CN114028554B - Rhodococcus erythropolis immunopotentiator and application thereof in poultry vaccine - Google Patents

Rhodococcus erythropolis immunopotentiator and application thereof in poultry vaccine Download PDF

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CN114028554B
CN114028554B CN202111196852.9A CN202111196852A CN114028554B CN 114028554 B CN114028554 B CN 114028554B CN 202111196852 A CN202111196852 A CN 202111196852A CN 114028554 B CN114028554 B CN 114028554B
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immunopotentiator
rhodococcus
rhodococcus erythropolis
vaccine
poultry
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CN114028554A (en
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严悌昆
王文勇
张毓金
谢秉超
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Guangzhou Keli Biotechnology Co ltd
Guangdong Yuyue Biotechnology Co ltd
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Guangzhou Keli Biotechnology Co ltd
Guangdong Yuyue Biotechnology Co ltd
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Abstract

The invention belongs to the technical field of biological products for poultry, and particularly relates to a red rhodococcus immunopotentiator and application thereof in poultry vaccines. The immunopotentiator is a culture of rhodococcus erythropolis, and the preparation method of the culture of rhodococcus erythropolis 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 vaccine prepared by adopting the culture of rhodococcus erythropolis as an immunopotentiator is safe and efficient, and has wide application range and long antibody duration.

Description

Rhodococcus erythropolis immunopotentiator and application thereof in poultry vaccine
Technical Field
The invention belongs to the technical field of biological products for poultry, and particularly relates to a red rhodococcus immunopotentiator and application thereof in poultry vaccines.
Background
With the continuous improvement of the standardized degree of the livestock breeding industry in China, particularly the intensive breeding of poultry, the mixed infection, secondary infection and concurrent infection layers of poultry diseases are endlessly generated due to the large breeding density, and the prevention and control of poultry epidemic diseases are extremely challenging. It is undeniable that in the livestock and poultry raising process, the use of antibiotics promotes the growth and development of animals, improves the production performance, plays a great role in preventing and treating diseases, controlling infection and the like, but in the veterinary clinical process, the problems of drug poisoning, drug residue, teratogenesis, mutagenesis, drug resistance and the like are increasingly prominent due to the abuse and overdose use of antibiotics, even environmental pollution is caused, and the health of human beings is seriously endangered along with the entry of biological chains into the human body. An immunopotentiator is a substance which can improve the immunity of the poultry by improving the resistance of the poultry body to antigen specific reaction through a certain way.
Although various types of immunopotentiators exist in the prior art and are applied to poultry vaccines, the existing immunopotentiators have the problems of poor immune effect, short antibody duration and the like.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a red rhodococcus immunopotentiator and application thereof in poultry vaccines. The invention adopts the culture of rhodococcus erythropolis as an immunopotentiator, and the addition of the rhodococcus erythropolis into the oil emulsion inactivated vaccine for poultry can obviously promote the vaccine to induce the generation of animal antibodies, and the duration of the antibodies is long. In addition to this. The vaccine prepared by the immunopotentiator is safe and efficient.
The technical scheme of the invention is as follows:
an immune enhancer of rhodococcus erythropolis, which is a culture of rhodococcus erythropolis, and the preparation method of the culture of rhodococcus erythropolis 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 rhodococcus erythropolis immunopotentiator 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 mass ratio of oxalic acid to methionine is 3:14.
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 an oil emulsion inactivated vaccine for poultry.
An inactivated vaccine of oil emulsion for poultry comprises the red rhodococcus immunopotentiator.
The preparation method of the red rhodococcus immunopotentiator comprises the following steps:
s1, taking a rhodococcus erythropolis immunopotentiator, inactivating the rhodococcus erythropolis immunopotentiator by high-pressure steam at 121 ℃ for 30min, sterilizing, and preserving at 4 ℃ for later use when the temperature is reduced to room temperature;
s2, quantifying the red rhodococcus immunopotentiator to obtain a quantitative red rhodococcus immunopotentiator;
s3, adding the quantitative red rhodococcus immunopotentiator obtained in the step S2 into the mixed anti-water phase according to an emulsification formula, uniformly stirring and performing conventional emulsification to obtain an initial oil emulsion inactivated vaccine for poultry;
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 inactivated vaccine after verification.
Further, the quantitative process of the red rhodococcus immunopotentiator in the step S1 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: adding 2mg of quantitative red rhodococcus immunopotentiator into antigen liquid, adding 5mL of Tween-80, uniformly mixing, and preparing an antigen water phase with the total volume of 100mL after full mixing and dissolution; 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 avian influenza H9 subtype, newcastle disease, infectious bursal disease, avian adenovirus, li Moshi bacillus duck disease and novel duck reovirus.
Further, in the step S4, commercial animal immunodetection is performed by using commercial chickens, ducks and geese to perform animal experiments, and the simulated clinic is used for evaluating vaccine effects.
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 the culture of rhodococcus erythropolis as an immunopotentiator, and the culture of rhodococcus erythropolis prepared by adopting 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 antibody induced by the vaccine when being added into the oil emulsion inactivated vaccine for poultry, and the duration of the antibody is long. The invention adopts the culture of rhodococcus erythropolis as an immunopotentiator for the preparation process of the oil emulsion inactivated vaccine for poultry, and can obtain vaccines aiming at different viruses according to different selected antigens, thereby having wide application range. The poultry oil emulsion inactivated vaccine prepared by using the rhodococcus rhodochrous culture as an immunopotentiator has high safety, and no obvious residue exists in the local injection part. The vaccine prepared by the immunopotentiator is safe and efficient.
Compared with the prior art, the invention has the following advantages:
(1) According to the invention, the culture of the rhodococcus erythropolis is used as an immunopotentiator, and the rhodococcus erythropolis is cultured in the fermentation tank to obtain the culture of the rhodococcus erythropolis, so that the culture process is simple, the fermentation time is short, and the large-scale acquisition of the culture of the rhodococcus erythropolis is facilitated.
(2) The culture of rhodococcus erythropolis obtained by the invention is used as an immunopotentiator for preparing the oil emulsion inactivated vaccine for poultry, and can be used for preparing vaccines with different functions according to different selected antigens, and has the advantages of wide application range and strong specificity.
(3) The culture of rhodococcus erythropolis obtained by the invention is used as an immunopotentiator for preparing the oil emulsion inactivated vaccine for poultry, has high safety, and has no obvious residue in local injection. In addition, the culture of rhodococcus erythropolis obtained by the invention is used as an immunopotentiator for preparing the oil emulsion inactivated vaccine for poultry, so that the generation of animal antibodies induced by the vaccine can be obviously promoted, and the duration of the antibodies is long. The vaccine prepared by the immunopotentiator is safe and efficient.
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. 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 immunopotentiator is a culture of rhodococcus erythropolis, and the preparation method of the culture of rhodococcus erythropolis 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 immunopotentiator is a culture of rhodococcus erythropolis, and the preparation method of the culture of rhodococcus erythropolis 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 immunopotentiator is a culture of rhodococcus erythropolis, and the preparation method of the culture of rhodococcus erythropolis 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 rhodococcus erythropolis immunopotentiator 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 for poultry oil emulsion
The preparation method of the poultry oil emulsion inactivated vaccine comprises the following steps:
I. sample sterilization treatment and quantification of red rhodococcus immunopotentiator
Taking a plurality of red rhodococcus immunopotentiator liquid samples prepared in the example 3, inactivating the liquid samples by high-pressure steam at 121 ℃ for 30min, and preserving the sterilized samples at 4 ℃ for later use when the temperature of the sterilized samples is reduced to room temperature.
The red rhodococcus immunopotentiator sample was 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 an inactivated vaccine of an oil emulsion for poultry added with an immune enhancer of rhodococcus erythropolis
(1) Preparation of antigen aqueous phase
2mg of quantitative red rhodococcus immunopotentiator is added into the mixed antigen liquid of Newcastle disease and H9 (Newcastle disease antigen: H9 subtype avian influenza antigen=1:1, the antigen stock liquid is prepared by using Newcastle disease antigen and H9 subtype avian influenza antigen), 5mL of Tween-80 is added, uniformly mixed, fully mixed and dissolved, and the total volume is 100mL finally, so that an antigen water phase is prepared. Meanwhile, a newcastle disease and H9 mixed antigen control group without red rhodococcus immunopotentiator is established, the difference between the control group and the test group is only whether the red rhodococcus immunopotentiator is added or not, and 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 prepared antigen water phase, slowly adding the antigen water phase into 200mL of 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 inactivated vaccine of the oil emulsion for poultry.
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 an inactivated vaccine of the oil emulsion for poultry;
and (3) detecting physical properties, safety detection, efficacy test detection and commodity animal immunity detection of the initial oil emulsion inactivated vaccine for poultry, and obtaining the oil emulsion inactivated vaccine for poultry after verification.
Comparative example 1, an immunopotentiator of Rhodococcus erythropolis
The immunopotentiator is a culture of rhodococcus erythropolis, and the preparation method of the culture of rhodococcus erythropolis 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 1:1; the pH of the medium was 7.2.
Comparative example 2, an immunopotentiator for Red coccus
The immunopotentiator is a culture of rhodococcus erythropolis, and the preparation method of the culture of rhodococcus erythropolis 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 is methionine.
Comparative example 3 preparation method of inactivated vaccine of oil emulsion for poultry
The preparation method of the poultry oil emulsion inactivated vaccine 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 poultry
The preparation method of the poultry oil emulsion inactivated vaccine 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 one, determination of lipid and polysaccharide content
Taking the culture of rhodococcus erythropolis obtained in example 3, comparative example 1 and comparative example 2, adding an equal amount of 5% KOH solution, treating in a water bath at 95 ℃ for 35min, breaking bacterial cell walls, and freeze-drying to obtain cell wall skeleton powder.
(1) Determination of lipid content
50mg of cell wall skeleton powder is weighed, 2mL of 2.5% NaOH solution is added, the mixture is refluxed and hydrolyzed in 20mL of methanol to benzene (1:1, v/v) for 2h, the mixture is neutralized by 1mol/L HCl, the mixture is extracted three times by diethyl ether, the extract is concentrated, and the lipid compound content of the extract is measured by refining by absolute ethyl alcohol.
(2) Determination of polysaccharide content
Weighing cell wall skeleton powder 50mg in test tube, adding 1mol/L H 2 SO 4 5mL, sealing the tube, hydrolyzing in water bath at 100deg.C for 8 hr, extracting polysaccharide from the liquid layer of the hydrolysate, and measuring polysaccharide content.
The results of the measurement of the lipid and polysaccharide contents are shown in Table 1.
Table 1: determination of lipid and polysaccharide content
Measurement item Example 3 Comparative example 1 Comparative example 2
Lipid compound (%) 20.3 16.4 12.7
Polysaccharide (%) 51.6 43.5 37.2
As can be seen from Table 1, the lipid and polysaccharide contents in the cultures of rhodococcus erythropolis obtained by the method of the present invention were significantly higher than those of comparative examples 1 and 2.
Test case two, physical property detection of oil emulsion inactivated vaccine for poultry
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 33.4cp, the viscosity of the vaccine in the control group is 43.3cp, 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 III safety test of inactivated vaccine for poultry oil emulsion
The vaccine prepared in the test group of example 4 was used to immunize 21-day-old SPF chickens, 2 mL/chicken, 10 animals per group were immunized with the vaccine, and the health status of the chickens was observed daily for 14 days.
The test result shows that SPF chicken has no death and good health condition, so that the vaccine prepared by the immunopotentiator of the invention is qualified and safe.
Test example four, efficacy test of oil emulsion inactivated vaccine for poultry
The vaccine prepared in the test group and the control group in example 4 is used for immunizing 21-day-old SPF chickens, 0.3 mL/chicken, each group is used for immunizing 10 chickens, each chicken is marked with a foot number, blood is collected and serum is separated every week after 1 week after vaccine injection for HI detection of ND and H9 subtype avian influenza, continuous detection is carried out for 3 weeks, and a blank group is additionally arranged. The test results are shown in tables 1 and 2.
Table 2: efficacy comparison test of New stream bivalent vaccine H9 antibody assay results
Table 3: new stream bivalent vaccine efficacy comparison test ND antibody level comparison
From tables 2 and 3, it can be seen that the levels of H9 antibody and ND antibody of the test group of the vaccine prepared by the immunopotentiator of the invention are significantly higher than those of the control group 21 days after immunization, and the immunopotentiator of the invention can play a role in obviously enhancing the immunity of the organism.
Test example five, commercial chicken immunization test
Commercially available commercial chickens were used, vaccine immunization was performed at about 8 days of age using the vaccines prepared in the test group of example 4, the vaccine prepared in the comparative example 3, the vaccine prepared in the comparative example 4, and the vaccine prepared in the control group of example 4, 0.3 mL/chicken were immunized with 20 vaccine, each chicken was labeled with a foot number, and after 1 week of vaccine injection, serum was collected and separated every week for HI detection of ND and H9 subtype avian influenza, and continuous detection was performed for 8 weeks, and the test results are shown in Table 4.
Table 4: results of commercial chicken immunization test
As can be seen from Table 4, the vaccine prepared by using the immunopotentiator of the present invention has significantly better immune effect than that of the control group.
In conclusion, the vaccine prepared by the immunopotentiator is safe and efficient.

Claims (6)

1. The rhodococcus erythropolis immunopotentiator is characterized by being a rhodococcus erythropolis culture, and the preparation method of the rhodococcus erythropolis 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 is prepared from 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 rhodococcus rhodochrous was purchased from the collection of microbial strains, GDMCC No.:1.819; the fermentation auxiliary agents are oxalic acid and methionine; the mass ratio of oxalic acid to methionine is 2-4:10-15.
2. The rhodococcus erythropolis immunopotentiator of claim 1, wherein the culture medium is prepared from 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%.
3. The red rhodococcus immunopotentiator of claim 1, wherein the mass ratio of oxalic acid to methionine is 3:14.
4. Use of the red rhodococcus immunopotentiator according to any one of claims 1 to 3 for the preparation of an inactivated vaccine for poultry oil emulsion; the poultry oil emulsion inactivated vaccine is a newcastle disease avian influenza duplex vaccine; the antigen is newcastle disease antigen: h9 subtype avian influenza antigen=1:1.
5. An inactivated vaccine for poultry oil emulsion, consisting of the red rhodococcus immunopotentiator of any one of claims 1 to 3; the poultry oil emulsion inactivated vaccine is a newcastle disease avian influenza duplex vaccine; the antigen is newcastle disease antigen: h9 subtype avian influenza antigen=1:1.
6. The method for preparing the inactivated vaccine for poultry oil emulsion according to claim 5, comprising the following steps:
s1, taking a rhodococcus erythropolis immunopotentiator, inactivating the rhodococcus erythropolis immunopotentiator by high-pressure steam at 121 ℃ for 30min, sterilizing, and preserving at 4 ℃ for later use when the temperature is reduced to room temperature;
s2, quantifying the red rhodococcus immunopotentiator to obtain a quantitative red rhodococcus immunopotentiator;
s3, adding the quantitative red rhodococcus immunopotentiator obtained in the step S2 into the mixed anti-water phase according to an emulsification formula, uniformly stirring and performing conventional emulsification to obtain an initial oil emulsion inactivated vaccine for poultry;
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 inactivated vaccine after verification.
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Publication number Priority date Publication date Assignee Title
CN1735431A (en) * 2002-09-06 2006-02-15 伦敦大学 Whole bacterial cells as immune modulator
CN107684621A (en) * 2017-07-25 2018-02-13 华南农业大学 Application of the corynebacterium diphtheroides as immunologic adjuvant in fowl oil emulsion inactivated vaccine
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
CN110935017A (en) * 2019-12-26 2020-03-31 广州科力生物科技有限公司 Application of nocardia immunopotentiator in preparation of poultry vaccine

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1735431A (en) * 2002-09-06 2006-02-15 伦敦大学 Whole bacterial cells as immune modulator
CN107684621A (en) * 2017-07-25 2018-02-13 华南农业大学 Application of the corynebacterium diphtheroides as immunologic adjuvant in fowl oil emulsion inactivated vaccine
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
CN110935017A (en) * 2019-12-26 2020-03-31 广州科力生物科技有限公司 Application of nocardia immunopotentiator in preparation of poultry vaccine

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