CN106490361A - A kind of preparation method of the CpG ODN for having immune-enhancing activity to pig - Google Patents
A kind of preparation method of the CpG ODN for having immune-enhancing activity to pig Download PDFInfo
- Publication number
- CN106490361A CN106490361A CN201610938491.3A CN201610938491A CN106490361A CN 106490361 A CN106490361 A CN 106490361A CN 201610938491 A CN201610938491 A CN 201610938491A CN 106490361 A CN106490361 A CN 106490361A
- Authority
- CN
- China
- Prior art keywords
- cpg odn
- pig
- immune
- preparation
- enhancing activity
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
-
- 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
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Mycology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to veterinary biological product technical field, and in particular to a kind of preparation method of the CpG ODN for having immune-enhancing activity to pig；Comprise the following steps：(1) CpG ODN volume productions (2) coating agent (3) dispensing；The present invention can induce body and produce panimmunity effect, which is promoted to secrete cytokine profiles, improve the special and nonspecific immune reaction ability of body, it is added in feedstuff and can effectively improves the immunity of pig itself, there is good immune effect to the great animal epidemic such as current highly pathogenic PRRS, high pathogenic avian influenza, foot and mouth disease, swine fever, the survival rate of pig is effectively improved, the generation of disease is reduced, aquaculture cost is reduced.
Description
Technical field
The present invention relates to veterinary biological product technical field, and in particular to a kind of CpG for having immune-enhancing activity to pig
The preparation method of ODN.
Background technology
Cytosine-guanine oligodeoxynucleotide (CpGoligodeoxymieleotides, CpG ODN), also referred to as exempts from
Epidemic disease stimulate DNA sequence, be a class with non-methylated cytidylic acid and guanylic acid the specific nucleotide as core
Sequence, can induce body and produce panimmunity effect (such as activating immune cell), promote which to secrete cytokine profiles, improve
The special and nonspecific immune reaction ability of body.Functional study find, CpG ODN can activating B cell, NK cells, monokaryon/
The immunocytes such as macrophage, dendritic cell, T cell, induction produce the cytokine profiles such as IL-12, TNF-d, INF- γ,
Promote the expression of mhc class ii molecule and costimulating factor CD40, CD80, CD86.Importantly, it is capable of adding for enhancement antigen
Work is offered, and induction immunne response is changed to Th1 types, produces stronger specific cellular immunity and humoral immunization.Therefore, CpG
ODN is not only a kind of effective immunostimulant, and preferably Th1 type immunological adjuvants, is a kind of for traditional vaccine
Adjuvant better than aluminium porcelain enamelling.
The great animal epidemics such as current highly pathogenic PRRS, high pathogenic avian influenza, foot and mouth disease, swine fever are threatened always
The sound development of Yongning pastoral industry, strengthens vaccine virus for the research of CpG ODN concentrates on which as immunological adjuvant at present
In the immunological enhancement of strain, and CpG ODN are rarely reported as the application of feed additive, this research further will be promoted
Applications of the new immunostimulant CpG ODN in swine diseasess immune protection, provides new method and thinking for swine diseasess preventing and treating.
Content of the invention
The present invention is solution above-mentioned technical problem, there is provided a kind of preparation of the CpG ODN for having immune-enhancing activity to pig
Method.
Realize particular by technical scheme below：
A kind of preparation method of the CpG ODN for having immune-enhancing activity to pig, comprises the following steps：
(1) CpG ODN volume productions, comprise the following steps：
A, fermentation：CpG ODN plasmids are proceeded in e.colistraindh5α, 35～36 DEG C, 250～280rpm concussion trainings
2～2.5h is supported, Escherichia coli bacteria liquid is obtained, the volume ratio by 1: 70～80 is inoculated in fermentation medium, fermentation culture 15
～20h.
B, extraction：Bacterium solution concussion stirring after fermenting in step a, subsequently by the bacteriolyze for adding 50～60 μ g per ml bacterium solutions
Enzyme Standard entertion, stirs, and under 35.2～35.8 DEG C of water bath with thermostatic control, stirs 8～12min, subsequently, adds by every ml bacterium solutions
Enter the LiCl of 0.2～0.3ml, 6～8min is centrifuged under the rotating speed of 10000r/min, takes supernatant.
C, heating：Supernatant in stepb, concussion 15～20min of stirring are placed in 20～25min of heating in boiling water bath, cold
But the isopropanol of the quality such as supernatant, sedimentation and centrifugation to 20～25 DEG C, is added to take precipitation.
D, sterilizing：1～1.5 times of precipitation quality of ethanol is added, 20～30min is stood, vacuum distillation is reclaimed ethanol and is
Can.
(2) coating agent：The distilled water of 20～30 parts of beta-schardinger dextrin -s, 6～8 parts of ethyl celluloses and 35～40 parts is mixed
Compound is obtained, stirring in water bath to volume concentration to mixture volume 1/3 at 100 DEG C, after being cooled to room temperature, adds 5～7 parts
Oleum menthae, 2.5～3 parts of phosphoric acid, 20～30min of stirring in water bath at 70 DEG C.
(3) dispensing：Coating agent in step (2) is coated in after the sterilizing in step (1) by 1～1.5 μm of thickness
In precipitation, at 35～40 DEG C, 3～4min is vibrated under the frequency of vibration of 400～500 times/min, obtain microcapsule, 100
Add 0.05～0.1 part of microcapsule in part daily ration, mix thoroughly.
Further, described fermentation medium, is made up of following raw material by weight：Beef extract 4～5, albumen
Peptone 1～2, glucose 2.5～4, agar 1～2, Fructus Schisandrae Chinensis 10～15, Herba Epimedii 4～7, Semen Trigonellae 8～12, the Cortex Eucommiae 2～4, third
Ketone is some.
Further, described fermentation medium, be Fructus Schisandrae Chinensis, Herba Epimedii, Semen Trigonellae, the Cortex Eucommiae are weighed in proportion after, smash
Broken 20～25min, adds 5～6 times of Chinese medicine gross mass of acetone, impregnates 38～42h, and vacuum distillation is reclaimed acetone, obtained remaining
Liquid, adds beef extract, peptone, glucose and agar, mixes thoroughly, and it is 25 DEG C that heating adjusts the temperature of culture medium.
Further, described LiCl, its concentration are 2.5～3.1mol/l.
Further, described ethanol, its concentration are 15～20%.
In sum, the beneficial effects of the present invention is：CpG ODN can induce body and produce panimmunity effect, promote
Enter which and secrete cytokine profiles, improve the special and nonspecific immune reaction ability of body, being added in feedstuff can be effective
Raising pig itself immunity, great to current highly pathogenic PRRS, high pathogenic avian influenza, foot and mouth disease, swine fever etc.
Animal epidemic has good immune effect, effectively improves the survival rate of pig, reduces the generation of disease, reduces aquaculture cost.
Specific embodiment
Below the specific embodiment of the present invention is described in further detail, but the invention is not limited in these realities
Mode is applied, any improvement or replacement on the present embodiment essence spirit still falls within the claims in the present invention required for protection
Scope.
Embodiment 1
A kind of preparation method of the CpG ODN for having immune-enhancing activity to pig, comprises the following steps：
(1) CpG ODN volume productions, comprise the following steps：
A, fermentation：CpG ODN plasmids are proceeded in escherichia coli DH5a bacterial strain, 35 DEG C, 250rpm concussion and cultivates 2h are obtained greatly
Enterobacteria bacterium solution, the volume ratio by 1: 70 are inoculated in fermentation medium, fermentation culture 15h.
B, extraction：Bacterium solution concussion stirring after fermenting in step a, subsequently by the lysozyme mark for adding 50 μ g per ml bacterium solutions
Quasi- addition, stirs, and under 35.2 DEG C of water bath with thermostatic control, stirs 8min, subsequently, by the LiCl that every ml bacterium solutions add 0.2ml,
6min is centrifuged under the rotating speed of 10000r/min, supernatant is taken.
C, heating：Supernatant in stepb, concussion stirring 15min, is placed in heating 20min in boiling water bath, is cooled to 20
DEG C, add the isopropanol of the quality such as supernatant, sedimentation and centrifugation to take precipitation.
D, sterilizing：1 times of precipitation quality of ethanol is added, 20min is stood, ethanol is reclaimed in vacuum distillation.
(2) coating agent：The distilled water of 20g beta-schardinger dextrin -s, 6g ethyl celluloses and 35g is mixed to obtain compound, 100
Stirring in water bath to volume concentration to mixture volume 1/3 at DEG C, after being cooled to room temperature, adds Oleum menthae, the phosphorus of 2.5g of 5g
Acid, stirring in water bath 20min at 70 DEG C.
(3) dispensing：Coating agent in step (2) is coated in the precipitation after the sterilizing in step (1) by 1 μm of thickness
On, at 35 DEG C, 3min is vibrated under the frequency of vibration of 400 times/min, obtain microcapsule, add 0.05g in 100g daily rations
Microcapsule, mix thoroughly.
Described fermentation medium, is made up of following raw material by weight：Beef extract 4g, peptone 1g, Fructus Vitis viniferae
Sugared 2.5g, agar 1g, Fructus Schisandrae Chinensis 10g, Herba Epimedii 4g, Semen Trigonellae 8g, Cortex Eucommiae 2g, acetone are some.
Described fermentation medium, be Fructus Schisandrae Chinensis, Herba Epimedii, Semen Trigonellae, the Cortex Eucommiae are weighed in proportion after, smash 20min to pieces,
5～6 times of Chinese medicine gross mass of acetone is added, 38h is impregnated, vacuum distillation is reclaimed acetone, obtains residual solution, add beef leaching
Cream, peptone, glucose and agar, mix thoroughly, and it is 25 DEG C that heating adjusts the temperature of culture medium.
Described LiCl, its concentration are 2.5mol/l.
Described ethanol, its concentration are 15%.
Embodiment 2
A kind of preparation method of the CpG ODN for having immune-enhancing activity to pig, comprises the following steps：
(1) CpG ODN volume productions, comprise the following steps：
A, fermentation：CpS ODN plasmids are proceeded in e.colistraindh5α, 36 DEG C, 280rpm concussion and cultivates 2.5h are obtained
Escherichia coli bacteria liquid, the volume ratio by 1: 80 are inoculated in fermentation medium, fermentation culture 20h.
B, extraction：Bacterium solution concussion stirring after fermenting in step a, subsequently by the lysozyme mark for adding 60 μ g per ml bacterium solutions
Quasi- addition, stirs, and under 35.8 DEG C of water bath with thermostatic control, stirs 12min, subsequently, adds 0.3ml's by every ml bacterium solutions
LiCl, under the rotating speed of 10000r/min is centrifuged 8min, takes supernatant.
C, heating：Supernatant in stepb, concussion stirring 20min, is placed in heating 25min in boiling water bath, is cooled to 25
DEG C, add the isopropanol of the quality such as supernatant, sedimentation and centrifugation to take precipitation.
D, sterilizing：1.5 times of precipitation quality of ethanol is added, 30min is stood, ethanol is reclaimed in vacuum distillation.
(2) coating agent：The distilled water of 30g beta-schardinger dextrin -s, 8g ethyl celluloses and 40g is mixed to obtain compound, 100
Stirring in water bath to volume concentration to mixture volume 1/3 at DEG C, after being cooled to room temperature, adds Oleum menthae, the phosphoric acid of 3g of 7g,
Stirring in water bath 30min at 70 DEG C.
(3) dispensing：Coating agent in step (2) is coated in the precipitation after the sterilizing in step (1) by 1.5 μm of thickness
On, at 40 DEG C, 4min is vibrated under the frequency of vibration of 500 times/min, obtain microcapsule, add 0.1g's in 100g daily rations
Microcapsule, mixes thoroughly.
Described fermentation medium, is made up of following raw material by weight：Beef extract 5g, peptone 2g, Fructus Vitis viniferae
Sugared 4g, agar 2g, Fructus Schisandrae Chinensis 15g, Herba Epimedii 7g, Semen Trigonellae 12g, Cortex Eucommiae 4g, acetone are some.
Described fermentation medium, be Fructus Schisandrae Chinensis, Herba Epimedii, Semen Trigonellae, the Cortex Eucommiae are weighed in proportion after, smash 25min to pieces,
5～6 times of Chinese medicine gross mass of acetone is added, 42h is impregnated, vacuum distillation is reclaimed acetone, obtains residual solution, add beef leaching
Cream, peptone, glucose and agar, mix thoroughly, and it is 25 DEG C that heating adjusts the temperature of culture medium.
Described LiCl, its concentration are 3.1mol/l.
Described ethanol, its concentration are 20%.
Embodiment 3
A kind of preparation method of the CpG ODN for having immune-enhancing activity to pig, comprises the following steps：
(1) CpG ODN volume productions, comprise the following steps：
A, fermentation：CpG ODN plasmids are proceeded in e.colistraindh5α, 35.5 DEG C, 270rpm concussion and cultivates 2.3h,
Escherichia coli bacteria liquid is obtained, the volume ratio by 1: 75 is inoculated in fermentation medium, fermentation culture 18h.
B, extraction：Bacterium solution concussion stirring after fermenting in step a, subsequently by the lysozyme mark for adding 55 μ g per ml bacterium solutions
Quasi- addition, stirs, and under 35.5 DEG C of water bath with thermostatic control, stirs 10min, subsequently, adds 0.25ml's by every ml bacterium solutions
LiCl, under the rotating speed of 10000r/min is centrifuged 7min, takes supernatant.
C, heating：Supernatant in stepb, concussion stirring 18min, is placed in heating 23min in boiling water bath, is cooled to 23
DEG C, add the isopropanol of the quality such as supernatant, sedimentation and centrifugation to take precipitation.
D, sterilizing：1.3 times of precipitation quality of ethanol is added, 25min is stood, ethanol is reclaimed in vacuum distillation.
(2) coating agent：The distilled water of 25g beta-schardinger dextrin -s, 7g ethyl celluloses and 38g is mixed to obtain compound, 100
Stirring in water bath to volume concentration to mixture volume 1/3 at DEG C, after being cooled to room temperature, adds Oleum menthae, the phosphorus of 2.8g of 6g
Acid, stirring in water bath 25min at 70 DEG C.
(3) dispensing：Coating agent in step (2) is coated in the precipitation after the sterilizing in step (1) by 1.3 μm of thickness
On, at 38 DEG C, 3.5min is vibrated under the frequency of vibration of 450 times/min, obtain microcapsule, add in 100g daily rations
The microcapsule of 0.08g, mixes thoroughly.
Described fermentation medium, is made up of following raw material by weight：Beef extract 4.5g, peptone 1.5g,
Glucose 3g, agar 1.5g, Fructus Schisandrae Chinensis 13g, Herba Epimedii 5g, Semen Trigonellae 10g, Cortex Eucommiae 3g, acetone are some.
Described fermentation medium, be Fructus Schisandrae Chinensis, Herba Epimedii, Semen Trigonellae, the Cortex Eucommiae are weighed in proportion after, smash 23min to pieces,
5～6 times of Chinese medicine gross mass of acetone is added, 40h is impregnated, vacuum distillation is reclaimed acetone, obtains residual solution, add beef leaching
Cream, peptone, glucose and agar, mix thoroughly, and it is 25 DEG C that heating adjusts the temperature of culture medium.
Described LiCl, its concentration are 2.8mol/l.
Described ethanol, its concentration are 18%.
Embodiment 4
A kind of preparation method of the CpG ODN for having immune-enhancing activity to pig, comprises the following steps：
(1) CpG ODN volume productions, comprise the following steps：
A, fermentation：CpG ODN plasmids are proceeded in e.colistraindh5α, 36 DEG C, 250rpm concussion and cultivates 2h are obtained greatly
Enterobacteria bacterium solution, the volume ratio by 1: 80 are inoculated in fermentation medium, fermentation culture 15h.
B, extraction：Bacterium solution concussion stirring after fermenting in step a, subsequently by the lysozyme mark for adding 60 μ g per ml bacterium solutions
Quasi- addition, stirs, and under 35.2 DEG C of water bath with thermostatic control, stirs 12min, subsequently, adds 0.2ml's by every ml bacterium solutions
LiCl, under the rotating speed of 10000r/min is centrifuged 6min, takes supernatant.
C, heating：Supernatant in stepb, concussion stirring 20min, is placed in heating 20min in boiling water bath, is cooled to 25
DEG C, add the isopropanol of the quality such as supernatant, sedimentation and centrifugation to take precipitation.
D, sterilizing：1 times of precipitation quality of ethanol is added, 30min is stood, ethanol is reclaimed in vacuum distillation.
(2) coating agent：The distilled water of 20g beta-schardinger dextrin -s, 6g ethyl celluloses and 40g is mixed to obtain compound, 100
Stirring in water bath to volume concentration to mixture volume 1/3 at DEG C, after being cooled to room temperature, adds Oleum menthae, the phosphorus of 2.5g of 5g
Acid, stirring in water bath 30min at 70 DEG C.
(3) dispensing：Coating agent in step (2) is coated in the precipitation after the sterilizing in step (1) by 1 μm of thickness
On, at 35 DEG C, 3min is vibrated under the frequency of vibration of 500 times/min, obtain microcapsule, add 0.05g in 100g daily rations
Microcapsule, mix thoroughly.
Described fermentation medium, is made up of following raw material by weight：Beef extract 5g, peptone 1g, Fructus Vitis viniferae
Sugared 2.5g, agar 2g, Fructus Schisandrae Chinensis 15g, Herba Epimedii 7g, Semen Trigonellae 8g, Cortex Eucommiae 2g, acetone are some.
Described fermentation medium, be Fructus Schisandrae Chinensis, Herba Epimedii, Semen Trigonellae, the Cortex Eucommiae are weighed in proportion after, smash 20min to pieces,
5～6 times of Chinese medicine gross mass of acetone is added, 38h is impregnated, vacuum distillation is reclaimed acetone, obtains residual solution, add beef leaching
Cream, peptone, glucose and agar, mix thoroughly, and it is 25 DEG C that heating adjusts the temperature of culture medium.
Described LiCl, its concentration are 3.1mol/l.
Described ethanol, its concentration are 15%.
Embodiment 5
A kind of preparation method of the CpG ODN for having immune-enhancing activity to pig, comprises the following steps：
(1) CpG ODN volume productions, comprise the following steps：
A, fermentation：CpG ODN plasmids are proceeded in e.colistraindh5α, 36 DEG C, 250rpm concussion and cultivates 2h are obtained greatly
Enterobacteria bacterium solution, the volume ratio by 1: 70 are inoculated in fermentation medium, fermentation culture 20h.
B, extraction：Bacterium solution concussion stirring after fermenting in step a, subsequently by the lysozyme mark for adding 60 μ g per ml bacterium solutions
Quasi- addition, stirs, and under 35.2 DEG C of water bath with thermostatic control, stirs 12min, subsequently, adds 0.2ml's by every ml bacterium solutions
LiCl, under the rotating speed of 10000r/min is centrifuged 8min, takes supernatant.
C, heating：Supernatant in stepb, concussion 15～20min of stirring, is placed in heating 25min in boiling water bath, is cooled to
20 DEG C, the isopropanol of the quality such as supernatant, sedimentation and centrifugation is added to take precipitation.
D, sterilizing：1 times of precipitation quality of ethanol is added, 20min is stood, ethanol is reclaimed in vacuum distillation.
(2) coating agent：The distilled water of 30g beta-schardinger dextrin -s, 6g ethyl celluloses and 35g is mixed to obtain compound, 100
Stirring in water bath to volume concentration to mixture volume 1/3 at DEG C, after being cooled to room temperature, adds Oleum menthae, the phosphorus of 2.5g of 7g
Acid, stirring in water bath 30min at 70 DEG C.
(3) dispensing：Coating agent in step (2) is coated in the precipitation after the sterilizing in step (1) by 1 μm of thickness
On, at 35 DEG C, 3min is vibrated under the frequency of vibration of 500 times/min, obtain microcapsule, add 0.1g's in 100g daily rations
Microcapsule, mixes thoroughly.
Described fermentation medium, is made up of following raw material by weight：Beef extract 5g, peptone 1g, Fructus Vitis viniferae
Sugared 2.5g, agar 2g, Fructus Schisandrae Chinensis 15g, Herba Epimedii 4g, Semen Trigonellae 8g, Cortex Eucommiae 4g, acetone are some.
Described fermentation medium, be Fructus Schisandrae Chinensis, Herba Epimedii, Semen Trigonellae, the Cortex Eucommiae are weighed in proportion after, smash 20min to pieces,
5～6 times of Chinese medicine gross mass of acetone is added, 42h is impregnated, vacuum distillation is reclaimed acetone, obtains residual solution, add beef leaching
Cream, peptone, glucose and agar, mix thoroughly, and it is 25 DEG C that heating adjusts the temperature of culture medium.
Described LiCl, its concentration are 2.5mol/l.
Described ethanol, its concentration are 20%.
Experimental example
The some heads of pig of healthy growth, disease-free, all ages and classes are randomly selected, two groups are randomly divided into, first group in day
Add the present invention to be fed in grain, second group without, Oestrus rate and the litter size to the sow in two groups, piglet is deposited
Motility rate and litter weight, the daily gain and meat feed ratio of growing and fattening pigs, are tested and assessed, as a result as shown in table 1, to pig blue-ear disease, bird flu, mouth
Fever aphthous, the antibody level of serum of swine fever are evaluated and tested, as a result as shown in table 2：
Table 1
| Project | First group | Second group |
| The Oestrus rate of sow | 75～80% | 60～65% |
| Litter size | 12～14 | 10～12 |
| The survival rate of piglet | 86～90% | 75～79% |
| The weight of averagely every piglet | 2025.3g | 1350.1g |
| The daily gain of growing and fattening pigs | 552.3～613.2g | 431.2～479.8g |
| The meat feed ratio of growing and fattening pigs | 2.15～2.37: 1 | 1.55～1.75: 1 |
Table 2
Claims (5)
1. a kind of preparation method of the CpG ODN for having immune-enhancing activity to pig, it is characterised in that comprise the following steps：
(1) CpG ODN volume productions, comprise the following steps：
A, fermentation：CpG ODN plasmids are proceeded in e.colistraindh5α, 35～36 DEG C, 250～280rpm concussion and cultivates 2
～2.5h, obtains Escherichia coli bacteria liquid, and the volume ratio by 1: 70～80 is inoculated in fermentation medium, and fermentation culture 15～
20h；
B, extraction：Bacterium solution concussion stirring after fermenting in step a, subsequently by the lysozyme mark for adding 50～60 μ g per ml bacterium solutions
Quasi- addition, stirs, and under 35.2～35.8 DEG C of water bath with thermostatic control, stirs 8～12min, subsequently, adds by every ml bacterium solutions
The LiCl of 0.2～0.3ml, under the rotating speed of 10000r/min is centrifuged 6～8min, takes supernatant；
C, heating：Supernatant in stepb, concussion 15～20min of stirring, is placed in 20～25min of heating in boiling water bath, is cooled to
20～25 DEG C, the isopropanol of the quality such as supernatant, sedimentation and centrifugation is added to take precipitation；
D, sterilizing：1～1.5 times of precipitation quality of ethanol is added, 20～30min is stood, ethanol is reclaimed in vacuum distillation；
(2) coating agent：The distilled water of 20～30 parts of beta-schardinger dextrin -s, 6～8 parts of ethyl celluloses and 35～40 parts is mixed and must be mixed
Material is closed, stirring in water bath to volume concentration to mixture volume 1/3 at 100 DEG C, after being cooled to room temperature, adds 5～7 parts thin
Lotus oil, 2.5～3 parts of phosphoric acid, 20～30min of stirring in water bath at 70 DEG C；
(3) dispensing：Coating agent in step (2) is coated in the precipitation after the sterilizing in step (1) by 1～1.5 μm of thickness
On, at 35～40 DEG C, 3～4min is vibrated under the frequency of vibration of 400～500 times/min, obtain microcapsule, 100 parts of days
Add 0.05～0.1 part of microcapsule in grain, mix thoroughly.
2. the preparation method of the CpG ODN of immune-enhancing activity is had to pig as claimed in claim 1, it is characterised in that described
Fermentation medium, be made up of following raw material by weight：Beef extract 4～5, peptone 1～2, glucose 2.5～4,
Agar 1～2, Fructus Schisandrae Chinensis 10～15, Herba Epimedii 4～7, Semen Trigonellae 8～12, the Cortex Eucommiae 2～4, acetone are some.
3. the preparation method of the CpG ODN of immune-enhancing activity is had to pig as claimed in claim 1 or 2, it is characterised in that institute
The fermentation medium that states, be Fructus Schisandrae Chinensis, Herba Epimedii, Semen Trigonellae, the Cortex Eucommiae are weighed in proportion after, smash 20～25min to pieces, in addition
The acetone of 5～6 times of medicine gross mass, impregnate 38～42h, vacuum distillation reclaim acetone, obtain residual solution, add beef extract,
Peptone, glucose and agar, mix thoroughly, and it is 25 DEG C that heating adjusts the temperature of culture medium.
4. the preparation method of the CpG ODN of immune-enhancing activity is had to pig as claimed in claim 1, it is characterised in that described
LiCl, its concentration be 2.5～3.1mol/l.
5. the preparation method of the CpG ODN of immune-enhancing activity is had to pig as claimed in claim 1, it is characterised in that described
Ethanol, its concentration be 15～20%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610938491.3A CN106490361A (en) | 2016-10-31 | 2016-10-31 | A kind of preparation method of the CpG ODN for having immune-enhancing activity to pig |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610938491.3A CN106490361A (en) | 2016-10-31 | 2016-10-31 | A kind of preparation method of the CpG ODN for having immune-enhancing activity to pig |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106490361A true CN106490361A (en) | 2017-03-15 |
Family
ID=58319042
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610938491.3A Pending CN106490361A (en) | 2016-10-31 | 2016-10-31 | A kind of preparation method of the CpG ODN for having immune-enhancing activity to pig |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106490361A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110042103A (en) * | 2019-03-25 | 2019-07-23 | 华南农业大学 | A kind of pair of pig has CpG-ODN and its application of specific immunity stimulation |
| CN112080505A (en) * | 2020-07-23 | 2020-12-15 | 杭州华缔集团有限公司 | CG island oligonucleotide with piglet immunostimulation capacity and application thereof in feed additive |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1267519A (en) * | 1999-03-19 | 2000-09-27 | 浅间化成株式会社 | Immune regulator, immune regulator food and immune regulator fodder |
| CN1554359A (en) * | 2003-12-24 | 2004-12-15 | 广东省农业科学院农业生物技术研究所 | Process for preparing cytosine suanine dinuleotide sequence molecule immunopotentiator |
| CN102399784A (en) * | 2010-09-10 | 2012-04-04 | 中国科学院海洋研究所 | CpG oligonucleotide possessing immune enhancement activity to aquatic livestock, its preparation method and its application |
| CN104686845A (en) * | 2015-03-10 | 2015-06-10 | 浙江皇冠科技有限公司 | Nature feed additive for microorganism-derived aquatic animals, and application of nature feed additive |
-
2016
- 2016-10-31 CN CN201610938491.3A patent/CN106490361A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1267519A (en) * | 1999-03-19 | 2000-09-27 | 浅间化成株式会社 | Immune regulator, immune regulator food and immune regulator fodder |
| CN1554359A (en) * | 2003-12-24 | 2004-12-15 | 广东省农业科学院农业生物技术研究所 | Process for preparing cytosine suanine dinuleotide sequence molecule immunopotentiator |
| CN102399784A (en) * | 2010-09-10 | 2012-04-04 | 中国科学院海洋研究所 | CpG oligonucleotide possessing immune enhancement activity to aquatic livestock, its preparation method and its application |
| CN104686845A (en) * | 2015-03-10 | 2015-06-10 | 浙江皇冠科技有限公司 | Nature feed additive for microorganism-derived aquatic animals, and application of nature feed additive |
Non-Patent Citations (2)
| Title |
|---|
| 李娇清等: ""CpG- ODN不同免疫途径对仔猪免疫增强效果的研究"", 《广东农业科学》 * |
| 蔡津生等: "《中国食药用菌工程学》", 31 January 2015, 上海科学技术文献出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110042103A (en) * | 2019-03-25 | 2019-07-23 | 华南农业大学 | A kind of pair of pig has CpG-ODN and its application of specific immunity stimulation |
| CN110042103B (en) * | 2019-03-25 | 2023-01-17 | 华南农业大学 | CpG-ODN with specific immunostimulation effect on pigs and application thereof |
| CN112080505A (en) * | 2020-07-23 | 2020-12-15 | 杭州华缔集团有限公司 | CG island oligonucleotide with piglet immunostimulation capacity and application thereof in feed additive |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109576180B (en) | Rhodococcus ruber and application thereof as immunologic adjuvant in preparation of vaccine | |
| Gross et al. | Some effects of feeding corticosterone to chickens | |
| CN109666609A (en) | A kind of Rhodococcus ruber fermentation process and its application as adjuvant in animal vaccine | |
| CN1108575A (en) | Oil-based and water-based adjuvant mixture | |
| CN106490361A (en) | A kind of preparation method of the CpG ODN for having immune-enhancing activity to pig | |
| Roos et al. | The immune modulation of Bacillus cereus var. Toyoi in mice immunized with experimental inactivated Bovine Herpesvirus Type 5 vaccine | |
| CN102579339A (en) | Oil emulsion vaccine for broilers and preparation method thereof | |
| Yuan et al. | Sunflower seed oil combined with ginseng stem-leaf saponins as an adjuvant to enhance the immune response elicited by Newcastle disease vaccine in chickens | |
| CN106232805A (en) | Novel phage and comprise its compositions | |
| CN106615640A (en) | Biologically fermented feed and preparation method thereof | |
| WO2009014497A1 (en) | Use of cordyceps species or extract thereof in the egg production | |
| CN103083659B (en) | Preparation method and application of novel oil-free adjuvant | |
| CN110042103B (en) | CpG-ODN with specific immunostimulation effect on pigs and application thereof | |
| KR101873682B1 (en) | Novel Lactobacillus acidophilus KNU-02 having immune enhancing activity and use thereof | |
| CN107115365A (en) | One kind preventing and treating diarrhea of pigs biological agent and preparation method thereof | |
| Rahman et al. | Enhancement of Th1-biased protective immunity against avian influenza H9N2 virus via oral co-administration of attenuated Salmonella enterica serovar Typhimurium expressing chicken interferon-α and interleukin-18 along with an inactivated vaccine | |
| CN102836431B (en) | Method for preparing inactivated vaccine of anti-Listonella-anguillarum egg yolk antibody | |
| Echeverry et al. | Yeast cell wall polysaccharides enhanced expression of T helper type 1 and 2 cytokines profile in chicken B lymphocytes exposed to LPS challenge and enzyme treatment | |
| CN103396958B (en) | Preparation method of mycobacterium phlei and immune enhancer thereof | |
| Cheraghi et al. | Effects of dietary levels of yeast extracted β-glucans and α-mannans (Alphamune TM) on performance of broiler chicken raised in normal and thermal-stressed conditions | |
| CN103828765B (en) | A kind of pseudo-classic chicken-raising method healthy, the most efficient | |
| CN106509429A (en) | Preparation method of IFN-gamma (interferon-gamma) having immunity enhancing activity for pigs | |
| TWI378144B (en) | Recombinant plasmic containing multicopy cpg motifs and transformant thereof for applying on dna adjuvant in avian vaccines | |
| CN102631675A (en) | High-efficiency CpG preparation, preparation method thereof and application | |
| CN105497885A (en) | Subunit vaccine, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170315 |
|
| RJ01 | Rejection of invention patent application after publication |