CN108079289A - The microcapsule preparation method of oral killed transmissible gastro-enteritis virus - Google Patents
The microcapsule preparation method of oral killed transmissible gastro-enteritis virus Download PDFInfo
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- 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
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- A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
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Abstract
The microcapsule preparation method of oral killed transmissible gastro-enteritis virus, it is related to the microcapsule preparation method of oral killed transmissible gastro-enteritis virus, the present invention is in order to solve the technical deficiency in Current Domestic prevention and control transmissible gastro-enteritis virus prevention and control in oral inactivation vaccine, the present invention is based on inactivation transmissible gastro-enteritis virus and oral Micro-Encapsulation Technique, for the microcapsule preparation method of oral killed transmissible gastro-enteritis virus.Results of animal shows, serum neutralize antibody titers testing result illustrates that the micro-capsule vaccine can induce piglet mucosa-immune and systemic immunity response, and generate the serum and mucosal antibodies of higher level, while it is objective illustrate the micro-capsule vaccine have certain slow release effect.Micro-capsule vaccine antigen substance after Piglet by Oral, can be induced the expression of piglet specific antibody IgG and IgA, and add resistance of the piglet to TGEV, reduce the piglet death rate, attack malicious protective rate higher than control group 60 75%.
Description
Technical field
The present invention relates to the inactivation technology of transmissible gastro-enteritis virus and Micro-Encapsulation Technique methods.
Background technology
Infection transmissible gastro-enteritis virus (TGEV) is one of the main reason for causing pig virus diarrhoea (TGEV), special
It levies as serious enteritis, vomiting, watery diarrhea and weight loss etc..Occurred all year round for the disease, especially serious in winter,
The swinery of various age levels easily infects, but maximum to the harm of piglet, case fatality rate highest.The serious piglet death rate makes
Obtaining TGEV infection becomes a big factor of Chinese pig breeding industry loss.The Life Cycles of coronavirus start from the existing cell of invasion.It completes
After absorption, virus is needed in next step into the cytoplasm matter of host cell.Virus may be inserted into the concurrent sick poison of cell membrane with
The film fusion of cell is helped, enters the lesion that cytoplasm causes Small Intestine of Piglets epithelial cell so as to the genome of releasing virus.
Although serum antibody titer can be used as TGE serological diagnostic methods, effective intestinal mucosa local immunity is defence
Wherein, secretory IgA special TGEV (SIgA) antibody level is that alimentary canal mucous membrane is immunized to the key of TGEV invasions and infection
Importance.By oral route after immune swine, Specific IgA antibody can detect TGEV in intestinal juice and serum, and parenterally
Immunization route is but not easy to detect.It is believed that the IgA antibody in serum is probably derived from enteron aisle, it can be as TGE active immunities
Index.In addition, the IgA antibody secretory cell quantity of TGEV highly-wetting liquids induction is discrepant.There is scholar's report, TGEV is malicious by force
Strain can stimulate gut associated lymphoid tissue (GALT) to generate a large amount of IgA antibody secretory cells, and attenuated vaccine only induces a small amount of IgA
Antibody secreting cell generates.In addition to Local Humoral Immunity, cell-mediated immune response also plays important in TGEV infection is resisted
Effect.Lymphproliferation response, spontaneous cell-mediated cytotoxicity experiment, the examination of antibody dependent cellular mediation cytotoxicity
Testing etc. proves cellular immunity in the rehabilitation of TGEV infected pigs or resists and infect may play a role again.
It is extremely important that passive immunity resists TGEV infection for newborn piglet.Piglet obtains passive protect by sucking colostrum
Shield, Immunity Globulin of Colostrums is mainly IgG.Nursing period, with the decline of IgG levels in milk, IgA started into after a week
For main protection antibody.IgG has played important protective effect in milk, but generally believes that IgA classes TGEV antibody carries in milk
The protection of bigger effect is supplied, possible cause includes：(1) IgA titres in milk are higher：(2) proteolytic enzyme can be resisted：
(3) can be combined with enterocyte choice.SIgA is that gut-derived cell is secreted in breast tissue, is thought that IgA immunocytes
Breast localization is transferred to after antigen sensibilization in the intestine, and IgA is secreted into milk.There is scholar to be proposed in TGEV infection
" intestines-mammary gland " Immune Axis concept has great importance to designing rational immune programme.Therefore, as can disease sufficient amount
Virion is effectively presented to Small Intestine of Piglets mucous membrane, degrades from the digestion of hydrochloric acid in gastric juice and casing slime, antigenic activation piglet mucous membrane is exempted from
Epidemic disease system, induction generate the mucosal antibodies Ig of sufficient amount, and the prevention and the development of oral vaccine infect the disease of TGEV provide
New thinking.
The research of micro-capsule technology of preparing start from the 1930s, report for the first time the sixties carry bioactive substance it is micro-
The research of capsule prepares micro-capsule by core of bioactive substances such as protein, hormone, enzymes, and gained micro-capsule product is known as
" bio-microcapsule ".Then, research of the microcapsule technology in each field is gradually carried out, and rapidly develops.The application master of microcapsule technology
There are following characteristics：1) stability and resistant storage properties of application product are improved；2) for the defects of making up traditional handicraft, solve to pass
The technical barrier that system technique faces saves cost；3) insulating properties being applied between substance and substance or substance and surrounding medium；
4) it is applied to industry, the slow release of drug, biological products, achievees the purpose that microcapsule controlled-release or controlled release；5) some liquid are reduced
Volatility, change its dispersity, solve its incompatibility between surrounding dielectric material.At present, microcapsule technology is wide
It is general to be applied to the fields such as food industry, biological products and pharmaceutical sector, cosmetics industry, feed processing and production, textile industry, it is special
It is not in biological products and field of medicaments, the pharmaceutical dosage form prepared using the technology has very high superiority.However dynamic
The prevention and control of object disease and application is upper also rarely has research, especially in the disease and prevention and control of pig, are wrapped up merely with microcapsule formulation
In the TGEV of escherichia coli prokaryotic expression and antigens c OE albumen.There is no utilization microcapsule formulation packages to inactivate microorganism, including
Pig inactivates pathogenic bacteria and virus.
The content of the invention
For the technical deficiency in oral inactivation vaccine in Current Domestic prevention and control transmissible gastro-enteritis virus prevention and control, sheet
Research is based on inactivation transmissible gastro-enteritis virus technology and oral Micro-Encapsulation Technique, for oral killed pig transmissible
The microcapsule preparation method of marcy agent.
The present invention oral killed transmissible gastro-enteritis virus microcapsule preparation method, it be according to following steps into
Capable：First, transmissible gastro-enteritis virus strain secondary culture will be infected, 70~80% cell occurs thin after secondary culture
Born of the same parents' lesion effect collects the virus stain of culture, multigelation 3 times in -80 DEG C of ultra low temperature freezers, room temperature is placed in, so that carefully
Born of the same parents fully crack, and then 2000~4000r/min centrifuges 30min, draw supernatant, are transmissible gastro-enteritis virus liquid；
2nd, the infection transmissible gastro-enteritis virus liquid of step 1 is taken to be added in sodium alginate solution, abundant mixing；With perseverance
Fixed speed is added dropwise in the mixed liquor of calcium chloride, chitosan acetic acid；Its film formation reaction is made with the stirring of 300~400r/min constant speed
Fully, it is stored at room temperature suspensions of the 30~60min to get calcium alginate-chitosan microcapsules；Suspension is separated by filtration and uses steaming
Distilled water is cleaned repeatedly, then by it in -80 DEG C of 2~3h of precooling, vacuum freeze drying is to get oral killed transmissible gastroenteritis of swine disease
The microcapsules of poison.
The present invention includes following advantageous effect：
The present invention solves gastrointestinal tract virus oral vaccine immunization route by oral administration, and one kind is provided for the manufacture craft of vaccine
New method and new immunization route and method.The technology has filled up this domestic and international field blank.
The microcapsule formulation product of the present invention, which is stablized, is convenient for storage and transport, easy to use, cost-effective.With sustained release or
Controlled release, its incompatibility between surrounding dielectric material of determining.
The present invention is fabricated to fire extinguishing virus using transmissible gastro-enteritis virus for the first time, and mouth is fabricated to using microcapsule method
Formulation stimulates piglet intestinal mucosa to generate mucosa-immune, and generates specific antibody IgA and IgG, effectively neutralizes virus precaution sense
Dye.
After the microcapsules oral formulations piglet immunological, experimental group attacks malicious protective rate higher than control group 60-75%.
The present invention utilizes special wall material and core, and internal antigens only just can be slowly discharged under alkaline environment, are avoided
It degrades inside hydrochloric acid in gastric juice viral antigen, from the effect of the digestion degradation of hydrochloric acid in gastric juice and intestinal digestion liquid.Effectively antigen submission is given
Antigen presenting cell and Dendritic Cells.
The present invention solves virus and is immunized by oral administration, keeps the antigen submission of viral antigen structure and sufficient amount to intestinal mucosa table
Face, generates enough specific mucosal immunities and systemic immunity antibody, and protection piglet avoids viral infection.
The present invention is for the technology in oral inactivation vaccine in Current Domestic prevention and control transmissible gastro-enteritis virus prevention and control
Deficiency, this research are based on inactivation transmissible gastro-enteritis virus technology and oral Micro-Encapsulation Technique, for oral killed
The microcapsule preparation method of transmissible gastro-enteritis virus.Results of animal shows serum neutralize antibody titers testing result
Illustrate that the micro-capsule vaccine can induce piglet mucosa-immune and systemic immunity response, and generate the serum of higher level and mucous membrane resists
Body, at the same it is objective illustrate the micro-capsule vaccine have certain slow release effect.Micro-capsule vaccine antigen substance is by after Piglet by Oral, energy
The expression of piglet specific antibody IgG and IgA are enough induced, and adds resistance of the piglet to TGEV, reduces piglet death
Rate attacks malicious protective rate higher than control group 60-75%.
Description of the drawings
Fig. 1 is influence figure of the different sodium alginate concentrations to TGEV microencapsulation rates；
Fig. 2 is influence figure of the different chitosan concentrations to TGEV microencapsulation rates；
Fig. 3 is influence figure of the different calcium chloride concentrations to TGEV microencapsulation rates；
Fig. 4 is influence figure of the different vaccine additions to TGEV microencapsulation rates；
Fig. 5 is the influence figure that different vaccine additions carry TGEV micro-capsules protein content；
Fig. 6 is Piglet by Oral not same amount IgG vaccine specifics antibody test figure；
Fig. 7 is Piglet by Oral not same amount IgA vaccine specifics antibody test figure；
Fig. 8 attacks malicious protecting effect evaluation figure for Piglet by Oral microcapsule formulation；Wherein, A is experimental group, and B is control group.
Specific embodiment
Specific embodiment one：The microcapsules preparation side of the oral killed transmissible gastro-enteritis virus of present embodiment
Method, it is followed the steps below：First, transmissible gastro-enteritis virus strain secondary culture will be infected, in secondary culture
70~80% cells showed cytopathic effect afterwards collects the virus stain of culture, is placed in -80 DEG C of ultra low temperature freezers, room
Middle benefit gas multigelation 3 times, so that cell fully cracks, then 2000~4000r/min centrifuges 30min, draws supernatant, is pig
Infectious gastroenteritis virus liquid；
2nd, the infection transmissible gastro-enteritis virus liquid of step 1 is taken to be added in sodium alginate solution, abundant mixing；With perseverance
Fixed speed is added dropwise in the mixed liquor of calcium chloride, chitosan acetic acid；Its film formation reaction is made with the stirring of 300~400r/min constant speed
Fully, it is stored at room temperature suspensions of the 30~60min to get calcium alginate-chitosan microcapsules；Suspension is separated by filtration and uses steaming
Distilled water is cleaned repeatedly, then by it in -80 DEG C of 2~3h of precooling, vacuum freeze drying is to get oral killed transmissible gastroenteritis of swine disease
The microcapsules of poison.
Specific embodiment two：The present embodiment is different from the first embodiment in that：Infect gastroenteritis virus of swine disease
Poison strain HLJ2017 strains.It is other same as the specific embodiment one.
Specific embodiment three：The present embodiment is different from the first embodiment in that：Gastroenteritis virus of swine will be infected
Virus stain secondary culture refers to the continuous passage culture on ST cells by HLJ2017 strains.Other and specific embodiment one
It is identical.
Specific embodiment four：The present embodiment is different from the first embodiment in that：It is added dropwise to constant speed
Calcium chloride, chitosan acetic acid mixed liquor in refer to：Calcium chloride, chitosan acetic acid are added dropwise to the speed of 60-90 drops/min
In mixed liquor.It is other same as the specific embodiment one.
Specific embodiment five：The present embodiment is different from the first embodiment in that：75% after secondary culture
Cells showed cytopathic effect.It is other same as the specific embodiment one.
Specific embodiment six：The present embodiment is different from the first embodiment in that：It is centrifuged with 4000r/min
30min draws supernatant, as infects gastroenteritis virus of swine virus liquid.It is other same as the specific embodiment one.
Specific embodiment seven：The present embodiment is different from the first embodiment in that：It is stirred with 300r/min constant speed
Make its film formation reaction abundant, be stored at room temperature suspensions of the 30min to get calcium alginate-chitosan microcapsules.Other and specific implementation
Mode one is identical.
Specific embodiment eight：The present embodiment is different from the first embodiment in that：The pig transmissible stomach of step 1
The mass concentration of enteritis disease venom and sodium alginate solution is 1.2%.It is other same as the specific embodiment one.
Specific embodiment nine：The present embodiment is different from the first embodiment in that：Calcium chloride and shell in mixed liquor
Glycan acetic acid, wherein calcium chloride mass concentration are 1%, and chitosan quality of acetic acid concentration is 3.5.Other and specific embodiment one
It is identical.
Specific embodiment ten：The present embodiment is different from the first embodiment in that：- 80 DEG C of precooling 2.5h.It is other
It is same as the specific embodiment one.
Present invention is not limited only to the content of the respective embodiments described above, the group of one of them or several specific embodiments
Contract sample can also realize the purpose of invention.
Beneficial effects of the present invention are verified by following embodiment：
Embodiment 1
The Micro-Encapsulation Technique specific steps of oral killed transmissible gastro-enteritis virus
Isolated strain HLJ2017 on ST cells was continuously passed into for 10 generations, cell is observed under inverted microscope, works as 70-80%
Cell when there is CPE (cytopathic effect), the algebraically of harvest viral material and labeled virus.The viral material of harvest is put
Multigelation is placed in -80 DEG C of ultra low temperature freezers, room temperature 3 times so that cell fully cracks, and 4000r/min centrifugation 30min are drawn
Supernatant is TGEV virus liquids.The virus multiplication titre of P1, P2, P4, P6, P8, PIO are measured, is calculated using Reed-Muench methods
The TCID50 of virus.
The result shows that with the increase of passage number, virus titer gradually rises, and stablizes, and can reach 107.5TCID50/
ML or so.With the separated TGEV of ST cell proliferations, viral level is prepared higher than 107.5The TGEC semi-finished product antigens of TCID50/mL,
TGEV inactivation antigens are prepared with the inactivation of 5ml/L for 24 hours and carry out inactivation inspection.
It is prepared by one-step method：Suitable vaccine is taken to be added in sodium alginate solution, abundant mixing.With medical No. 9 needle injections
It is added dropwise to constant speed (about 60-90 drops/min) in the mixed liquor of calcium chloride, chitosan acetic acid by device.With 300r/min
Constant speed stirring makes its film formation reaction abundant, is stored at room temperature suspensions of the 30min to get calcium alginate-chitosan microcapsules.Filtering point
It is cleaned repeatedly from and with distilled water.By it in -80 DEG C of precooling 1-2h, vacuum freeze drying.
Embodiment 2
Influence of the different sodium alginate concentrations to TGEV microencapsulation rates
With 1 other preparation conditions of embodiment it is constant in the case of, when sodium alginate concentration increases, microencapsulation takes the lead in
It is to gradually increase and then be gradually reduced；When sodium alginate concentration is 1.5%, microencapsulation rate highest (referring to Fig. 1).
Embodiment 3
Influence of the different chitosan concentrations to TGEV microencapsulation rates
With 1 other preparation conditions of embodiment it is constant in the case of, when chitosan concentration increases, microencapsulation takes the lead in being
It gradually increases and then is gradually reduced；When chitosan concentration is 1.2%, microencapsulation rate highest (referring to Fig. 2).
Embodiment 4
Influence of the different calcium chloride concentrations to TGEV microencapsulation rates
In the case where other preparation conditions are constant, when calcium chloride concentration increases, microencapsulation takes the lead in being to gradually increase
Then it is gradually reduced；When calcium chloride concentration is 3.5%, microencapsulation rate highest (referring to Fig. 3).
Embodiment 5
Influence of the different vaccine additions to TGEV microencapsulation rates
In the case where other preparation conditions are constant, when vaccine, which adds in volume, to be increased, microencapsulation rate is gradually reduced (in detail
See Fig. 4)；Analyze the micro-capsule load protein content when, with add in vaccine volume increase when, micro-capsule carry protein content keep substantially
Constant (referring to Fig. 5).Therefore, under conditions of micro-capsule vaccine maximum envelop rate is ensured, vaccine addition is preferably protected in preparation process
It holds in 1~2mL or so, to ensure the stabilization of microencapsulation rate.
Embodiment 6
Piglet by Oral not same amount vaccine specific antibody test
Piglet blood and intestinal mucosa liquid are detected by ELISA, specific antibody IgA in casing slime in different oral dose groups
Antibody OD490 is respectively for 0.97 and 1.49.The antibody titer of specific antibody IgG is 1 in different oral dose group serum：
1000 and 1：1750.Comprehensive analysis two above difference oral dose group the result shows that, experimental group -2 can play apparent anti-
Body protecting effect, difference is extremely significantly (referring to Fig. 6 and Fig. 7).
Embodiment 7
Piglet by Oral microcapsule formulation attacks malicious protecting effect evaluation
The optimal experimental group -2 of selection antibody effects carry out it is immune after attack poison, challenge viral dosage the result shows that, TGEV attacks poison 6
Afterwards, the immune group death rate is less than the control group death rate (referring to Fig. 8).It is computed, piglet infectious gastroenteritis virus micro-capsule vaccine
Relative immunity protection be 80%.
Claims (10)
1. the microcapsule preparation method of oral killed transmissible gastro-enteritis virus, it is characterised in that it be according to following steps into
Capable：
First, transmissible gastro-enteritis virus strain secondary culture will be infected, 70~80% cell occurs thin after secondary culture
Born of the same parents' lesion effect collects the virus stain of culture, multigelation 3 times in -80 DEG C of ultra low temperature freezers, room temperature is placed in, so that carefully
Born of the same parents fully crack, and then 2000~4000r/min centrifuges 30min, draw supernatant, are transmissible gastro-enteritis virus liquid；
2nd, the infection transmissible gastro-enteritis virus liquid of step 1 is taken to be added in sodium alginate solution, abundant mixing；With constant
Speed is added dropwise in the mixed liquor of calcium chloride, chitosan acetic acid；Its film formation reaction is filled with the stirring of 300~400r/min constant speed
Point, it is stored at room temperature suspensions of the 30~60min to get calcium alginate-chitosan microcapsules；Suspension is separated by filtration and uses distillation
Water cleans repeatedly, then by it in -80 DEG C of 2~3h of precooling, vacuum freeze drying is to get oral killed transmissible gastro-enteritis virus
Microcapsules.
2. the microcapsule preparation method of oral killed transmissible gastro-enteritis virus according to claim 1, feature exist
In infection gastroenteritis virus of swine virus stain HLJ2017 strains.
3. the microcapsule preparation method of oral killed transmissible gastro-enteritis virus according to claim 1, feature exist
Refer to the continuous passage culture on ST cells by HLJ2017 strains in gastroenteritis virus of swine virus stain secondary culture will be infected.
4. the microcapsule preparation method of oral killed transmissible gastro-enteritis virus according to claim 1, feature exist
Refer in being added dropwise to constant speed in the mixed liquor of calcium chloride, chitosan acetic acid：It is added dropwise to the speed of 60-90 drops/min
Calcium chloride, chitosan acetic acid mixed liquor in.
5. the microcapsule preparation method of oral killed transmissible gastro-enteritis virus according to claim 1, feature exist
In after secondary culture 75% cells showed cytopathic effect.
6. the microcapsule preparation method of oral killed transmissible gastro-enteritis virus according to claim 1, feature exist
In centrifuging 30min with 4000r/min, supernatant is drawn, as infects gastroenteritis virus of swine virus liquid.
7. the microcapsule preparation method of oral killed transmissible gastro-enteritis virus according to claim 1, feature exist
In making its film formation reaction abundant with the stirring of 300r/min constant speed, 30min is stored at room temperature to get calcium alginate-chitosan microcapsules
Suspension.
8. the microcapsule preparation method of oral killed transmissible gastro-enteritis virus according to claim 1, feature exist
In step 1 transmissible gastro-enteritis virus liquid and sodium alginate solution mass concentration be 1.2%.
9. the microcapsule preparation method of oral killed transmissible gastro-enteritis virus according to claim 1, feature exist
Calcium chloride and chitosan acetic acid in mixed liquor, wherein calcium chloride mass concentration are 1%, and chitosan quality of acetic acid concentration is 3.5.
10. the microcapsule preparation method of oral killed transmissible gastro-enteritis virus according to claim 1, feature exist
In -80 DEG C of precooling 2.5h.
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| CN1704120A (en) * | 2004-04-05 | 2005-12-07 | 丛繁滋 | Compound biological product conposition and preparation method thereof |
| CN101491673A (en) * | 2008-12-26 | 2009-07-29 | 天津瑞普生物技术股份有限公司 | Triple vaccine of pig transmissible gastroenteritis, pig epidemic diarrhea and pig rotavirus |
| CN102397541A (en) * | 2010-09-17 | 2012-04-04 | 江苏省家禽科学研究所 | Preparation method for microencapsulated oral live vaccine of gosling plague |
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