CN106176634A - A kind of transmissible gastroenteritis of swine epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution freeze-dried products and preparation method thereof - Google Patents
A kind of transmissible gastroenteritis of swine epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution freeze-dried products and preparation method thereof Download PDFInfo
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Abstract
A kind of transmissible gastroenteritis of swine epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution freeze-dried products and preparation method thereof, belongs to veterinary biologics technical field.Every 1000mlPBS solution contains following components: rhodioside 5 10g, chlorogenic acid 2 6g, Hericium Erinaceus Polysaccharide 5 10g, liposome 6 12g, sucrose 10 15g and mannitol 10 15g.Liposome dilution freeze-dried products prepared by the present invention can extend antigen action time in vivo, increases the immanoprotection action time.Liposome dilution freeze-dried products prepared by the present invention can promote to induce immune response in animal body, and then increases substantially the antibody horizontal of transmissible gastro-enteritis virus epidemic diarrhea virus, strengthens the immune effect of vaccine.
Description
Technical field
The invention belongs to veterinary biologics technical field, be specifically related to a kind of transmissible gastroenteritis of swine-epidemic diarrhea
Bi-combined attenuated Vaccine liposome dilution freeze-dried products and preparation method thereof.
Background technology
Transmissible gastroenteritis of swine sick (Transmissible gastroenteritis of swine, TGE) and popularity
Diarrhoea (Porcine epidemic diarrhea, PED) is the infectious disease of two kinds of serious harm pig industrys, and they are right
Pig, within especially making 2 week old, piglet causes vomiting, severe diarrhea, dehydration and high mortality (up to 100%) to be principal character；5
Pig more than week old infects these two kinds poor growths after being ill, and efficiency of feed utilization drastically declines, and causes to whole world pig industry
Huge economic loss.Still lacking effective clinical medicine at present, vaccine immunity is the major measure that prevention both diseases infect
One of.
Liposome is that one has a same biomembrane kin phospholipid bilayer structure carrier, and Chinese medicine liposome system
The spherical drug carriers formulations of a kind of superminiature referring to that middle the effective elements of the medicine is encapsulated in lipoids bilayer and make.This medicine
Thing carrier has an advantage of targeting, sustained-release administration, and liposome is the most degradable, avirulence and non-immunogenicity, and it is made
It is possible not only to improve drug therapeutic indices for a kind of pharmaceutical carrier, additionally it is possible to reduce drug toxicity and reduce poisonous side effect of medicine etc.
Advantage.
Rhodioside is the Natural antioxidant being found in Radix Rhodiolae, has pharmacological action widely, such as neuroprotective
Effect, cardiovascular protective effect etc., and there is good adjuvant sample effect, body's immunity can be improved, make immunologic function low
Under (such as myocarditis or aging etc.) animal specificity immunologic function and non-specific immunity recover, also can be made by protection
The function of hemocytoblast, improves body's immunity.
Polysaccharide is one of active ingredient of Chinese herbs, and numerous studies show, polysaccharide and saccharide complex are not only work in vivo
For energy resource and constituent material, it is often more important that it is present in all membrane structures, participate in cell in biosis
Various activities.Polysaccharose substance is the important component part of all Living organisms, has removing free radical, improves antioxidase
Activity and the ability of anti-lipid peroxidation
One of most important index of veterinary biologics performance rating is exactly immune period, but weak in actual production process
Though it is fast that poison vaccine has generation antibody, but is difficult to control to the length of its guard time, present inventive concept is that solving to extend pig passes
Metachromia marcy agent-epidemic diarrhea virus antigen action time in vivo, the immune response of induced animal body,
And then also increase substantially the immune effect of vaccine.
Summary of the invention
The problem existed for prior art, it is an object of the invention to design provides a kind of transmissible gastroenteritis of swine-stream
The technical scheme of row diarrhoea Bi-combined attenuated Vaccine liposome dilution freeze-dried products and preparation method thereof.
Described a kind of transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution freeze-dried products,
It is characterized in that every 1000mlPBS solution contains following components: rhodioside 5-10g, chlorogenic acid 2-6g, Hericium Erinaceus Polysaccharide 5-
10g, liposome 6-12g, sucrose 10-15g and mannitol 10-15g.
Described a kind of transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution freeze-dried products,
It is characterized in that every 1000mlPBS solution contains following components: rhodioside 6-8g, chlorogenic acid 3-5g, Hericium Erinaceus Polysaccharide 6-8g,
Liposome 8-10g, sucrose 12-14g and mannitol 12-14g.
Described a kind of transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution freeze-dried products,
It is characterized in that described liposome is prepared by following steps: by soybean lecithin, cholesterol ultrasonic power in PBS solution
300-350W, processes 5-10min, adds ethanol, then sample is put into supercritical CO after vibration mix homogeneously2In extraction kettle, so
After extraction kettle is put in 40 DEG C of waters bath with thermostatic control, be first passed through air in gas purge still before reaction, be forced into 25MPa, face super
Hatching 10-60min under boundary's state, slowly release pressure i.e. obtains liposome solutions, is prepared as liposome with refiner after dehydration.
Described a kind of transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution freeze-dried products,
It is characterized in that in 200ml PBS solution, after sonic oscillation mix homogeneously, add soybean lecithin 4-6g, cholesterol 2-6g
500ml ethanol.
Described a kind of transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution freeze-dried products,
It is characterized in that described PBS solution is obtained by following steps: take sodium chloride 6-10g, potassium chloride 0.05-0.5g, phosphoric acid hydrogen
Disodium 1-1.2g, potassium dihydrogen phosphate 0.05-0.5g, calcium chloride 0.05-0.2g, magnesium chloride 0.05-0.2g containing 6 water of crystallization；
After above each composition is mixed, it is dissolved in 1000ml distilled water, through 116 DEG C, autoclaving 30 minutes and get final product.
Described a kind of transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution freeze-dried products
Preparation method, it is characterised in that comprise the following steps: after each component of described weight being sufficiently mixed, join 1000ml pH
Vibration aquation in 7 PBS solution, forms uniform solution；After the filter of 0.22um filters, first it is positioned over-40 DEG C of low temperature
Refrigerator quick freezing, room temperature is slowly melted, and is repeated four times, then is placed in-40 DEG C of pre-freeze 4-8h；Last in vacuum freeze-drying machine
It is dried, i.e. obtains transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution freeze-dried products.
Described a kind of transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution freeze-dried products
Preparation method, it is characterised in that described PBS solution is obtained by following steps: take sodium chloride 6-10g, potassium chloride 0.05-
0.5g, disodium hydrogen phosphate 1-1.2g, potassium dihydrogen phosphate 0.05-0.5g, calcium chloride 0.05-0.2g, magnesium chloride containing 6 water of crystallization
0.05-0.2g；After above each composition is mixed, it is dissolved in 1000ml distilled water, through 116 DEG C, autoclaving 30 minutes and get final product.
In the present invention, rhodioside is by Wuhan Synchallenge Unipharm Inc.'s production and sales；Chlorogenic acid is stepped by Shanghai
The production and sales of riel chemical technology company limited；Hericium Erinaceus Polysaccharide is by Hubei Yuancheng Saichuang Technology Co., Ltd.'s production and sales.
The method have the advantages that
1. the liposome dilution freeze-dried products prepared by the present invention can extend antigen action time in vivo, increases immunity
The protective effect time.
2. the liposome dilution freeze-dried products prepared by the present invention can promote to induce immunne response in animal body
Reaction, and then increase substantially the antibody horizontal of transmissible gastro-enteritis virus-epidemic diarrhea virus, strengthen the immunity of vaccine
Effect.
Detailed description of the invention
In order to make the present invention easier to understand, below in conjunction with specific embodiment, the application reason of the present invention is expanded on further
Solving, these embodiments are merely to illustrate the present invention rather than limit the scope of the present invention, NM tool in the following example
Body experimental technique, experimental technique is carried out the most routinely.
Prepared by embodiment 1:PBS solution
Take sodium chloride 6-10g, potassium chloride 0.05-0.5g, disodium hydrogen phosphate 1-1.2g, potassium dihydrogen phosphate 0.05-0.5g, calcium chloride
0.05-0.2g, magnesium chloride 0.05-0.2g containing 6 water of crystallization；After above each composition is mixed, it is dissolved in 1000ml distilled water,
Through 116 DEG C, autoclaving 30 minutes and get final product.
Embodiment 2: prepared by liposome
By soybean lecithin 6g, cholesterol 2g ultrasonic power 300W in the PBS solution (embodiment 1 prepares) of 200ml, process
5min, adds the ethanol of 500ml, then sample is placed in supercritical CO after vibration mix homogeneously2In extraction kettle, then by extraction kettle
Put in 40 DEG C of waters bath with thermostatic control, be first passed through air in gas purge still before reaction, be forced into 25MPa, incubate in the supercritical state
Changing 40min, slowly release pressure i.e. obtains liposome solutions, is prepared as liposome with refiner after dehydration.
Embodiment 3: prepared by liposome
It is 320W by soybean lecithin 4g, cholesterol 6g ultrasonic power in the PBS solution (embodiment 1 prepares) of 200ml, processes
6min, adds the ethanol of 500ml, then is placed in by sample in SCF-CO 2 still, then by extraction kettle after vibration mix homogeneously
Put in 40 DEG C of waters bath with thermostatic control, be first passed through air in gas purge still before reaction, be forced into 25MPa, incubate in the supercritical state
Changing 40min, slowly release pressure i.e. obtains liposome solutions, is prepared as liposome with refiner after dehydration.
Embodiment 4: a kind of transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution freeze-dried products
Preparation
Take rhodioside 5g, chlorogenic acid 2g, Hericium Erinaceus Polysaccharide 5g, liposome (embodiment 2 prepares) 6g, sucrose 10g, mannitol
10g；
After above-mentioned each component being sufficiently mixed, join vibration in 1000ml pH 7 phosphate buffer (embodiment 1 prepares)
Aquation, forms uniform solution；
After the filter of 0.22um filters, being first positioned over-40 DEG C of cryogenic refrigerator quick freezing, room temperature is slowly melted, instead
Carry out again four times, then be placed in-40 DEG C of pre-freeze 6h；
Finally it is dried in vacuum freeze-drying machine, i.e. obtains transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine fat
Plastid diluent freeze-dried products.
Embodiment 5: a kind of transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution freeze-dried products
Preparation
Take rhodioside 6g, chlorogenic acid 4g, Hericium Erinaceus Polysaccharide 8g, liposome (embodiment 3 prepares) 8g, sucrose 12g, mannitol
12g；
After above-mentioned each component being sufficiently mixed, join vibration in 1000ml pH 7 phosphate buffer (embodiment 1 prepares)
Aquation, forms uniform solution；
After the filter of 0.22um filters, being first positioned over-40 DEG C of cryogenic refrigerator quick freezing, room temperature is slowly melted, instead
Carry out again four times, then be placed in-40 DEG C of pre-freeze 4h；
Finally it is dried in vacuum freeze-drying machine, i.e. obtains transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine fat
Plastid diluent freeze-dried products.
Embodiment 6: a kind of transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution freeze-dried products
Preparation
Take rhodioside 10g, chlorogenic acid 6g, Hericium Erinaceus Polysaccharide 10g, liposome (embodiment 3 prepares) 12g, sucrose 15g, manna
Alcohol 15g；
After above-mentioned each component being sufficiently mixed, join vibration in 1000ml pH 7 phosphate buffer (embodiment 1 prepares)
Aquation, forms uniform solution；
After the filter of 0.22um filters, being first positioned over-40 DEG C of cryogenic refrigerator quick freezing, room temperature is slowly melted, instead
Carry out again four times, then be placed in-40 DEG C of pre-freeze 8h；
Finally it is dried in vacuum freeze-drying machine, i.e. obtains transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine fat
Plastid diluent freeze-dried products.
The application of test example 1 present invention
1 material
1.1 transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution freeze-dried products: real by the present invention
Execute example 4-6 to prepare
1.2 transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine
1.3 experimental animals: 14～21 age in days sodium selenites 20, TGE, PED antibody test is negative, and (NAT is equal
Not higher than 1:4).
2 methods
The mensuration of 2.1 envelop rates
Take freeze-dried lipidosome and the lipidosome freeze-dried powder of entrapped drug of blank respectively, redissolve in phosphate buffer,
Ultracentrifugation, takes after supernatant dilutes by a certain percentage, measures its absorbance (with reference to veterinary drug allusion quotation), and average each sample is surveyed 3 times, calculates
Its meansigma methods.
2.2 morphologic observations and particle diameter
Liposome dilution lyophilized powder, after PBS redissolves, is observed under transmission electron microscope (× 20000)
2.3 immune effects measure
The liposome dilution freeze-dried products of preparation in embodiment of the present invention 4-6 is respectively placed in the phosphate of a certain amount of PH7
Buffer is completely dissolved, as vaccine diluent.60 21 age in days piglets being divided into four groups, often group 15, A group uses this
The liposome dilution freeze-dried products of bright embodiment 4 preparation carries out vaccine dilution；B group uses the embodiment of the present invention 5 to prepare lipid
Body diluent freeze-dried products carries out vaccine dilution；The C group employing embodiment of the present invention 6 is prepared liposome dilution freeze-dried products and is carried out
Vaccine dilutes；D group, test control group, use vaccine to carry diluent freeze-dried products and carry out vaccine dilution.After immunity in 7d,
14d, 21d, 28d, 35d, 42d, 49d take a blood sample respectively, separate serum, measure the NAT of TGE, PED.
3 results
3.1 envelop rate results
According to formula: envelop rate (%)=system is encapsulated and non-encapsulated total dose (A2) in dose (the A1)/system of encapsulating
× 100%, the results are shown in Table 1.
Table 1 entrapment efficiency determination result
3.2 morphologic observations and particle size determination
Liposome dilution freeze-dried products is multilamelar liposome, and granularity rule, for circle, particle diameter is basically identical, the results are shown in Table 2.
Table 2 morphologic observation and particle size determination result
3.2 measure immune effect
Table 3 TGE neutralizing antibody measurement result
Table 4 PED neutralizing antibody measurement result
4 conclusions
In sum, transmissible gastroenteritis of swine of the present invention-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution freeze-dried products
Processing technology there is good repeatability, this liposome dilution freeze-dried products be possible not only to extend transmissible gastroenteritis of swine-
The epidemic diarrhea Bi-combined attenuated Vaccine protection period, it is also possible to increase substantially transmissible gastroenteritis of swine-porcine epizootic diarrhea bigeminy
The NAT of attenuated vaccine.
Claims (7)
1. transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution freeze-dried products, its feature exists
Following components is contained: rhodioside 5-10g, chlorogenic acid 2-6g, Hericium Erinaceus Polysaccharide 5-10g, liposome in every 1000mlPBS solution
6-12g, sucrose 10-15g and mannitol 10-15g.
2. a kind of transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution as claimed in claim 1
Freeze-dried products, it is characterised in that every 1000mlPBS solution contains following components: rhodioside 6-8g, chlorogenic acid 3-5g, Hericium erinaceus (Bull. Ex Fr.) Pers.
Polysaccharide 6-8g, liposome 8-10g, sucrose 12-14g and mannitol 12-14g.
3. a kind of transmissible gastroenteritis of swine as claimed in claim 1 or 2-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution
Liquid freeze-dried products, it is characterised in that described liposome is prepared by following steps: soybean lecithin, cholesterol is molten at PBS
Ultrasonic power 300-350W in liquid, processes 5-10min, adds ethanol, then sample is put into supercritical CO after vibration mix homogeneously2
In extraction kettle, then extraction kettle is put in 40 DEG C of waters bath with thermostatic control, be first passed through air in gas purge still before reaction, be forced into
25MPa, hatches 10-60min in the supercritical state, and slowly release pressure i.e. obtains liposome solutions, by homogenate mechanism after dehydration
Standby one-tenth liposome.
4. a kind of transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution as claimed in claim 3
Freeze-dried products, it is characterised in that by equal for soybean lecithin 4-6g, cholesterol 2-6g sonic oscillation mixing in 200ml PBS solution
500ml ethanol is added after even.
5. a kind of transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome as described in claim 1,2 or 4 is dilute
Release liquid freeze-dried products, it is characterised in that described PBS solution is obtained by following steps: take sodium chloride 6-10g, potassium chloride
0.05-0.5g, disodium hydrogen phosphate 1-1.2g, potassium dihydrogen phosphate 0.05-0.5g, calcium chloride 0.05-0.2g, containing 6 water of crystallization
Magnesium chloride 0.05-0.2g；After above each composition is mixed, it is dissolved in 1000ml distilled water, through 116 DEG C, autoclaving 30 minutes
Obtain.
6. a kind of transmissible gastroenteritis of swine as claimed in claim 1 or 2-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution
The preparation method of liquid freeze-dried products, it is characterised in that comprise the following steps: after each component of described weight being sufficiently mixed, adds
Vibration aquation in 1000ml pH 7 PBS solution, forms uniform solution；After the filter of 0.22um filters, first put
Being placed in-40 DEG C of cryogenic refrigerator quick freezing, room temperature is slowly melted, and is repeated four times, then is placed in-40 DEG C of pre-freeze 4-8h；Finally
Vacuum freeze-drying machine is dried, i.e. obtains transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution
Liquid freeze-dried products.
7. a kind of transmissible gastroenteritis of swine-epidemic diarrhea Bi-combined attenuated Vaccine liposome dilution as claimed in claim 6
The preparation method of freeze-dried products, it is characterised in that described PBS solution is obtained by following steps: take sodium chloride 6-10g, chlorination
Potassium 0.05-0.5g, disodium hydrogen phosphate 1-1.2g, potassium dihydrogen phosphate 0.05-0.5g, calcium chloride 0.05-0.2g, containing 6 water of crystallization
Magnesium chloride 0.05-0.2g；After above each composition is mixed, it is dissolved in 1000ml distilled water, through 116 DEG C, autoclaving 30 points
Clock and get final product.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108060139A (en) * | 2017-08-31 | 2018-05-22 | 浙江美保龙生物技术有限公司 | A kind of isolated culture method of Porcine epidemic diarrhea virus |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108060139A (en) * | 2017-08-31 | 2018-05-22 | 浙江美保龙生物技术有限公司 | A kind of isolated culture method of Porcine epidemic diarrhea virus |
| CN108060139B (en) * | 2017-08-31 | 2021-07-20 | 浙江美保龙生物技术有限公司 | Isolation culture method of porcine epidemic diarrhea virus |
| CN109620962A (en) * | 2017-10-09 | 2019-04-16 | 中牧实业股份有限公司 | A kind of thinner for vaccine and the preparation method and application thereof |
| CN109620962B (en) * | 2017-10-09 | 2021-11-26 | 中牧实业股份有限公司 | Vaccine diluent and preparation method and application thereof |
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