CN108904795A - A kind of preparation method of the oral vaccine of Porcine epidemic diarrhea virus - Google Patents
A kind of preparation method of the oral vaccine of Porcine epidemic diarrhea virus Download PDFInfo
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Abstract
The invention belongs to Vet Biotechnology application fields, and in particular to a kind of preparation method of effective prevention and control Porcine epidemic diarrhea virus new oral vaccine, including：Virus attenuated strain freeze-drying；By the freeze-dried powder centrifugal granulating of virus, the pellet for being mounted with virus attenuated strain is obtained；Pellet obtained is coated with isolation coat；The pellet coating enteric coating of isolation coat will be coated with to get oral vaccine.The present invention prepares a kind of PEDV new oral vaccine for being resistant to gastric acid and effective stimulus pig Intestinal Mucosal Immunity, for producing upper prevention and control PED, can mix with feed and carry out feeding pig, operate easier；Greatly reduce other immunization ways as caused by injection stress, the anti-scattered malicious risk raised etc., it is safer.
Description
Technical field
The invention belongs to Vet Biotechnology application fields, and in particular to a kind of effective prevention and control Porcine epidemic diarrhea virus is new
The preparation method of type oral vaccine.
Background technique
Pig epidemic diarrhea (Porcine epidemic diarrhea, PED) is by Porcine epidemic diarrhea virus
Acute, the highly contagious disease of pig caused by (Porcine epidemic diarrhea virus, PEDV).Infect PEDV
Newborn piglet there is diarrhea, dehydration, vomiting, case fatality rate is high.In recent years, PED is worldwide widely current, China, beauty
The successive epidemic outbreaks of the country such as state, South Korea, Canada seriously endanger the development of global pig breeding industry.There are 29 provinces and cities' autonomies in China
Area occurs and prevalence PED, can lead to the newborn piglet death rate and is up to 100%, causes huge economic damage to China's pig breeding industry
It loses.The disease there is no effective treatment method at present, can only be by based on vaccine immunity prevention and control.It is main after PEDV is oral, nose infects pig
The intestinal epithelial cell for invading pig causes cellular damage, and intestinal villus atrophy falls off, and small intestine absorption area is caused significantly to subtract
It is few, can not normal absorption nutriment, finally cause grice diarrhoea, dehydration and it is dead.Since PEDV mainly passes through intrusion chitling road
Body is infected, body mucosal immune response is particularly important for prevention and control PEDV, and the high-affinity sIgA that mucosa-immune generates can be with
It effectively prevents cause of disease from adsorbing mucous membrane and neutralizes cause of disease.Therefore, how pig Intestinal Mucosal Immunity is effectively excited, prevention and control PEDV is felt
It contaminates most important.
So far, common PED vaccine include inactivated vaccine, attenuated vaccine, Transgenic Plant Vaccines, with bacterium, disease
Poison is recombinant vaccine and subunit vaccine of carrier preparation etc..Wherein, commercialized vaccine packet currently in use in production
Before the pig epidemic diarrhea-transmissible gastroenteritis of swine-rotavirus trigeminal live vaccine and the Wuhan section that include Harbin dimension section biology
Pig epidemic diarrhea-transmissible gastroenteritis of swine bivalent inactivated vaccine of Biological Co., Ltd.'s preparation；Both commercialization epidemic diseases
Seedling immunization ways are parenteral.In addition, production on also frequently by directly return raise morbidity piglet intestinal contents infection cherish
It is immune to generate milk for pregnant sow, and then gives newborn piglet and provide passive protection.But the PED vaccine used in production at present, no
Pipe is attenuated live vaccines or inactivated vaccine is all made of Parenteral immunization mode, and the effect of excitation pig Intestinal Mucosal Immunity has very much
Limit.In addition, returning by the excrement that PEDV is infected to pig or the intestinal contents for the piglet that falls ill and raising artificial challenge farrowing sow, can have
Effect stimulation body generates the maternal antibody for being directed to PEDV, after newborn piglet is sucked the breast, is protected, it is dead to greatly reduce newborn piglet
Rate is died, while shortening the fashionable colors of PED, significant effect, but returns feeding and there is serious scattered malicious risk, may cause bigger economy
Loss.And if being easy PEDV attenuated live vaccines Direct-fed pig by its internal gastric acid destruction, and then influence immune effect.
Therefore, the PEDV new oral vaccine that preparation can be effective and good safety is extremely urgent.
Summary of the invention
In consideration of it, it is necessary to provide a kind of effective prevention and control Porcine epidemic diarrhea virus new oral vaccine regarding to the issue above
Preparation method.The present invention destroys to overcome existing vaccine not can avoid gastric acid, and chitling road cannot be stimulated to generate foot well
The shortcomings that enough PEDV specific mucosal immunities, prepares a kind of gastric acid and effective stimulus pig Intestinal Mucosal Immunity of being resistant to
PEDV new oral vaccine lays the foundation to produce upper prevention and control PED, and also for prevention and control, other pig enteric infectious diseases provide new approaches.
The present invention is achieved by the following technical solutions：
A kind of preparation method of effective prevention and control Porcine epidemic diarrhea virus oral vaccine, including：
1), virus attenuated strain freeze-drying；
2), by the freeze-dried powder centrifugal granulating of virus, the pellet for being mounted with virus attenuated strain is obtained；
3) pellet made from step 2), is coated with isolation coat；
4) step 3), is coated with the pellet coating enteric coating of isolation coat to get oral vaccine.
Further, in step 1), the operation of virus attenuated strain freeze-drying is：
1-1, mixing will be sufficiently stirred with isometric aseptic freeze-dried protective agent after the virus attenuated strain filtering that culture obtains；
1-2, freeze dryer progress pre-freeze is opened, to extremely -50 DEG C of plate temperature drop, the virus liquid mixed in 1-1 is put into freeze-drying
Machine keeps -50 DEG C after inlet, maintains 2h；
1-3, successively unlatching rear cabinet cools down, vacuumizes, opens big butterfly valve, opens lobe pump, and carriage body vacuum degree is made to be down to 20pa simultaneously
Maintain 1h；
1-4：200min is heated after 1h to be made sample temperature rise to -7 DEG C and maintains at least 1h；Then pass through reheating
120min takes out after so that all samples is warming up to 15 DEG C, and obtained viral freeze-dried powder, which vacuumizes, is sealed and placed in 4 DEG C of preservations.
Further, in step 1-1, the freeze drying protectant can reduce freezing and drying process to the shadow of virus protein
It rings, composition includes：10% skimmed milk power, 5% sucrose and 85% water.
Further, granulation operations include in the step 2)：By viral freeze-dried powder with etc. quality cornstarch it is abundant
Mixing is used as material, and using 500-3000g capsule core as parent nucleus, 50-100ml adhesive carries out centrifugation in centrifugal granulator and makes
Grain.
Further, freeze-dried powder applied sample amount is the 1/10 of capsule core；Binder dosage is about the 1/10 of capsule core.
Further, the capsule core is cane sugar core, starch capsule core, lactose capsule core, mannitol capsule core, microcrystalline cellulose vegetable pill
Core etc., the preferably sucrose capsule core in terms of preparation effect and to the protection of virus.
Further, described adhesive is pure water or HPMC.
Further, concrete operations are granulated in the step 2) is：Centrifugal granulator is opened, capsule core is put into, turntable is set
Revolving speed 200r/m (revolving speed/minute), air inlet 0~3000r/m of wind speed (preferably 700r/m), 0~3000r/m of air draft wind speed are (preferably
900r/m), 25~45 DEG C of temperature of charge (preferably 28 DEG C) open 1KW heating, then turn in atomizing pressure 0.15mpa, peristaltic pump
Adhesive is sprayed onto capsule core surface under conditions of fast 10r/m, upper powder is added in viral freeze-dried powder after capsule core sufficiently soaks
In cylinder, the upper powder for starting viral powder shakes feeding, maintains all parameter settings of instrument, is all adhered to capsule core table to viral powder
Face can stop wriggling and be atomized, and continue to keep turntable rotation and air inlet, air draft wind speed, about 15min is dried to get arriving
Load virulent pellet.
Further, the operation of coating isolation coat includes in the step 3)：It is (preferably hemizygous to weigh isolation coat material
At polymer hydroxypropyl methyl cellulose (HPMC)), the isolation coat solution of configuration concentration 4-6% is taken water as a solvent, is then opened
Fluidized-bed coating machine is opened, the pellet for being mounted with virus attenuated strain that step 2) centrifugal granulating obtains is poured into, setting rotation speed of fan is 0
~2500r/m (preferably 1700r/m), temperature of charge are 25~45 DEG C (preferably 28 DEG C), open 3KW heating, atomizing pressure is arranged
0.15mpa opens peristaltic pump, steps up revolving speed and be pumped into isolated coating liquid to 20r/m when temperature of charge is stablized at 28 DEG C
Bottom spray coating is carried out to pellet in fluidized bed；Stop peristaltic pump after coating weight gain about 5% to wriggle, instrument other parameters is kept to set
Set constant, 30 DEG C are continued dry 20min.
Further, the operation of coating enteric coating is in the step 4)：
4-1, preparation enteric coating liquid；
The pellet for being coated with isolation coat that 4-2, the coating step 3) of the coating solution made from 4-1 obtain.
Further, the enteric coating liquid is the aqueous dispersion polymerization object configured using talcum powder as antitackiness agent, including：
Talcum powder amount of polymers 25-50%, triethyl citrate amount of polymers 10%, the weight gain of L30D-55 (Utech) aqueous dispersion
30%, water surplus.
Further, the operation of the step 4-2 is：It is arranged 0~2500r/m of rotation speed of fan (preferably 1700r/m), material
Temperature is 25~35 DEG C (preferably 28 DEG C), and inlet air temperature is 30-32 DEG C, opens 3KW heating, atomizing pressure 0.17mpa is arranged, to
Temperature of charge stablize at 28 DEG C, open peristaltic pump, step up revolving speed enteric coating liquid is pumped into 30r/m it is right in fluidized bed
Pellet carries out bottom spray coating；Stop peristaltic pump after coating weight gain about 30% to wriggle, 30 DEG C of fluidized drying 30min.
Further, the preparation-obtained PEDV oral vaccine of a kind of the method for the present invention, partial size be mainly distributed on for
700-900um。
Beneficial effect of the present invention：
Compared with prior art, PEDV new oral vaccine prepared by the present invention has the advantages that three aspects：
If 1) production is upper selects Direct-fed PEDV virus liquid oral immunity, vulnerable to the immune effect of gastric acid destroying infection in pig body
Fruit, and PEDV low virulent strain is loaded in cane sugar core by PEDV new oral vaccine prepared by the present invention, and is added and can be resisted
The enteric-coating material of gastric acid is handled by external simulated gastric fluid, and is confirmed by feeding pig experiment in vivo, present invention preparation
PEDV new oral vaccine be resistant to gastric acid, destruction of the effective protection PEDV low virulent strain from gastric acid in pig body；And it can
With effective stimulus chitling mucous membrane immune response.
2) the PEDV vaccine used in production at present is mostly Parenteral immunization, chitling road can not be stimulated to generate enough glutinous
Film immune response, PEDV new oral vaccine peroral immunity prepared by the present invention can effectively reach chitling road, and then sufficiently pierce
Swash enteron aisle and mucosal immune response generation sIgA occurs.
3) it is compared with returning feeding, the present invention can according to need selection morbid pig in preparation PEDV new oral vaccination process
It corresponding PEDV prevalence strain and can be caused weak, there is accuracy and controllability, avoid and directly return feeding PEDV
It existing blindness and raises velogen strain bring due to returning and dissipates malicious risk.
In addition, PEDV new oral vaccine prepared by the present invention can be mixed with feed carries out feeding pig, greatly reduce
Other immunization ways stress as caused by injection.
Detailed description of the invention
Fig. 1 is PEDV low virulent strain freeze-dried powder.
Fig. 2 is the preparation technology flow chart of Porcine epidemic diarrhea virus new oral vaccine of the invention.
Fig. 3 is electron microscope observation PEDV new oral vaccine and particle size determination as a result, wherein：A：Blank sucrose ball
Core；B：PEDV oral vaccine；C：PEDV oral vaccine partial size is mainly distributed on as 700-900um.
Fig. 4 is PEDV new oral vaccine acidproof situation in vitro；Wherein：A：Epidemic disease is taken orally by the PEDV that artificial gastric acid is handled
Seedling；B：In the PEDV oral vaccine of pH 6.8DMEM cell culture medium dissolution release；C：PEDV freeze-dried powder is without artificial gastric acid
Handle TCID50=106.5, TCID is handled by artificial gastric acid50=0；PEDV oral vaccine is handled and is passed through without artificial gastric acid
Cross artificial gastric acid treated virus TCID50Without significant difference.
Fig. 5 is acidproof situation in PEDV new oral vaccine body：PEDV oral vaccine is immunized after weanling pig the 6th day,
Toxin expelling occurs for 100% (8/8)；Toxin expelling occurs for directly oral only 25% (2/8) of virus liquid.
Specific embodiment
The problem of solved in order to better illustrate the present invention, used technical solution and effect achieved, are now tied
It closes specific embodiment and related data is further described.It should be noted that the content of present invention is including but not limited to following implementation
Example and combinations thereof embodiment.
Particular technique or condition are not specified in the embodiment of the present invention, according to the literature in the art described technology or
Condition is carried out according to product description.Reagents or instruments used without specified manufacturer, being can be by commercially available etc.
The conventional products that approach obtains.
The preparation of one present invention of embodiment effectively prevention and control Porcine epidemic diarrhea virus new oral vaccine
Including following operation：
1, virus freeze-drying：By the Porcine epidemic diarrhea virus (PEDV) of African green monkey kidney (Vero) cell mass propgation
Low virulent strain is abundant with isometric aseptic freeze-dried protective agent (containing 10% skimmed milk power, 5% sucrose) after being filtered with 4 layers of sterile gauze
It stirs and evenly mixs.Then, it opens freeze dryer and carries out pre-freeze, to plate temperature drop to -50 DEG C, the virus liquid mixed is put into freeze dryer support
Disk keeps -50 DEG C of maintenance 2h after inlet；Rear cabinet is successively opened later to cool down, vacuumize, open big butterfly valve, open lobe pump；Make carriage body
Vacuum degree is down to 20pa and maintains 1h；200min is heated after 1h to be made sample temperature rise to -7 DEG C and is maintained for more than 1h, is then passed through
Reheating 120min takes out after so that all samples is warming up to 15 DEG C.Obtained PEDV freeze-dried powder, which vacuumizes, is sealed and placed in 4 DEG C
Refrigerator saves, is spare.
2, centrifugal granulating：By PEDV freeze-dried powder with etc. the cornstarch of quality be sufficiently mixed as material, with cane sugar core
As parent nucleus, pure water carries out centrifugal granulating as adhesive in centrifugal granulator.Steps are as follows：Centrifugal granulator is opened, is put
Enter cane sugar core, be arranged rotary speed 200r/m (revolving speed/minute), enters the wind wind speed 700r/m, air draft wind speed 900r/m, material temperature
28 DEG C of degree opens 1KW heating, is then sprayed onto pure water under conditions of atomizing pressure 0.15mpa, wriggling revolution speed 10r/m
Viral freeze-dried powder is added in upper powder cylinder after capsule core sufficiently soaks for cane sugar core surface, starts the upper powder shake of viral powder
Feeding maintains all parameter settings of instrument, and wriggling and atomization can be stopped by being all adhered to cane sugar core surface to viral powder,
Continue to keep turntable rotation and air inlet, air draft wind speed, about 15min is dried to get to the pellet for being mounted with PEDV.
3, isolation coat：Enteric-coating material generally in acidity, causes to lose to avoid viral powder from directly contacting enteric material
Living, the pellet of centrifugal granulating preparation needs to be coated with one layer of isolation coat before being coated with enteric-coating material, thus by virus and intestines
Molten coating is sufficiently spaced from.In the present invention, isolation coat selects semi synthetic polymer hydroxypropyl methyl cellulose (HPMC), and makes
It is coated with fluidized-bed coating machine.Steps are as follows：Weigh a certain amount of HPMC as required, take water as a solvent configuration 4% concentration every
From Coating Solution, fluidized-bed coating machine is then opened, what centrifugal granulating obtained is mounted with the pellet of PEDV before pouring into, and wind is arranged
Machine revolving speed is 1700r/m, and temperature of charge is 28 DEG C, opens 3KW heating, atomizing pressure 0.15mpa is arranged, stablize to temperature of charge
At 28 DEG C, peristaltic pump is opened, revolving speed is stepped up and isolated coating liquid is pumped into fluidized bed to pellet progress bottom spray to 20r/m
Coating.Stop peristaltic pump after coating weight gain about 5% to wriggle, is kept for constant 30 DEG C of other settings of instrument continue dry 20min.
4, enteric coating：It weighs talcum powder polymer 7.5g, triethyl citrate polymer 3g, height in 129.5g water is added
Fast 1500r/m stirs 10min, and then slowly 100g L30D-55 is added in (liquid does not splash out as standard in stirring when pouring into)
The weight gain of (Utech) aqueous dispersion continues middling speed 500-800r/m stirring to get the enteric coating liquid for being 12.5% to concentration.It will
The pellet continuation that previous step is coated with isolation coat is coated with enteric coating on fluidized-bed coating machine.Steps are as follows：Blower is set
Revolving speed 1700r/m, temperature of charge are 28 DEG C, and inlet air temperature is 30-32 DEG C, open 3KW heating, atomizing pressure 0.17mpa is arranged,
When temperature of charge is stablized at 28 DEG C, peristaltic pump is opened, revolving speed is stepped up and enteric coating liquid is pumped into fluidized bed to 30r/m
Bottom spray coating is carried out to pellet.Stop peristaltic pump after coating weight gain about 30% to wriggle, 30 DEG C of fluidized drying 30min are completed
Entirely oral pellet preparation process obtains PEDV new oral vaccine.
Vaccine Function detection made from two the method for the present invention of embodiment
2.1 external acidproof detections：
Weigh 2g NaCl, 3.2g pepsin (12000U) is completely dissolved in configuration in 900ml distilled water and obtains artificial stomach
Dense HCL is added later and adjusts pH value to 1.2, and is settled to 1L for liquid.Using small-radius curve track (paddle method) by the novel mouth of the PEDV prepared
It takes vaccine to be placed in intelligent dissolution tester cuvette, is subsequently added into 100mL simulated gastric fluid, 37 DEG C, 100r/m stirring 2h.After 2h
Simulated gastric fluid is outwelled, is cleaned to be transferred to for one time with pure water and is dissolved in pH 6.8DMEM cell culture medium and carry out live virus drop
Degree measurement, is as a result shown in Fig. 4, and wherein PEDV freeze-dried powder handles TCID without artificial gastric acid50=106.5, handled by artificial gastric acid
TCID50=0；PEDV oral vaccine without artificial gastric acid handle with by artificial gastric acid treated virus TCID50Without significant
Difference (C referring to fig. 4).
The measurement of 2.2 virus titers：
By well-grown Vero cell dissociation, by 104A cell/hole 100uL/ is inoculated in 96 porocyte culture plates.It is secondary
Day will be dissolved in pH6.8DMEM cell culture medium by the PEDV new oral vaccine of artificial gastric acid processing, make 10 times of multiple proportions
Titre dilution, each dilution make 8 repetitions, then remove the cell culture fluid in 96 orifice plates, and PBS will dilution after cleaning 2 times
Good virus liquid sequentially adds in cell according to the hole 100uL/ from high dilution to the sequence of low dilution, while being arranged without people
The pellet of work gastric juice processing, the PEDV freeze-dried powder handled through artificial gastric acid are control.Tissue culture plate is placed in 5%CO later2
Incubator, 37 DEG C are cultivated 3-5 days.Culture plate is placed in microscopically observation cytopathy after 3-5 days, records the hole of all lesions
Number calculates TCID according to Karber method50, so that it is determined that PEDV new oral vaccine is after simulated gastric fluid is handled with the presence or absence of work
Virion.The results show that the method for the present invention preparation PEDV new oral vaccine through artificial gastric acid processing after, virus titer with
Pellet without the processing of artificial gastric acid shows that PEDV new oral vaccine is resistant to artificial gastric acid without significant difference.
Acidproof detection in 2.3 bodies：
According to table 1 by 32 health without uniform disconnected of diarrhea (acquisition anus swab, qRT-PCR detection PEDV are negative), weight
Milk piglet is randomly divided into 4 groups and is immunized.
1 experimental animal of table is grouped and is immunized
Weanling pig the 1st, 3,5,6,7 day acquisition anus swab after oral detects PEDV toxin expelling feelings also with qRT-PCR
Condition, to evaluate PEDV new oral vaccine in the intracorporal stomach juice-resistant implementations of weanling pig.
Quantitative fluorescent PCR (qRT-PCR) detects toxin expelling situation:
1) viral DNA/RNA is extracted
It takes the sterile 1 × PBS of 1mL to be added in anus swab, sufficiently vibrates, is uniformly mixed in turbula shaker, if not having to temporarily
It can freeze in -80 DEG C；If extracting viral RNA immediately, it is centrifuged 1min by 12000r/m room temperature, takes supernatant spare；Then according to virus
RNA/DNA extracts kit extracts viral genome, finally takes 50 μ L Nuclease Free Water to be eluted, will elute
Obtained viral RNA/DNA freeze in -80 DEG C, it is spare.
2) qRT-PCR is detected
Obtained RNA will be extracted according to PrimeScriptTMRT-PCR Kit kit carries out reverse transcription and obtains cDNA.It takes
4 μ L RNA, 0.5 μ L dNTP Mixture and 0.5 μ L Random 6mers are added in PCR pipe, according to 65 DEG C/5min of program, 4
DEG C be stored in PCR instrument be denaturalized, annealing reaction；25 × PrimeScript of μ L are sequentially added in above-mentioned reaction solution later
Buffer、0.25μL RNase Inhibitor、0.25μL PrimeScript RTase、2.5μL RNase Free dH2O,
Then in PCR instrument, according to following response procedures：30 DEG C/10min, 42 DEG C/1h, 95 DEG C/5min, 4 DEG C of preservations carry out reverse transcription
Reaction.The cDNA that reverse transcription is obtained carries out PCR amplification according to table 2, and PCR is reacted in Applied Biosystem 7500
It is carried out in Fast instrument PCR instrument；
2 PCR amplification system of table
| Ingredient | Volume (μ L) |
| Thunderbird Probe qPCR Mix | 10 |
| ddH2O | 7.46 |
| 50×Rox reference dye | 0.04 |
| Probe | 0.1 |
| Upstream primer | 0.2 |
| Downstream primer | 0.2 |
| cDNA | 2 |
| Total | 20 |
PCR response procedures are：95 DEG C of initial denaturation 10min；95 DEG C of denaturation 15s, 60 DEG C of annealing/extension 40s, totally 40 are followed
Ring.Wherein qRT-PCR specific primer is：
Upstream primer:5'-GAATTCCCAAGGGCGAAAA-3',
Downstream primer：5'-TTTTCGACAAATTCCGCATCT-3',
Probe is：5'-FAM-CGTAGCAGGCTTGCTTCGGACCCA-BHQ-3'.
It is synthesized by Invitrogen company.It is compareed simultaneously using PEDV, PRRSV as positive and negative.Finally determined according to Ct value
Anus swab whether there is PEDV genome, so that it is determined that toxin expelling situation, as a result referring to Fig. 5.
Embodiment three
Referring to the preparation method of the prevention and control Porcine epidemic diarrhea virus new oral vaccine of embodiment one, difference is：
Step 2 respectively by viral freeze-dried powder with etc. cornstarch, lactose, dextrin, the mannitol of quality be sufficiently mixed, other step operations
With embodiment one.Observe the state and subsequent technique situation of each freeze-drying powder mixture.
In the present invention program, freeze-dried powder is because be easy to moisture absorption dampness into bulk, cornstarch fineness containing sucrose milk powder
Very carefully, hygroscopicity is strong and freeze-dried powder mixing can play dilution and moisture absorption, keeps freeze-dried powder flow of powder state, and
In the enteric stage, starch can play disintegrating agent and virus is accelerated to discharge in enteron aisle.And with powder high income on cornstarch, using cream
The powder such as sugar, dextrin, mannitol and freeze-dried powder mixing, it is all good that the flow regime of cornstarch is not added, and these powder are thin
It is thin to spend inadequate starch, upper powder yield is low after mixing.
Example IV
Referring to the preparation method of the prevention and control Porcine epidemic diarrhea virus new oral vaccine of embodiment one, difference is：
Cane sugar core, starch capsule core and microcrystalline cellulose pellet is respectively adopted as parent nucleus, the same embodiment of other step operations in step 2
One.It is spare that oral vaccine is made respectively.
Consistent 24 health of upgrowth situation is chosen without diarrhea (acquisition anus swab, qRT-PCR detection PEDV are negative), weight
Uniform weanling pig is randomly divided into 3 groups, is made respectively using above-mentioned using cane sugar core, starch capsule core and microcrystalline cellulose pellet
It is immunized for oral vaccine made from parent nucleus by identical immune programme.As the result is shown：
Oral vaccine made of cane sugar core：TCID50=106.0；Oral vaccine made of starch capsule core：TCID50=
104.0；Oral vaccine made of microcrystalline cellulose pellet：TCID50=0.
In present invention process, temperature and wind speed directly affect the efficiency of granulation and coating, and theoretically, temperature and wind speed are got over
Height, liquid evaporation is faster, and the time for completing oral vaccine preparation process needs is shorter.But temperature is too high, rises to virus
To deactivation, temperature is too low, and the speed of hydrojet cannot be too fast to cause a preparation process time too long；Wind speed is too small not
Capsule core adhesion is be easy to cause conducive to liquid evaporation, and freeze-dried powder loss is big when wind speed excessive granulation, and when coating may cause coating spray
Mist is dry to influence coating effect.Summary, temperature of the present invention and wind speed are all most useful for holding viral viability and to improve process efficiency
Parameter.
What coating weight gain was mainly determined by effect acidproof after being coated.Coating weight gain cannot resist the digestion of gastric acid when inadequate,
But weight gain also will increase greatly very much the time of dissolution.Experimental data shows when weight gain is 10%, the artificial stomach of the oral vaccine of preparation
After liquid processing, TCID50=0；Increase weight 20% when, preparation oral vaccine simulated gastric fluid processing after, TCID50=103.5；Weight gain
When 30%, after the oral vaccine simulated gastric fluid processing of preparation, TCID50=106.0, with the oral vaccine handled without simulated gastric fluid
Without significant difference, therefore the present invention selects enteric coating weight gain 30%.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of preparation method of Porcine epidemic diarrhea virus oral vaccine, which is characterized in that step includes：
1), virus attenuated strain freeze-drying；
2), by the freeze-dried powder centrifugal granulating of virus, the pellet for being mounted with virus attenuated strain is obtained；
3) pellet made from step 2), is coated with isolation coat；
4) step 3), is coated with the pellet coating enteric coating of isolation coat to get oral vaccine.
2. preparation method according to claim 1, which is characterized in that in the step 1), virus attenuated strain freeze-drying
Operation be：
1-1, mixing will be sufficiently stirred with isometric aseptic freeze-dried protective agent after the virus attenuated strain filtering that culture obtains；
1-2, freeze dryer progress pre-freeze is opened, to extremely -50 DEG C of plate temperature drop, the virus liquid mixed in 1-1 is put into freeze dryer, into
- 50 DEG C are kept after case, maintain 2h；
1-3, rear cabinet cooling is successively opened, is vacuumized and so that carriage body vacuum degree is down to 20pa and maintain 1h；
1-4：200min is heated after 1h to be made sample temperature rise to -7 DEG C and maintains at least 1h；Then made by reheating 120min
All samples take out after being warming up to 15 DEG C, and obtained viral freeze-dried powder, which vacuumizes, is sealed and placed in 4 DEG C of preservations.
3. preparation method according to claim 2, which is characterized in that in step 1-1, the freeze drying protectant composition：
10% skimmed milk power, 5% sucrose and 85% water.
4. preparation method according to claim 1, which is characterized in that granulation operations include in the step 2)：It will be viral
Freeze-dried powder with etc. the cornstarch of quality be sufficiently mixed as material, using capsule core as parent nucleus, addition adhesive is in centrifugal granulating
Centrifugal granulating is carried out in machine.
5. the preparation method according to claim 4, which is characterized in that the freeze-dried powder applied sample amount is the 1/10 of capsule core；It is viscous
Mixture dosage is about the 1/10 of capsule core.
6. the preparation method according to claim 4, which is characterized in that being granulated concrete operations in the step 2) is：It opens
Centrifugal granulator is put into capsule core, and turntable is arranged, and enters the wind 0~3000r/m of wind speed, air draft 0~3000r/m of wind speed, temperature of charge 25
~45 DEG C；Heating, is then sprayed onto capsule core surface for pure water, and upper powder cylinder is added in viral freeze-dried powder after capsule core sufficiently soaks
In, the upper powder for starting viral powder shakes feeding, maintains all parameter settings of instrument, is all adhered to cane sugar core to viral powder
Surface can stop wriggling and be atomized, and continue to keep turntable rotation and air inlet, air draft wind speed, be dried to get loading has been arrived
Virulent pellet.
7. preparation method according to claim 1, which is characterized in that the operation packet of coating isolation coat in the step 3)
It includes：Configuration isolation Coating Solution, then opens fluidized-bed coating machine, pour into that step 2) centrifugal granulating obtains to be mounted with virus weak
The pellet of strain, setting rotation speed of fan are 0~2500r/m, and temperature of charge is 25~45 DEG C, and atomizing pressure is arranged, to object in heating
When material temperature degree is stablized, peristaltic pump is opened, revolving speed is stepped up and is pumped into fluidized bed to by isolated coating liquid to pellet progress bottom spray
Coating；Stop peristaltic pump after coating weight gain about 5% to wriggle, keeps instrument other parameter settings constant, continue drying.
8. preparation method according to claim 1, which is characterized in that the operation of coating enteric coating in the step 4)
For：
4-1, preparation enteric coating liquid；
The pellet for being coated with isolation coat that 4-2, the coating step 3) of the coating solution made from 4-1 obtain.
9. preparation method according to claim 8, which is characterized in that the operation of the step 4-2 is：Rotation speed of fan is set
1700r/m, temperature of charge are 28 DEG C, and inlet air temperature is 30-32 DEG C, opens heating, and atomizing pressure is arranged, and are stablized to temperature of charge
At 28 DEG C, peristaltic pump is opened, revolving speed is stepped up and enteric coating liquid is pumped into fluidized bed to the spray coating of pellet progress bottom；To
Stop peristaltic pump after coating weight gain about 30% to wriggle, fluidized drying.
10. preparation method according to any one of claims 1 to 9, which is characterized in that the enteric coating liquid composition packet
It includes：Talcum powder amount of polymers 25-50%, triethyl citrate amount of polymers 10%, the weight gain of L30D-55 (Utech) aqueous dispersion
30%, water surplus.
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