CN114381374A - Haemophilus parasuis animal source-free freeze-drying protective agent and preparation method and application thereof - Google Patents
Haemophilus parasuis animal source-free freeze-drying protective agent and preparation method and application thereof Download PDFInfo
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- 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
- C12N1/04—Preserving or maintaining viable microorganisms
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- 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
- A61K39/02—Bacterial antigens
- A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- 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
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Abstract
The invention discloses a haemophilus parasuis non-animal source freeze-drying protective agent, a preparation method and application thereof, wherein the haemophilus parasuis non-animal source freeze-drying protective agent comprises the following components in percentage by weight: sucrose: 2% -8%, mannitol: 0.5% -4%, polyvinylpyrrolidone: 0.25% -3%, dextran: 2 to 6 percent of ultrapure water, and the balance of ultrapure water. Dissolving polyvinylpyrrolidone and dextran in a part of water, and sterilizing at high temperature to obtain a solution A; then dissolving the cane sugar and the mannitol in the rest water, and filtering and sterilizing to obtain a solution B; and finally, uniformly mixing the solution A and the solution B to obtain the freeze-drying protective agent. The freeze-drying protective agent disclosed by the invention is low in production cost, does not contain animal-derived components, and reduces the occurrence of adverse reactions. In addition, the freeze-dried live Haemophilus parasuis vaccine of the protective agent has good heat resistance and storage stability.
Description
Technical Field
The invention relates to the technical field of microbial preservation, in particular to a haemophilus parasuis animal origin-free freeze-drying protective agent and a preparation method and application thereof.
Background
Haemophilus parasuis, a opportunistic pathogen that colonizes the upper respiratory tract of pigs, causes multiple serositis, arthritis and meningitis in pigs, causing significant economic losses to the swine industry (Brockmeier SL, Long CL, Mullins MA, Register KB, Nicholson TL, Wiseman BS, Baker RB, Kehli ME Jr. Virulence, transmission, and biotechnology protection of four isocyanates of Haemophilus parasus [ J ]. Clin Vaccine Immunol,2013,20: 1466-. Weak live vaccination is an important means for the prevention and control of infectious diseases, and not only is it relatively inexpensive, but it also stimulates the body to produce a more comprehensive immune response (Liu H, Xue Q, Zeng Q, Zhao Z. Haemophilus parasquas vaccines [ J ]. Vet immunological Immunopathol,2016,180: 53-58). However, live vaccines are relatively poorly stable, are largely controlled by external conditions, and are prone to losing protective efficacy during transport and storage (Price DN, Kunda NK, Ellis R, Muttil P.design and optimization of a temperature-stable dry powder BCG vaccine [ J ]. Pharm Res,2019,37: 11). Therefore, stability becomes an important indicator of commercialization of live vaccines.
Freeze-drying is a common microbial preservation technology, which not only can prolong the preservation time of products, but also can reduce the transportation cost and the requirement of storage conditions, and is widely applied to the development of live vaccines for livestock (Hansen LJJ, Daoussi R, Vervaet C, Remon JP, De Beer TRM. Freeze-drying of live viruses: A review [ J ] Vaccine,2015,33: 5507-. However, the stresses associated with freezing and drying often lead to decreased cell viability and death, which in turn affects immune performance, and thus, the freeze-drying process requires the addition of suitable lyoprotectants to reduce cell damage (Friess W, Winter G.meeting the challenge in freeze-drying of pharmaceuticals and biologicals [ J ]. Eur J Pharm biopharmarm, 2013,85: 161).
The lyoprotectant components can be classified into sugars, alcohols, amino acids, macromolecular polymers, etc., each of which includes a variety of specific substances (Izutsu KI. applications of freezing and freezing-drying in pharmaceutical formulations [ J ] Adv Exp Med Biol,2018,1081: 371) 383), depending on the nature of the substance, such as:
1) the saccharide includes sucrose, trehalose, lactose, etc.
2) Alcohols include sorbitol, mannitol, glycerol, and the like.
3) Amino acids include arginine, glutamic acid, glycine and the like.
4) The macromolecular polymer comprises dextran, polyvinylpyrrolidone, polyethylene glycol, 2-hydroxypropyl-beta-cyclodextrin and the like.
Because the protection mechanisms of different protective agents are different, a single component cannot meet the requirement of microorganisms on resisting severe environment, and the freeze-drying protective agent usually needs compatibility and combination of multiple components.
At present, aiming at the research of the haemophilus parasuis freeze-drying protective agent, only the laboratory of the inventor discloses a specific formula component (Qin embryo. screening and optimizing the haemophilus parasuis freeze-drying protective agent) [ master's academic thesis ]. Wuhan: library of university of agriculture in Huazhong, 2015), namely 9.12% of skim milk, 2.87% of trehalose, 2.96% of glycerol, and 60.30% of freeze-drying survival rate; however, the protective agent has the following problems:
1) the protective agent contains animal source substance, i.e. skimmed milk powder, in a large proportion, and can be used as allergen to cause adverse reaction of animals.
2) Secondly, the protective agent contains trehalose in a large proportion, so that the production cost is greatly increased.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an animal source-free haemophilus parasuis freeze-drying protective agent, a preparation method and application thereof. In addition, the freeze-dried live Haemophilus parasuis vaccine has good heat resistance.
In order to achieve the aim, the invention designs a haemophilus parasuis animal origin-free freeze-drying protective agent, which comprises the following components in percentage by weight: sucrose: 2% -8%, mannitol: 0.5% -4%, polyvinylpyrrolidone: 0.25% -3%, dextran: 2 to 6 percent of ultrapure water, and the balance of ultrapure water.
Further, the freeze-drying protective agent comprises the following components in percentage by weight: sucrose: 2% -4%, mannitol: 1% -2%, polyvinylpyrrolidone: 0.25% -1%, dextran: 2 to 4 percent of ultrapure water, and the balance of ultrapure water.
Still further, the freeze-drying protective agent comprises the following components in percentage by weight: sucrose: 2%, mannitol: 2%, polyvinylpyrrolidone: 0.5%, dextran: 3% and ultrapure water: 92.5 percent.
The invention also provides a preparation method of the animal source-free haemophilus parasuis freeze-drying protective agent, which comprises the following steps:
1) weighing sucrose, mannitol, polyvinylpyrrolidone, dextran and ultrapure water according to the volume weight ratio;
2) dissolving polyvinylpyrrolidone and dextran in a part of water, and sterilizing at high temperature to obtain solution A;
3) dissolving sucrose and mannitol in the rest water, filtering and sterilizing to obtain solution B;
4) and uniformly mixing the solution A and the solution B to obtain the freeze-drying protective agent.
Further, in the step 2), the high-temperature sterilization temperature is 110-121 ℃; the time is 10-30 min;
in the step 3), the aperture of the filter membrane for filtration sterilization is 0.22 μm.
The invention also provides an application of the freeze-drying protective agent in preparation of animal-source-free freeze-dried live vaccine products of haemophilus parasuis.
The invention also provides a preparation method of the animal source-free freeze-dried live vaccine product of haemophilus parasuis, and the vaccine product is obtained by mixing the freeze-drying protective agent and haemophilus parasuis bacterial liquid according to the volume ratio of 2-4: 1 and freeze-drying.
Further, the haemophilus parasuis bacterial liquid is cultured by the following method:
1) the freeze-dried haemophilus parasuis species that are cryopreserved are activated by resuscitation with TSA medium (with serum and NAD addition);
2) inoculating the single colony in TSB liquid culture medium for culture, and further performing amplification culture; selecting a bacterium solution at the last logarithm stage for centrifugation and re-suspension; obtaining the haemophilus parasuis bacterial liquid, wherein the viable count of the haemophilus parasuis bacterial liquid is 9-15 multiplied by 109CFU/mL。
Still further, the lyophilization procedure is:
firstly, pre-freezing the mixture for 3-4 hours at the temperature of-30 ℃ to-45 ℃; then, under the condition that the vacuum pressure is 0.07-0.15 mbar, selecting a plurality of temperature points to carry out gradient sublimation drying gradually within the temperature range of-25-0 ℃, wherein the total drying time is 11-16 h; and then, gradually desorbing and drying by selecting a plurality of temperature points in a gradient manner within the temperature range of 10-35 ℃ under the vacuum pressure of 0.02-0.05 mbar, wherein the total drying time is 7-9 hours.
Still further, the sublimation drying procedure is as follows:
drying for 2-4 h at the temperature of-25 ℃ to-20 ℃; drying for 5-8 h at the temperature of less than-25 ℃ and less than or equal to-10 ℃, and drying for 2-4 h at the temperature of less than-10 ℃ and less than or equal to 0 ℃.
The desorption drying procedure was as follows:
drying for 2-4 h at the temperature of 10-25 ℃, and drying for 5-7 h at the temperature of less than 25 ℃ and less than or equal to 35 ℃.
The invention has the beneficial effects that:
1) the invention has simple formula and easy preparation, and is suitable for large-scale production.
2) The invention does not contain expensive components such as trehalose and the like, thereby greatly reducing the production cost.
3) The invention does not contain animal-derived substances such as skimmed milk powder, gelatin and the like, and reduces the occurrence of adverse reaction of animals.
4) The method greatly shortens the time required by freeze drying preparation of the product through interval freeze drying, and further saves the production cost.
5) The freeze-dried haemophilus parasuis live vaccine has good heat resistance, and almost no live bacteria loss exists when the freeze-dried haemophilus parasuis live vaccine is stored for 10 days at room temperature.
Drawings
FIG. 1 is a graph of the growth of Haemophilus parasuis;
in the figure, FIG. 1A is OD600Graph of values versus incubation time; FIG. 1B is a graph showing the relationship between the number of viable bacteria and the culture time;
FIG. 2 is a graph of vacuum freeze-drying;
FIG. 3 is a diagram showing a comparison of the freeze-dried form of a Haemophilus parasuis freeze-dried live vaccine preparation;
in the figure, figure 3A is a state diagram of a lyophilized live vaccine product 1 after lyophilization;
FIG. 3B is a diagram of the lyophilized live vaccine preparation 2 after lyophilization;
FIG. 3C is a diagram showing the lyophilized state of the lyophilized live vaccine preparation 3;
FIG. 3D is a diagram of the lyophilized live vaccine preparation 4 after lyophilization;
fig. 3E is a diagram of the lyophilized live vaccine preparation 5 after lyophilization.
Detailed Description
The invention is described in further detail below with reference to the figures and specific embodiments for the understanding of those skilled in the art.
Example 1
The preparation method of the haemophilus parasuis animal origin-free freeze-drying protective agent 1 comprises the following steps:
1) weighing the following components in percentage by volume: 6%, mannitol: 2%, polyvinylpyrrolidone: 0.5%, dextran: 4% of ultrapure water: 87.5 percent;
2) dissolving polyvinylpyrrolidone and dextran in a part of water, and sterilizing at 116 deg.C for 20min to obtain solution A;
3) dissolving sucrose and mannitol in the rest water, and filtering with filter membrane with pore diameter of 0.22 μm for sterilization to obtain solution B;
4) and uniformly mixing the solution A and the solution B to obtain the freeze-drying protective agent 1.
Example 2
The preparation method of the haemophilus parasuis animal origin-free freeze-drying protective agent 2 comprises the following steps:
1) weighing the following components in percentage by volume: 2%, mannitol: 2%, polyvinylpyrrolidone: 0.5%, dextran: 3% of ultrapure water: 92.5 percent;
2) dissolving polyvinylpyrrolidone and dextran in a part of water, and sterilizing at 116 deg.C for 20min to obtain solution A;
3) dissolving sucrose and mannitol in the rest water, and filtering with filter membrane with pore diameter of 0.22 μm for sterilization to obtain solution B;
4) and uniformly mixing the solution A and the solution B to obtain the freeze-drying protective agent 2.
Example 3
The preparation method of the haemophilus parasuis animal origin-free freeze-drying protective agent 3 comprises the following steps:
1) weighing the following components in percentage by volume: 4%, mannitol: 3%, polyvinylpyrrolidone: 0.25%, dextran: 3% of ultrapure water: 89.75 percent;
2) dissolving polyvinylpyrrolidone and dextran in a part of water, and sterilizing at 116 deg.C for 20min to obtain solution A;
3) dissolving sucrose and mannitol in the rest water, and filtering with filter membrane with pore diameter of 0.22 μm for sterilization to obtain solution B;
4) and uniformly mixing the solution A and the solution B to obtain the freeze-drying protective agent 3.
Comparative example 1
The haemophilus parasuis freeze-drying protective agent 4 provided by the comparative example comprises the following components in percentage by weight: 9.12 percent of skimmed milk powder, 2.87 percent of trehalose, 2.96 percent of glycerol and the balance of ultrapure water; the preparation method comprises the following steps:
dissolving skimmed milk powder and glycerol in ultrapure water, and sterilizing at 116 deg.C for 20min to obtain solution A; dissolving trehalose in ultrapure water, and filtering with a filter membrane with the pore diameter of 0.22 μm to obtain a solution B; and uniformly mixing the solution A and the solution B to obtain the freeze-drying protective agent 4.
Comparative example 2
The traditional freeze-drying protective agent 5 comprises the following components in percentage by weight: 10% of skimmed milk powder, 5% of cane sugar and the balance of ultrapure water; the preparation method comprises the following steps:
dissolving skimmed milk powder in ultrapure water, and sterilizing at 116 deg.C for 20min to obtain solution A; dissolving sucrose in ultrapure water, and then filtering with a filter membrane with the pore diameter of 0.22 μm to obtain a solution B; and uniformly mixing the solution A and the solution B to obtain the freeze-drying protective agent 5.
And (3) safety inspection of the freeze-drying protective agent:
respectively inoculating 5mL of each of 1-3 parts of freeze-drying protective agent and 4-5 parts of freeze-drying protective agent to 5 healthy weaned susceptible piglets of 28-35 days old, carrying out temperature detection and symptom observation once every 6 hours, and continuously observing for 24 hours, wherein the results are shown in Table 1.
TABLE 1 Freeze-drying protective agent safety test results
As can be seen from Table 1, after the healthy weaned susceptible piglets are inoculated with the freeze-drying protective agent 1-3, the body temperature does not exceed 40.5 ℃, and abnormal clinical symptoms do not appear. After the healthy weaned susceptible piglet is inoculated with the freeze-drying protective agent 4, the body temperature of the 2/5 healthy weaned susceptible piglet is increased to be higher than 40.5 ℃, and the 1/5 healthy weaned susceptible piglet vomits; after the healthy weaned susceptible piglets are inoculated with the freeze-drying protective agent 5, the body temperature of the 2/5 healthy weaned susceptible piglets is increased to be over 40.5 ℃, and abnormal clinical symptoms do not appear. Therefore, compared with 4-5 parts of freeze-drying protective agents, 1-3 parts of freeze-drying protective agents have good safety.
Example 4
Preparation method of animal-source-free freeze-dried live vaccine product 1-3 of haemophilus parasuis
1) The haemophilus parasuis bacterial liquid is cultured by the following method:
a. inoculating the freeze-dried haemophilus parasuis strain which is frozen and preserved at-20 ℃ into a TSA culture medium (added with 5% serum by volume and 10 mu g/mL NAD), and culturing in a constant temperature box at 37 ℃ for 48h for resuscitation and activation;
b. selecting a single colony, inoculating the single colony in 5mL of TSB liquid medium (added with serum with the volume ratio of 5% and NAD of 10 mu g/mL), and performing shake culture for 7-9 h at the temperature of 37 ℃ and under the condition of 185 r/min; transferring the strain to a TSB liquid medium in a transfer ratio of 1:1,000 for amplification culture, centrifuging the strain liquid cultured to the late logarithm, adding PBS for re-suspension to obtain the haemophilus parasuis strain liquid, wherein the viable count of the haemophilus parasuis strain liquid is 9 multiplied by 109CFU/mL (FIG. 1).
2) Respectively mixing the freeze-drying protective agents 1-3 in the embodiments 1-3 with haemophilus parasuis bacterial liquid according to the volume ratio of 3:1, sucking 2mL of uniformly mixed bacterial suspension, subpackaging the mixture into a sterile 7mL tube glass bottle, and placing the bottle in a freeze dryer for freeze-drying to obtain freeze-dried live vaccine products 1-3; storing at-20 deg.C; wherein, the freeze-drying procedure: firstly, pre-freezing the mixture at the lowest temperature of-32 ℃ for 4 hours;
then, under the condition that the vacuum pressure is 0.10mbar, the drying temperature is-25 ℃, the drying time lasts for 3 hours, the drying temperature is-15 ℃, the drying time lasts for 6 hours, the drying temperature is-5 ℃ and the drying time lasts for 3 hours, and the vacuum pressure is controlled to be 0.10 mbar;
finally, under the condition that the vacuum pressure is 0.05mbar, the desorption drying temperature is 10 ℃, and the time lasts for 3 hours; the temperature was 28 ℃ and the time was 6h (FIG. 2).
Comparative example 3
Preparation method of haemophilus parasuis freeze-dried live vaccine product 4-5
1) The haemophilus parasuis bacterial liquid is cultured by the following method:
a. inoculating the freeze-dried haemophilus parasuis strain which is frozen and preserved at-20 ℃ into a TSA culture medium (added with 5% serum by volume and 10 mu g/mL NAD), and culturing in a constant temperature box at 37 ℃ for 48h for resuscitation and activation;
b. selecting a single colony, inoculating the single colony in 5mL of TSB liquid medium (added with serum with the volume ratio of 5% and NAD of 10 mu g/mL), and performing shake culture for 7-9 h at the temperature of 37 ℃ and under the condition of 185 r/min; transferring the strain to a TSB liquid medium in a transfer ratio of 1:1,000 for amplification culture, centrifuging the strain liquid cultured to the late logarithm, adding PBS for re-suspension to obtain the haemophilus parasuis strain liquid, wherein the viable count of the haemophilus parasuis strain liquid is 9 multiplied by 109CFU/mL。
2) Respectively mixing the freeze-drying protective agents 4-5 in the comparative examples 1-2 with haemophilus parasuis bacterial liquid according to the volume ratio of 1:1, sucking 2mL of uniformly mixed bacterial suspension, subpackaging the mixture into sterile 7mL tubular glass bottles, and placing the bottles in a freeze dryer for freeze-drying to obtain freeze-dried live vaccine products 4-5; storing at-20 deg.C; wherein, the freeze-drying procedure: firstly, pre-freezing the mixture at the lowest temperature of-80 ℃ for 8 hours; taking out, immediately putting into a vacuum freeze dryer for freeze drying, setting the vacuum degree of freeze drying conditions to be 0.37mbar, setting the temperature of a condenser to be-55 ℃, setting the vacuum drying time to be 25h, taking out the freeze-dried sample, and putting into a refrigerator at-20 ℃ for storage for later use.
Respectively carrying out quality standard inspection on the freeze-dried live vaccine products 1-3 and the freeze-dried live vaccine products 4-5, and specifically comprising the following steps:
1. determination of physical property, residual moisture, vacuum degree and rehydration time of vaccine
TABLE 2 determination of physical Properties, residual moisture, vacuum, and rehydration time of the vaccine
Note: the water content is expressed in%.
As shown in Table 2, the freeze-dried live vaccine products 1 to 3 were white sponge-like aggregates in appearance, and the freeze-dried live vaccine products 4 to 5 were pale yellow sponge-like aggregates in appearance (FIG. 3). The vacuum degree detection results of all groups of freeze-dried products are qualified, the rehydration time has no obvious difference, the water content result meets the requirement of 'Chinese pharmacopoeia' 2020 edition, and the water content is not higher than 3%.
2. Freeze-drying survival assay for vaccines
And adding 2mL of PBS into the freeze-dried live vaccine products 1-3 and the freeze-dried vaccine products 4-5 to restore the volume of the bacterial suspension before freeze-drying, standing for 5min to fully rehydrate the bacterial suspension, counting viable bacteria by adopting a concentration gradient dilution method, and calculating the freeze-drying survival rate according to the average value of counting before and after freeze-drying, wherein the specific result is shown in Table 3.
And counting bacteria by adopting a concentration gradient dilution method, carrying out serial dilution on the original bacteria liquid by multiple times, selecting bacteria liquid with proper dilution times (the number of bacterial colonies is between 30 and 300) and inoculating the bacteria liquid to a TSA culture medium for counting, thereby calculating the bacterial quantity of the original liquid. Detailed operation: 2mL of the tube was added with 900. mu.L of physiological saline and labeled (10)–1～10–n). Adding 100 mu L of original bacteria liquid into the 1 st centrifugal tube to obtain 10–1Dilution factor. Taking 100 μ L of 10–1Adding the diluent into the 2 nd centrifugal tube to obtain 10–2Dilution factor according toAnd by analogy, diluting the mixture by multiple times to a proper dilution multiple of 10–n. Taking 100 μ L of 10–nInoculating the diluent into a TSA culture medium, placing the TSA culture medium in a constant-temperature incubator at 37 ℃, standing and culturing for 48 hours, counting, and further calculating the bacterial count of the original bacterial liquid.
The freeze-drying survival rate calculation method comprises the following steps: freeze-drying survival rate ═ live bacteria before freeze-drying (CFU/mL)/live bacteria after freeze-drying (CFU/mL) × 100%.
TABLE 3 Freeze-drying survival assay results for vaccines
Note: the unit of the number of bacteria is 109CFU/mL; the freeze-drying survival rate unit is%.
As is clear from Table 3, the viable cell loss of the Haemophilus parasuis lyophilized live vaccine 4 prepared in comparative example 1 was 1.46X 109CFU/mL, lyophilized survival rate of 62.01%; the viable bacteria loss of the haemophilus parasuis freeze-dried live vaccine 5 prepared in the comparative example 2 is 2.17 multiplied by 109CFU/mL, the freeze-drying survival rate is 43.54%; compared with 4-5 of haemophilus parasuis freeze-drying protective agent, 1-3 of the freeze-drying protective agent has better freeze-drying protection effect on haemophilus parasuis, and the loss of viable bacteria of 1-3 of the prepared freeze-dried live vaccine is less than 1.55 multiplied by 109CFU/mL, the freeze-drying survival rate is greater than or equal to 63.21%, wherein the freeze-drying protective agent 2 has the best freeze-drying protective effect on Haemophilus parasuis, and the freeze-drying survival rate is 72.58%.
3. Experimental determination of vaccine resistance to aging
Taking 1-3 animal source-free freeze-dried live vaccines of haemophilus parasuis and 4-5 freeze-dried live vaccines of haemophilus parasuis, placing the products at room temperature and 37 ℃, respectively carrying out sampling detection 7 days and 10 days after placing, extracting 3 bottles of freeze-dried vaccines of each group for viable bacteria counting, and the specific results are shown in table 4.
TABLE 4. determination results of the aging resistance experiment of the Haemophilus parasuis live vaccine
Note: bacteria count unit 109CFU/mL。
The results in Table 4 show that compared with 4-5 of the haemophilus parasuis freeze-dried live vaccine, the haemophilus parasuis animal source-free freeze-dried live vaccine products 1-3 have obviously better storage stability no matter under the room temperature condition or the 37 ℃; wherein, the heat-resistant property of the haemophilus parasuis animal source-free freeze-dried live vaccine product 2 prepared by the freeze-drying protective agent 2 is better.
Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (10)
1. The animal source-free haemophilus parasuis freeze-drying protective agent is characterized in that: the freeze-drying protective agent comprises the following components in percentage by weight: sucrose: 2% -8%, mannitol: 0.5% -4%, polyvinylpyrrolidone: 0.25% -3%, dextran: 2 to 6 percent of ultrapure water, and the balance of ultrapure water.
2. The animal origin-free freeze-drying protective agent for haemophilus parasuis according to claim 1, which is characterized in that: the freeze-drying protective agent comprises the following components in percentage by weight: sucrose: 2% -4%, mannitol: 1% -2%, polyvinylpyrrolidone: 0.25% -1%, dextran: 2 to 4 percent of ultrapure water, and the balance of ultrapure water.
3. The animal origin-free freeze-drying protective agent for haemophilus parasuis according to claim 1 or 2, which is characterized in that: the freeze-drying protective agent comprises the following components in percentage by weight: sucrose: 2%, mannitol: 2%, polyvinylpyrrolidone: 0.5%, dextran: 3% and ultrapure water: 92.5 percent.
4. A preparation method of the animal origin-free haemophilus parasuis freeze-drying protective agent according to any one of claims 1 to 3, which is characterized by comprising the following steps: the method comprises the following steps:
1) weighing sucrose, mannitol, polyvinylpyrrolidone, dextran and ultrapure water according to the volume weight ratio;
2) dissolving polyvinylpyrrolidone and dextran in a part of water, and sterilizing at high temperature to obtain solution A;
3) dissolving sucrose and mannitol in the rest water, filtering and sterilizing to obtain solution B;
4) and uniformly mixing the solution A and the solution B to obtain the freeze-drying protective agent.
5. The method for preparing the animal origin-free haemophilus parasuis freeze-drying protective agent according to claim 4, wherein the animal origin-free freeze-drying protective agent comprises the following steps: in the step 2), the high-temperature sterilization temperature is 110-121 ℃; the time is 10-30 min;
in the step 3), the aperture of the filter membrane for filtration sterilization is 0.22 μm.
6. The application of the cryoprotectant of claims 1-2 in the preparation of animal-free lyophilized live vaccine products of haemophilus parasuis.
7. A preparation method of animal source-free freeze-dried live vaccine products of haemophilus parasuis is characterized by comprising the following steps: mixing the freeze-drying protective agent as claimed in any one of claims 1 to 3 with haemophilus parasuis bacterial liquid according to a volume ratio of 2-4: 1, and freeze-drying to obtain a vaccine product.
8. The method of claim 7, wherein: the haemophilus parasuis bacterial liquid is cultured by the following method:
1) the freeze-dried haemophilus parasuis strain preserved by freezing is activated by recovering with a TSA culture medium;
2) inoculating the single colony in TSB liquid culture medium for culture, and further enlarging cultureCultivating; selecting a bacterium solution at the last logarithm stage for centrifugation and re-suspension; obtaining the haemophilus parasuis bacterial liquid, wherein the viable count of the haemophilus parasuis bacterial liquid is 9-15 multiplied by 109CFU/mL。
9. The method for preparing the animal origin-free freeze-dried live Haemophilus parasuis vaccine preparation according to claim 7, wherein the animal origin-free freeze-dried live vaccine preparation comprises the following steps: the procedure for the lyophilization was:
firstly, pre-freezing the mixture for 3-4 hours at the temperature of-30 ℃ to-45 ℃; then, under the condition that the vacuum pressure is 0.07-0.15 mbar, selecting a plurality of temperature points to carry out gradient sublimation drying gradually within the temperature range of-25-0 ℃, wherein the total drying time is 11-16 h; and then, gradually desorbing and drying by selecting a plurality of temperature points in a gradient manner within the temperature range of 10-35 ℃ under the vacuum pressure of 0.02-0.05 mbar, wherein the total drying time is 7-9 hours.
10. The method for preparing the animal origin-free freeze-dried live vaccine preparation of haemophilus parasuis according to claim 9, wherein the animal origin-free freeze-dried live vaccine preparation comprises the following steps: the sublimation drying procedure was as follows:
drying for 2-4 h at the temperature of-25 ℃ to-20 ℃; drying for 5-8 h at the temperature of less than minus 25 ℃ and less than or equal to minus 10 ℃, and drying for 2-4 h at the temperature of less than minus 10 ℃ and less than or equal to 0 ℃;
the desorption drying procedure was as follows:
drying for 2-4 h at the temperature of 10-25 ℃, and drying for 5-7 h at the temperature of less than 25 ℃ and less than or equal to 35 ℃.
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| CN101912608A (en) * | 2010-08-17 | 2010-12-15 | 北京海淀中海动物保健科技公司 | Method for producing paratyphus living vaccine for piglets |
| CN105999284A (en) * | 2016-05-06 | 2016-10-12 | 江苏省农业科学院 | Heat-resisting cryoprotectant for classical swine fever live vaccines and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101912608A (en) * | 2010-08-17 | 2010-12-15 | 北京海淀中海动物保健科技公司 | Method for producing paratyphus living vaccine for piglets |
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| CN115369040A (en) * | 2022-09-27 | 2022-11-22 | 福建傲农生物科技集团股份有限公司 | Preservation reagent, kit and preservation method for haemophilus parasuis strain |
| CN115369040B (en) * | 2022-09-27 | 2024-04-02 | 福建傲农生物科技集团股份有限公司 | Preservation reagent, kit and preservation method for haemophilus parasuis strain |
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