CN108904795B - Preparation method of oral vaccine for porcine epidemic diarrhea virus - Google Patents
Preparation method of oral vaccine for porcine epidemic diarrhea virus Download PDFInfo
- Publication number
- CN108904795B CN108904795B CN201810997606.5A CN201810997606A CN108904795B CN 108904795 B CN108904795 B CN 108904795B CN 201810997606 A CN201810997606 A CN 201810997606A CN 108904795 B CN108904795 B CN 108904795B
- Authority
- CN
- China
- Prior art keywords
- coating
- virus
- freeze
- oral vaccine
- pedv
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5026—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5073—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals having two or more different coatings optionally including drug-containing subcoatings
- A61K9/5078—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals having two or more different coatings optionally including drug-containing subcoatings with drug-free core
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
-
- 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- 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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Virology (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention belongs to the field of veterinary biotechnology application, and particularly relates to a preparation method of a novel oral vaccine for effectively preventing and controlling porcine epidemic diarrhea virus, which comprises the following steps: freezing and drying virus attenuated strains; centrifugally granulating the freeze-dried powder of the virus to obtain a pellet loaded with virus low virulent strains; coating the prepared pellets with an isolation coating; coating the pellet coated with the isolation coating with enteric coating to obtain the oral vaccine. The novel PEDV oral vaccine capable of tolerating gastric acid and effectively stimulating the mucous membrane immunity of the intestinal tract of a pig, prepared by the invention, is used for preventing and controlling PED in production, can be mixed with feed for feeding pigs, and is simpler and more convenient to operate; greatly reduces the stress caused by other immunization modes such as injection, the toxin dispersing risk of reverse feeding and the like, and is safer.
Description
Technical Field
The invention belongs to the field of veterinary biotechnology application, and particularly relates to a preparation method of a novel oral vaccine for effectively preventing and controlling porcine epidemic diarrhea virus.
Background
Porcine Epidemic Diarrhea (PED) is an acute, highly contagious disease of pigs caused by Porcine Epidemic Diarrhea Virus (PEDV). The newborn piglets infected with PEDV have diarrhea, dehydration and vomiting with high fatality rate. In recent years, PEDs have been widely spread throughout the world, and countries such as china, the united states, korea, and canada have been in outbreak of epidemic situations, which seriously jeopardize the development of the global pig industry. 29 provinces and cities of China occur and prevail PED in autonomous regions, so that the death rate of newborn piglets can reach 100 percent, and huge economic loss is caused to the pig raising industry in China. At present, no effective treatment method exists for the disease, and the disease can be mainly prevented and controlled only by vaccine immunization. After the PEDV is infected by the oral and nasal administration of pigs, the PEDV mainly invades epithelial cells of small intestines of the pigs to cause cell damage, atrophy and shedding of intestinal villi, so that the absorption area of the small intestines is greatly reduced, nutrient substances cannot be normally absorbed, and finally, piglets are diarrhea and dehydrated to die. Because PEDV mainly infects organisms through invading intestinal tracts of pigs, mucosal immune response of the organisms is particularly important for preventing and controlling PEDV, and high-affinity sIgA generated by mucosal immunity can effectively prevent pathogens from adsorbing the mucosa and neutralizing the pathogens. Therefore, how to effectively stimulate porcine intestinal mucosal immunity is crucial to preventing and controlling PEDV infection.
So far, common PED vaccines include inactivated vaccines, attenuated vaccines, transgenic plant vaccines, genetic engineering vaccines prepared by using bacteria and viruses as vectors, subunit vaccines, and the like. Wherein, the commercial vaccines in production comprise porcine epidemic diarrhea-porcine transmissible gastroenteritis-rotavirus triple live vaccines of Harbin Vitaceae organisms and porcine epidemic diarrhea-porcine transmissible gastroenteritis double inactivated vaccines prepared by Wuhan's department of biology GmbH; both commercial vaccines were immunized by parenteral injection. In addition, the pregnant sow is often infected by directly feeding the intestinal contents of the sick piglets back in production to generate milk immunity, so that the newborn piglets are provided with passive protection. However, PED vaccines used in the current production adopt an parenteral immunization mode regardless of weak-toxin live vaccines or inactivated vaccines, and the effect of stimulating the immunization of porcine intestinal mucosa is very limited. In addition, the feces of the pigs infected by PEDV or the intestinal contents of the sick piglets are fed back to artificially infect the pregnant sows, so that the organisms can be effectively stimulated to generate maternal antibodies aiming at the PEDV, the newborn piglets are protected after sucking the milk, the death rate of the newborn piglets is greatly reduced, the epidemic time of the PED is shortened, the effect is obvious, but the serious toxicity dispersing risk exists in the feed back, and more economic loss can be caused. And if the PEDV attenuated live vaccine is directly fed to the pig, the PEDV attenuated live vaccine is easily damaged by gastric acid in the pig, so that the immune effect is influenced. Therefore, the preparation of novel oral vaccines of PEDV which are effective and safe is urgent.
Disclosure of Invention
In view of the above, there is a need to provide a method for preparing a novel oral vaccine for effectively preventing and controlling porcine epidemic diarrhea virus. In order to overcome the defects that the existing vaccine cannot avoid gastric acid damage and cannot well stimulate the porcine intestinal tract to generate sufficient PEDV specific mucosal immunity, the invention prepares the novel PEDV oral vaccine which can tolerate gastric acid and effectively stimulate the porcine intestinal tract mucosal immunity, lays a foundation for PED prevention and control in production and provides a new idea for other porcine intestinal infectious diseases.
The invention is realized by the following technical scheme:
a method for preparing an oral vaccine for effectively preventing and controlling porcine epidemic diarrhea virus comprises the following steps:
1) freezing and drying virus attenuated strains;
2) centrifuging and granulating the freeze-dried powder of the virus to obtain a pellet loaded with virus low virulent strains;
3) coating the micro-pills prepared in the step 2) with an isolation coating;
4) and (3) coating the pellet coated with the isolating coating in the step 3) with an enteric coating to obtain the oral vaccine.
Further, in the step 1), the operation of freeze-drying the virus attenuated strain comprises the following steps:
1-1, filtering the virus low virulent strain obtained by culture, and then fully stirring and uniformly mixing the filtered virus low virulent strain with an equal volume of sterile freeze-drying protective agent;
1-2, starting a freeze dryer for pre-freezing, putting the mixed virus liquid in the step 1-1 into the freeze dryer when the temperature of the plate is reduced to-50 ℃, keeping the temperature at-50 ℃ after the mixed virus liquid is put into a box, and maintaining for 2 hours;
1-3, opening the rear box in sequence to cool, vacuumize, open a large butterfly valve and a Roots pump, reducing the vacuum degree of the compartment body to 20pa and maintaining for 1 h;
1-4: heating for 200min after 1h to raise the temperature of the sample to-7 ℃ and maintaining for at least 1 h; and heating all samples to 15 ℃ by secondary heating for 120min, taking out, vacuumizing and sealing the obtained virus freeze-dried powder, and storing at 4 ℃.
Further, in step 1-1, the lyoprotectant can reduce the influence of the freezing and drying processes on the viral proteins, and comprises: 10% skimmed milk powder, 5% sucrose and 85% water.
Further, the granulation operation in step 2) comprises: the virus freeze-dried powder and corn starch with equal mass are fully mixed to be used as materials, 500-3000g of pill cores are used as mother cores, and 50-100ml of adhesive is used for centrifugal granulation in a centrifugal granulator.
Furthermore, 1/10 of the pill core is loaded on the freeze-dried powder; the binder was used at about 1/10 of the pellet core.
Furthermore, the pill cores are sucrose pill cores, starch pill cores, lactose pill cores, mannitol pill cores, microcrystalline cellulose pill cores and the like, and the sucrose pill cores are preferred from the aspects of preparation effect and virus protection.
Further, the adhesive is pure water or HPMC.
Further, the granulation in the step 2) is specifically operated as follows: opening a centrifugal granulator, putting a pellet core, setting the rotating speed of a turntable to be 200r/m (rotating speed/min), the air inlet speed to be 0-3000 r/m (preferably 700r/m), the air exhaust speed to be 0-3000 r/m (preferably 900r/m), the material temperature to be 25-45 ℃ (preferably 28 ℃), starting 1KW for heating, spraying an adhesive on the surface of the pellet core under the conditions of the atomizing pressure of 0.15mpa and the rotating speed of a peristaltic pump to be 10r/m, adding virus freeze-dried powder into a powder feeding barrel after the pellet core is fully wetted, starting powder feeding and shaking feeding of the virus powder, maintaining all parameter settings of the instrument, stopping creeping and atomizing after the virus powder is completely adhered to the surface of the pellet core, continuously keeping the rotating speed of the turntable and the air inlet and air exhaust speeds, and drying for about 15min to obtain the pellet loaded with the virus.
Further, the step 3) of coating the release coating comprises the following steps: weighing an isolation coating material (preferably a semi-synthetic polymer hydroxypropyl methylcellulose (HPMC)), preparing an isolation coating solution with the concentration of 4-6% by taking water as a solvent, starting a fluidized bed coating machine, pouring the isolation coating solution into the pellets loaded with the virus attenuated strains obtained in the step 2) through centrifugal granulation, setting the rotating speed of a fan to be 0-2500 r/m (preferably 1700r/m), the material temperature to be 25-45 ℃ (preferably 28 ℃), starting 3KW for heating, setting the atomization pressure to be 0.15mpa, starting a peristaltic pump when the material temperature is stabilized at 28 ℃, gradually increasing the rotating speed to 20r/m, pumping the isolation coating solution into the fluidized bed, and performing bottom spray coating on the pellets; stopping peristaltic pump when the weight of the coating is increased by about 5%, keeping other parameters of the apparatus unchanged, and continuously drying for 20min at 30 ℃.
Further, the step 4) of coating the enteric coating comprises the following steps:
4-1, preparing enteric coating liquid;
4-2, coating the micropellets coated with the isolation coating obtained in the step 3) with the coating solution prepared in the step 4-1.
Further, the enteric coating solution is an aqueous dispersion polymer prepared by taking talcum powder as an anti-sticking agent, and comprises: 25-50% of talcum powder polymer, 10% of triethyl citrate polymer, 30% of L30D-55 (Ewing) water dispersion weight gain, and the balance of water.
Further, the operation of step 4-2 is: setting the rotating speed of a fan to be 0-2500 r/m (preferably 1700r/m), the material temperature to be 25-35 ℃ (preferably 28 ℃), the air inlet temperature to be 30-32 ℃, starting a 3KW heating, setting the atomization pressure to be 0.17mpa, starting a peristaltic pump when the material temperature is stabilized at 28 ℃, gradually increasing the rotating speed to 30r/m, pumping the enteric coating liquid into a fluidized bed, and carrying out bottom spray coating on the pellets; stopping peristaltic pump when the weight of the coating is increased by about 30%, and fluidized drying at 30 deg.C for 30 min.
Further, the particle size of the PEDV oral vaccine prepared by the method is mainly distributed in the range of 700-900 um.
The invention has the beneficial effects that:
compared with the prior art, the novel PEDV oral vaccine prepared by the invention has three advantages:
1) if the PEDV virus liquid is directly fed for oral immunization in production, the immune effect is easily influenced by the damage of gastric acid in a pig body, the PEDV novel oral vaccine prepared by the invention loads the PEDV attenuated strain on a sucrose pill core, and is added with an enteric coating material capable of resisting gastric acid, the in-vitro artificial gastric juice treatment is carried out, and the in-vivo experiment of the pig is fed for verification, the PEDV novel oral vaccine prepared by the invention can resist gastric acid, and the PEDV attenuated strain is effectively protected from the damage of the gastric acid in the pig body; and can effectively stimulate the immune response of the intestinal mucosa of the pig.
2) Most of PEDV vaccines used in the prior production are parenteral immunizations, which cannot stimulate the intestinal tract of pigs to generate enough mucosal immune response.
3) Compared with the back feeding, the PEDV vaccine can select the PEDV epidemic strain corresponding to a sick pig farm according to the needs in the process of preparing the PEDV novel oral vaccine, can be attenuated, has accuracy and controllability, and avoids the blindness existing in the direct back feeding of the PEDV and the virus scattering risk caused by the back feeding of virulent strains.
In addition, the novel PEDV oral vaccine prepared by the invention can be mixed with feed to feed pigs, so that the stress caused by other immunization modes such as injection is greatly reduced.
Drawings
Figure 1 is a lyophilized powder of PEDV attenuated strain.
FIG. 2 is a flow chart of the preparation process of the novel oral vaccine for porcine epidemic diarrhea virus of the invention.
Fig. 3 shows the results of the observation of the novel PEDV oral vaccine under an electron microscope and the measurement of the particle size, wherein: a: a blank sucrose pellet core; b: PEDV oral vaccine; c: the particle size of the PEDV oral vaccine is mainly distributed at 700-900 um.
FIG. 4 is an in vitro acid tolerance profile of the novel oral vaccine against PEDV; wherein: a: PEDV oral vaccine treated with artificial gastric acid; b: dissolving the released PEDV oral vaccine in DMEM cell culture medium pH 6.8; c: the PEDV freeze-dried powder is not subjected to TCID treatment by artificial gastric acid50＝106.5TCID treated with artificial gastric acid 500; the PEDV oral vaccine is not treated by artificial gastric acid and is subjected toVirus TCID after artificial gastric acid treatment50There was no significant difference.
Figure 5 is the in vivo acid tolerance of the novel oral vaccine against PEDV: 100% (8/8) of weaned pigs immunized by the PEDV oral vaccine have toxin expulsion on the 6 th day; only 25% (2/8) of the virus liquid is directly taken orally to expel toxin.
Detailed Description
To better illustrate the problems addressed by the present invention, the technical solutions adopted and the effects achieved, reference will now be made to the following detailed description and related information. It should be noted that the present disclosure includes, but is not limited to, the following examples and combinations thereof.
The specific techniques or conditions not specified in the examples of the present invention are performed according to the techniques or conditions described in the literature in the art or according to the product specification. The reagents or instruments used are not indicated by manufacturers, and are all conventional products which can be obtained by commercial purchase and the like.
Example one preparation of the novel oral vaccine for effectively preventing and controlling porcine epidemic diarrhea virus of the present invention
The method comprises the following operations:
1. freezing and drying the virus: the Porcine Epidemic Diarrhea Virus (PEDV) low virulent strain cultured in a large amount by African green monkey kidney (Vero) cells is filtered by 4 layers of sterile gauze and then fully stirred and uniformly mixed with an equal volume of sterile freeze-drying protective agent (containing 10 percent of skimmed milk powder and 5 percent of cane sugar). Then, starting a freeze dryer for pre-freezing, putting the mixed virus liquid into a tray of the freeze dryer when the temperature of the plate is reduced to-50 ℃, and keeping the temperature at-50 ℃ for 2 hours after the mixed virus liquid is put into a box; then opening the rear box for cooling, vacuumizing, opening a large butterfly valve and a Kirotz pump in sequence; reducing the vacuum degree of the compartment body to 20pa and maintaining for 1 h; after 1h, the sample is heated for 200min to raise the temperature to-7 ℃ and maintain the temperature for more than 1h, and then all the samples are taken out after being heated to 15 ℃ by secondary heating for 120 min. The obtained PEDV freeze-dried powder is vacuumized, sealed and stored in a refrigerator at 4 ℃ for later use.
2. And (3) centrifugal granulation: and (3) fully mixing PEDV freeze-dried powder with corn starch with equal mass to obtain a material, taking a sucrose pill core as a mother core, and taking pure water as an adhesive to carry out centrifugal granulation in a centrifugal granulator. The method comprises the following steps: opening a centrifugal granulator, putting a sucrose pellet core, setting the rotating speed of a turntable to be 200r/m (rotating speed/min), the air inlet speed to be 700r/m, the air exhaust speed to be 900r/m, the material temperature to be 28 ℃, starting 1KW for heating, then spraying pure water on the surface of the sucrose pellet core under the conditions that the atomizing pressure is 0.15mpa and the rotating speed of a peristaltic pump is 10r/m, adding virus freeze-dried powder into a powder feeding barrel after the pellet core is fully wetted, starting powder feeding and shaking and feeding of the virus powder, maintaining all parameter settings of an instrument, stopping creeping and atomizing after the virus powder is completely adhered to the surface of the sucrose pellet core, continuously keeping the rotating speed of the turntable and the air inlet and air exhaust speeds, and drying for about 15min to obtain the pellet loaded with PEDV.
3. Isolation coating: the enteric coating material is generally acidic, and in order to avoid inactivation caused by direct contact of virus powder with the enteric coating material, the pellet prepared by centrifugal granulation needs to be coated with a layer of isolating coating before the enteric coating material is coated, so that the virus and the enteric coating are fully isolated. In the present invention, the barrier coating is selected from the semi-synthetic polymer hydroxypropyl methylcellulose (HPMC) and is coated using a fluid bed coater. The method comprises the following steps: weighing a certain amount of HPMC according to requirements, preparing 4% concentration isolation coating solution by using water as a solvent, starting a fluidized bed coating machine, pouring the pellets loaded with PEDV obtained by centrifugal granulation, setting the rotating speed of a fan to be 1700r/m, setting the material temperature to be 28 ℃, starting 3KW for heating, setting the atomizing pressure to be 0.15mpa, and when the material temperature is stabilized to be 28 ℃, starting a peristaltic pump, gradually increasing the rotating speed to 20r/m, pumping the isolation coating solution into the fluidized bed to perform bottom spray coating on the pellets. And stopping the peristaltic pump after the weight of the coating is increased by about 5 percent, and keeping the other settings of the equipment unchanged at 30 ℃ for continuously drying for 20 min.
4. Enteric coating: weighing 7.5g of talcum powder polymer and 3g of triethyl citrate polymer, adding 129.5g of water, stirring at a high speed of 1500r/m for 10min, slowly (taking the standard that liquid does not spill out during pouring as the standard) adding 100g L30D-55 (Ewing) aqueous dispersion for weight increment, and continuously stirring at a medium speed of 500r/m to obtain the enteric coating solution with the concentration of 12.5 percent. And (4) continuously coating the pellets coated with the isolation coating in the last step with enteric coating on a fluidized bed coating machine. The method comprises the following steps: setting the rotation speed of a fan at 1700r/m, the material temperature at 28 ℃, the air inlet temperature at 30-32 ℃, starting 3KW for heating, setting the atomization pressure at 0.17mpa, starting a peristaltic pump when the material temperature is stabilized at 28 ℃, gradually increasing the rotation speed to 30r/m, pumping the enteric coating liquid into a fluidized bed, and carrying out bottom spray coating on the pellets. And stopping the peristaltic pump after the weight of the coating is increased by about 30%, and performing fluidized drying at 30 ℃ for 30min to complete the whole preparation process of the oral pellet to obtain the novel PEDV oral vaccine.
Example two functional assays of vaccines prepared according to the method of the invention
2.1 in vitro acid resistance detection:
weighing 2g NaCl and 3.2g pepsin (12000U), fully dissolving in 900ml distilled water to obtain artificial gastric juice, adding concentrated HCl to adjust pH value to 1.2, and metering to 1L. The prepared novel PEDV oral vaccine is placed in a small cup of an intelligent dissolution tester by a small cup method (paddle method), then 100mL of artificial gastric juice is added, and the mixture is stirred for 2 hours at 37 ℃ and 100 r/m. After 2h, the artificial gastric juice was decanted, washed once with purified water, transferred to DMEM cell culture medium at pH6.8 for solubilization and live virus titer determination, the results are shown in FIG. 4, where the PEDV lyophilized powder was not TCID treated with artificial gastric acid50＝106.5TCID treated with artificial gastric acid 500; virus TCID of PEDV oral vaccine without artificial gastric acid treatment and after artificial gastric acid treatment50There was no significant difference (see fig. 4C).
2.2 virus titer assay:
well-grown Vero cells were digested and treated as 104Cells/100 uL/well were plated in 96-well cell culture plates. The next day, PEDV novel oral vaccine treated by artificial gastric acid is dissolved in pH6.8DMEM cell culture medium, 10-fold specific titer dilution is carried out, each dilution is carried out for 8 times, then cell culture solution in a 96-well plate is removed, PBS is washed for 2 times, diluted virus solution is sequentially added into cells according to the sequence from high dilution to low dilution of 100 uL/hole, and meanwhile, pellets which are not treated by artificial gastric acid and PEDV freeze-dried powder which is treated by artificial gastric acid are set as a contrast. The cell culture plate was then placed in 5% CO2Culturing in an incubator at 37 deg.C for 3-5 days. After 3-5 days, the culture plate is placed under a microscope to observe cytopathic effect, and all pathological changes are recordedThe number of holes of (D) was calculated by the Karber method50Thereby determining whether live virus particles exist after the novel PEDV oral vaccine is treated by the artificial gastric juice. The result shows that after the novel PEDV oral vaccine prepared by the method is treated by artificial gastric acid, the virus titer is not obviously different from that of pellets which are not treated by the artificial gastric acid, and the novel PEDV oral vaccine can resist the artificial gastric acid.
2.3 in vivo acid resistance detection:
32 healthy, diarrhea-free (anal swab collected, PEDV negative by qRT-PCR assay) and weight-uniform weaned piglets were randomly divided into 4 groups for immunization according to Table 1.
TABLE 1 grouping and immunization of experimental animals
Collecting anal swabs of weaned piglets at days 1, 3, 5, 6 and 7 after oral administration, and detecting the PEDV detoxification condition by using qRT-PCR (quantitative reverse transcription-polymerase chain reaction) so as to evaluate the gastric acid resistance condition of the novel PEDV oral vaccine in the weaned piglets.
Fluorescent quantitative PCR (qRT-PCR) for toxin expelling:
1) viral DNA/RNA extraction
Adding 1mL of sterile 1 XPBS into the anus swab, fully oscillating in a vortex oscillator, and uniformly mixing, wherein the anus swab can be frozen at-80 ℃ if the anus swab is not frozen temporarily; if the virus RNA is extracted immediately, centrifuging for 1min at the room temperature of 12000r/m, and taking the supernatant for later use; then extracting virus genome according to virus RNA/DNA extraction kit, finally eluting with 50 μ L of nucleic Free Water, freezing the eluted virus RNA/DNA at-80 deg.C for use.
2) qRT-PCR detection
The extracted RNA was expressed according to PrimeScriptTMAnd carrying out reverse transcription by using the RT-PCR Kit to obtain cDNA. Add 4. mu.L of RNA, 0.5. mu.L of dNTP mix and 0.5. mu.L of Random 6mers into the PCR tubeStoring the mixture on a PCR instrument for denaturation and annealing at the temperature of 4 ℃ according to the program of 65 ℃/5 min; then, 2. mu.L of 5 XPrimeScript Buffer, 0.25. mu.L of RNase Inhibitor, 0.25. mu.L of PrimeScript RTase and 2.5. mu.L of RNase Free dH were added to the above reaction solution in this order2O, followed by the following reaction procedure on a PCR instrument: 30 ℃/10min,42 ℃/1h,95 ℃/5min, and 4 ℃ for reverse transcription reaction. Performing PCR amplification on the cDNA obtained by reverse transcription according to the following table 2, wherein the PCR reaction is performed on an Applied Biosystem 7500 Fast instrument PCR instrument;
TABLE 2 PCR amplification System
| Composition (A) | 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 |
| |
20 |
The PCR reaction program is: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15s and annealing/elongation at 60 ℃ for 40s for 40 cycles. Wherein the qRT-PCR specific primers are as follows:
the upstream primer is 5'-GAATTCCCAAGGGCGAAAA-3', and the upstream primer is,
a downstream primer: 5'-TTTTCGACAAATTCCGCATCT-3' the flow of the air in the air conditioner,
the probe is as follows: 5 '-FAM-CGTAGCAGGCTTGCTTCGGACCCA-BHQ-3'.
Synthesized by Invitrogen corporation. Meanwhile, PEDV and PRRSV are used as negative and positive controls. And finally, determining whether the PEDV genome exists in the anal swab according to the Ct value so as to determine the detoxification condition, wherein the result is shown in figure 5.
EXAMPLE III
Referring to the preparation method of the novel oral vaccine for preventing and controlling the porcine epidemic diarrhea virus in the first embodiment, the difference is that: and 2, fully mixing the virus freeze-dried powder with corn starch, lactose, dextrin and mannitol with equal mass respectively, and performing other steps as same as the first embodiment. And observing the state of each freeze-dried powder mixture and the subsequent process condition.
In the scheme of the invention, the freeze-dried powder is very easy to absorb moisture and wet into blocks because of containing cane sugar, the corn starch has very fine fineness and strong moisture absorption, can play a role in diluting and absorbing moisture when being mixed with the freeze-dried powder, keeps the flowing state of the freeze-dried powder, and can play a role of a disintegrating agent to accelerate the release of viruses in intestinal tracts in an enteric-coated stage. The yield of the powder on the corn starch is high, the powder is mixed with the freeze-dried powder by adopting lactose, dextrin, mannitol and other powder, the fluidity state of the powder without adding the corn starch is good, the fineness of the powder is not enough, the starch is fine, and the yield of the powder on the mixed powder is low.
Example four
Referring to the first embodiment, the method for preparing the novel oral vaccine for preventing and controlling the porcine epidemic diarrhea virus is different in that: and step 2, respectively adopting the sucrose pellet core, the starch pellet core and the microcrystalline cellulose pellet core as mother cores, and performing other steps as in the first embodiment. Respectively preparing oral vaccine for later use.
Selecting 24 healthy and diarrhea-free (anal swab collection, PEDV negative detection by qRT-PCR) weaned piglets with consistent growth conditions and uniform weight, randomly dividing the weaned piglets into 3 groups, and immunizing according to the same immunization program by respectively using the oral vaccines prepared by taking the sucrose pill core, the starch pill core and the microcrystalline cellulose pill core as mother nuclei. The results show that:
oral vaccine prepared from sucrose pill core: TCID50＝106.0(ii) a Oral vaccine prepared from starch pill core: TCID50＝104.0(ii) a Oral vaccine prepared from microcrystalline cellulose pill core: TCID50＝0。
In the process of the invention, the temperature and the wind speed directly influence the granulation and coating efficiency, and theoretically, the higher the temperature and the wind speed, the faster the liquid evaporates, and the shorter the time required for completing the preparation process of the once oral vaccine. But the temperature is too high, the virus can be inactivated, and the temperature is too low, the liquid spraying speed cannot be too fast, so that the time of a one-time preparation process is too long; too low wind speed is not beneficial to liquid evaporation and is easy to cause pellet core adhesion, too high wind speed is large for freeze-dried powder loss during granulation, and coating spray drying is possibly caused during coating to influence the coating effect. In summary, the temperature and wind speed of the present invention are the parameters that are most beneficial to maintain the virus activity and improve the process efficiency.
The weight gain of the coating is mainly determined by the acid resistance effect after coating. Insufficient weight gain of the coating will not resist digestion by gastric acid, but too much weight gain will also increase dissolution time. The experimental data show that when the weight gain is 10%, the TCID is obtained after the artificial gastric juice of the prepared oral vaccine is treated500; when the weight is increased by 20 percent, the TCID is obtained after the artificial gastric juice of the prepared oral vaccine is treated50＝103.5(ii) a When the weight is increased by 30 percent, TCID is obtained after the artificial gastric juice of the prepared oral vaccine is treated50＝106.0And the weight of the enteric coating is increased by 30 percent because the enteric coating has no obvious difference with the oral vaccine which is not treated by artificial gastric juice.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (4)
1. The preparation method of the porcine epidemic diarrhea virus oral vaccine is characterized by comprising the following steps:
1) freezing and drying virus attenuated strains;
2) centrifuging and granulating the freeze-dried powder of the virus to obtain a pellet loaded with virus low virulent strains; the granulation is to fully mix virus freeze-dried powder and corn starch with equal mass as a material, take a pill core as a parent nucleus, add an adhesive and carry out centrifugal granulation in a centrifugal granulator; the core is a sucrose core; the granulation parameters are that the rotating speed of the turntable is 200r/m, the air inlet speed is 700r/m, the air outlet speed is 900r/m, the material temperature is 28 ℃, the atomization pressure is 0.15mpa, and the rotating speed of the peristaltic pump is 10 r/m;
3) coating the micro-pills prepared in the step 2) with an isolation coating; the operation of coating the barrier coating comprises: preparing a separation coating solution, wherein the separation coating solution takes hydroxypropyl methylcellulose as a separation coating and takes water as a solvent to prepare a 4% concentration separation coating solution; then starting a fluidized bed coating machine, pouring the pellets loaded with the virus attenuated strains obtained by centrifugal granulation in the step 2), setting the rotating speed of a fan to be 1700r/m, the material temperature to be 28 ℃, starting 3kw for heating, setting the atomization pressure to be 0.15mpa, starting a peristaltic pump when the material temperature is stabilized at 28 ℃, gradually increasing the rotating speed to 20r/m, pumping the isolation coating liquid into a fluidized bed, and carrying out bottom spray coating on the pellets; stopping the peristaltic pump from creeping after the weight of the coating is increased by 5 percent, keeping other parameter settings of the instrument unchanged, and continuously drying for 20min at 30 ℃;
4) coating the pellet coated with the isolation coating in the step 3) with an enteric coating to obtain the oral vaccine;
the operation of coating the enteric coating is as follows:
4-1, preparing enteric coating liquid; weighing 7.5g of talcum powder polymer and 3g of triethyl citrate polymer, adding 129.5g of water, stirring at a high speed of 1500r/m for 10min, slowly adding 100g L30D-55 water dispersion for weight increment, and continuously stirring at a medium speed of 800r/m to obtain an enteric coating solution with the concentration of 12.5 percent;
4-2, coating the micropellets coated with the isolation coating obtained in the step 3) with the coating solution prepared in the step 4-1; the specific operation is as follows: setting the rotating speed of a fan at 1700r/m, the material temperature at 28 ℃, the air inlet temperature at 30-32 ℃, starting 3kw for heating, setting the atomization pressure at 0.17mpa, starting a peristaltic pump when the material temperature is stabilized at 28 ℃, gradually increasing the rotating speed to 30r/m, pumping the enteric coating liquid into a fluidized bed, and carrying out bottom spray coating on the pellets; stopping peristaltic pump when the weight of the coating is increased by 30%, and fluidized drying at 30 deg.C for 30 min.
2. The method according to claim 1, wherein the step 1) of freeze-drying the attenuated virus strain comprises the steps of:
1-1, filtering the virus low virulent strain obtained by culture, and then fully stirring and uniformly mixing the filtered virus low virulent strain with an isometric sterile freeze-drying protective agent;
1-2, starting a freeze dryer for pre-freezing, putting the mixed virus liquid in the step 1-1 into the freeze dryer when the temperature of the plate is reduced to-50 ℃, keeping the temperature at-50 ℃ after the mixed virus liquid is put into a box, and maintaining for 2 hours;
1-3, opening the rear box in sequence, cooling, vacuumizing to reduce the vacuum degree of the compartment body to 20pa, and maintaining for 1 hour;
1-4: heating for 200min after 1h to raise the temperature of the sample to-7 ℃ and maintaining for at least 1 h; and heating all samples to 15 ℃ by secondary heating for 120min, taking out, vacuumizing and sealing the obtained virus freeze-dried powder, and storing at 4 ℃.
3. The method of claim 2, wherein the lyoprotectant consists of, in step 1-1: 10% skimmed milk powder, 5% sucrose and 85% water.
4. The preparation method of claim 1, wherein the lyophilized powder is loaded in an amount of 1/10 for pellet core; the binder was used in an amount of 1/10 parts of the pellet core.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810997606.5A CN108904795B (en) | 2018-08-29 | 2018-08-29 | Preparation method of oral vaccine for porcine epidemic diarrhea virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810997606.5A CN108904795B (en) | 2018-08-29 | 2018-08-29 | Preparation method of oral vaccine for porcine epidemic diarrhea virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108904795A CN108904795A (en) | 2018-11-30 |
| CN108904795B true CN108904795B (en) | 2022-07-05 |
Family
ID=64406132
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810997606.5A Active CN108904795B (en) | 2018-08-29 | 2018-08-29 | Preparation method of oral vaccine for porcine epidemic diarrhea virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108904795B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101283985A (en) * | 2008-06-05 | 2008-10-15 | 天津市水产研究所 | Enteric oral vaccine for fishing gear and preparation method thereof |
| CN105821006A (en) * | 2016-03-15 | 2016-08-03 | 华中农业大学 | Attenuated strain YN150 of variant porcine epidemic diarrhea virus and applications thereof |
| CN106554944A (en) * | 2015-09-28 | 2017-04-05 | 普莱柯生物工程股份有限公司 | The vaccine combination and application of pig epidemic diarrhea virus attenuated strain and its preparation |
| WO2017066134A1 (en) * | 2015-10-16 | 2017-04-20 | Merck Sharp & Dohme Corp. | Processes for preparing formulations for gastrointestinal-targeted therapies |
| CN108024955A (en) * | 2015-06-12 | 2018-05-11 | 瓦克萨特公司 | Preparation for the delivering of the small intestine of RSV and norovirus antigen |
| CN108114277A (en) * | 2018-02-07 | 2018-06-05 | 姜新鹏 | The microcapsule preparation method of oral killed Porcine epidemic diarrhea virus |
-
2018
- 2018-08-29 CN CN201810997606.5A patent/CN108904795B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101283985A (en) * | 2008-06-05 | 2008-10-15 | 天津市水产研究所 | Enteric oral vaccine for fishing gear and preparation method thereof |
| CN108024955A (en) * | 2015-06-12 | 2018-05-11 | 瓦克萨特公司 | Preparation for the delivering of the small intestine of RSV and norovirus antigen |
| CN106554944A (en) * | 2015-09-28 | 2017-04-05 | 普莱柯生物工程股份有限公司 | The vaccine combination and application of pig epidemic diarrhea virus attenuated strain and its preparation |
| WO2017066134A1 (en) * | 2015-10-16 | 2017-04-20 | Merck Sharp & Dohme Corp. | Processes for preparing formulations for gastrointestinal-targeted therapies |
| CN105821006A (en) * | 2016-03-15 | 2016-08-03 | 华中农业大学 | Attenuated strain YN150 of variant porcine epidemic diarrhea virus and applications thereof |
| CN108114277A (en) * | 2018-02-07 | 2018-06-05 | 姜新鹏 | The microcapsule preparation method of oral killed Porcine epidemic diarrhea virus |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108904795A (en) | 2018-11-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104894075A (en) | Method for preparing vaccine by editing pseudorabies virus genomes based on CRISPR/Cas9 and Cre/lox systems and application of method | |
| CN106636012B (en) | One boar lid his virus stain, vaccine combination and its preparation method and application | |
| CN102732545B (en) | Duck plague virus transfer vector pUC-deltagC-EGFP, and gC-deleted recombinant strain DPV-deltagC-EGFP | |
| CN109182278A (en) | Seneca Valley virus strain and its application | |
| CN104498599B (en) | One group of microsporidian molecule universal detector primer and kit thereof | |
| CN109868262B (en) | Canine distemper attenuated strain and application thereof | |
| CN110144332A (en) | One plant of pig Delta Coronavirus Strain and its application | |
| CN110747175B (en) | Pig delta coronavirus and application thereof | |
| CN112079904B (en) | Recombinant H7N9 subtype avian influenza virus-like particle and preparation method and application thereof | |
| CN111073862B (en) | Bovine viral diarrhea type2 attenuated strain and application thereof | |
| CN105506175A (en) | Mutiplex PCR detection kit for detecting PRRSV, PCV2, PRV and CSFV | |
| CN112458064A (en) | Gatasavir full-length infectious clone, replicon system, preparation and application thereof | |
| CN111073863B (en) | Porcine epidemic diarrhea and porcine delta coronavirus bivalent attenuated vaccine and preparation method thereof | |
| CN116426487A (en) | Porcine epidemic diarrhea virus strain and application thereof in vaccine preparation | |
| CN108904795B (en) | Preparation method of oral vaccine for porcine epidemic diarrhea virus | |
| CN112195161B (en) | Canine parvovirus vaccine strain, vaccine and preparation method thereof | |
| CN108796124A (en) | A kind of the multiple PCR detection primer group and kit of quick differentiation PCV1, PCV2 and PCV3 | |
| CN101235363B (en) | Pig transmissible gastroenteritis virus vaccine strain and application thereof | |
| CN112063596A (en) | Pigeon paramyxovirus type 1 PPMV-1/BJ-C strain and application thereof | |
| CN110527684A (en) | Application of the nanosizing RNAi preparation in PVY prevention and treatment | |
| CN107267467B (en) | Method for separating porcine reproductive and respiratory syndrome virus | |
| CN114015598B (en) | Pediococcus acidilactici separated from Tibetan mushroom and application of pediococcus acidilactici in prevention and treatment of rotavirus infection | |
| CN106916832B (en) | O-type foot-and-mouth disease virus recombinant nucleic acid, recombinant vaccine strain, preparation method and application thereof | |
| CN109022371A (en) | One plant of Porcine epidemic diarrhea virus strain and its preparing the application in pig epidemic diarrhea vaccine | |
| CN108531511A (en) | A kind of expression African swine fever virus E183L gene recombinant adenovirus vectors, construction method and recombined adhenovirus preparation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |