CN109223716B - Quadruple yolk antibody soluble powder for resisting porcine epidemic diarrhea, swine fever, pseudorabies and transmissible gastroenteritis and preparation method thereof - Google Patents
Quadruple yolk antibody soluble powder for resisting porcine epidemic diarrhea, swine fever, pseudorabies and transmissible gastroenteritis and preparation method thereof Download PDFInfo
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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
Abstract
The invention belongs to the technical field of immunology and antibody preparation, and particularly discloses soluble quadruple yolk antibody powder for resisting porcine epidemic diarrhea, swine fever, pseudorabies and transmissible gastroenteritis and a preparation method thereof. The invention takes the dry dextrin and the maltodextrin as carriers, and the tetragenous egg yolk antibody which is added with the fulvic acid, the sucrose, the potassium sorbate and the sodium azide and is used for resisting swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis is sprayed on the surface of the carrier to prepare the soluble powder which has high purity, high stability, high curative effect and easy preservation, is a preparation which has specificity, good treatment effect and quick curative effect for the swine fever, the porcine pseudorabies, the porcine epidemic diarrhea and the porcine transmissible gastroenteritis, and can effectively prevent and treat the four infectious diseases of the pigs.
Description
Technical Field
The invention belongs to the technical field of immunology and antibody preparation, and particularly relates to soluble quadruple yolk antibody powder for resisting porcine epidemic diarrhea, swine fever, pseudorabies and transmissible gastroenteritis and a preparation method thereof.
Background
Hog cholera is commonly called as 'gastrointestinal plague', and is an acute, fever and contact infectious disease caused by hog cholera virus. Pigs of various ages can become infected and can occur throughout the year. Is highly contagious and lethal. Is one of the main infectious diseases threatening the pig industry. Porcine pseudorabies is an infectious disease caused by pseudorabies virus. Pseudorabies virus can infect pigs of various ages, and suckling piglets are most sensitive. After the pigs are infected with the pseudorabies virus, the sow is caused with oestrus disorder, abortion, stillbirth, mummy and weak piglets; pseudorabies congenital or early infection causes neurological symptoms and high mortality in suckling piglets; the pseudorabies virus of the growing and fattening pigs is an important pathogen of immune syndrome of a respiratory system, and causes respiratory system symptoms and growth retardation of growing and fattening pigs; once the pig farm shows the positive pseudorabies, huge economic loss is caused. Porcine transmissible gastroenteritis and epidemic diarrhea are highly contagious intestinal diseases in pigs. Highly contagious enteric infections characterized by vomiting, severe diarrhea and dehydration, respectively, caused by transmissible gastroenteritis virus and epidemic diarrhea virus.
Currently, no effective treatment medicine for swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis exists, vaccination prevention is an important means for controlling the occurrence of the diseases, but the long-term recessive infection and continuous latent infection of a swinery are caused by immunosuppression, feed mildew, various stresses, non-standard immunity, uneven vaccine quality, insufficient immune strength and immune density and the like, so that the health of the swinery is threatened all the time. When a swine individual or a pig farm with missed immunization or failed immunity is infected with the four viruses, antiviral medicines and antibiotic medicines are mainly clinically used for symptomatic treatment and prevention of complications, but when a newborn piglet cannot obtain a sufficient amount of maternal antibodies from breast milk, once the newborn piglet is infected with the four viruses, the treatment effect of the treatment method is not ideal due to acute morbidity, high mortality and the like. Therefore, a specific and specific treatment preparation with a quick treatment effect aiming at the four common infectious diseases of the pigs is urgently needed to be developed, and the preparation can be suitable for treating diseases of newborn piglets.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide the soluble quadruple egg yolk antibody powder for resisting swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis and the preparation method thereof, which can effectively prevent and treat the four infectious diseases and have the advantages of good stability at normal temperature, long shelf life and convenient transportation and storage.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
the invention provides a quadruple yolk antibody soluble powder for resisting swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis.
Further, the preparation method of the tetrad yolk antibody soluble powder comprises the following steps:
(1) preparing a quadruple yolk antibody solution for resisting swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis;
(2) adding fulvic acid, sucrose, potassium sorbate and sodium azide into the tetrad yolk antibody solution obtained in the step (1) to obtain a tetrad yolk antibody combined solution;
(3) and (3) spraying the tetrad yolk antibody combination solution obtained in the step (2) on the carrier.
Further, in the step (2), the addition mass ratios of the fulvic acid, the sucrose, the potassium sorbate and the sodium azide to the volume ratio of the quadruple yolk antibody solution are respectively as follows: 3-5%, 15-20%, 0.01-0.03% and 0.01-0.02%;
preferably, the volume ratios are 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 to the carrier is 1:4-1: 5.
Further, in the carrier, the mass ratio of the dry dextrin to the maltodextrin is 2:1-1:2, and preferably 1: 1.
Further, the step (1) comprises the steps of:
s1, immunizing laying peak young chickens with the vaccines of swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis, and then taking high-immunity eggs laid by the chickens;
s2, separating the yolk of the hyperimmune egg, removing oil by a poloxamer precipitation method, and collecting the supernatant containing yolk antibody;
and S3, taking the supernatant obtained in the step S2, and performing ultrafiltration concentration by a concentration separation ultrafiltration system to obtain the compound.
Preferably, the immunization strategy in S1 is: the swine fever attenuated vaccine, the porcine pseudorabies attenuated vaccine and the porcine epidemic diarrhea-porcine transmissible gastroenteritis bivalent attenuated vaccine are respectively used for immunizing 3 times, and then the porcine epidemic diarrhea-porcine transmissible gastroenteritis bivalent inactivated vaccine is used for immunizing 2 times.
The invention further provides a preparation method of the quadruple yolk antibody soluble powder for resisting swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis, which comprises the following steps:
(1) preparing a quadruple yolk antibody solution for resisting swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis;
(2) adding fulvic acid, sucrose, potassium sorbate and sodium azide into the tetrad yolk antibody solution obtained in the step (1) to obtain a tetrad yolk antibody combined solution;
(3) and (3) spraying the tetrad yolk antibody combination solution obtained in the step (2) on the carrier.
Further, the addition mass ratios of the fulvic acid, the sucrose, the potassium sorbate and the sodium azide to the volume ratio of the quadruple yolk antibody solution are respectively as follows: 3-5%, 15-20%, 0.01-0.03% and 0.01-0.02%; preferably, the volume ratios are respectively as follows: 5%, 15%, 0.02% and 0.02%;
and/or the presence of a gas in the gas,
the mass ratio of the tetrad yolk antibody combination solution to the carrier is 1:4-1: 5;
and/or the presence of a gas in the gas,
in the carrier, the mass ratio of the dry dextrin to the maltodextrin is 2:1-1:2, and preferably 1: 1.
More preferably, the step (1) includes the steps of:
s1, immunizing laying peak young chickens with the vaccines of swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis, and then taking high-immunity eggs laid by the chickens;
the preferred immunization strategy is: the swine fever attenuated vaccine, the porcine pseudorabies attenuated vaccine and the porcine epidemic diarrhea-porcine transmissible gastroenteritis bivalent attenuated vaccine are respectively used for immunizing 3 times, and then the porcine epidemic diarrhea-porcine transmissible gastroenteritis bivalent inactivated vaccine is used for immunizing 2 times;
s2, separating the yolk of the hyperimmune egg, removing oil by a poloxamer precipitation method, and collecting the supernatant containing yolk antibody;
and S3, taking the supernatant obtained in the step S2, and performing ultrafiltration concentration by a concentration separation ultrafiltration system to obtain the compound.
Optionally, vaccines S1, namely the swine fever live vaccine and the porcine pseudorabies live vaccine, are purchased from Shandong Ludu Biotech limited; the porcine transmissible gastroenteritis and porcine epidemic diarrhea bigeminal live vaccine is purchased from Dabei nong; the bivalent inactivated vaccine for the transmissible gastroenteritis and the porcine epidemic diarrhea is purchased from Wuhan pre-biological GmbH.
Preferably, S2 is specifically: stirring the separated egg yolk and deionized water until the egg yolk and the deionized water are completely mixed to obtain an egg yolk aqueous solution; adding poloxamer solution to make the concentration of poloxamer in the mixed solution not exceed 1.0 w/v% -1.2 w/v%, stirring, standing to precipitate yolk oil, and separating and extracting supernatant; filtering the supernatant to remove impurities, adding poloxamer solution again to make the poloxamer concentration in the mixed solution be 0.5 w/v%, stirring, standing, separating residual oil from the egg yolk again to obtain crude separated egg yolk antibody solution, i.e. supernatant containing egg yolk antibody.
Further, the environmental temperature of standing after the poloxamer solution is added for the first time is not more than 10 ℃, and the standing time is not less than 8 hours.
More preferably, the step of removing oil and fat by using poloxamer precipitation and collecting the supernatant containing the egg yolk antibody comprises the following steps:
a) preparing a 10% (w/v) poloxamer solution with deionized water, for example, adding 1 kg of poloxamer into 10L of deionized water, and stirring for dissolving;
b) mixing the separated yolk and deionized water according to the proportion of 1: 6, and stirring forcefully to completely mix the yolk and water to obtain a yolk aqueous solution;
c) adding the 10% (w/v) poloxamer solution prepared in the step a) into the yolk aqueous solution obtained in the step b), enabling the concentration of poloxamer in the mixed solution to be 1% (w/v), and stirring forcefully to obtain a yolk and poloxamer aqueous solution A; standing at 4-10 deg.C for 8-12 hr to precipitate yolk oil, and separating and extracting supernatant;
d) filtering the supernatant obtained in the step c) by four layers of medical gauze to remove impurities, thereby obtaining a yolk antibody solution;
e) adding 10% (w/v) poloxamer solution into the egg yolk antibody solution obtained in the step d) to enable the concentration of poloxamer in the mixed solution to be 0.5% (w/v), stirring forcefully and uniformly to obtain egg yolk and poloxamer aqueous solution B; standing for 2-4 hours at the temperature of 4-10 ℃, and separating residual oil from the precipitated egg yolk again;
the specific operation can be as follows: and d) removing the uppermost layer of grease by using kitchen paper, and filtering the solution according to the method in the step d) to obtain a crude separated egg yolk antibody solution.
Preferably, S3 is obtained by collecting supernatant obtained from S2, adding into a liquid storage tank of an ultrafiltration concentration separation system, using an ultrafiltration membrane with molecular weight cutoff of 20000Da, and controlling the operation pressure of 0.05-0.15MPa and the flow rate of concentrate reflux at 15-20m3And (4) carrying out ultrafiltration concentration separation under the condition of/h to obtain a refined tetrad yolk antibody aqueous solution.
After the preparation of the quadruple antibody solution for resisting swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis in the technical scheme is finished, the specific antibody titer of the product is determined, for example, by using an ELISA method, and the titer of the product is determined by using swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis kits respectively.
It will be appreciated that the above preferred conditions, in accordance with common knowledge in the art, may be combined to arrive at specific embodiments.
The invention has the beneficial effects that:
the soluble quadruple yolk antibody powder for resisting swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis, prepared by the method, is a preparation with specificity, good treatment effect and quick treatment effect on the swine fever, the porcine pseudorabies, the porcine epidemic diarrhea and the porcine transmissible gastroenteritis, and can effectively prevent and treat the four infectious diseases of the pigs.
The invention takes dry dextrin and maltodextrin as carriers, and sprays the tetragenous egg yolk antibody which is added with fulvic acid, sucrose, potassium sorbate and sodium azide and is used for resisting swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis on the surface of the carrier to prepare soluble powder which has high purity, high stability and high curative effect and is easy to store, has the advantages of convenient transportation and low requirement on storage environment temperature compared with liquid egg yolk antibody, and only needs to be placed in ventilated and cool space; compared with the yolk antibody freeze-dried powder, the yolk antibody freeze-dried powder does not need freeze drying equipment, has low cost and is suitable for large-scale production; compared with antibiotics and antiviral drugs, the product of the invention has the advantages of no residue, no side effect and the like, and is suitable for large-scale popularization and use.
Drawings
FIG. 1 shows the change in antibody titer at room temperature for different periods of time in Experimental example 1 of the present invention.
Detailed Description
The present invention is further illustrated by the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The vaccines used in the examples, namely the classical swine fever live vaccine and the porcine pseudorabies live vaccine, are purchased from Shandong Ludu Biotech limited; the porcine transmissible gastroenteritis and porcine epidemic diarrhea bigeminal live vaccine is purchased from Dabei nong; the bivalent inactivated vaccine for the transmissible gastroenteritis and the porcine epidemic diarrhea is purchased from Wuhan pre-biological GmbH.
Example 1
This example illustrates the preparation of a tetra-immune egg for swine fever, pseudorabies, epidemic diarrhea and transmissible gastroenteritis by the following specific method:
(1) healthy and non-immunized laying peak young chickens are selected to be fed in an isolation mode, and feed nutrition and environmental sanitation are enhanced to prevent pathogenic infection;
(2) immunizing the young high-yield laying hens treated in the step (1) by using the vaccines of swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine gastroenteritis, and specifically comprising the following steps:
for the first time: immunizing a porcine epidemic diarrhea-transmissible gastroenteritis bivalent attenuated vaccine, a bivalent inactivated vaccine and a swine fever live vaccine, immunizing chickens according to the injection amount of 1/5 porcine doses (namely immunizing 100 chickens by 20 parts of the porcine vaccine), wherein the injection way is the neck subcutaneous injection of the porcine epidemic diarrhea-transmissible gastroenteritis bivalent inactivated vaccine, and the thigh intramuscular injection of the porcine epidemic diarrhea-transmissible gastroenteritis bivalent attenuated vaccine and the swine fever attenuated vaccine; and (3) for the second time: after 2 weeks, the epidemic diarrhea-transmissible gastroenteritis bivalent attenuated vaccine, the swine fever attenuated vaccine and the pseudorabies attenuated vaccine of the immunized pig are respectively injected into thigh muscles according to the dose of 1/5 pigs; and thirdly: injecting swine fever and swine pseudorabies attenuated vaccine into thigh muscle of each chicken according to 1/5 pig dose after 2 weeks; fourth time: and injecting 1/5 pig dose of the porcine epidemic diarrhea-transmissible gastroenteritis combined inactivated vaccine subcutaneously at the neck after 2 weeks, and injecting the porcine pseudorabies attenuated vaccine intramuscularly at the thigh according to 1/5 pig dose.
(3) And (4) after immunizing the high-yield young laying hens, collecting the high-immunity eggs.
Example 2
This example is intended to illustrate the preparation of a quadruple yolk antibody solution using the hyperimmune egg obtained in example 1, by the following specific method:
(1) separating the high-immunity egg yolk:
specificity of antigen production in egg yolk for the corresponding vaccine was assayed for samples taken every 7 days starting 1 week after the second immunization of each vaccine as described in example 1Antibody titers were compared to control with the yolk of the immunized egg. When the egg yolk OD is not immune450The value is less than 0.2, and the specific antibody OD of egg yolk antibody of the immune vaccine450Non-immune egg yolk OD450When the ratio is more than 5.0, collecting the separated egg yolk, wherein the specific method for separating the egg yolk comprises the following steps: placing the high-immunity eggs obtained in the example 1 into a closed container or a house, fumigating and sterilizing the eggs for 30 minutes by peroxyacetic acid, and then sending the eggs into a yolk separator for yolk separation;
(2) preparing a refined egg yolk antibody:
mixing the yolk separated in the step (1) with a mixture of 1: adding deionized water according to the proportion (the amount of the deionized water in the proportion also comprises the volume of poloxamer solution added in the subsequent operation), and violently stirring to fully and uniformly mix the egg yolk and the water to obtain an egg yolk aqueous solution; adding 10% poloxamer solution into the obtained yolk aqueous solution to ensure that the poloxamer concentration in the yolk solution and the poloxamer mixed solution is 1% (W/V), immediately stirring and fully mixing, weighing 1 kg of poloxamer in the 10% (W/V) poloxamer solution, adding 10L of deionized water into the solution, and stirring and fully dissolving to obtain the yolk aqueous solution; standing the obtained poloxamer egg yolk aqueous solution at 4-8 ℃ for 8-12 hours, firstly removing a layer of grease and floating foam on the surface of the egg yolk aqueous solution by using kitchen paper, then filtering supernatant by using 4 layers of medical gauze, adding 10% (W/V) poloxamer aqueous solution into the filtered egg yolk aqueous solution to ensure that the poloxamer concentration in the mixed solution of egg yolk and poloxamer is 0.5% (W/V), fully and uniformly mixing, standing at 4-8 ℃ for 2-4 hours, then removing upper layer of grease, and filtering the supernatant by using 4 layers of medical gauze; subjecting yolk antibody water solution to remove oil by poloxamer precipitation twice to ultrafiltration membrane with cut-off molecular weight of 20000Da under operating pressure of 0.05-0.15Mpa and flow rate of concentrated reflux liquid of 15-20m3And (4) carrying out ultrafiltration concentration separation under the condition of/h to obtain a tetrad yolk antibody solution.
Example 3
This example illustrates the preparation of a quadruple yolk antibody combination solution.
5% (w/v) fulvic acid, 15% (w/v) sucrose, 0.02% (w/v) potassium sorbate and 0.02% (w/v) sodium azide were added to the tetrad yolk antibody solution obtained in example 2, and the mixture was sufficiently stirred.
Example 4
This example illustrates the preparation of soluble powder of tetragenous egg-yolk antibody using dry dextrin and maltodextrin as carriers, which comprises the following steps:
(1) adding dry dextrin and maltodextrin into spray drying equipment according to the weight ratio of 1:1, adjusting the temperature of an air inlet to 70-80 ℃, the temperature of an air outlet to 50-60 ℃, and preheating and mixing the dextrin for 10 min;
(2) the tetrad yolk antibody composite solution obtained in the example 3 is sprayed on mixed dextrin at the flow rate of 150-.
Comparative example 1
This comparative example differs from example 4 in that: the tetrad yolk antibody combination solution used was: and (3) adding 5% (w/v) fulvic acid, 15% (w/v) sucrose and 0.02% (w/v) potassium sorbate into the tetrad yolk antibody solution obtained in example 2, and fully stirring to obtain the yolk antibody.
Comparative example 2
This comparative example differs from example 4 in that: the tetrad yolk antibody combination solution used was: and (3) adding 5% (w/v) fulvic acid, 15% (w/v) sucrose and 0.02% (w/v) sodium azide into the tetrad yolk antibody solution obtained in the example 2, and fully stirring to obtain the yolk antibody.
Comparative example 3
This comparative example differs from example 4 in that: the tetrad yolk antibody combination solution used was: 15% (w/v) sucrose, 0.02% (w/v) potassium sorbate and 0.02% (w/v) sodium azide were added to the tetrad yolk antibody solution obtained in example 2, and the mixture was sufficiently stirred.
Comparative example 4
This comparative example differs from example 4 in that: the tetrad yolk antibody combination solution used was: 5% (w/v) fulvic acid, 0.02% (w/v) potassium sorbate and 0.02% (w/v) sodium azide were added to the tetrad yolk antibody solution obtained in example 2, and the mixture was sufficiently stirred.
Comparative example 5
This comparative example differs from example 4 in that: the quadruple yolk antibody combination solution obtained in example 3 was replaced with the quadruple yolk antibody solution obtained in example 2, and no other components were added.
Experimental example 1
This example is intended to illustrate the product performance and quality testing results for example 4 and comparative examples 1-5.
(1) The detected material is as follows: the tetrad yolk antibody soluble powders prepared in example 4 (experimental group) and comparative examples 1 to 5 (corresponding groups in order: comparative groups 1 to 5).
(2) The test method comprises the following steps: placing the product in the step (1) at normal temperature (20-25 ℃) for 18 months, weighing samples at the time points after the 1 st month, the 2 nd month, the 3 rd month, the 6 th month, the 9 th month, the 12 th month, the 15 th month and the 18 th month respectively, adding deionized water to prepare 30% (w/v) antibody solution (S), simultaneously diluting the egg yolk antibody solution which is produced in the same batch and is not treated with any treatment into 6% (v/v) egg yolk solution as a control (C), and detecting the antibody titer by an ELISA method. Retention rate of antibody titer: by OD450S/OD450C100% for calculation.
(3) And (3) test results:
test results of normal temperature preservation: the retention rate of antibody titer was plotted on the ordinate and the sampling time on the abscissa to generate different antibody titer attenuation maps, and the results are shown in FIG. 1.
In fig. 1, it is shown that when the soluble powder (dry powder) is stored at room temperature for 18 months, the antibody titer of the test group is retained by more than 85%, the reduction is the slowest, and the reduction speed is successively the control group 3, the control group 2, the control group 4, the control group 1 and the control group 5 from slow to fast. From the test results it can be seen that the stability of the formulation used in example 4 is the best.
Quality standard:
(1) the characteristics are as follows: a yellowish or yellow homogeneous powder;
(2) solubility: is easily soluble in water;
(3) and (3) detection of mycoplasma: the method is carried out according to Chinese veterinary pharmacy, and mycoplasma does not grow;
(4) and (3) detecting the exogenous viruses: the method is carried out according to the Chinese animal pharmacy, and no exogenous virus pollution exists;
(5) and (3) mould detection: the method is carried out according to Chinese animal pharmacy, and meets the standard;
(6) shelf life: according to the result of detecting the titer of the antibody at normal temperature, the antibody can be stored for at least 18 months at normal temperature.
(7) And (3) safety inspection: selecting 7-day-old SPF chickens, 10 SPF chickens, and keeping the weight of each SPF chicken at 45 g/kg (3 times of the usage amount), continuously using for 3 days, observing for 21 days, keeping normal spirit, and keeping food intake and drinking water normal, and keeping the chickens alive.
Experimental example 2
This example describes the efficacy assay of the products of example 4 and comparative examples 1-5 for classical swine fever.
The products tested in this example were tetrad yolk antibody soluble powders prepared in example 4 (test group) and comparative examples 1-5 (corresponding groups in order: comparative groups 1-5).
The experimental animals select 80 healthy piglets of 7 days old.
The test method comprises the following steps: selecting 7-day-old clean-grade Guangxi Bama miniature pigs, carrying out isolated feeding observation for 1 week, randomly dividing the pigs into 8 groups, and carrying out isolated feeding on 10 pigs in each group. 8 groups were blank control groups, respectively, without any product; the virus challenge group, the test group and the comparison group were administered 3-5 ml of normal saline 24 hours after each pig was injected intramuscularly with the diluted classical swine fever virus solution, the test group was administered 3-5 ml of the product prepared in example 4 dissolved in normal saline, the comparison group 1-5 was administered 3-5 ml of the product prepared in comparative example 1-5 dissolved in normal saline sequentially, the dry powder products used in the test group and the comparison group 1-5 were administered at a weight of 15 g/kg for 3 days continuously, and the observation was carried out for 7 days, and the results are shown in Table 1.
Evaluation criteria of efficacy:
and (3) curing: normal spirit, normal drinking and eating, normal feces and no symptoms.
The method has the following advantages: listlessness, incomplete recovery of food intake, and alleviation of clinical symptoms.
And (4) invalidation: listlessness, convulsions, fever, severe decline of food intake or no food intake eventually lead to death.
TABLE 1 therapeutic effect of different groups
| Group of | Number of onset (head) | Number of cure (head) | Effective number (head) | Cure rate (%) |
| Test group | 8 | 7 | 1 | 88 |
| Toxin counteracting group | 10 | 1 | 1 | 10 |
| Comparative group 1 | 10 | 3 | 2 | 30 |
| Comparative group 2 | 10 | 6 | 3 | 60 |
| Comparative group 3 | 10 | 6 | 4 | 60 |
| Comparative group 4 | 10 | 5 | 3 | 50 |
| Comparative group 5 | 10 | 2 | 2 | 20 |
| Blank control | 0 | / | / | / |
As can be seen from Table 1, the curative ratio of the test group to the swine fever can reach 88%, the curative ratio of the comparison groups 1-5 to the swine fever is within the range of 20-60%, and statistical analysis shows that the curative effect of the test group to the swine fever is obviously higher than that of the comparison groups 1-5(p < 0.01).
Experimental example 3
This example describes the efficacy assay of the products of example 4 and comparative examples 1-5 for treatment of porcine pseudorabies.
The products tested in this example were tetrad yolk antibody soluble powders prepared in example 4 (test group) and comparative examples 1-5 (corresponding groups in order: comparative groups 1-5).
The experimental animals select 80 healthy piglets of 7 days old.
The test method comprises the following steps: selecting 7-day-old clean-grade Guangxi Bama miniature pigs, carrying out isolated feeding observation for 1 week, randomly dividing the pigs into 8 groups, and carrying out isolated feeding on 10 pigs in each group. 8 groups were blank control groups, respectively, without any product; the challenge group, the test group and the comparison group were administered 3-5 ml of physiological saline 24 hours after each pig was injected intramuscularly with diluted porcine pseudorabies virus solution, the test group was administered 3-5 ml of the product prepared in example 4 dissolved in physiological saline, the comparison group 1-5 was sequentially administered 3-5 ml of the product prepared in comparative examples 1-5 dissolved in physiological saline, the dry powder products used in the test group and the comparison group 1-5 were administered at a weight of 15 g/kg for 3 days continuously, and the observation was carried out for 7 days, and the results are shown in Table 2.
Evaluation criteria of efficacy:
and (3) curing: normal spirit, normal eating and drinking, normal body temperature and no symptoms.
The method has the following advantages: lassitude, gradually increased food intake, decreased body temperature, no syncope, and no vomiting.
And (4) invalidation: listlessness, syncope, persistent high fever, no appetite, vomiting and death.
TABLE 2 therapeutic effect of different groups
As can be seen from Table 2, the cure rate of the test group on the porcine pseudorabies can reach 90 percent, and is within the range of 30 to 70 percent compared with the cure rate of the test group 1 to 5 on the porcine pseudorabies. Statistical analysis shows that the therapeutic effect of the test group on the porcine pseudorabies is remarkably higher than that of the control group 1-5(P < 0.01).
Experimental example 4
This example describes an assay of the efficacy of the products of example 4 and comparative examples 1-5 in the treatment of porcine epidemic diarrhea.
The products tested in this example were tetrad yolk antibody soluble powders prepared in example 4 (test group) and comparative examples 1-5 (corresponding groups in order: comparative groups 1-5).
The experimental animals select 80 healthy piglets of 7 days old.
The test method comprises the following steps: selecting 7-day-old clean-grade Guangxi Bama miniature pigs, carrying out isolated feeding observation for 1 week, randomly dividing the pigs into 8 groups, and carrying out isolated feeding on 10 pigs in each group. 8 groups were blank control groups, respectively, without any product; the challenge group, the test group and the comparison group were administered 3-5 ml of physiological saline 24 hours after each pig was injected intramuscularly with the diluted porcine epidemic diarrhea virus solution, the test group was administered 3-5 ml of the product prepared in example 4 dissolved with physiological saline, the comparison groups 1-5 were sequentially administered 3-5 ml of the products prepared in comparative examples 1-5 dissolved with physiological saline, the dry powder products used in the test group and the comparison groups 1-5 were administered at 15 g/kg body weight for 3 days, and the results were observed for 7 days and shown in table 3.
Evaluation criteria of efficacy:
and (3) curing: normal spirit, normal drinking and eating, normal body temperature and normal feces and has no any symptom.
The method has the following advantages: the spirit is not vibrating, the food intake is gradually increased, and the symptoms of diarrhea and vomiting are improved.
And (4) invalidation: listlessness, high body temperature, water-sampling, foul stool, no food intake, continuous vomiting and death.
TABLE 3 therapeutic effect of different groups
As can be seen from Table 3, the cure rate of the experimental group on the porcine epidemic diarrhea reaches 90%, and the cure rate of the comparative groups 1 to 5 on the porcine epidemic diarrhea is 20 to 70%. Statistical analysis shows that the curative effect of the experimental group on the porcine epidemic diarrhea is obviously higher than that of the comparative group 1-5(P < 0.01).
Experimental example 5
This example describes the efficacy assay of the products of example 4 and comparative examples 1-5 in transmissible gastroenteritis of swine.
The products tested in this example were tetrad yolk antibody soluble powders prepared in example 4 (test group) and comparative examples 1-5 (corresponding groups in order: comparative groups 1-5).
The experimental animals select 80 healthy piglets of 7 days old.
The test method comprises the following steps: selecting 7-day-old clean-grade Guangxi Bama miniature pigs, carrying out isolated feeding observation for 1 week, randomly dividing the pigs into 8 groups, and carrying out isolated feeding on 10 pigs in each group. 8 groups were blank control groups, respectively, without any product; the challenge group, the test group and the comparison group were administered 3-5 ml of physiological saline 24 hours after the injection of the diluted transmissible gastroenteritis virus solution into the muscle of each pig, the test group was administered 3-5 ml of the product prepared in example 4 dissolved in physiological saline, the comparison groups 1-5 were sequentially administered 3-5 ml of the products prepared in comparative examples 1-5 dissolved in physiological saline, the dry powder products used in the test group and the comparison groups 1-5 were administered at a weight of 15 g/kg for 3 days continuously, and the observation was carried out for 7 days, with the results shown in Table 4.
Evaluation criteria of efficacy:
and (3) curing: normal spirit, normal drinking and eating, normal body temperature and normal feces and has no any symptom.
The method has the following advantages: the spirit is not vibrating, the food intake is gradually increased, the symptoms of diarrhea and vomiting are obviously improved, and the body temperature tends to be normal.
And (4) invalidation: listlessness, increased body temperature, diarrhea and vomiting, no ingestion and death.
TABLE 4 therapeutic effects of different groups
As can be seen from Table 4, the cure rate of the test group on transmissible gastroenteritis of swine reaches 89%, and the cure rate of the comparative groups 1-5 on porcine epidemic diarrhea reaches 30-70%. Statistical analysis shows that the curative effect of the experimental group on the porcine epidemic diarrhea is obviously higher than that of the comparative group 1-5(P < 0.01).
According to the experimental results of the experimental examples 2-5, the soluble powder of the quadruple yolk antibody prepared in the example 4 of the invention has obvious curative effects on swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis, namely, the curative effects and the stability of the yolk antibody are obviously improved by using fulvic acid, potassium sorbate, sucrose and sodium azide in a matching way.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (14)
1. The soluble powder of the quadruple yolk antibody for resisting swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis is characterized in that dry dextrin and maltodextrin are used as carriers, the quadruple yolk antibody for resisting swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis is used as an active ingredient, and fulvic acid, sucrose, potassium sorbate and sodium azide are used as auxiliary agents of the active ingredient.
2. The soluble quadruple yolk antibody powder according to claim 1, wherein the preparation method comprises the following steps:
(1) preparing a quadruple yolk antibody solution for resisting swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis;
(2) adding fulvic acid, sucrose, potassium sorbate and sodium azide into the tetrad yolk antibody solution obtained in the step (1) to obtain a tetrad yolk antibody combined solution;
(3) and (3) spraying the tetrad yolk antibody combination solution obtained in the step (2) on the carrier.
3. The soluble powder of quadruple yolk antibodies according to claim 2, wherein in the step (2), the addition mass ratios of the fulvic acid, the sucrose, the potassium sorbate and the sodium azide relative to the volume ratio of the quadruple yolk antibody solution are respectively as follows: 3-5%, 15-20%, 0.01-0.03% and 0.01-0.02%.
4. The soluble powder of quadruple yolk antibodies according to claim 3, wherein in the step (2), the addition mass ratios of the fulvic acid, the sucrose, the potassium sorbate and the sodium azide relative to the volume ratio of the quadruple yolk antibody solution are respectively as follows: 5%, 15%, 0.02% and 0.02%.
5. The soluble quadruple yolk antibody powder of any one of claims 2 to 4, wherein in the step (3), the mass ratio of the quadruple yolk antibody combination solution to the carrier is 1:4-1: 5.
6. The tetrad yolk antibody soluble powder of any one of claims 1 to 4, wherein the carrier comprises the dry dextrin and the maltodextrin in a mass ratio of 2:1 to 1: 2.
7. The tetrad yolk antibody soluble powder as claimed in claim 6, wherein the carrier has a mass ratio of dry dextrin to maltodextrin of 1: 1.
8. The tetrad yolk antibody soluble powder according to any one of claims 1 to 4, wherein the step (1) comprises the steps of:
s1, immunizing laying peak young chickens with the vaccines of swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis, and then taking high-immunity eggs laid by the chickens;
s2, separating the yolk of the hyperimmune egg, removing oil by a poloxamer precipitation method, and collecting the supernatant containing yolk antibody;
and S3, taking the supernatant obtained in the step S2, and performing ultrafiltration concentration by a concentration separation ultrafiltration system to obtain the compound.
9. The soluble powder of quadruple yolk antibodies according to claim 8, wherein the immunization strategy in S1 is: the swine fever attenuated vaccine, the porcine pseudorabies attenuated vaccine and the porcine epidemic diarrhea-porcine transmissible gastroenteritis bivalent attenuated vaccine are respectively used for immunizing 3 times, and then the porcine epidemic diarrhea-porcine transmissible gastroenteritis bivalent inactivated vaccine is used for immunizing 2 times.
10. A preparation method of a tetrad yolk antibody soluble powder for resisting swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis is characterized by comprising the following steps:
(1) preparing a quadruple yolk antibody solution for resisting swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis;
(2) adding fulvic acid, sucrose, potassium sorbate and sodium azide into the tetrad yolk antibody solution obtained in the step (1) to obtain a tetrad yolk antibody combined solution;
(3) and (3) spraying the tetrad yolk antibody combination solution obtained in the step (2) on the carrier.
11. The preparation method according to claim 10, wherein the addition mass ratios of the fulvic acid, the sucrose, the potassium sorbate and the sodium azide to the tetragenous egg-yolk antibody solution are respectively: 3-5%, 15-20%, 0.01-0.03% and 0.01-0.02%;
and/or the presence of a gas in the gas,
the mass ratio of the tetrad yolk antibody combination solution to the carrier is 1:4-1: 5;
and/or the presence of a gas in the gas,
in the carrier, the mass ratio of the dry dextrin to the maltodextrin is 2:1-1: 2.
12. The preparation method according to claim 11, wherein the addition mass ratios of the fulvic acid, the sucrose, the potassium sorbate and the sodium azide to the tetragenous egg yolk antibody solution are respectively: 5%, 15%, 0.02% and 0.02%;
and/or the presence of a gas in the gas,
in the carrier, the mass ratio of the dry dextrin to the maltodextrin is 1: 1.
13. The production method according to claim 11 or 12, wherein the step (1) comprises the steps of:
s1, immunizing laying peak young chickens with the vaccines of swine fever, porcine pseudorabies, porcine epidemic diarrhea and porcine transmissible gastroenteritis, and then taking high-immunity eggs laid by the chickens;
s2, separating the yolk of the hyperimmune egg, removing oil by a poloxamer precipitation method, and collecting the supernatant containing yolk antibody;
and S3, taking the supernatant obtained in the step S2, and performing ultrafiltration concentration by a concentration separation ultrafiltration system to obtain the compound.
14. The method of claim 13, wherein the immunization strategy of step S1 is: the swine fever attenuated vaccine, the porcine pseudorabies attenuated vaccine and the porcine epidemic diarrhea-porcine transmissible gastroenteritis bivalent attenuated vaccine are respectively used for immunizing 3 times, and then the porcine epidemic diarrhea-porcine transmissible gastroenteritis bivalent inactivated vaccine is used for immunizing 2 times.
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| 三联卵黄抗体口服液防控仔猪腹泻临床效果观察;文贤周;《中国动物保健》;20151231;第17卷(第6期);全文 * |
| 抗猪传染性胃肠炎病毒与猪流行性腹泻病毒卵黄抗体制备及其临床应用研究;崔焕忠 等;《中国畜牧兽医》;20121231;第39卷(第6期);全文 * |
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