CN113975383B - Rhodococcus erythropolis immunopotentiator and application thereof in vaccine for pigs - Google Patents
Rhodococcus erythropolis immunopotentiator and application thereof in vaccine for pigs Download PDFInfo
- Publication number
- CN113975383B CN113975383B CN202111196854.8A CN202111196854A CN113975383B CN 113975383 B CN113975383 B CN 113975383B CN 202111196854 A CN202111196854 A CN 202111196854A CN 113975383 B CN113975383 B CN 113975383B
- Authority
- CN
- China
- Prior art keywords
- rhodococcus
- culture
- immunopotentiator
- vaccine
- chitosan oligosaccharide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- 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
- A61K39/12—Viral antigens
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55583—Polysaccharides
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55588—Adjuvants of undefined constitution
- A61K2039/55594—Adjuvants of undefined constitution from bacteria
-
- 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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Public Health (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Mycology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of biological products for pigs, and particularly relates to a red rhodococcus immunopotentiator and application thereof in a vaccine for pigs. The red rhodococcus immunopotentiator comprises a red rhodococcus culture, chitosan oligosaccharide and coriolus versicolor polysaccharide, wherein the mass ratio of the red rhodococcus culture to the chitosan oligosaccharide to the coriolus versicolor polysaccharide is 8-10:3-4:1. The lard emulsion inactivated vaccine prepared by the immunopotentiator of the invention is safe and efficient, and has long antibody duration and good immunoprotection effect.
Description
Technical Field
The invention belongs to the technical field of biological products for pigs, and particularly relates to a red rhodococcus immunopotentiator and application thereof in a vaccine for pigs.
Background
In recent years, pig industry is faced with more complicated and changeable pig diseases than before, and not only the types of diseases are more and more increased, but also the prevention and the control are more and more difficult. This phenomenon is mainly caused by the introduction of various foreign diseases and the update of virus mutation. With the popularity of infectious viral diseases such as porcine reproductive and respiratory syndrome, porcine circovirus type 2, swine fever, porcine pseudorabies and other epidemic diseases, new vaccines on the market have been developed. At present, the occurrence probability of immunosuppressive diseases of various diseases in China is generally increased, and serious harm is generated. In this case, control of swine epidemic disease becomes more important and emphasis is placed on overall control of immunosuppressive disease. In swine disease control, the use of immunopotentiators is critical.
Although the immunopotentiator applied to the vaccine for pigs exists in the prior art, the immunopotentiator applied to the vaccine for pigs has the problems of poor immunoprotection effect and the like. Therefore, there is an urgent need to develop a highly effective immunopotentiator for pigs.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a red rhodococcus immunopotentiator and application thereof in pig vaccines. The lard emulsion inactivated vaccine prepared by the immunopotentiator of the invention is safe and efficient, and has long antibody duration and good immunoprotection effect.
The technical scheme of the invention is as follows:
an immune enhancer for rhodococcus erythropolis comprises a culture of rhodococcus erythropolis, chitosan oligosaccharide and Coriolus versicolor polysaccharide.
Further, the mass ratio of the culture of the rhodococcus erythropolis, the chitosan oligosaccharide and the coriolus versicolor polysaccharide is 8-10:3-4:1.
Further, the mass ratio of the culture of rhodococcus erythropolis, the chitosan oligosaccharide and the coriolus versicolor polysaccharide is 9:3:1.
Further, the preparation method of the rhodococcus rhodochrous culture comprises the following steps:
placing rhodococcus erythropolis in a 30L fermentation tank added with a culture medium for culturing for 24-36h, and inactivating at high temperature and high pressure to obtain the rhodococcus erythropolis; the culture medium comprises the following components in percentage by mass: 1-2% of glucose, 2-4% of yeast powder, 0.01-0.02% of dipotassium hydrogen phosphate, 0.4-0.7% of magnesium sulfate, 3-5% of tryptone, 0.3-0.5% of fermentation auxiliary agent and 87.78-92.29% of purified water.
Further, the culture medium comprises the following components in percentage by mass: glucose 1%, yeast powder 3%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate 0.5%, tryptone 4%, fermentation auxiliary agent 0.4% and purified water 91.09%.
Further, the fermentation aid comprises oxalic acid and methionine.
Further, the mass ratio of oxalic acid to methionine is 2-4:10-15.
Further, the pH of the culture medium is 7.0-7.2.
Further, the conditions for culturing rhodococcus erythropolis in a 30L fermenter with the addition of a medium were: the stirring speed is 200r/min, the aeration rate is 1:1.0, the inoculation amount is 6%, the liquid loading amount is 70% of the tank volume, and the temperature is 30+/-1 ℃.
Further, the specific operation of the high-temperature high-pressure inactivation is 121 ℃,101kPa, and the treatment is carried out for 20 minutes.
The invention also aims to provide the application of the red rhodococcus immunopotentiator in preparing the oil emulsion inactivated vaccine for pigs.
An inactivated vaccine of oil emulsion for pigs comprises the red rhodococcus immunopotentiator.
The preparation method of the oil emulsion inactivated vaccine for pigs comprises the following steps:
s1, taking a rhodococcus rhodochrous culture, chitosan oligosaccharide and corious versicolor polysaccharide, inactivating the rhodococcus rhodochrous culture, chitosan oligosaccharide and corious versicolor polysaccharide by adopting high-pressure steam at 121 ℃ for 30min, sterilizing, and preserving the rhodococcus rhodochrous culture at 4 ℃ for later use when the rhodochrous culture is cooled to room temperature;
s2, quantifying the culture of the rhodococcus erythropolis to obtain a quantified culture of the rhodococcus erythropolis;
s3, adding the quantitative rhodococcus ruber culture, chitosan oligosaccharide and corious versicolor polysaccharide obtained in the step S2 into an anti-water phase according to an emulsification formula, uniformly stirring and performing conventional emulsification to obtain an initial oil emulsion inactivated vaccine for pigs;
s4, carrying out physical property detection, safety detection, efficacy test detection and commodity animal immunity detection on the initial oil emulsion inactivated vaccine obtained in the step S3, and obtaining the vaccine after qualified verification.
Further, the quantifying process of the culture of rhodococcus erythropolis in the step S2 is as follows: the 30mL immunopotentiator sample is aseptically removed and divided into 3 15mL centrifuge tubes, the mixture is centrifuged at 12000r/min for 5min at a high speed, the supernatant is removed, and the mixture is placed in a drying oven at 103 ℃ for full drying for 2 hours, so that the dry matter weight of the sample is measured.
Further, the specific operation of step S3 is as follows: 2.5mg of quantitative culture of rhodococcus erythropolis, chitosan oligosaccharide and corious versicolor polysaccharide are added into antigen liquid, 5mL of Tween-80 is added and uniformly mixed, and after full mixing and dissolution, the final total volume is 100mL, and antigen water phase is prepared; according to the antigen water phase: oil phase = 1:2 ratio, emulsified at high speed for 5min using an IKA T25 emulsifier, 12000 r/min.
Further, the antigen in the step S3 comprises a highly pathogenic blue-ear virus antigen, a porcine pseudorabies virus antigen, a porcine circovirus type 2 antigen, a mycoplasma hyopneumoniae, a swine fever virus antigen, a haemophilus parasuis antigen, porcine infectious pleuropneumonia, a porcine blue-ear virus GP5 antigen expressed by adopting genetic engineering, a porcine circovirus Cap protein expressed by adopting genetic engineering, and a swine fever E2 protein expressed by adopting genetic engineering.
Further, in the step S4, commercial animal immunodetection is performed by using commercial pigs for animal experiments, and the vaccine effect evaluation is performed in a simulated clinic.
The safety and high efficiency of a vaccine are important indicators for evaluating the quality of the vaccine, and the selection of a proper immunopotentiator is important for the safety and efficacy of the vaccine. The invention adopts red rhodococcus immunopotentiator composed of red rhodococcus culture, chitosan oligosaccharide and corious versicolor polysaccharide specific components in mass ratio, and the components are synergistic, so that the immunogenicity of various antigens can be enhanced, the duration of antibodies can be prolonged, and the immune effect of vaccine can be improved.
The rhodococcus erythropolis culture prepared by the specific culture medium has high content of lipid compounds, polysaccharide and other components, can non-specifically stimulate the cellular immune function, can obviously promote the secretion of various cytokines, and can obviously promote the generation of animal antibodies induced by the vaccine when being added into the oil emulsion inactivated vaccine for pigs, and the duration of the antibodies is long.
The immunopotentiator is used in the preparation process of the oil emulsion inactivated vaccine for pigs, and can obtain vaccines aiming at different viruses according to different selected antigens, so that the application range is wide. The inactivated vaccine of the oil emulsion for pigs prepared by the immunopotentiator has high safety, no obvious residue in the local injection of the vaccine, safety and high efficiency.
Compared with the prior art, the invention has the following advantages:
the invention adopts specific components and qualities of a culture of rhodococcus erythropolis, chitosan oligosaccharide and coriolus versicolor polysaccharide
The red rhodococcus immunopotentiator formed by the quantitative ratio has the advantages that the components are synergistic, the immunogenicity of various antigens can be enhanced, the immune effect of the vaccine is improved, and the immune protection effect is good.
The culture of rhodococcus erythropolis of the present invention is obtained by culturing rhodococcus erythropolis in a fermenter
The obtained rhodococcus erythropolis has simple culture process and short fermentation time, and is beneficial to large-scale acquisition of the rhodococcus erythropolis culture.
(3) The immunopotentiator is used in the preparation process of the oil emulsion inactivated vaccine for pigs, and can obtain vaccines aiming at different viruses according to different selected antigens, so that the immunopotentiator has the advantages of wide application range and strong specificity.
(4) The immunopotentiator of the invention is used for preparing the oil emulsion inactivated vaccine for pigs, has high safety,
the vaccine injection part has no obvious residue, and the antibody has long duration and good immune protection effect.
Detailed Description
The invention is further illustrated by the following description of specific embodiments, which are not intended to be limiting, and various modifications or improvements can be made by those skilled in the art in light of the basic idea of the invention, but are within the scope of the invention without departing from the basic idea of the invention.
The raw materials used in the present invention are commercially available unless otherwise specified. For example, chitosan oligosaccharide is available from Zhejiang Kangxing biotechnology Co., ltd., product number: k6; coriolus versicolor polysaccharide is available from Siam Rayleigh Biotechnology Inc., cat#: REL1056; rhodococcus rhodochrous of the present invention is available from the collection of microorganism strains, GDMCC No.: GDMCC 1.819.
Example 1, an immunopotentiator of Rhodococcus erythropolis
The red rhodococcus immunopotentiator comprises a red rhodococcus culture, chitosan oligosaccharide and coriolus versicolor polysaccharide; the mass ratio of the culture of the rhodococcus erythropolis, the chitosan oligosaccharide and the coriolus versicolor polysaccharide is 8:4:1.
The preparation method of the rhodococcus rhodochrous culture comprises the following steps: rhodococcus erythropolis was placed in a 30L fermenter with the addition of a medium for 24 hours under the following conditions: stirring at a rotation speed of 200r/min, wherein the aeration rate is 1:1.0, the inoculation amount is 6%, the liquid loading amount is 70% of the tank volume, the temperature is 30 ℃, the high-temperature and high-pressure inactivation is carried out, the specific operation of the high-temperature and high-pressure inactivation is 121 ℃,101kPa, and the treatment is carried out for 20min, thus obtaining the high-temperature and high-pressure inactivation;
the culture medium comprises the following components in percentage by mass: glucose 1%, yeast powder 2%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate 0.4%, tryptone 3%, fermentation auxiliary agent 0.3%, purified water 92.29%; the fermentation auxiliary agent consists of oxalic acid and methionine according to the mass ratio of 2:15; the pH of the medium was 7.0.
Example 2, an immunopotentiator of Rhodococcus erythropolis
The red rhodococcus immunopotentiator comprises a red rhodococcus culture, chitosan oligosaccharide and coriolus versicolor polysaccharide; the mass ratio of the culture of the rhodococcus erythropolis, the chitosan oligosaccharide and the coriolus versicolor polysaccharide is 10:3:1.
The preparation method of the rhodococcus rhodochrous culture comprises the following steps: rhodococcus erythropolis was placed in a 30L fermenter with the addition of a medium for 36 hours under the following conditions: stirring at a rotation speed of 200r/min, wherein the aeration rate is 1:1.0, the inoculation amount is 6%, the liquid loading amount is 70% of the tank volume, the temperature is 30 ℃, the high-temperature and high-pressure inactivation is carried out, the specific operation of the high-temperature and high-pressure inactivation is 121 ℃,101kPa, and the treatment is carried out for 20min, thus obtaining the high-temperature and high-pressure inactivation;
the culture medium comprises the following components in percentage by mass: 2% of glucose, 4% of yeast powder, 0.02% of dipotassium hydrogen phosphate, 0.7% of magnesium sulfate, 5% of tryptone, 0.5% of fermentation auxiliary agent and 87.78% of purified water; the fermentation auxiliary agent consists of oxalic acid and methionine according to the mass ratio of 4:10; the pH of the medium was 7.2.
Example 3A red rhodococcus immunopotentiator
The red rhodococcus immunopotentiator comprises a red rhodococcus culture, chitosan oligosaccharide and coriolus versicolor polysaccharide; the mass ratio of the culture of the rhodococcus erythropolis, the chitosan oligosaccharide and the coriolus versicolor polysaccharide is 9:3:1.
The preparation method of the rhodococcus rhodochrous culture comprises the following steps: rhodococcus erythropolis was placed in a 30L fermenter with the addition of a medium for 30 hours under the following conditions: stirring at a rotation speed of 200r/min, wherein the aeration rate is 1:1.0, the inoculation amount is 6%, the liquid loading amount is 70% of the tank volume, the temperature is 30 ℃, the high-temperature and high-pressure inactivation is carried out, the specific operation of the high-temperature and high-pressure inactivation is 121 ℃,101kPa, and the treatment is carried out for 20min, thus obtaining the high-temperature and high-pressure inactivation;
the culture medium comprises the following components in percentage by mass: glucose 1%, yeast powder 3%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate 0.5%, tryptone 4%, fermentation auxiliary agent 0.4% and purified water 91.09%; the fermentation auxiliary agent consists of oxalic acid and methionine according to the mass ratio of 3:14; the pH of the medium was 7.2.
Example 4 preparation method of an inactivated vaccine of oil emulsion for pigs
The preparation method of the oil emulsion inactivated vaccine for pigs comprises the following steps:
I. sample sterilization treatment and quantification of red rhodococcus immunopotentiator
Taking a plurality of red rhodococcus immunopotentiator samples of example 3, including a culture of red rhodococcus, chitosan oligosaccharide and coriolus versicolor polysaccharide, inactivating the red rhodococcus immunopotentiator samples by using high-pressure steam at 121 ℃ for 30min, and preserving the sterilized samples at 4 ℃ for standby when the temperature of the sterilized samples is reduced to room temperature.
The culture samples of rhodococcus erythropolis were quantified prior to use. The quantitative process is as follows: the 30mL immunopotentiator sample is aseptically removed and divided into 3 15mL centrifuge tubes, the mixture is centrifuged at 12000r/min for 5min at a high speed, the supernatant is removed, and the mixture is placed in a drying oven at 103 ℃ for full drying for 2 hours, so that the dry matter weight of the sample is measured.
II, preparation of oil emulsion inactivated vaccine added with rhodococcus erythropolis immunopotentiator
(1) Preparation of antigen aqueous phase
2.5mg of quantitative culture of rhodococcus erythropolis, chitosan oligosaccharide and corious versicolor polysaccharide are added into highly pathogenic blue ear disease virus antigen, 5mL of Tween-80 is added, and the mixture is uniformly mixed and dissolved, and the final total volume is 100mL, so that an antigen water phase is prepared. Meanwhile, a highly pathogenic blue-ear virus antigen control group without red rhodococcus immunopotentiator is established, and the difference between the control group and the test group is only whether the red rhodococcus immunopotentiator is added or not, and the other groups are treated in the same way.
Note that the antigen aqueous phase should be prepared by first preparing a control antigen aqueous phase without the red rhodococcus immunopotentiator and then preparing a test antigen aqueous phase containing the red rhodococcus immunopotentiator.
(2) Emulsion preparation of vaccine
Taking 100mL of the prepared antigen water phase, slowly adding the antigen water phase into 200mL of an oil phase prepared according to an emulsification formula, wherein the preparation method of the oil phase comprises the following steps: adding span-80 into mineral oil, mixing, adding aluminum stearate, mixing, and sterilizing; the mineral oil (V/mL): span-80 (V/mL): aluminum stearate (W/g) 94:6:1.5; emulsifying by using an IKA T25 emulsifying machine, wherein the emulsifying machine is required to be stirred at a low speed of 5000r/min in the process of adding the antigen water phase, and the rotating speed of the emulsifying machine is regulated to 12000r/min for high-speed shearing and emulsifying for 5min after the antigen water phase is completely added into the oil phase. And subpackaging the vaccine prepared after emulsification, and keeping the vaccine at 4 ℃ for standby to obtain the initial oil emulsion inactivated vaccine for pigs.
Note that the vaccine should be prepared by preparing a control vaccine without the red rhodococcus immunopotentiator and then preparing a test vaccine containing the red rhodococcus immunopotentiator.
(3) Detecting the lard emulsion inactivated vaccine;
and (3) carrying out physical property detection, safety detection, efficacy test detection and commercial animal immunity detection on the obtained initial oil emulsion inactivated vaccine for pigs, and obtaining the oil emulsion inactivated vaccine for pigs after verification is qualified.
Comparative example 1, an immunopotentiator of Rhodococcus erythropolis
The rhodococcus erythropolis immunopotentiator comprises a rhodococcus erythropolis culture and chitosan oligosaccharide; the mass ratio of the culture of the rhodococcus erythropolis to the chitosan oligosaccharide is 9:3.
The preparation method of the rhodococcus rhodochrous culture comprises the following steps: rhodococcus erythropolis was placed in a 30L fermenter with the addition of a medium for 30 hours under the following conditions: stirring at a rotation speed of 200r/min, wherein the aeration rate is 1:1.0, the inoculation amount is 6%, the liquid loading amount is 70% of the tank volume, the temperature is 30 ℃, the high-temperature and high-pressure inactivation is carried out, the specific operation of the high-temperature and high-pressure inactivation is 121 ℃,101kPa, and the treatment is carried out for 20min, thus obtaining the high-temperature and high-pressure inactivation;
the culture medium comprises the following components in percentage by mass: glucose 1%, yeast powder 3%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate 0.5%, tryptone 4%, fermentation auxiliary agent 0.4% and purified water 91.09%; the fermentation auxiliary agent consists of oxalic acid and methionine according to the mass ratio of 3:14; the pH of the medium was 7.2.
Comparative example 2, an immunopotentiator for Red coccus
The rhodococcus erythropolis immunopotentiator comprises a rhodococcus erythropolis culture and chitosan oligosaccharide; the mass ratio of the culture of the rhodococcus erythropolis to the chitosan oligosaccharide is 1:1.
The preparation method of the rhodococcus rhodochrous culture comprises the following steps: rhodococcus erythropolis was placed in a 30L fermenter with the addition of a medium for 30 hours under the following conditions: stirring at a rotation speed of 200r/min, wherein the aeration rate is 1:1.0, the inoculation amount is 6%, the liquid loading amount is 70% of the tank volume, the temperature is 30 ℃, the high-temperature and high-pressure inactivation is carried out, the specific operation of the high-temperature and high-pressure inactivation is 121 ℃,101kPa, and the treatment is carried out for 20min, thus obtaining the high-temperature and high-pressure inactivation;
the culture medium comprises the following components in percentage by mass: glucose 1%, yeast powder 3%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate 0.5%, tryptone 4%, fermentation auxiliary agent 0.4% and purified water 91.09%; the fermentation auxiliary agent consists of oxalic acid and methionine according to the mass ratio of 3:14; the pH of the medium was 7.2.
Comparative example 3 preparation method of inactivated vaccine of oil emulsion for pigs
The preparation method of the oil emulsion inactivated vaccine for pigs is similar to that of the example 4;
the difference from example 4 is that the red rhodococcus immunopotentiator was the red rhodococcus immunopotentiator prepared in comparative example 1.
Comparative example 4, preparation method of inactivated vaccine of oil emulsion for pigs
The preparation method of the oil emulsion inactivated vaccine for pigs is similar to that of the example 4;
the difference from example 4 is that the red rhodococcus immunopotentiator was the red rhodococcus immunopotentiator prepared in comparative example 2.
Test example I, physical property detection of oil emulsion inactivated vaccine for pigs
Taking the vaccine prepared and packaged in example 4, and after the vaccine is warmed to room temperature, the following physical properties are detected respectively:
A. the vaccine viscosity was measured with a rotational viscometer using 10mL samples from each group of vaccines.
B. Slowly dripping the vaccine onto the standing water surface, and observing the diffusion condition of the vaccine.
C. Each group is filled with 30mL of vaccine into 3 10mL cone bottom centrifuge tubes, and the mixture is centrifuged for 15min at 3000r/min, and whether layering demulsification phenomenon exists or not is observed. If demulsification and layering are carried out, the vaccine preparation is unsuccessful, and the vaccine needs to be prepared again.
D. The centrifuged vaccine is respectively placed at 37 ℃ after being marked, and is preserved for 1 month at the room temperature and the temperature of 4 ℃ to observe whether demulsification is carried out.
The test results are as follows:
A. the viscosity of the vaccine in the test group is 35cp, the viscosity of the vaccine in the control group is 41cp, and both the vaccines are qualified.
B. The test group vaccine and the control group vaccine are qualified.
C. The test group vaccine and the control group vaccine have no demulsification, and are qualified.
D. The test group vaccine and the control group vaccine have no demulsification, and are qualified.
Test example II, safety test of oil emulsion inactivated vaccine for pigs
The vaccine prepared in the test group of example 4 was used to immunize 21-day-old weaned pigs (African swine fever antigen antibody negative, porcine reproductive and respiratory syndrome antigen antibody double negative), 2.0 mL/pig, 5 pigs were immunized with each vaccine group, and the health status of the pigs was observed daily for 14 days continuously.
The test result shows that the piglets are not dead and have good health condition, so that the vaccine prepared by the immunopotentiator is qualified and safe.
Test example III, efficacy test of oil emulsion inactivated vaccine for pigs
The vaccine prepared in the test group, the comparative example 3, the comparative example 4 and the comparative example are respectively used for immunizing 21-day-old weaned pigs (African swine fever antigen antibody negative and porcine reproductive and respiratory syndrome antigen antibody double negative), 1.0 mL/vaccine is adopted for immunizing 5 vaccine groups, each vaccine group is provided with an ear tag, the vaccine is boosted once after being injected for 21 days, and blood is taken for measuring the serum neutralization antibody titer of 14 days and 21 days after the secondary immunization. A blank group was additionally provided and the test results are shown in table 1.
Table 1: determination result of high pathogenicity blue-ear disease inactivated vaccine antibody
As can be seen from Table 1, the vaccine provided by the invention shows a significant difference between the level of the neutralizing antibody and the level of the neutralizing antibody after only 21 days of secondary immunization of pigs. The serum neutralizing antibody titer of the vaccine prepared by the immunopotentiator is obviously superior to that of a control group, a comparative example 3 group and a comparative example 4 group after 14 days and 21 days of secondary immunization. The result shows that the vaccine prepared by the immunopotentiator has high-efficiency protective effect on the porcine reproductive and respiratory syndrome.
Claims (6)
1. An immune enhancer for rhodococcus erythropolis, which is characterized by comprising a culture of rhodococcus erythropolis, chitosan oligosaccharide and coriolus versicolor polysaccharide; the mass ratio of the culture of the rhodococcus erythropolis, the chitosan oligosaccharide and the coriolus versicolor polysaccharide is 8-10:3-4:1;
the preparation method of the rhodococcus rhodochrous culture comprises the following steps:
placing rhodococcus erythropolis in a 30L fermentation tank added with a culture medium for culturing for 24-36h, and inactivating at high temperature and high pressure to obtain the rhodococcus erythropolis; the culture medium comprises the following components in percentage by mass: 1-2% of glucose, 2-4% of yeast powder, 0.01-0.02% of dipotassium hydrogen phosphate, 0.4-0.7% of magnesium sulfate, 3-5% of tryptone, 0.3-0.5% of fermentation auxiliary agent and 87.78-92.29% of purified water;
the fermentation auxiliary agent comprises oxalic acid and methionine;
the rhodococcus rhodochrous was purchased from the collection of microorganism strains, GDMCC No. 1.819, cantonese province.
2. The red rhodococcus immunopotentiator of claim 1, wherein the mass ratio of the culture of red rhodococcus, chitosan oligosaccharide and coriolus versicolor polysaccharide is 9:3:1.
3. The red rhodococcus immunopotentiator of claim 1, wherein the mass ratio of oxalic acid to methionine is 2-4:10-15.
4. Use of the red coccus immunopotentiator according to any one of claims 1 to 3 for the preparation of an inactivated vaccine for lard comprising highly pathogenic blue-ear virus antigens.
5. An inactivated vaccine for swine oil emulsion comprising the red rhodococcus immunopotentiator of any one of claims 1-3 and a highly pathogenic blue ear disease virus antigen.
6. The method for preparing the lard emulsion inactivated vaccine as claimed in claim 5, wherein the method comprises the following steps:
s1, taking a rhodococcus rhodochrous culture, chitosan oligosaccharide and corious versicolor polysaccharide, inactivating the rhodococcus rhodochrous culture, chitosan oligosaccharide and corious versicolor polysaccharide by adopting high-pressure steam at 121 ℃ for 30min, sterilizing, and preserving the rhodococcus rhodochrous culture at 4 ℃ for later use when the rhodochrous culture is cooled to room temperature;
s2, quantifying the culture of the rhodococcus erythropolis to obtain a quantified culture of the rhodococcus erythropolis;
s3, adding the quantitative rhodococcus ruber culture, chitosan oligosaccharide and corious versicolor polysaccharide obtained in the step S2 into an anti-water phase according to an emulsification formula, uniformly stirring and performing conventional emulsification to obtain an initial oil emulsion inactivated vaccine for pigs;
s4, carrying out physical property detection, safety detection, efficacy test detection and commodity animal immunity detection on the initial oil emulsion inactivated vaccine obtained in the step S3, and obtaining the vaccine after qualified verification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111196854.8A CN113975383B (en) | 2021-10-14 | 2021-10-14 | Rhodococcus erythropolis immunopotentiator and application thereof in vaccine for pigs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111196854.8A CN113975383B (en) | 2021-10-14 | 2021-10-14 | Rhodococcus erythropolis immunopotentiator and application thereof in vaccine for pigs |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113975383A CN113975383A (en) | 2022-01-28 |
| CN113975383B true CN113975383B (en) | 2023-10-20 |
Family
ID=79738592
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111196854.8A Active CN113975383B (en) | 2021-10-14 | 2021-10-14 | Rhodococcus erythropolis immunopotentiator and application thereof in vaccine for pigs |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113975383B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104623654A (en) * | 2015-01-08 | 2015-05-20 | 中国医学科学院医学生物学研究所 | Application of chitosan oligosaccharide and vaccine containing chitosan oligosaccharide |
| CN105797153A (en) * | 2016-05-02 | 2016-07-27 | 浙江农林大学 | Veterinary vaccine immunologic adjuvant as well as preparation and application method thereof |
| CN106620689A (en) * | 2016-11-29 | 2017-05-10 | 浙江美保龙生物技术有限公司 | Dilution for live vaccine of porcine reproductive and respiratory syndrome |
| CN107373674A (en) * | 2017-08-25 | 2017-11-24 | 西宝生物科技（上海）股份有限公司 | Alimentation composition and its application after a kind of oncotherapy |
| CN108743923A (en) * | 2018-06-28 | 2018-11-06 | 河南省帝方生物制药有限公司 | A kind of compound preparation and preparation method thereof for the antiviral raising immunity of livestock and poultry |
| CN109576180A (en) * | 2018-12-17 | 2019-04-05 | 北京利昂盛生物技术有限公司 | One Rhodococcus ruber and its application in vaccine is being prepared as immunologic adjuvant |
| CN109666609A (en) * | 2019-01-14 | 2019-04-23 | 付家栋 | A kind of Rhodococcus ruber fermentation process and its application as adjuvant in animal vaccine |
| CN110101721A (en) * | 2019-05-21 | 2019-08-09 | 福建省微生物研究所 | A kind of purposes of Rhodococcus ruber extract |
| CN110742211A (en) * | 2019-11-21 | 2020-02-04 | 天津农学院 | Grouper feed accelerant and using method thereof |
-
2021
- 2021-10-14 CN CN202111196854.8A patent/CN113975383B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104623654A (en) * | 2015-01-08 | 2015-05-20 | 中国医学科学院医学生物学研究所 | Application of chitosan oligosaccharide and vaccine containing chitosan oligosaccharide |
| CN105797153A (en) * | 2016-05-02 | 2016-07-27 | 浙江农林大学 | Veterinary vaccine immunologic adjuvant as well as preparation and application method thereof |
| CN106620689A (en) * | 2016-11-29 | 2017-05-10 | 浙江美保龙生物技术有限公司 | Dilution for live vaccine of porcine reproductive and respiratory syndrome |
| CN107373674A (en) * | 2017-08-25 | 2017-11-24 | 西宝生物科技（上海）股份有限公司 | Alimentation composition and its application after a kind of oncotherapy |
| CN108743923A (en) * | 2018-06-28 | 2018-11-06 | 河南省帝方生物制药有限公司 | A kind of compound preparation and preparation method thereof for the antiviral raising immunity of livestock and poultry |
| CN109576180A (en) * | 2018-12-17 | 2019-04-05 | 北京利昂盛生物技术有限公司 | One Rhodococcus ruber and its application in vaccine is being prepared as immunologic adjuvant |
| CN109666609A (en) * | 2019-01-14 | 2019-04-23 | 付家栋 | A kind of Rhodococcus ruber fermentation process and its application as adjuvant in animal vaccine |
| CN110101721A (en) * | 2019-05-21 | 2019-08-09 | 福建省微生物研究所 | A kind of purposes of Rhodococcus ruber extract |
| CN110742211A (en) * | 2019-11-21 | 2020-02-04 | 天津农学院 | Grouper feed accelerant and using method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113975383A (en) | 2022-01-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109666609B (en) | Rhodococcus ruber fermentation method and application of Rhodococcus ruber fermentation method as adjuvant in animal vaccine | |
| TWI449533B (en) | Vaccine for protection against lawsonia intracellularis, mycoplasma hyopneumoniae and porcine circo virus | |
| CN103263666B (en) | Porcine circovirus 2 type, porcine mycoplasmal pneumonia bivalent inactivated vaccine and preparation method thereof | |
| JP2006528882A (en) | Lawsonia intracellularis of European origin and its vaccine, diagnostic agent and method of use | |
| CN105744951B (en) | Bacterial vaccines and methods of making the same | |
| CN107375922B (en) | Water-soluble composite immunologic adjuvant and porcine circovirus disease vaccine | |
| KR20170094186A (en) | Process for ready-to-use pcv2/m.hyo combination vaccine | |
| CN110935017A (en) | Application of nocardia immunopotentiator in preparation of poultry vaccine | |
| CN113975383B (en) | Rhodococcus erythropolis immunopotentiator and application thereof in vaccine for pigs | |
| CN109550046B (en) | Combined vaccine for adsorbing acellular pertussis-poliomyelitis-b haemophilus influenzae and preparation method thereof | |
| CN113957012A (en) | Mycoplasma synoviae culture medium and preparation method thereof | |
| CN102727883B (en) | Combined live vaccine against porcine reproductive and respiratory syndrome and swine fever, and application thereof | |
| CN105087501B (en) | Porcine circovirus type 2 strain and inactivated vaccine prepared therefrom and application | |
| CN110777160A (en) | Preparation method of foot-and-mouth disease virus-like particle antigen, foot-and-mouth disease virus-like particle antigen prepared by preparation method and application of foot-and-mouth disease virus-like particle antigen | |
| CN110540579A (en) | avibacterium paragallinarum antigen protein, vaccine composition containing avibacterium paragallinarum antigen, and preparation method and application thereof | |
| CN114028554B (en) | Rhodococcus erythropolis immunopotentiator and application thereof in poultry vaccine | |
| CN102793920B (en) | Compound immunopotentiator, inactivated vaccine for poultry, and preparation method thereof | |
| CN111349619A (en) | Duck circovirus tissue inactivated vaccine and preparation method of yolk antibody thereof | |
| CN112159814A (en) | CpG oligodeoxynucleotide, preparation and application thereof | |
| CN106511995A (en) | Water-in-oil-in-water type vaccine adjuvant and application thereof | |
| CN113975384B (en) | Rhodococcus erythropolis immunopotentiator and application thereof in fish vaccine | |
| CN113648407A (en) | Application of nocardia immunopotentiator in fish vaccine | |
| CN105920595B (en) | Porcine circovirus 2 type inactivated vaccine and preparation method thereof | |
| CN112062836B (en) | High-titer positive serum of porcine Seneca valley virus and preparation method thereof | |
| CN104324370B (en) | Vaccine composition, preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |