CN110025781B - Vaccine composition and preparation method thereof - Google Patents

Vaccine composition and preparation method thereof Download PDF

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CN110025781B
CN110025781B CN201910338040.XA CN201910338040A CN110025781B CN 110025781 B CN110025781 B CN 110025781B CN 201910338040 A CN201910338040 A CN 201910338040A CN 110025781 B CN110025781 B CN 110025781B
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antigen
pcv2
ppv
prv
prrsv
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CN110025781A (en
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李遥
杨彩娟
易太江
张丰昌
陈晓威
吴程雨
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Guangdong Provincial Animal Epidemic Prevention Material Reserve Center
Hangzhou Muda Intelligent Technology Co ltd
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Guangdong Provincial Animal Epidemic Prevention Material Reserve Center
Hangzhou Muda Intelligent Technology Co ltd
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A—HUMAN NECESSITIES
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    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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    • A61P31/12—Antivirals
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
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    • A61K2039/55588—Adjuvants of undefined constitution
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Abstract

The invention belongs to the technical field of animal vaccines, and particularly discloses a vaccine composition which comprises, by weight, 20-25 parts of PCV2 antigen, 20-25 parts of PRV antigen, 20-25 parts of PPV antigen, 20-25 parts of PRRSV antigen, 0.05-0.1 part of bovine serum albumin, 0.5-1 part of polyvinylpyrrolidone, 0.2-0.5 part of glycine, 0.2-0.5 part of L-sodium glutamate and 0.1-0.2 part of sucrose. The vaccine composition is prepared by collecting the antigen, the lyophilized composition stock solution and the lyophilized composition stock solution, ensures the effective efficacies of the four vaccines PCV2, PRV, PPV and PRRSV, can better prevent diseases caused by PCV2, PRV, PPV and PRRSV, and is convenient for the mass production of vaccine conjugates.

Description

Vaccine composition and preparation method thereof
Technical Field
The invention belongs to the technical field of animal vaccines, and particularly relates to a vaccine composition and a preparation method thereof.
Background
Porcine circovirus disease, a disease caused by porcine circovirus and having a variety of clinical symptoms, such as anorexia, dyspnea or bradykinesia. Porcine Circovirus (PCV) is the smallest animal virus found to date. PCV is now known of two serotypes, PCV1 and PCV 2. Among them, PCV2 is a pathogenic virus and is the main cause of Postweaning Multisystemic Wasting Syndrome (PMWS). The pig has strong susceptibility to PCV2, and infected pigs can discharge viruses from nasal fluid, excrement and other wastes and infect pigs of different ages through oral and respiratory pathways. After the pregnant sow is infected with PCV2, the pregnant sow can vertically spread through placenta to infect piglets.
Many studies have shown that PCV2 infection alone does not cause the symptoms typical of postweaning multisystemic wasting syndrome, whereas activation of the immune system and mixed infection with other pathogens can contribute to the onset of the symptoms. The most common mixed infection is porcine pseudorabies virus (PRV), Porcine Parvovirus (PPV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and the death rate of infected pigs is greatly improved due to double infection or multiple infection of some of the mixed infections.
In the market, a single vaccine only aiming at one disease is mostly adopted for prevention and control of the diseases, the immunization process needs to be carried out for multiple times, the problems of high cost and complex operation procedure exist, meanwhile, the infection risk is increased, the stress response of the swinery is increased, the immunity of part of swineries is failed, and a large amount of manpower and material resources are wasted.
In order to effectively solve the problem of mixed infection of PCV2 with PRV, PPV, PRRSV, there is a need in the art for a vaccine composition containing four pathogenic antigenic components to prevent four diseases simultaneously. However, the biggest problem that needs to be solved for combination vaccines at present is the recently identified possibility that one vaccine may have an adverse effect on another vaccine in the combination vaccine, i.e. "antigen interference effect". It was found by the skilled person that one or both of the vaccines of the combination vaccine may lose potency when simply mixing the two existing vaccines (see patent application CN 101035559A).
Therefore, providing a combination vaccine that maintains the efficacy of the original vaccine is a technical problem that those skilled in the art continue to solve.
Disclosure of Invention
Aiming at the defects in the prior art, the first purpose of the invention is to provide a vaccine composition which contains four antigens of PCV2, PRV, PPV and PRRSV, so that the effective efficacy of each vaccine is ensured, diseases caused by PCV2, PRV, PPV and PRRSV can be well prevented, and the clinical stress response is reduced.
It is a second object of the present invention to provide a method for preparing a vaccine composition, which is simple and convenient to operate and facilitates mass production of vaccine conjugates.
In order to achieve the first object, the invention provides the following technical scheme:
the vaccine composition comprises, by weight, 20-25 parts of PCV2 antigen, 20-25 parts of PRV antigen, 20-25 parts of PPV antigen, 20-25 parts of PRRSV antigen, 0.05-0.1 part of bovine serum albumin, 0.5-1 part of polyvinylpyrrolidone, 0.2-0.5 part of glycine, 0.2-0.5 part of L-sodium glutamate and 0.1-0.2 part of sucrose.
Preferably, the vaccine composition comprises 25 parts by weight of PCV2 antigen, 20 parts by weight of PRV antigen, 20 parts by weight of PPV antigen, 20 parts by weight of PRRSV antigen, 0.08 part by weight of bovine serum albumin, 0.8 part by weight of polyvinylpyrrolidone, 0.3 part by weight of glycine, 0.3 part by weight of L-sodium glutamate and 0.2 part by weight of sucrose.
By adopting the technical scheme, the vaccine composition contains four antigens of PCV2 antigen, PRV antigen, PPV antigen and PRRSV antigen, can effectively prevent and treat diseases caused by porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV), Porcine Parvovirus (PPV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) under the assistance of bovine serum albumin, polyvinylpyrrolidone, glycine, L-sodium glutamate and sucrose, has small side effect, high serum antibody titer, long immune period, less seeding times and small influence on pigs compared with the effect of single seedlings, simplifies immune procedures, reduces the breeding cost and the disease prevention cost of the pigs, and has good practicability.
In addition, most vaccines are not heat-resistant, so that the vaccines need to be carried out at a lower temperature in the processes of production, transportation, storage, sale and use, namely, the vaccines are called as 'cold chains', so that the cost of cold chain operation is extremely high due to the fact that the cold chains are connected in a ring-by-ring mode, the vaccine composition can greatly reduce the cost of cold chain operation, and has remarkable superiority.
Among them, PCV2 antigen is preferably a whole virus antigen of porcine circovirus type 2, in inactivated, modified live or attenuated form, a chimeric virus containing an immunogenic amino acid sequence of porcine circovirus type 2, a polypeptide or subunit component containing an immunogenic amino acid sequence of porcine circovirus type 2.
The PRV antigen is preferably a porcine pseudorabies whole virus antigen in an inactivated, modified live or attenuated form, a chimeric virus comprising a porcine pseudorabies immunogenic amino acid sequence, a polypeptide or subunit component comprising a porcine pseudorabies immunogenic amino acid sequence.
The PPV antigen is preferably a porcine parvovirus antigen in an inactivated, modified live or attenuated form, a chimeric virus comprising a porcine parvoimmunogenic amino acid sequence, a polypeptide or subunit component comprising a porcine parvoimmunogenic amino acid sequence.
The PRRSV antigen is preferably a porcine reproductive and respiratory syndrome virus antigen in an inactivated, modified live or attenuated form, a chimeric virus comprising an immunogenic amino acid sequence of porcine reproductive and respiratory syndrome, a polypeptide or subunit component comprising an immunogenic amino acid sequence of porcine reproductive and respiratory syndrome.
Preferably, the PCV2 antigen is derived from PCV2 SH strain.
Preferably, the PRV antigen is derived from Bartha-K61 strain.
Preferably, the PPV antigen is derived from HN-2011 strain.
Preferably, the PRRSV antigen is derived from the TJM strain.
By adopting the technical scheme, the vaccine composition provided by the invention selects PCV2 SH strain, Bartha-K61 strain, HN-2011 strain and NVDC-JXA1 strain, and has the characteristics of high antigen content, strong immunogenicity, good safety, maternal antibody interference resistance, good immune effect and the like. Meanwhile, when the four antigens are adopted, the mutual antigen interference effect is small, so that the excellent effective efficacies of the four antigens are ensured.
Preferably, the vaccine composition further comprises 0.2-0.4 parts of propolis.
Preferably, the vaccine composition further comprises 0.3 parts of propolis.
By adopting the technical scheme, the propolis is a colloidal solid substance with aromatic odor formed by mixing the resin collected by bees from tree buds, barks or buds of other plants with substances such as palatine gland secretion, beeswax and the like, has the effects of resisting bacteria and viruses, enhancing the immune function of an organism and the like, and can reduce the endotoxicity of the vaccine composition to a certain degree. The propolis particle can be crosslinked with antigen to form an immunostimulating complex structure, has no antigenicity, but can play a role of an immunoadjuvant, promote the generation of antibodies, and enhance the phagocytosis capability of macrophages and the activity of natural killer cells.
In order to achieve the second object, the invention provides the following technical scheme:
a method of preparing a vaccine composition comprising the steps of:
firstly, collecting the antigen
Respectively proliferating PCV2, PRV, PPV and PRRSV, inactivating the PCV2, PRV, PPV and PRRSV, and collecting PCV2 antigen, PRV antigen, PPV antigen and PRRSV antigen;
② preparing a composition stock solution
Taking the PCV2 antigen according to a proportion, sequentially adding bovine serum albumin, polyvinylpyrrolidone, glycine, L-sodium glutamate, sucrose and other processing auxiliary agents, uniformly mixing, sequentially adding the PRV antigen, the PPV antigen and the PRRSV antigen according to a proportion, and fully and uniformly mixing to obtain a composition stock solution;
③ Freeze-drying composition stock solution
Precooling the composition stock solution prepared in the step (II) at the temperature of minus 20 ℃ for 20-30min, and then freeze-drying the composition stock solution at the temperature of minus 80 ℃ to obtain the vaccine composition.
By adopting the technical scheme, bovine serum albumin, polyvinylpyrrolidone, glycine, sodium L-glutamate, sucrose and other processing auxiliary agents are added into PCV2 antigen, all the components are mutually matched and act synergistically to ensure the effective efficacy of PCV2 antigen, and PRV antigen, PPV antigen and PRRSV antigen are sequentially added for freeze-drying, so that the antigen interference effect is reduced, the vaccine composition can effectively prevent and treat diseases caused by porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV), Porcine Parvovirus (PPV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and the vaccine composition has the characteristics of strong pertinence, high antibody titer, and convenience in transportation and storage, and is convenient for mass production of the vaccine composition.
Preferably, the vaccine composition provided by the invention can be better applied to the preparation of medicines for preventing and treating porcine circovirus disease, porcine pseudorabies virus disease, porcine parvovirus disease and porcine reproductive and respiratory syndrome virus disease, and has a good application prospect.
In conclusion, the invention has the following beneficial effects:
according to the invention, PCV2 antigen, PRV antigen, PPV antigen, PRRSV antigen and various processing auxiliary agents are compounded, and the effective effects of the four antigens are ensured by adjusting the components and the dosage of the auxiliary agents, so that diseases caused by PCV2, PRV, PPV and PRRSV can be well prevented, and the clinical stress response is reduced;
2. the preparation method has the advantages of simple process and convenient operation through the preparation steps of collecting the antigen, preparing the composition stock solution and freeze-drying the composition stock solution, so that the vaccine composition is convenient to store and transport, is suitable for large-scale industrial production, and has good application prospect.
Drawings
Figure 1 is a process flow diagram for preparing a vaccine composition.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
In the specific embodiment of the invention, PCV2, PRV, PPV and PRRSV are used as national standard strains, and the invention is specifically illustrated by PCV2 SH strain, Bartha-K61 strain, HN-2011 strain and TJM strain.
Example 1
Preparation and test of PCV2-PRV-PPV-PRRSV tetrad inactivated vaccine composition
1. Collection of antigens
1.1 preparation of production seed Virus
1.1.1 preparation of PCV2 SH Strain:
diluting PCV2 SH strain virus strain with virus diluent (serum-free MEM cell maintenance liquid), inoculating 0.01MOI (multiplicity of infection) in full monolayer PK-15 cell culture, adsorbing at 37 deg.C for 30min, adding MEM cell maintenance liquid containing 4% (v/v) calf serum and 2mmol/L D-glucosamine hydrochloride, culturing at 37 deg.C for 4 days, freezing for 2-3 times, collecting SH strain virus liquid, and storing at-20 deg.C for no more than 2 months.
1.1.2 preparation of Bartha-K61 Strain:
diluting Bartha-K61 strain virus strain with virus diluent (serum-free MEM cell maintenance solution), inoculating 0.01MOI (multiplicity of infection) to full monolayer PK-15 cell culture, adsorbing at 37 deg.C for 30min, adding MEM cell maintenance solution containing 4% (v/v) calf serum and 2mmol/L D-glucosamine hydrochloride, culturing at 37 deg.C for 4 days, freezing for 2-3 times, collecting Bartha-K61 strain virus solution, and storing at-20 deg.C for no more than 2 months.
1.1.3 preparation of HN-2011 Strain
Diluting PPV HN-2011 strain virus strain with virus diluent (i.e. serum-free MEM cell maintenance solution), inoculating 0.01MOI (multiplicity of infection) to full monolayer ST cell culture, adsorbing at 37 deg.C for 30min, adding MEM cell maintenance solution containing 1% (v/v) calf serum and 2mmol/L D-glucosamine hydrochloride, culturing at 37 deg.C for 4 days, freezing for 2-3 times, harvesting HN-2011 strain virus solution, and storing at-20 deg.C for less than 2 months.
1.1.4 preparation of TJM Strain
Diluting TJM strain virus strain with virus diluent (serum-free MEM cell maintenance solution), inoculating 0.01MOI (multiplicity of infection) to full monolayer ST cell culture, adsorbing at 37 deg.C for 30min, adding MEM cell maintenance solution containing 1% (v/v) calf serum and 2mmol/L D-glucosamine hydrochloride, culturing at 37 deg.C for 4 days, freezing for 2-3 times, collecting TjM strain virus solution, and storing at-20 deg.C for no more than 2 months.
1.2 treatment of the Virus fluid
The four virus solutions in 1.1 were filtered through hollow fiber filter columns (pore size 10 μm and 0.45 μm) to remove cell debris.
1.3 concentration of the Virus solution
The virus liquid of PCV2 SH strain, Bartha-K61 strain, HN-2011 strain and TJM strain which are subjected to cell debris removal at 1.2 is respectively concentrated by an ultrafiltration concentration system, and PCV2 active antigen, PRV active antigen, PPV active antigen and PRRSV active antigen are correspondingly obtained.
1.4 determination of antigen content
1.4.1 measurement of PCV2 active antigen content
The PCV2 active antigen harvested at 1.3 was serially diluted 10-fold with MEM cell maintenance medium, and 10 cells were sampled -5 、10 -6 、10 -7 Three dilutions, each dilution was inoculated to a monolayer of PK-15 cells in 96 wells of culture plate (0.1 mL per well) with normal cell control at 37 deg.C and 5% CO 2 The cells were cultured in the incubator for 24 hours, and the MEM cell maintenance medium containing 2mM D-glucosamine hydrochloride was replaced and the cells were cultured for 24 hours; cells were fixed with cold acetone, the number of wells containing PCV2 positive cells (in green) at each dilution was determined by the indirect immunofluorescence assay (IFA), and the virus TCID was calculated according to the Karber method 50 。
1.4.2 measurement of PRV active antigen content
The PRV active antigen harvested at 1.3 times is serially diluted by 10 times, and 10 times of the dilution is taken -5 、10 -6 、10 -7 、10 -8 、10 -9 Five dilutions were plated in 96-well PK-15 cell monolayers in cell culture plates, each dilution being made in 4 wells, 100 μ L per well. Setting normal cell contrast at 37 deg.C and 5% CO 2 After 24 hours of incubation in an incubator, cytopathic effect (CPE) was observed and viral TCID was calculated according to Karber's method 50 。
1.4.3 measurement of PPV active antigen content
The PPV active antigen harvested at 1.3 is serially diluted 10 times, 10 are taken -5 、10 -6 、10 -7 、10 -8 、10 -9 Five dilutions were plated in 96-well cell culture plates of ST cell monolayer, each dilution being made in 4 wells, 100 μ L per well. Setting normal cell contrast at 37 deg.C and 5% CO 2 Cultured in an incubator for 5 days, cytopathic effect (CPE) was observed, and virus TCID was calculated according to Karber's method 50 。
1.4.4 determination of PRRSV active antigen content
The PRRSV active antigen harvested at 1.3 is diluted in 10 times series, 10 are taken -5 、10 -6 、10 -7 、10 -8 、10 -9 Five dilutions were plated in 96-well cell culture plates of ST cell monolayer, each dilution being made in 4 wells, 100 μ L per well. Setting normal cell contrast at 37 deg.C and 5% CO 2 Cultured in an incubator for 5 days, cytopathic effect (CPE) was observed, and virus TCID was calculated according to Karber's method 50 。
1.4.5 measurement results of the content of four Virus antigens after concentration
PCV2 active antigen, PRV active antigen, PPV active antigen and PRRSV active antigen were measured according to the measurement method of 1.4.1-1.4.4, and the measurement results are shown in the following table.
TABLE-concentrated content of four viral antigens
Viral antigens Content before fire extinguishment (TCID) 50 /mL)
PCV2 SH strain 10 8.0
Bartha-K61 strain 10 9.0
HN-2011 Strain 10 9.0
TJM strain 10 8.9
1.5 inactivation of antigen
1.5.1 inactivation of PCV2 antigen
Adding formaldehyde solution into PCV2 antigen harvested at 1.3 for inactivation to enable the final concentration of the formaldehyde solution to be 0.2% (V/V), then fully shaking up and heating up, starting timing when the temperature rises to 37 ℃, keeping 18 hours for inactivation, shaking and uniformly mixing once every 6 hours, and storing at 2-8 ℃ for no more than 1 month.
1.5.2 inactivation of PRV antigens
Adding formaldehyde solution into PRV antigen harvested at 1.3 for inactivation to enable the final concentration of the formaldehyde solution to be 0.15% (V/V), then fully shaking and raising the temperature, starting timing when the temperature is raised to 37 ℃, keeping for 24 hours until inactivation is finished, shaking and uniformly mixing once every 6 hours, and storing at 2-8 ℃ until the time does not exceed 1 month.
1.5.3 inactivation of PPV antigen
Adding formaldehyde solution into the PPV antigen harvested at 1.3 for inactivation to enable the final concentration of the formaldehyde solution to be 0.10% (V/V), then fully shaking and raising the temperature, starting timing when the temperature is raised to 37 ℃, keeping for 24 hours until inactivation is finished, shaking and uniformly mixing once every 6 hours, and storing at 2-8 ℃ for no more than 1 month.
1.5.4 inactivation of PRRSV antigen
Adding formaldehyde solution into the PRRSV antigen harvested in 1.3 for inactivation to enable the final concentration of the formaldehyde solution to be 0.15% (V/V), then fully shaking up and heating up, starting timing when the temperature rises to 37 ℃, keeping for 24 hours until inactivation is finished, oscillating and uniformly mixing once every 6 hours during the period, and storing at 2-8 ℃ for less than 1 month.
1.6 antigen inactivation test
1.6.1PCV2 antigen inactivation assay
Inoculating a small amount of PCV2 antigen to PK-15 cells which grow into a monolayer, adsorbing at 37 ℃ for 1 hour, then abandoning virus liquid, adding new MEM cell maintenance liquid, culturing at 37 ℃ for 2 days without CPE, continuously conducting blind propagation for 3 times, changing the cell maintenance liquid after growing into a cell monolayer, culturing at 37 ℃ for 2 days, and detecting by an IFA method without producing green PCV2 positive cells.
1.6.2PRV antigen inactivation assay
Inoculating PK-15 cells grown into monolayer with small amount of PRV antigen, adsorbing at 37 deg.C for 1 hr, discarding virus solution, adding new MEM cell maintenance solution, culturing at 37 deg.C for 2 days, and continuously performing blind culture for 3 times without CPE.
1.6.3PPV antigen inactivation assay
Inoculating small amount of PPV antigen to the ST cells which have grown into a monolayer, adsorbing at 37 deg.C for 1 hr, discarding virus solution, adding new MEM cell maintenance solution, culturing at 37 deg.C for 2 days, and continuously performing blind transfer for 3 times without CPE.
1.6.4PRRSV antigen inactivation test
Inoculating small amount of PRRSV antigen to the ST cell grown into monolayer, adsorbing at 37 deg.c for 1 hr, discarding virus liquid, adding new MEM cell maintaining liquid, culturing at 37 deg.c for 2 days, and continuous blind transferring for 3 times without CPE.
1.6.5 inactivation results
Detecting the inactivation condition of the antigen according to the method of 1.6.1-1.6.4, wherein the detection result is as follows: PCV2 antigen has no green PCV2 positive cells, PRV antigen has no CPE, PPV antigen has no CPE, and PRRSV antigen has no CPE.
2. Preparing a stock solution of the composition
Step 1.5 is used for obtaining inactivated PCV2 antigen, PRV antigen, PPV antigen and PRRSV antigen as antigen material.
Taking 20g of PCV2 antigen, sequentially adding 0.05g of bovine serum albumin, 0.5g of polyvinylpyrrolidone, 0.2g of glycine, 0.2g L-sodium glutamate, 0.1g of sucrose and 0.2g of propolis, uniformly mixing, sequentially adding 20g of PRV antigen, 20g of PPV antigen and 20g of PRRSV antigen, and fully and uniformly mixing to obtain a PRRSV composition stock solution.
3. Stock solution of lyophilized composition
Pre-cooling the composition stock solution obtained in the step 2 at-20 ℃ for 20-30min, and freeze-drying at-80 ℃ to obtain the vaccine composition.
Examples 2 to 5 and comparative example 1
Examples 2-5 and comparative example 1 the components and amounts of the vaccine composition were adjusted based on the method of example 1, and the specific adjustment and the components and amounts of example 1 are shown in the following table two.
TABLE two Components and amounts of vaccine compositions of examples 1-5 and comparative example 1
Figure BDA0002039768480000101
Detection of immunopotency
In order to evaluate the immune efficacy of different components and contents of the vaccine composition, the vaccine compositions prepared in examples 1-5 and comparative example 1 were named vaccine a, vaccine b, vaccine c, vaccine d, vaccine e and vaccine f in sequence, and the 6 vaccines were subjected to the following animal experimental design.
1. Vaccine immunization
Selecting 70 healthy susceptible piglets of 28 days old, dividing into 14 groups, each group comprises 5 heads, injecting 2ml vaccine a into the head and neck of each head and neck of the 1 st and 2 nd groups, injecting 2ml vaccine b into the 3 rd and 4 th groups, injecting 2m vaccine c into the 5 th and 6 th groups, injecting 2ml vaccine d into the 7 th and 8 th groups, injecting 2ml vaccine e into the 9 th and 10 th groups, injecting 2ml vaccine f into the 11 th and 12 th groups, and inoculating for the 2 nd time according to the same way and dose after two weeks; the group 13 was used as the first control (non-immune, non-toxic) and the group 14 was used as the second control (non-immune, non-toxic) and both were kept separately.
2. Counteracting toxic substances
2.1 preparing a toxin counteracting liquid:
PCV2 SH strain, Bartha-K61 strain, HN-2011 strain and TJM strain are mixed according to the weight ratio of 1:1:1:1 to obtain a mixed virus toxin-attacking solution (containing 10) 7.0 TCID 50 /mL)。
2.2 counteracting toxic materials with liquid:
and (3) collecting blood for piglets of 1 st, 3 rd, 5 th, 7 th, 9 th, 11 th and 13 th groups 120 th day after first immunization, separately feeding the piglets by using the mixed virus-attacking solution of the step 2.1 with 1mL of nasal drops and intramuscular injection with 2mL, continuously observing for 25 days after attacking, weighing the piglets for blood collection, killing and biopsy after the piglets are weighed on the 25 th day, and judging according to the body temperature, the relative daily gain and the detection result of the virus antigen.
2.3 Virus potency assay
The remaining non-challenged piglets were bled at 14, 30, 120 and 160 days after priming and tested for HI antibody titers for PCV2, PRV, PPV and PRRSV in the serum.
3. Results of efficacy testing
3.1 counteracting toxic effects
Immunoprotection results for Epimetrivaccine a-vaccine f
Figure BDA0002039768480000111
Note: the pathological changes include lung excess, swollen lymph nodes, bad dead points of kidney, mild swelling of spleen, and intestinal tympanites.
Change of body weight: the 7 groups of piglets are weighed before challenge and when slaughtered respectively, the average relative daily gain of each group is calculated, statistical analysis is carried out by using statistical software SPSS17.0, and the result shows that the relative daily gain of the pigs in the vaccine immune group is similar to that in the blank control group (P is 0.5 and is more than 0.05), but is obviously higher than that in the non-immune challenge control group (P is 0.015 and is less than 0.05).
The immune protection result and the weight change result of the third table show that the virus attack protection rate of the vaccine a-vaccine d to PCV2, PRV, PPV and PRRSV after 120d immunization reaches 100 percent; the virus attack protection rate of the vaccine e to PCV2, PRV, PPV and PRRSV is 80%; the vaccine composition prepared by directly mixing the four antigens has the virus attack protection rate of 40% on PCV2, PRV, PPV and PRRSV. Therefore, the vaccine composition can generate better immune protection for piglets.
3.2 results of antibody level comparison for duration of immunization
TABLE four PCV2 immune duration antibody level comparison results
Figure BDA0002039768480000121
Table five comparison of antibody levels for duration of PRV immunity
Figure BDA0002039768480000122
TABLE six PPV immune duration antibody level comparison results
Figure BDA0002039768480000123
TABLE seven PRRSV immune duration antibody level comparison results
Figure BDA0002039768480000124
Referring to tables four to seven, the detection results of the vaccine a-vaccine f are compared, and the results show that the antibody level of each group of vaccines is detected in 14 days after immunization, the antibody level reaches a peak value in 30 days after immunization, the antibody level of each group is reduced in 120 days after immunization, the antibody levels of the vaccines a to d are still maintained at a higher level, the vaccine c is optimal, and the antibody level of the vaccine f is obviously lower than that of the vaccine a-vaccine f.
Therefore, the vaccine composition prepared by the invention ensures the effective efficacies of the four antigens, namely PCV2, PRV, PPV and PRRSV, can better prevent diseases caused by PCV2, PRV, PPV and PRRSV, reduces clinical stress response, is suitable for large-scale industrial production, and has good application prospect.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.

Claims (5)

1. A vaccine composition is characterized by comprising, by weight, 20-25 parts of PCV2 antigen, 20-25 parts of PRV antigen, 20-25 parts of PPV antigen, 20-25 parts of PRRSV antigen, 0.05-0.1 part of bovine serum albumin, 0.5-1 part of polyvinylpyrrolidone, 0.2-0.5 part of glycine, 0.2-0.5 part of L-sodium glutamate, 0.1-0.2 part of sucrose and 0.2-0.4 part of propolis; the PCV2 antigen is derived from PCV2 SH strain, the PRV antigen is derived from Bartha-K61 strain, the PPV antigen is derived from HN-2011 strain, and the PRRSV antigen is derived from TJM strain.
2. The vaccine composition according to claim 1, comprising 25 parts by weight of PCV2 antigen, 20 parts by weight of PRV antigen, 20 parts by weight of PPV antigen, 20 parts by weight of PRRSV antigen, 0.08 part by weight of bovine serum albumin, 0.8 part by weight of polyvinylpyrrolidone, 0.3 part by weight of glycine, 0.3 part by weight of L-sodium glutamate and 0.2 part by weight of sucrose.
3. The vaccine composition of claim 1, wherein the propolis is 0.3 parts.
4. A process for the preparation of a vaccine composition according to any one of claims 1 to 3, comprising the steps of:
firstly, collecting the antigen
Respectively proliferating PCV2, PRV, PPV and PRRSV, inactivating the PCV2, PRV, PPV and PRRSV, and collecting PCV2 antigen, PRV antigen, PPV antigen and PRRSV antigen;
② preparing a composition stock solution
Taking the PCV2 antigen according to a certain proportion, sequentially adding bovine serum albumin, polyvinylpyrrolidone, glycine, L-sodium glutamate, sucrose and propolis, uniformly mixing, sequentially adding the PRV antigen, the PPV antigen and the PRRSV antigen according to a certain proportion, and fully and uniformly mixing to obtain a composition stock solution;
③ Freeze-drying composition stock solution
Pre-cooling the composition stock solution prepared in the step II at the temperature of-20 ℃ for 20-30min, and freeze-drying at the temperature of-80 ℃ to obtain the vaccine composition.
5. A vaccine composition according to any one of claims 1 to 3, for use in the preparation of a medicament for the prophylaxis and treatment of porcine circovirus, porcine pseudorabies, porcine parvovirus and porcine reproductive and respiratory syndrome.
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