Culture medium with low serum content, inactivated vaccine with low serum content and preparation method of inactivated vaccine
By using low serum content culture medium and inactivated vaccine preparation methods, the problems of high serum content, severe side effects, difficult antigen preparation, and high production costs of whole-culture inactivated mycoplasma vaccines have been solved, thus improving safety and economy.
Patent Information
- Application Number
- CN202511804727.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-03
- Publication Date
- 2026-01-20
AI Technical Summary
Existing mycoplasma whole culture inactivated vaccines suffer from problems such as high serum content, severe side effects, difficulty in antigen preparation, weak immunogenicity, and complex and costly production processes, resulting in insufficient safety, efficacy, and economy.
The culture medium with low serum content and the inactivated vaccine preparation method are adopted. By rationally proportioning raw materials, the serum content is reduced, yeast peptone and L-cysteine are added to provide comprehensive nutrition and improve the growth efficiency of mycoplasma. The inactivation is carried out by water-in-oil-in-water biphasic oil emulsion, which simplifies the production process.
It significantly reduces serum content in the culture medium, minimizes side effects, increases viable bacterial titers, lowers production costs, provides a sufficient antigenic basis, and improves vaccine quality and safety.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of biological agents, and particularly relates to a low-serum-content culture medium, a low-serum-content inactivated vaccine and a preparation method thereof. BACKGROUND
[0002] Mycoplasma pneumonia and other diseases have a huge impact on the breeding industry, and vaccination is a key means of prevention and control. At present, the existing mycoplasma inactivated vaccines on the market are mainly divided into tissue toxin products and whole culture products. Among them, the whole culture vaccine has several insurmountable technical bottlenecks and inherent defects in production and application, which seriously restrict its immune effect and popularization and application, which are specifically as follows: 1. Low vaccine purity, severe side effects, and prominent safety hazards: Whole culture vaccines are usually prepared in complex culture medium, which inevitably contains a large amount of animal serum (such as bovine serum) and other biological raw materials with unknown components. These exogenous proteins have small molecular weights and various types, and are extremely difficult to completely remove during vaccine purification. After vaccination, these residual heterologous proteins not only easily cause acute allergic reactions (such as shock, swelling, fever, etc.) in livestock and poultry, leading to decreased production performance; but also can interfere with the body's production of high-titer specific antibodies, and have the potential risk of introducing other exogenous pathogens.
[0003] 2. Difficulty in antigen preparation, weak immunogenicity, and substandard protection: Mycoplasma is a kind of small prokaryotic organism lacking cell wall, and its nutritional requirements are harsh, which is much more difficult to culture in vitro than ordinary bacteria, resulting in high cost and poor stability of antigen production. More importantly, mycoplasma is prone to loss of key protective antigens or insufficient expression during artificial culture medium passage, which leads to low content and uneven proportion of effective immune antigen components in the final vaccine. This directly leads to low antibody titer and insufficient neutralizing activity of the body after vaccination, which cannot provide solid immune protection. The animal population that fails to be immunized is still vulnerable to wild virus attacks, often leading to outbreaks and causing devastating economic losses to the breeding industry.
[0004] 3. Complex and lengthy production process, difficult quality control, and high cost: The production of whole culture vaccine covers multiple complex procedures such as strain propagation, large-scale fermentation, centrifugal concentration, chemical inactivation, deep purification, and adjuvant emulsification. Each step faces challenges: unstable fermentation titer, inactivation agent may damage antigen conformation, purification step may cause loss of target antigen while removing impurities. The whole process is time-consuming and labor-intensive, requiring high-quality production equipment and quality control systems, ultimately leading to high vaccine production costs. This not only increases the economic burden of breeders, but also limits the popularization of vaccines in the industry, which is not conducive to the overall purification of the vaccine.
[0005] In summary, the existing mycoplasma whole culture inactivated vaccine has significant deficiencies in safety, effectiveness and economy, and it is urgent to develop a high-efficiency, safe and inexpensive vaccine to meet the needs of the breeding industry. SUMMARY
[0006] Therefore, the present application aims to provide a low serum content culture medium, a low serum content inactivated vaccine and a preparation method thereof.
[0007] To achieve the above-mentioned purpose, the present application provides the following technical solutions. In a first aspect, the present application provides a low serum content culture medium, comprising the following raw materials by mass: Culture medium matrix 94-96 parts, animal serum 4-6 parts, yeast protein peptone CM03 0.05-0.15 parts, L-cysteine 0.05-0.15 parts.
[0008] As a preferred, the culture medium matrix comprises the following raw materials by mass: pplo broth 20-23 parts, sodium pyruvate 1.5-2.5 parts, glucose 1.5-2.5 parts, 1% phenol red solution 3.5-4 parts, 25% yeast extract 100 parts, water for injection 1000 parts.
[0009] As a preferred, the animal serum includes pig serum, bovine serum.
[0010] As a preferred, it can be used for culturing mycoplasma or virus.
[0011] In a second aspect, the present application provides a low serum content inactivated vaccine preparation method, comprising the following preparation steps: S1. The mycoplasma strain is obtained by primary seed culture and secondary seed culture to obtain secondary seed; S2. The secondary seed is fermented and cultured in the culture medium according to any one of claims 1-3 to obtain a fermentation broth; S3. The fermentation broth is concentrated to the target bacteria content, then inactivated, tested and vaccinated to obtain a low serum content inactivated vaccine.
[0012] As a preferred, the mycoplasma strain in S1 includes Mycoplasma pneumoniae, Mycoplasma filamentous capra subsp., Porcine circovirus type 2 and Mycoplasma hyopneumoniae.
[0013] As a preferred, the primary seed culture and secondary seed culture in S1 use a low serum content culture medium.
[0014] As preferred, the inoculation amount of the fermentation culture in S2 is 10%.
[0015] As preferred, 1% thiomersal solution is added according to 1% of the total amount of antigens in the antigen mixture in S3; the oil phase component is 206 oil adjuvant; the liquid phase component and the oil phase component are emulsified in a ratio of 1:1 to form a water-in-oil emulsion inactivated vaccine.
[0016] In a third aspect, the present application provides a low serum content inactivated vaccine prepared according to the preparation method described above.
[0017] At least the following beneficial technical effects are achieved: The present application significantly reduces the serum content of the culture medium, and the addition of yeast protein peptone CM03 makes up for the lack of nitrogen source and vitamins after the reduction of serum content, provides comprehensive nutrition for mycoplasma, reduces its dependence on complex nutritional ingredients in serum, and supports growth and reproduction in a low serum environment; improves the viable bacterial titer: the small molecule peptides and growth factors contained therein can be efficiently utilized by ovine mycoplasma to promote rapid proliferation of bacterial cells, help maintain or improve the viable bacterial titer in the culture medium, and provide sufficient antigen basis for vaccine production and other scenarios.
[0018] L-cysteine is added to provide suitable amino acids, providing essential material basis for the growth, metabolism and protein synthesis of ovine mycoplasma, meeting the demand of sulfur-containing amino acids for its life activities, making up for the possible nutritional deficiency caused by the reduction of serum content, and assisting the normal reproduction of ovine mycoplasma in a low serum environment. When culturing ovine mycoplasma, increasing cysteine can solve the difficulty of in vitro culture of mycoplasma, reduce the culture time, and greatly reduce the time and cost pressure of purification. DETAILED DESCRIPTION
[0019] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.
[0020] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value in the stated range and any other stated value or intermediate value in the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.
[0021] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, unless indicated otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application.
[0022] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.
[0023] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed materials and methods.
[0024] As used herein, the terms "room temperature", "ambient temperature" are intended to mean 25 ± 2 °C, unless otherwise specified.
[0025] The raw materials or instruments used in the following examples of the present application are commercially available, unless otherwise specified.
[0026] Example 1 Preparation of inactivated vaccine of Mycoplasma capripneumoniae with low serum content I. Preparation of the preparation 1. Preparation of materials and reagents Raw material sources: PPLO broth (BD, USA), sodium pyruvate (Shanghai Maikelin Biochemical Technology Co., Ltd.), glucose (Chemical Reagent Co., Ltd. of China National Pharmaceutical Group), 1% phenol red solution (Chengdu Kolon Chemicals Co., Ltd.), 25% yeast extract (Angel Yeast Co., Ltd.), yeast peptone (Angel Yeast Co., Ltd. Item No: CM03), L-cysteine (Chengdu Kolon Chemicals Co., Ltd.).
[0027] Strains: The strains are Mycoplasma capripneumoniae MoGH3-3 strain and Mycoplasma filamentous goat subspecies M1601 strain, and the test strains are Mycoplasma capripneumoniae MoGH3-3 strain and Mycoplasma filamentous goat subspecies M87-2 strain. They are identified, preserved and supplied by Lanzhou Animal Husbandry Institute, Chinese Academy of Agricultural Sciences.
[0028] Preparation of low serum content medium: Culture medium matrix: pplo broth 21 g, sodium pyruvate 2 g, glucose 2 g, 1% phenol red solution 3.75 g, 25% yeast extract 100 g, water for injection up to 1000 g. Adjust pH to 7.60-7.80 with 1.0 mol / L sodium hydroxide solution, quantitatively sub-pack, sterilize at 116°C for 30 minutes, and store at 2-8°C for standby use.
[0029] Take 95 parts of the culture medium matrix, then add 5 parts of pig serum, 0.1 parts of yeast extract CM03, and 0.1 parts of L-cysteine.
[0030] Apparatus: sterile centrifuge tube, triangular flask, 10 ml graduated pipette, pipette gun, bacterial filter (0.22 μm pore size for removing miscellaneous bacteria), biological safety cabinet, constant temperature incubator, etc., all of which need to be sterilized.
[0031] II. Preparation of primary seed Take the stored Mycoplasma ovis freeze-dried strains MoGH3-3 and M1601 from the -80°C refrigerator, and in the biological safety cabinet, use a sterile pipette gun to take 5 mL of culture medium and add the freeze-dried strains to dissolve, then inoculate into a sterile test tube containing 5 mL of low serum content medium, shake gently, and place the inoculated test tube in a 37°C biochemical incubator for static culture. Mycoplasma grows by fermenting glucose in the culture medium to produce acid, and after 3 days of culture, when the ph drops to 6.8, it indicates that the strain has recovered and proliferated. Take the qualified recovered bacterial liquid and inoculate into low serum content medium (scale up culture at a ratio of 1:10), 37°C, culture for 3 days, and when the ph drops to 6.8, it is 1 generation. Repeat for 2 generations to make the strain grow stably (the number of generations should not be too many to avoid changes in strain virulence or characteristics), as the primary seed.
[0032] III. Preparation of secondary seed Take the qualified primary seed and inoculate into low serum content medium at a ratio of 1:10, and place in a 37°C biochemical incubator for static culture for 3 days. When the ph drops to 6.8, it is 1 generation, and continuously transmit for 2 generations as the secondary seed.
[0033] IV. Preparation of seed liquid After the prepared low serum content medium is sterilized at 116°C for 30 min and cooled to 37°C, add 5% pig serum, 0.1% yeast extract CM03, and 0.1% L-cysteine to the total amount of the medium, then adjust the ph to 7.70 with 2 mol / L sodium hydroxide solution, inoculate at a rate of 10%, and place in a 37°C static incubator. When the ph drops to 6.8, harvest, and take samples for purity test and viable count (CCU).
[0034] V. Concentration Concentrate the MoGH3-3 and M1601 fermentation bacterial liquids with an ultrafiltration system, respectively, so that the titers reach 6 x 108 ccu / ml.
[0035] Six, inactivation and inactivation test Formaldehyde solution (40%) was added to MoGH3-3 and M1601 concentrated bacterial solution respectively, so that the final concentration was 0.2%, and inactivated at 37℃ for 10 hours. After the inactivation time, two low serum content medium inoculated samples were taken, and 10-fold serial dilution was made into 10 -1 to 10 -5 , and cultured at 37℃ for 10 days. If there was no mycoplasma growth, it was considered to be completely inactivated.
[0036] Seven, vaccine preparation After the above tests are passed, the vaccine is prepared.
[0037] Liquid phase composition: inactivated MoGH3-3 and M1601 concentrated antigens are mixed in a ratio of 1:1, and 1% thiomersal solution (final concentration 0.01%) is added at 1% of the total antigen amount.
[0038] Oil phase composition: 206 oil adjuvant.
[0039] Emulsification method: liquid phase composition and oil phase composition are emulsified in a ratio of 1:1.
[0040] First, the 206 oil adjuvant is fed into the emulsifier, then the liquid phase composition is fed, and the mixture is fully mixed by circulating stirring under the pressure of 38MPa through the homogenizer, and emulsified into a slightly sticky water-in-oil-in-water (w / o / w) double-phase oil emulsion inactivated vaccine.
[0041] Eight, comparison of the culture medium prepared by the application and the traditional low serum content medium with 20% serum content.
[0042] Table 1 is a comparison of the time and semi-finished product effective antigen titer CCU of the sheep pneumonia mycoplasma MoGH3-3 strain cultured in the traditional low serum content medium with 20% serum content.
[0043] Table 1
[0044] Table 2 is a comparison of the time and semi-finished product effective antigen titer CCU of the sheep pneumonia mycoplasma MoGH3-3 strain cultured in the low serum content medium with 5% serum content of the application.
[0045]
[0046] Table 3 is a comparison of the time and semi-finished product effective antigen titer CCU of the sheep pneumonia mycoplasma M1601 strain cultured in the traditional low serum content medium with 20% serum content.
[0047] Table 3
[0048] Table 4 is the comparison of the time and semi-finished product effective antigen titer CCU of the low serum content culture medium of the 5% serum content of the application for culturing the M1601 strain of Mycoplasma pneumoniae of sheep.
[0049] Table 4
[0050] Table 5 is a total of 149 batches produced in 2024. One item of serum reduction reduces the direct production cost by 4.276 million yuan.
[0051] Table 5
[0052] As can be seen, the low serum content culture medium can improve product quality, reduce allergic reactions of immunized animals and reduce direct production costs; the serum proportion can be reduced by replacing the animal serum with unknown components with yeast protein components with known components, and the serum is reduced from the specified 20% serum to 5%.
[0053] The method of replacing serum with yeast protein for culture can also be used for other animal mycoplasma culture and virus culture with the same effect.
[0054] The above is only the preferred embodiment of the application, and it should be pointed out that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the application, and these improvements and refinements should also be considered within the protection scope of the application.
Claims
1. A low serum content medium, characterized in that, The raw materials include the following components by mass: Culture medium base 94-96 parts, animal serum 4-6 parts, yeast peptone CM03 0.05-0.15 parts, L-cysteine 0.05-0.15 parts.
2. The medium of claim 1, wherein, The culture medium base includes the following components by mass: pplo broth 20-23 parts, sodium pyruvate 1.5-2.5 parts, glucose 1.5-2.5 parts, 1% phenol red solution 3.5-4 parts, 25% yeast extract 100 parts, water for injection 1000 parts.
3. The medium of claim 1, wherein, The animal serum includes pig serum and cow serum.
4. The medium of claim 1, wherein, The mycoplasma or virus can be cultured.
5. A method for the preparation of an inactivated vaccine with low serum content, characterized in that, The preparation method includes the following steps: S1. The mycoplasma strain is obtained through primary seed culture and secondary seed culture; S2. The secondary seed is fermented using the culture medium according to any one of claims 1-3 to obtain a fermentation broth; S3. The fermentation broth is concentrated to a target bacteria content, and then inactivated, tested, and formulated to obtain an inactivated vaccine with low serum content.
6. The method of preparing an inactivated vaccine according to claim 3, wherein, The mycoplasma strain in S1 includes Mycoplasma pneumoniae, Mycoplasma filamentous caprae, porcine circovirus type 2, and Mycoplasma hyopneumoniae.
7. The method of preparing an inactivated vaccine according to claim 3, wherein, The primary seed culture and the secondary seed culture in S1 use a low serum content culture medium.
8. The method of preparing an inactivated vaccine according to claim 3, wherein, The inoculation amount for the fermentation culture in S2 is 10%.
9. The method of preparing an inactivated vaccine according to claim 3, wherein, In S3, 1% thiomersal solution is added according to 1% of the total antigen amount; the oil phase component is 206 oil adjuvant; the liquid phase component and the oil phase component are emulsified at a ratio of 1:1 to form a water-in-oil-in-water double-phase oil emulsion inactivated vaccine.
10. A low serum content inactivated vaccine, characterized in that, The preparation method is prepared according to any one of claims 5-9.