring and dissolving in ionized water；
B) firmly stirring is thoroughly mixed yolk and water after mixing isolated yolk and deionized water in 1: 6 ratio,
Obtain yolk aqueous solution；
C) 10% (w/v) Poloxamer solution that step a) is prepared is added in the yolk aqueous solution obtained into step b),
Make poloxamer concentration 1% (w/v) in mixed solution, firmly stir, obtains yolk and poloxamer water solution A；4 DEG C-
8-12 hours are stood at 10 DEG C, precipitates yolk grease, separation and Extraction supernatant；
D) supernatant for obtaining step c) obtains Yolk antibody solution by four layers of hospital gauze filtering removal impurity；
E) 10% (w/v) Poloxamer solution is added in the Yolk antibody solution obtained to step d), makes in mixed solution
Poloxamer concentration is 0.5% (w/v), and firmly stirring mixes well, and obtains yolk and poloxamer aqueous solution B；At 4 DEG C -10
2-4 hours are stood at DEG C, again remaining grease in precipitation and separation yolk；
Concrete operations can are as follows: is gone after most upper one layer of grease according to step d) method filtering solution with kitchen paper is viscous to get slightly mentioning
Isolated Yolk antibody solution.
Preferably, S3 is that the supernatant for taking S2 to obtain is added in the fluid reservoir of ultrafiltration concentrated separation system, uses retention
The ultrafiltration membrane of molecular weight 20000Da, in operating pressure 0.05-0.15Mpa and the control of concentrating return-flow flow velocity in 15-20m3/h
Under conditions of carry out ultrafiltration concentration separation, that is, obtain the tetrad Yolk antibody aqueous solution of purification.
Four len antibodies of anti-swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine in above technical scheme
Should also specific antibody titration be carried out to product after the completion of solution preparation, such as using ELISA method, use swine fever, pig respectively
Pseudo- mad dog, pig epidemic diarrhea and transmissible gastroenteritis of swine kit measurement its potency.
It should be appreciated that on the basis of common knowledge of the art, above-mentioned each optimum condition can be combined with each other, obtain each
Specific technical solution.
The beneficial effects of the present invention are:
Utilize the four of the anti-swine fever of the method for the present invention preparation, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine
Join Yolk antibody soluble powder, is that one kind is directed to swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine with spy
It is anisotropic, therapeutic effect is good, a kind of rapid preparation of curative effect, the above-mentioned four kinds of infectious diseases of pig can be effectively prevented.
The present invention, will be added with fulvic acid, sucrose, potassium sorbate and Sodium azide using dry dextrin and maltodextrin as carrier
Anti- swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine tetrad Yolk antibody be sprayed onto carrier surface, be made
High-purity, high stability, high curative effect, and the soluble powder for being easy to save have convenient transportation relative to liquid egg yolk antibody,
The not high advantage of Conservation environment temperature requirement, it is only necessary to be placed in ventilation, shady and cool space；Relative to Yolk antibody freeze-dried powder,
Equipment is drained without freezing, it is low in cost, it is suitable for being mass produced；Relative to antibiotic and antiviral drugs, the present invention is produced
Product have many advantages, such as noresidue, without side-effects to be suitable for large-scale promotion use.
Detailed description of the invention
Fig. 1 is room temperature different time sections antibody titer variation in experimental example 1 of the present invention.
Specific embodiment
Below with reference to embodiment the present invention will be further explained explanation.It will be appreciated that following embodiment provides
Merely to playing the purpose of explanation, it is not used to limit the scope of the present invention.Those skilled in the art is not
In the case where spirit of the invention and spirit, the present invention can be carry out various modifications and be replaced.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Vaccine live vaccines of hog cholera used in embodiment, pseudorabies disease live-vaccine, which are purchased from Shandong Green City biotechnology, to be had
Limit company；Transmissible gastroenteritis of swine and pig epidemic diarrhea bigeminal live vaccine are purchased from Da Bei Nong；Transmissible gastroenteritis of swine and pig are flowed
Row diarrhea bivalent inactivated vaccine is purchased from Wuhan Ke Qian Biological Co., Ltd..
Embodiment 1
The present embodiment is used to illustrate to prepare swine fever, pseudo- mad dog, epidemic diarrhea and transmissible gastroenteritis tetrad height and exempts from egg,
The specific method is as follows:
(1) the not immune peak youth's chicken isolated rearing of laying eggs of selection health reinforces feed nutrition and environmental sanitation in order to avoid disease
Original infection；
(2) with the blueness handled well in swine fever, pseudorabies, pig epidemic diarrhea and pig stomach enteritis vaccine immunity step (1)
Year high-yield egg chicken, specific steps are as follows:
For the first time: immune pig epidemic diarrhea-transmissible gastroenteritis Bi-combined attenuated Vaccine and bivalent inactivated vaccine and pig
Fever live vaccine, for injection volume by 1/5 pig dose immunization chicken (i.e. 100 chickens are immunized in 20 part pig vaccines), injecting pathway is that pig is popular
Property the subcutaneous injection of diarrhea-transmissible gastroenteritis bivalent inactivated vaccine neck, the pig epidemic diarrhea-weak poison of transmissible gastroenteritis bigeminy
Vaccine and swine fever attenuated vaccine carry out leg muscle injection；Second: pig epidemic diarrhea-transmissible gastroenteritis being immunized after 2 weeks
Bi-combined attenuated Vaccine, swine fever attenuated vaccine and pseudo- mad dog attenuated vaccine press the dosage injection leg muscle of 1/5 pig respectively；Third
Secondary: every chicken is by 1/5 pig dosage leg muscle injection swine fever and the pseudo- mad attenuated vaccine of pig after 2 weeks；4th time: the pig after 2 weeks again
1/5 pig dosage is subcutaneously injected in epidemic diarrhea-transmissible gastroenteritis bivalent inactivated vaccine neck, while equally pressing 1/5 pig dosage
Leg muscle injects pseudorabies attenuated vaccine.
(3) after high yield young layer being immunized, collection obtains height and exempts from egg.
Embodiment 2
The present embodiment prepares tetrad Yolk antibody solution, specific side for illustrating that the height obtained using embodiment 1 exempts from egg
Method is as follows:
(1) separation height exempts from egg yolk:
Start to correspond to epidemic disease in sampling Detection egg yolk every 7 days within 1 week after every kind of vaccine secondary immunity described in embodiment 1
The specific antibody titres that antigen generates in seedling, while being compared with egg yolk rather.As not immune egg yolk OD450Value
Less than 0.2, and immune vaccine egg yolk antibody specific antibody OD450/ egg yolk OD is not immunized450Ratio be greater than 5.0 with
Separation yolk was collected when upper, the separation yolk method particularly includes: the height that embodiment 1 obtains is exempted from into egg and places closed container
Or in house, it is sent into yolk seperator after Peracetic acid fumigation 30 minutes and carries out yolk separation；
(2) refined vitelline antibody is prepared:
Deionized water will be added with 1:6 ratio in the separated yolk of step (1), (amount of the ratio deionised water is also
Volume including the Poloxamer solution being added in subsequent operation) be vigorously stirred and makes yolk and water mixes well acquisition yolk
Aqueous solution；Add 10% Poloxamer solution in the yolk aqueous solution of acquisition, guarantees in yolk solution and poloxamer mixed solution
Poloxamer concentration 1% (W/V), stirring mixes well immediately, and described 10% (W/V) Poloxamer solution weighs 1 kilogram of pool Lip river
Sha Mu is stirred after 10 liters of deionized waters of addition sufficiently dissolve thereto to obtain the final product；By the poloxamer yolk aqueous solution of acquisition in 4-8
After DEG C standing 8-12 hour, after first going one layer of grease above and offscum with kitchen paper is viscous, then supernatant is by 4 layers of hospital gauze mistake
10% (W/V) poloxamer aqueous solution is added in filter in filtered yolk aqueous solution, guarantees that yolk and poloxamer mixing are molten
Poloxamer concentration 0.5% (W/V) in liquid glues after standing 2-4 hours at 4-8 DEG C after mixing well and removes upper layer grease, by supernatant
Solution is by 4 layers of hospital gauze filtering；It will be used by the Yolk antibody aqueous solution of the removal grease of the poloxamer precipitation method twice
The ultrafiltration membrane of molecular cut off 20000Da, in operating pressure 0.05-0.15Mpa and the control of concentrating return-flow flow velocity in 15-
20m3Ultrafiltration concentration separation is carried out under conditions of/h, obtains tetrad Yolk antibody solution.
Embodiment 3
The present embodiment is used to illustrate the preparation of tetrad Yolk antibody combination solution.
The fulvic acid of 5% (w/v), the sugarcane of 15% (w/v) are added in the tetrad Yolk antibody solution obtained to embodiment 2
Sugar, the Sodium azide of the potassium sorbate of 0.02% (w/v) and 0.02% (w/v), be sufficiently stirred to get.
Embodiment 4
The present embodiment for illustrating using dry dextrin and maltodextrin as the preparation of the tetrad Yolk antibody soluble powder of carrier,
Include the following steps:
(1) dry dextrin and maltodextrin are added in spray drying device in the ratio of 1: 1 (weight ratio), adjustment air inlet
70-80 DEG C of temperature of mouth, 50-60 DEG C of air outlet temperature, pre- hot mixing dextrin 10min；
(2) the tetrad Yolk antibody combination solution obtained in embodiment 3 is passed through into wriggling according to the ratio of 1:4 (weight ratio)
Pump, on the velocity spray of 150-250ml/min to mixing dextrin, spray drying device carries out 50-60 DEG C of dry 60-70min,
It crushes, is sieved to obtain the final product.
Comparative example 1
The difference of this comparative example and embodiment 4 is: used tetrad Yolk antibody combination solution are as follows: to embodiment 2
5% (w/v) fulvic acid, 15% (w/v) sucrose, 0.02% (w/v) sorbic acid are added in the tetrad Yolk antibody solution of acquisition
Potassium, be sufficiently stirred to get.
Comparative example 2
The difference of this comparative example and embodiment 4 is: used tetrad Yolk antibody combination solution are as follows: to embodiment 2
5% (w/v) fulvic acid of addition in the tetrad Yolk antibody solution of acquisition, 15% (w/v) sucrose, 0.02% (w/v) Sodium azide,
Be sufficiently stirred to get.
Comparative example 3
The difference of this comparative example and embodiment 4 is: used tetrad Yolk antibody combination solution are as follows: to embodiment 2
15% (w/v) sucrose, 0.02% (w/v) potassium sorbate are added in the tetrad Yolk antibody solution of acquisition, 0.02% (w/v) is folded
Nitrogen sodium, be sufficiently stirred to get.
Comparative example 4
The difference of this comparative example and embodiment 4 is: used tetrad Yolk antibody combination solution are as follows: to embodiment 2
5% (w/v) fulvic acid, 0.02% (w/v) potassium sorbate are added in the tetrad Yolk antibody solution of acquisition, 0.02% (w/v) is folded
Nitrogen sodium, be sufficiently stirred to get.
Comparative example 5
The difference of this comparative example and embodiment 4 is: the tetrad Yolk antibody combination solution that will be obtained in embodiment 3 replaces
It is changed to the tetrad Yolk antibody solution of the acquisition of embodiment 2, no longer addition other components.
Experimental example 1
This experimental example is used to illustrate the properties of product and quality measurements to embodiment 4 and comparative example 1-5.
(1) it is detected material: prepared by embodiment 4 (test group) and comparative example 1-5 (being corresponding in turn to group: contrast groups 1-5)
Tetrad Yolk antibody soluble powder.
(2) test method: placing room temperature (20-25 DEG C) for product described in step (1), and 18 months, respectively in January,
2 months, in March, in June, September in December, sample was weighed on the time point after 15 months, 18 months deionized water is added and be configured to 30% (w/v)
Antibody-solutions (S), while the same series-produced Yolk antibody solution without any processing is diluted to 6% with deionized water
(v/v) yolk solution detects antibody titer with ELISA method as control (C).Antibody titer retention rate: with OD450S/OD450C
× 100% calculates.
(3) test result:
It is stored at room temperature test result: using antibody titer retention rate as ordinate, being made using sample time as abscissa
Different other antibody titer decay patterns, the result is shown in Figure 1.
It is shown in Fig. 1, when soluble powder (dry powder) was stored at room temperature by 18 months, test group antibody titer retains 85%
More than, abatement is most slow, secondly cuts down speed and is followed successively by contrast groups 3, contrast groups 2, contrast groups 4, contrast groups 1 and comparison from slow to fast
Group 5.As can be seen from the test results when the formulation stability of the use of embodiment 4 is best.
Quality standard:
(1) character: faint yellow or yellow homogeneous powder；
(2) dissolubility: soluble easily in water；
(3) detection of mycoplasma: being carried out by " Chinese veterinary drug shop ", no mycoplasma growth；
(4) exogenous virus detects: carrying out by " Chinese veterinary drug shop ", no exogenous virus pollution；
(5) mould detects: carrying out, complies with standard by " Chinese veterinary drug shop "；
(6) antibody titer the shelf-life: is detected as a result, room temperature can at least be stored 18 months according to room temperature.
(7) safety examination: 7 age in days SPF chickens are selected, 10, every chicken presses 45 gs/kg of weight (3 times of usage amounts), even
It with 3 days, observes 21 days, spirit is normal, and feeding and drinking-water are normal, answers whole keys living.
Experimental example 2
This experimental example, which is described, tests the efficacy analysis of hog cholera the product of embodiment 4 and comparative example 1-5.
Institute's testing product is that embodiment 4 (test group) and comparative example 1-5 (are corresponding in turn to group: contrast groups in the present embodiment
1-5) the tetrad Yolk antibody soluble powder prepared.
Experimental animal selects 7 age in days sodium selenite 80.
Test method: 7 age in days clean grade Guangxi Bama Mini-pigs of selection are randomly divided into 8 after isolated rearing is observed 1 week
Group, every group 10, isolated rearing.8 groups are respectively blank control group, do not give any product；Malicious group is attacked, test group and contrast groups are every
Malicious group is attacked within 24 hours to 3-5 milliliters of physiological saline after hog cholera venom after head pig muscle injection dilution, and test group gavages 3-5 milliliters
Product is prepared with the embodiment 4 of physiological saline solution, contrast groups 1-5 successively gavages the 3-5 milliliters of comparisons for using physiological saline solution
Example 1-5 prepares product, and power-product used is used by 15 gs/kg of weight in test group and contrast groups 1-5, continuously gavages 3
It, observes 7 days, the results are shown in Table 1.
Curative effect evaluation standard:
Cure: spirit is normal, and feeding drinking-water is normal, and excrement is normal, without any symptom.
Effective: apathetic, feed intake is not restored completely, clinical symptom relief.
Invalid: apathetic, twitch, body temperature high fever search for food and degradation or do not feed and eventually lead to death.
The different group curative effect situations of table 1
| Group | It falls ill number (head) | It cures number (head) | Significant figure (head) | Cure rate (%) |
| Test group | 8 | 7 | 1 | 88 |
| Attack malicious group | 10 | 1 | 1 | 10 |
| Contrast groups 1 | 10 | 3 | 2 | 30 |
| Contrast groups 2 | 10 | 6 | 3 | 60 |
| Contrast groups 3 | 10 | 6 | 4 | 60 |
| Contrast groups 4 | 10 | 5 | 3 | 50 |
| Contrast groups 5 | 10 | 2 | 2 | 20 |
| Blank control | 0 | / | / | / |
As it can be seen from table 1 test group can reach 88% to swine fever cure rate, contrast groups 1-5 is to swine fever cure rate in 20-
In 60% range, statistical analysis shows that test group is higher than contrast groups 1-5 (p ﹤ 0.01) to the significant in efficacy of swine fever.
Experimental example 3
This experimental example, which is described, tests the efficacy analysis of porcine pseudorabies the product of embodiment 4 and comparative example 1-5.
Institute's testing product is that embodiment 4 (test group) and comparative example 1-5 (are corresponding in turn to group: contrast groups in the present embodiment
1-5) the tetrad Yolk antibody soluble powder prepared.
Experimental animal selects 7 age in days sodium selenite 80.
Test method: 7 age in days clean grade Guangxi Bama Mini-pigs of selection are randomly divided into 8 after isolated rearing is observed 1 week
Group, every group 10, isolated rearing.8 groups are respectively blank control group, do not give any product；Malicious group is attacked, test group and contrast groups are every
Malicious group is attacked within 24 hours to 3-5 milliliters of physiological saline after porcine pseudorabies venom after head pig muscle injection dilution, and test group gavages 3-5
The embodiment 4 of milliliter physiological saline solution prepares product, and contrast groups 1-5 successively gavages 3-5 milliliters with physiological saline solution
Comparative example 1-5 prepares product, and power-product used is used by 15 gs/kg of weight in test group and contrast groups 1-5, continuously gavages
It 3 days, observes 7 days, the results are shown in Table 2.
Curative effect evaluation standard:
Cure: spirit is normal, and feeding drinking-water is normal, and body temperature is normal, without any symptom.
Effective: lassitude, feed intake gradually increase, and body temperature is declined, no faintness, and vomiting stops.
It is invalid: it is apathetic, it faints, hyperpyrexia, not material feeding keeps vomiting, dead.
The different group curative effect situations of table 2
From table 2 it can be seen that test group can reach 90% to the cure rate of porcine pseudorabies, comparison group 1-5 is mad to pig puppet
The cure rate of dog disease is within the scope of 30-70%.Statistical analysis shows that test group is higher than comparison to the significant in efficacy of porcine pseudorabies
Group 1-5 (P ﹤ 0.01).
Experimental example 4
This experimental example, which is described, tests the efficacy analysis of pig epidemic diarrhea the product of embodiment 4 and comparative example 1-5.
Institute's testing product is that embodiment 4 (test group) and comparative example 1-5 (are corresponding in turn to group: contrast groups in the present embodiment
1-5) the tetrad Yolk antibody soluble powder prepared.
Experimental animal selects 7 age in days sodium selenite 80.
Test method: 7 age in days clean grade Guangxi Bama Mini-pigs of selection are randomly divided into 8 after isolated rearing is observed 1 week
Group, every group 10, isolated rearing.8 groups are respectively blank control group, do not give any product；Malicious group is attacked, test group and contrast groups are every
Malicious group is attacked within 24 hours to 3-5 milliliters of physiological saline after Porcine epidemic diarrhea virus liquid after head pig muscle injection dilution, and test group fills
It takes 3-5 milliliters and prepares product with the embodiment 4 of physiological saline solution, contrast groups 1-5 successively gavages 3-5 milliliters and uses physiological saline
The comparative example 1-5 of dissolution prepares product, and power-product used is used by 15 gs/kg of weight in test group and contrast groups 1-5, even
It is continuous to gavage 3 days, it observes 7 days, the results are shown in Table 3.
Curative effect evaluation standard:
Cure: spirit is normal, and feeding drinking-water is normal, and body temperature is normal, and excrement is normal, without any symptom.
Effective: lassitude, feed intake gradually increase, and diarrhea and symptoms of emesis are improved.
Invalid: apathetic, body temperature increases, and draws water sample stench excrement, and not material feeding keeps vomiting, dead.
The different group curative effect situations of table 3
From table 3 it can be seen that test group reaches 90% to the cure rate of pig epidemic diarrhea, contrast groups 1-5 is to pig prevalence
The cure rate of property diarrhea is in 20-70%.Statistical analysis shows that test group is significant in efficacy to Porcine Epidemic Diarrhea and is higher than contrast groups
1-5 (P ﹤ 0.01).
Experimental example 5
This experimental example, which is described, tries the efficacy analysis of transmissible gastroenteritis of swine the product of embodiment 4 and comparative example 1-5
It tests.
Institute's testing product is that embodiment 4 (test group) and comparative example 1-5 (are corresponding in turn to group: contrast groups in the present embodiment
1-5) the tetrad Yolk antibody soluble powder prepared.
Experimental animal selects 7 age in days sodium selenite 80.
Test method: 7 age in days clean grade Guangxi Bama Mini-pigs of selection are randomly divided into 8 after isolated rearing is observed 1 week
Group, every group 10, isolated rearing.8 groups are respectively blank control group, do not give any product；Malicious group is attacked, test group and contrast groups are every
Malicious group is attacked within 24 hours to 3-5 milliliters of physiological saline, test group after transmissible gastro-enteritis virus liquid after head pig muscle injection dilution
It gavages 3-5 milliliters and prepares product with the embodiment 4 of physiological saline solution, contrast groups 1-5 successively gavages 3-5 milliliters and uses physiology salt
The comparative example 1-5 of water dissolution prepares product, and power-product used is used by 15 gs/kg of weight in test group and contrast groups 1-5,
It continuously gavages 3 days, observes 7 days, the results are shown in Table 4.
Curative effect evaluation standard:
Cure: spirit is normal, and feeding drinking-water is normal, and body temperature is normal, and excrement is normal, without any symptom.
Effective: lassitude, feed intake gradually increase, and diarrhea and symptoms of emesis are clearly better, and body temperature tends to be normal.
Invalid: apathetic, body temperature increases, and has loose bowels and keeps vomiting, does not search for food, dead.
The different group curative effect situations of table 4
From table 4, it can be seen that test group reaches 89% to the cure rate of transmissible gastroenteritis of swine, contrast groups 1-5 flows pig
The cure rate of row diarrhea is in 30-70%.Statistical analysis shows that test group is significant in efficacy to Porcine Epidemic Diarrhea and is higher than comparison
Group 1-5 (P ﹤ 0.01).
According to experimental example 2-5's the experimental results showed that, the embodiment of the present invention 4 prepare tetrad Yolk antibody soluble powder pair
Swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine have significant curative effect, i.e., by fulvic acid, mountain in the present invention
Potassium sorbate, sucrose and Sodium azide are used cooperatively and significantly improve its curative effect and stability into Yolk antibody.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a kind of anti-swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine tetrad Yolk antibody soluble powder,
It is characterized in that, it is using dry dextrin and maltodextrin as carrier, with anti-swine fever, pseudorabies, pig epidemic diarrhea and pig biography
The tetrad Yolk antibody of metachromia gastroenteritis is as active constituent.
2. tetrad Yolk antibody soluble powder according to claim 1, which is characterized in that preparation method includes:
(1) the tetrad Yolk antibody solution of anti-swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine is prepared；
(2) fulvic acid, sucrose, potassium sorbate and Sodium azide are added into the resulting tetrad Yolk antibody solution of step (1), are obtained
Tetrad Yolk antibody combination solution；
(3) on the carrier by the resulting tetrad Yolk antibody combination solution spray of step (2).
3. tetrad Yolk antibody soluble powder according to claim 2, which is characterized in that in the step (2), the Huang
Rotten acid, sucrose, potassium sorbate and Sodium azide addition quality be respectively as follows: relative to the volume ratio of the tetrad Yolk antibody solution
3-5%, 15-20%, 0.01-0.03% and 0.01-0.02%；
Preferably volume ratio is respectively as follows: 5%, 15%, 0.02% and 0.02%.
4. tetrad Yolk antibody soluble powder according to claim 2 or 3, which is characterized in that in the step (3), institute
The mass ratio for stating tetrad Yolk antibody combination solution and the carrier is 1:4-1:5.
5. tetrad Yolk antibody soluble powder according to claim 1-4, which is characterized in that in the carrier,
The mass ratio of dry dextrin and maltodextrin is 2:1-1:2, preferably 1:1.
6. tetrad Yolk antibody soluble powder according to claim 1-5, which is characterized in that the step (1)
Include the following steps:
S1, peak youth chicken of being laid eggs with swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine vaccine immunity, and
The height for taking chicken group to produce afterwards exempts from egg；
S2, the separation height exempt from the yolk of egg, remove grease with the poloxamer precipitation method, collect the supernatant containing Yolk antibody
Liquid；
S3, take S2 obtain supernatant by concentration and separation ultrafiltration system be concentrated by ultrafiltration to get.
7. tetrad Yolk antibody soluble powder according to claim 6, which is characterized in that the immunization strategy in S1 are as follows: benefit
Respectively exempted from swine fever attenuated vaccine, pseudorabies attenuated vaccine and pig epidemic diarrhea-transmissible gastroenteritis of swine Bi-combined attenuated Vaccine
Epidemic disease 3 times, recycle pig epidemic diarrhea-transmissible gastroenteritis of swine bivalent inactivated vaccine 2 times immune.
8. a kind of anti-swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine tetrad Yolk antibody soluble powder
Preparation method, which comprises the steps of:
(1) the tetrad Yolk antibody solution of anti-swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine is prepared；
(2) fulvic acid, sucrose, potassium sorbate and Sodium azide are added into the resulting tetrad Yolk antibody solution of step (1), are obtained
Tetrad Yolk antibody combination solution；
(3) on the carrier by the resulting tetrad Yolk antibody combination solution spray of step (2).
9. preparation method according to claim 8, which is characterized in that the fulvic acid, sucrose, potassium sorbate and Sodium azide
Addition quality be respectively as follows: 3-5%, 15-20%, 0.01-0.03% relative to the volume ratio of the tetrad Yolk antibody solution
And 0.01-0.02%；Preferably volume ratio is respectively as follows: 5%, 15%, 0.02% and 0.02%；
And/or
The mass ratio of the tetrad Yolk antibody combination solution and the carrier is 1:4-1:5；
And/or
In the carrier, the mass ratio of dry dextrin and maltodextrin is 2:1-1:2, preferably 1:1.
10. preparation method according to claim 9, which is characterized in that the step (1) includes the following steps:
S1, peak youth chicken of being laid eggs with swine fever, pseudorabies, pig epidemic diarrhea and transmissible gastroenteritis of swine vaccine immunity, and
The height for taking chicken group to produce afterwards exempts from egg；
It is preferred that immunization strategy are as follows: utilize swine fever attenuated vaccine, pseudorabies attenuated vaccine and pig epidemic diarrhea-pig transmissible stomach
Enteritis Bi-combined attenuated Vaccine is each 3 times immune, recycles pig epidemic diarrhea-transmissible gastroenteritis of swine bivalent inactivated vaccine immune 2
It is secondary；
S2, the separation height exempt from the yolk of egg, remove grease with the poloxamer precipitation method, collect the supernatant containing Yolk antibody
Liquid；
S3, take S2 obtain supernatant by concentration and separation ultrafiltration system be concentrated by ultrafiltration to get.
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