CN109316603A - A kind of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector, preparation method and application - Google Patents
A kind of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector, preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotectors, preparation method and application, are made of the raw material for including following mass percent: placenta polypeptide 1 ~ 2%；Procyanidine 0.5 ~ 3%；Sorbierite 1 ~ 2%；Sucrose 2 ~ 4%；Trehalose 1 ~ 3%；Dipotassium hydrogen phosphate 0.057%；Dipotassium hydrogen phosphate 0.13%；Excess water.Preparation method, the following steps are included: by placenta polypeptide, procyanidine, sorbierite, sucrose, trehalose, dipotassium hydrogen phosphate, dipotassium hydrogen phosphate, selectively adding the black alcohol of nanometer plant, after mixing with water after weighing each raw material in proportion, cross 0.22 micron of filter membrane degerming.Heat-resisting cryoprotector solubility produced by the present invention is good, and cold-resistant anti-heat effects are significant, and allergy rate is low.
Description
Technical field
The invention belongs to technical field of vaccines, and in particular to a kind of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector,
Preparation method and application.
Background technique
Porcine mycoplasmal pneumonia is also known as swine enzootic pneumonia, be by mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae,
Mhp a kind of contact chronic respiratory infectious disease, is widely present in all over the world caused by).Removing after pig infection leads to feed
Conversion ratio reduces, outside retarded growth, and easily secondary virus or bacterium infection, causes the pig death rate to increase, leads to economic damage
It loses heavy.Vaccine immunity is the prevention most important means of this disease.
It the use of vaccine is the main means for preventing porcine mycoplasmal pneumonia, the porcine mycoplasmal pneumonia live vaccine listed at present
168 plants, RM48 plants of porcine mycoplasmal pneumonia live vaccine or porcine mycoplasmal pneumonia live vaccine Z 3 kinds of vaccines of strain, take in use
Obtained good effect, still, the common protective agent of above 3 kinds of live vaccines be mainly sucrose, milk, its protecting effect of gelatin compared with
Difference, although our company it is newly developed in recent years comprising yeast extract, lactoalbumin hydrolysate, polyvinylpyrrolidone protective agent,
Protective value is more excellent, but there are potential anaphylactogens, finds that allergy case is more in clinical seeded process, and its freeze-dried products
Solubility it is poor, seriously affect inoculation speed.
Summary of the invention
It is an object of the invention to overcome the deficiencies of existing technologies, a kind of porcine mycoplasmal pneumonia live-vaccine heat-proof freezing is provided
Protective agent, preparation method and application.
To achieve the goals above, the technical solution adopted by the present invention is as follows:
Technical solution one:
A kind of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector is made of the raw material for including following mass percent:
It further, further include the black alcohol 0.5~1.5% of nanometer plant.
Technical solution two
A kind of preparation method of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector will after weighing each raw material in proportion
Placenta polypeptide, sorbierite, sucrose, trehalose, dipotassium hydrogen phosphate, dipotassium hydrogen phosphate, selectively adds a nanometer plant at procyanidine
Black alcohol after mixing with water crosses 0.22 micron of filter membrane degerming.
Technical solution three
A kind of application of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector, the porcine mycoplasmal pneumonia being prepared is living
The heat-resisting cryoprotector of vaccine and porcine mycoplasmal pneumonia live vaccine bacterium solution, by 1:0.5~1.5 mass ratio after mixing, it is cold
It is lyophilized dry.
Further, the porcine mycoplasmal pneumonia bacterium solution is 168 plants of porcine mycoplasmal pneumonia live vaccine, porcine mycoplasmal pneumonia is lived
RM48 plants of vaccine or Z plants of porcine mycoplasmal pneumonia live vaccine of weak venom.
Compared with prior art, obtained by the present invention to have the beneficial effect that:
It can be effectively prevent in heat-resisting lyophilized protecting agent formula provided by the invention using sucrose and trehalose as freeze-dried
Or the formation of the crystallization water in refrigerating process is reduced, sorbierite can prevent effective component from distilling loss with vapor, and placenta is more
Peptide can play stable eucaryotic cell structure with the antigen binding of vaccine, protect the effect of antigen, and procyanidine can will be in vaccine
Mycoplasma is adjusted to active most stability region, maintains to play to activity of vaccine under the conditions ofs low temperature and high temperature etc. most important
Effect.In addition, the present invention proves the addition of the black alcohol of nanometer plant through a large number of experiments, the Heat-resistant stable of vaccine is effectively raised
Property.The present invention derives from pig placenta using placenta polypeptide, and homologous with pig, allergy rate is extremely low, and is free of other potential anaphylactogens, has
Effect reduces the allergic problem in theoretical clinical seeded process, and using the heat-resisting cryoprotector of the present invention, freeze dried vaccine solubility
Effect.
Specific embodiment
Further details of narration is carried out to the present invention with reference to embodiments.
In the present invention:
168 plants of porcine mycoplasmal pneumonia live vaccine are provided by Guizhou Foster Biotechnology Co., Ltd；
Procyanidine: it is purchased from Wuhan Yuancheng Technology Development Co., Ltd.；
The nanometer black alcohol of plant: it is purchased from Pu Rui biological medicine technology Co., Ltd also known as nano liposomes phytosterol, is one
The novel phytosterol nano liposome preparations of kind, wrap up the black alcohol of plant using nano material, and partial size is less than 30nm, solution
The water solubility problems of the black alcohol of plant of having determined.
The preparation method of pig placenta polypeptide used in the present invention:
Step 1: pig placenta is passed through virus examination, chooses and examine qualified pig placenta, after shredding, by the mass of 1:2~4
Tissue homogenate is carried out than physiological saline is added, obtains homogenate；
Step 2: by homogenate and protease mass ratio 100:1, be added into homogenate protease in 30 DEG C, pH be 6
4~5h of enzyme digestion reaction under part, enzyme digestion reaction are centrifuged with the revolving speed of 5000r/min, obtain centrifugate,
Step 3: pressing the volume ratio 2.5:1 of centrifugate water phase and extractant organic phase, carried out under the conditions of 35~45 DEG C
Extraction processing 45min, then using the KCl aqueous solution of 2.5mol/L as strip aqueous, by the organic phase and KCl in back extraction
Aqueous solution water phase volume ratio 2:1, is stripped 30min under the conditions of 35~45 DEG C, obtains crude extract；
Step 4: carrying out purifying removal protease to crude extract by ultrafiltration apparatus, molecular weight is collected 5000~10000
Polypeptide between dalton, i.e. pig placenta polypeptide.
The extractant uses hexanol and petroleum ether volume ratio for the mixed solution of 1:4, also includes in the mixed solution
5% surfactant, the surfactant press quality by AOT (2- ethylhexyl Disodium sulfosuccinate) and Arlacel-60
It is compounded than 2:1.
The protease is by trypsase, papain, pepsin and carboxypeptidase 1:1:1:1 preparation group in mass ratio
At.
Embodiment 1
A kind of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector is made of the raw material for including following mass percent:
Preparation method are as follows: after weighing each raw material in proportion, by placenta polypeptide, procyanidine, sorbierite, sucrose, seaweed
Sugar, dipotassium hydrogen phosphate, dipotassium hydrogen phosphate, with water after mixing, cross 0.22 micron of filter membrane degerming.
Its application method is, by the porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector being prepared and porcine mycoplasmal lung
Scorching live vaccine (168 plants) bacterium solution, by 1:1 mass ratio after mixing, freeze-drying.
Embodiment 2
A kind of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector is made of the raw material for including following mass percent:
Preparation method are as follows: with embodiment 1.
Its application method are as follows: with embodiment 1.
Embodiment 3
A kind of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector is made of the raw material for including following mass percent:
Preparation method are as follows: with embodiment 1.
Its application method are as follows: with embodiment 1.
Embodiment 4
A kind of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector is made of the raw material for including following mass percent:
Preparation method are as follows: after weighing each raw material in proportion, by placenta polypeptide, procyanidine, sorbierite, sucrose, seaweed
Sugar, the black alcohol of nanometer plant, dipotassium hydrogen phosphate, dipotassium hydrogen phosphate, with water after mixing, cross 0.22 micron of filter membrane degerming, i.e.,
It can.
Its application method is, with embodiment 1.
Comparative example 1
A kind of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector is made of the raw material for including following mass percent:
Preparation method are as follows: after weighing each raw material in proportion, by procyanidine, sorbierite, sucrose, trehalose, phosphoric acid hydrogen
Dipotassium, dipotassium hydrogen phosphate, with water after mixing, cross 0.22 micron of filter membrane degerming.
Its application method is, with embodiment 1.
Comparative example 2
A kind of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector is made of the raw material for including following mass percent:
Preparation method are as follows: after weighing each raw material in proportion, by placenta polypeptide, sorbierite, sucrose, trehalose, phosphoric acid hydrogen
Dipotassium, dipotassium hydrogen phosphate, with water after mixing, cross 0.22 micron of filter membrane degerming.
Its application method is, with embodiment 1.
Comparative example 3
A kind of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector is made of the raw material for including following mass percent:
Gelatin 1.5%；Trehalose 4%；Lactoalbumin hydrolysate 2%；Yeast extract 2%；Polyvinylpyrrolidone 1%；Phosphorus
Sour hydrogen dipotassium 0.0285%；Dipotassium hydrogen phosphate 0.0650%；Excess water.
Preparation method are as follows: after weighing each raw material in proportion, by gelatin, trehalose, lactoalbumin hydrolysate, yeast extract,
Polyvinylpyrrolidone, dipotassium hydrogen phosphate, dipotassium hydrogen phosphate, with water after mixing, cross 0.22 micron of filter membrane degerming, i.e.,
It can.
Its application method is, with embodiment 1.
Comparative example 4
A kind of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector is made of the raw material for including following mass percent:
Sucrose 4%, milk 2%, gelatin 2%, lactose 1%, dipotassium hydrogen phosphate 0.0285%；Dipotassium hydrogen phosphate
0.0650%；Excess water；
Preparation method are as follows: after weighing each raw material in proportion, by sucrose, milk, gelatin, lactose, polyvinylpyrrolidone,
Dipotassium hydrogen phosphate, dipotassium hydrogen phosphate, with water after mixing, cross 0.22 micron of filter membrane degerming.
Its application method is, with embodiment 1.
1 heat resistant test of effect example
Freeze-drying: the vaccine prepared is subjected to efficacy test first, is then dispensed into cillin bottle, every bottle of dispensed loading amount is
2mL, 10 part/bottles of vaccine specification, the vaccine dispensed is lyophilized according to the procedure below: -40 DEG C of pre-freeze 3h, temperature programmed control
Setting: -50 DEG C of 30min；- 8 DEG C of 15h, -13 DEG C of 1h10min；-28℃6h60min.
Heat stabilization test: by each 2 parts of freeze-dried live vaccine made from each embodiment and comparative example, 2~8 DEG C of placements 9 of difference,
12,15,18,24,30 months, 37 DEG C were placed 5,7,14,21,28 days, and the separately sampled progress effect inspection after freeze-drying before freeze-drying
It tests.As a result it see the table below.
Efficacy test method: CCU content assaying method is used；
Mycoplasma hyopneumoniae culture to be measured is subjected to 10 times of serial dilutions, i.e. 1.8mLKM2 culture medium with KM2 culture medium
It is inoculated with 0.2mL sample, successively 1:10 successively decreases serial dilution at 10-1、10-2.······10-11、10-12, separately set KM2 training
3 pipe of base control is supported, totally 15 pipe, makees 3 repetitions with batch, set 37 DEG C of cultures, observe and record culture medium color change and muddiness daily
The variation of degree is dyed up to the 10th day, and to culture smear, and microscopy checks for typical mycoplasma hyopneumoniae thalli morphology.
Dilution culture after, thalli growth, discoloration highest dilution be culture CCU content to be checked.
KM2 culture medium prescription and related experiment consumptive material are provided by Guizhou Foster Biotechnology Co., Ltd.
Solubility detection: taking 1 bottle of vaccine, be diluted with 10 times of amount physiological saline, the time required to observation is completely dissolved.Knot
Fruit see the table below.
By upper table it follows that heat resistant freeze drying agent solubility prepared by the present invention and cold-resistant anti-heat effects are best.
2 clinical test of effect example and hypersensitive test
(1) inoculation and hypersensitive test: choosing susceptible pig 1000 of 5~15 ages in days health, be divided into five groups, and every group 200, yin
Property control group be not immunized, respectively at being vaccinated in right lung, after injection in 30min, observe and mark and following symptom occur
Pig and count its quantity, symptom include: fall down to the ground suddenly, uneasy, forelimb swimming sample of yelping swings, spits out white foams, vomitting, hind leg
Numbness, gatism, muscular tremor, spasm, palpitating speed, blood pressure decline, breathing urge, suffer a shock in 15~60min
Or it is dead；Drug desensitization is given to the pig for above-mentioned symptom occur and carries out first aid, and observation improves and death condition.
For there is the pig of above-mentioned symptom 30 days after healing, the heat-resisting lyophilized protecting agent injected with vaccine dose (is free of
Live vaccine), after injection in 30min, observe and mark the pig for following symptom occur and count its quantity, symptom includes: unexpected
Fall down to the ground, uneasy, forelimb swimming sample of yelping swings, spits out white foams, vomitting, rear acroanesthesia, gatism, muscular tremor, spasm,
Palpitating speed, blood pressure decline, breathing urges, occurs suffering a shock in 15~60min or dead；It will repeat the pig of above-mentioned symptom,
It is determined as to heat-resisting cryoprotector allergy, and records its quantity.As a result it see the table below.
It is grouped situation:
Test one group: freeze dried vaccine made from injection embodiment 1；Separately do the freeze-dried products of heat-resisting lyophilized protecting agent, system
Preparation Method is identical as freeze dried vaccine (difference is only that without live vaccine), injects the heat resistant freeze drying to the pig that above-mentioned symptom occurs and protects
Protect the freeze-dried products of agent.
Test two groups: freeze dried vaccine made from injection embodiment 4；Separately do the freeze-dried products of heat-resisting lyophilized protecting agent, system
Preparation Method is identical as freeze dried vaccine (difference is only that without live vaccine), injects the heat resistant freeze drying to the pig that above-mentioned symptom occurs and protects
Protect the freeze-dried products of agent.
Compare one group: freeze dried vaccine made from injection comparative example 3；Separately do the freeze-dried products of heat-resisting lyophilized protecting agent, system
Preparation Method is identical as freeze dried vaccine (difference is only that without live vaccine), injects the heat resistant freeze drying to the pig that above-mentioned symptom occurs and protects
Protect the freeze-dried products of agent.
Compare two groups: freeze dried vaccine made from injection comparative example 4；Separately do the freeze-dried products of heat-resisting lyophilized protecting agent, system
Preparation Method is identical as freeze dried vaccine (difference is only that without live vaccine), injects the heat resistant freeze drying to the pig that above-mentioned symptom occurs and protects
Protect the freeze-dried products of agent.
Negative control group: without any vaccine injection.
(2) challenge test
Will be immune 70 days latter, choose the pig 10 and the pig of negative control group that above-mentioned symptom does not occur respectively from each group
The challenge test of 10 progress mycoplasma hyopneumoniaes, each immune group and control group are all made of CVC354 plants (purchased from Chinese veterinary drug
Product supervise institute, which is the i (mycoplasma hyopneumoniae) vaccine efficacy test strain of China Veterinery Drug Inspection Office's preservation), tracheae
5mL/ (100MID) is injected, is observed 30 days after attacking poison, cuts open and kills test porcine mycoplasmal pneumonia tuberculosis varying index scoring criteria to examination
The tuberculosis change for testing pig is scored, and carries out tuberculosis varying index variance analysis.
The pig of each test group is weighed before attacking poison, is weighed after observation, and being averaged at the end of extremely observing after attacking poison is calculated
Daily gain.As a result it see the table below.
As can be seen from the above table, vaccine allergy rate produced by the present invention is minimum, protective rate highest.
Embodiment described above is merely a preferred embodiment of the present invention, and the simultaneously exhaustion of the feasible implementation of non-present invention.It is right
For persons skilled in the art, any aobvious to made by it under the premise of without departing substantially from the principle of the invention and spirit and
The change being clear to should be all contemplated as falling within claims of the invention.
Claims (5)
1. a kind of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector, which is characterized in that by including following mass percent
Raw material is made:
Placenta polypeptide 1 ~ 2%；
Procyanidine 0.5 ~ 3%；
Sorbierite 1 ~ 2%；
Sucrose 2 ~ 4%；
Trehalose 1 ~ 3%；
Dipotassium hydrogen phosphate 0.057%；
Dipotassium hydrogen phosphate 0.13%；
Excess water.
2. a kind of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector according to claim 1, which is characterized in that also wrap
Include the black alcohol 0.5 ~ 1.5% of nanometer plant.
3. a kind of preparation method of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector according to claim 1, special
Sign is, after weighing each raw material in proportion, by placenta polypeptide, procyanidine, sorbierite, sucrose, trehalose, dipotassium hydrogen phosphate,
Dipotassium hydrogen phosphate selectively adds the black alcohol of nanometer plant, after mixing with water, crosses 0.22 micron of filter membrane degerming.
4. a kind of application of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector according to claim 1, feature exist
In by the porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector being prepared and porcine mycoplasmal pneumonia live vaccine bacterium solution, by 1:
0.5 ~ 1.5 mass ratio after mixing, freeze-drying.
5. a kind of application of porcine mycoplasmal pneumonia live-vaccine heat-proof cryoprotector according to claim 1, feature exist
In the porcine mycoplasmal pneumonia bacterium solution is 168 plants of porcine mycoplasmal pneumonia live vaccine, RM48 plants of porcine mycoplasmal pneumonia live vaccine or pig
Z plants of Eaton agent pneumonia live vaccine of weak venom.
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| CN114042167A (en) * | 2021-12-08 | 2022-02-15 | 山东滨州博莱威生物技术有限公司 | Newcastle disease and infectious bronchitis bivalent live vaccine heat-resistant protective agent and preparation method and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114042167A (en) * | 2021-12-08 | 2022-02-15 | 山东滨州博莱威生物技术有限公司 | Newcastle disease and infectious bronchitis bivalent live vaccine heat-resistant protective agent and preparation method and application thereof |
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